The Bispidinone Derivative 3,7-Bis-[2-(S)-amino-3-(1 H-indol-3-yl)-propionyl]-1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one Dihydrochloride Induces an Apoptosis-Mediated Cytotoxic Effect on Pancreatic Cancer Cells In Vitro

Abstract

Pancreatic cancer (PC) is a complex, heterogeneous disease with a dismal prognosis. Current therapies have failed to improve survival outcomes, urging the need for discovery of novel targeted treatments. Bispidinone derivatives have yet to be investigated as cytotoxic agents against PC cells. The cytotoxic effect of four bispidinone derivatives (BisP1: 1,5-diphenyl-3,7-bis(2-hydroxyethyl)-3,7-diazabicyclo[3.3.1]nonan-9-one; BisP2: 3,7-bis-(2-(S)-amino-4-methylsulfanylbutyryl)-1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one dihydrochloride; BisP3: [2-{7-[2-(S)-tert-butoxycarbonylamino-3-(1H-indol-3-yl)-propionyl]-9-oxo-1,5-diphenyl-3,7-diazabicyclo[3.3.1]non-3-yl}-1-(S)-(1H-indol-3-ylmethyl)-2-oxoethyl]-carbamic acid tertbutyl ester; BisP4: 3,7-bis-[2-(S)-amino-3-(1H-indol-3-yl)-propionyl]-1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one dihydrochloride) was assessed against PC cell lines (MiaPaca-2, CFPAC-1 and BxPC-3). Cell viability was assessed using a Cell Counting Kit-8 (CCK-8) colorimetric assay, while apoptotic cell death was confirmed using fluorescence microscopy and flow cytometry. Initial viability screening revealed significant cytotoxic activity from BisP4 treatment (1 µM⁻100 µM) on all three cell lines, with IC₅₀ values for MiaPaca-2, BxPC-3, and CFPAC-1 16.9 µM, 23.7 µM, and 36.3 µM, respectively. Cytotoxic treatment time-response (4 h, 24 h, and 48 h) revealed a 24 h treatment time was sufficient to produce a cytotoxic effect on all cell lines. Light microscopy evaluation (DAPI staining) of BisP4 treated MiaPaca-2 PC cells revealed dose-dependent characteristic apoptotic morphological changes. In addition, flow cytometry confirmed BisP4 induced apoptotic cell death induction of activated caspase-3/-7. The bispidinone derivative BisP4 induced an apoptosis-mediated cytotoxic effect on MiaPaca-2 cell lines and significant cytotoxicity on CFPAC-1 and BxPC-3 cell lines. Further investigations into the precise cellular mechanisms of action of this class of compounds are necessary for potential development into pre-clinical trials.

Keywords: Pancreatic cancer; apoptosis; bispidinone; cytotoxic; drug development; in vitro.

Figure 1

SwissADME (http://www.swissadme.ch) predicted properties of a selected series of bispidinone analogues. The introduction of N-substituents facilitated modulation of the fraction of sp3 hybridized carbon atoms (Fsp³) character, increased topological polar surface area (TPSA), and predicted no blood brain barrier (BBB) penetration.

Scheme 1

Synthesis of 1,5-diphenyl-3,7-bis(2-hydroxyethyl)-3,7-diazabicyclo[3.3.1]nonan-9-one (BisP1).

Scheme 2

Synthesis of BisP2, BisP3, and BisP4.

Figure 2

After 48 h treatment, derivative BisP4 had a statistically significant reduction in cell viability from all concentrations, except for 10 µM treatment on BxPC3 cells. Results are mean absorbance ± SD, n = 6. Values for variance (p) between control versus treatment and gemcitabine versus treatment are respectively denoted by symbols ****,^####, p < 0.0001, ***,^### p < 0.001, **,^## p < 0.01, *,^# p < 0.05, determined using a one-way ANOVA and Dunnett’s post-hoc test.

Figure 3

Characteristic apoptotic morphological changes in MiaPaca-2 cells after 24 h treatment with BisP4: (A,D,G) 0 µM; (B,E,H) 26 µM (C,F,I) 35 µM. The DAPI stain (A–F) revealed morphological changes indicative of apoptosis in a dose-dependent manner. BisP4 treated MiaPaca-2 cells display distinct apoptotic bodies (arrow), as a result of increased DAPI staining of fragmented nuclear bodies. At the higher concentration (35 µM) the cells show an intense fluorescence corresponding to chromatin condensation and fragmentation, synonymous with apoptosis. (G–I): Decreased live cells (green) and increased dead cells (red) following treatment with increasing doses of BisP4 (26 µM and 35 µM). The compromised plasma membrane integrity of the dead or dying cells allows uptake of ethidium homodimer-1 (EthD-1), which fluoresces red.

Figure 4

Caspase 3/7 analysis to assess apoptosis of MiaPaca-2 cells after 24h treatment with BisP4: (A) 0 µM (no compound); (B) 17.5 µM; and (C) 26 µM. The lower left quadrant depicts live cells not undergoing detectable apoptosis; the lower right quadrant shows cells in the early stages of apoptosis; the upper right quadrant shows cells in the late stage of apoptosis or dead by apoptotic mechanisms; and the upper left quadrant shows cells that died via necrosis but not through the apoptotic pathway.