The Bispidinone Derivative 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 Induces an Apoptosis-Mediated Cytotoxic Effect on Pancreatic Cancer Cells In Vitro

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.


Introduction
Pancreatic cancer (PC) remains a devastating disease; it is the fourth most common cause of cancer death in the Western world [1] and is projected to be the second leading cause of cancer death by 2030 [2]. Newly diagnosed PC patients have a median survival of six months and a five-year survival rate of less than 7% [1]. PC is an inherently metastatic disease, limiting the effectiveness of local therapies, such as surgery and radiation [3]. A major challenge of PC is that it is highly resistant to current chemotherapy drugs [4] and without effective chemotherapy options available for PC patients, a pressing need exists for the continued discovery of novel drug candidates.
Drug development from synthetic compounds and synthetic analogues of pharmacophores in natural products provides the benefit of structural modification of drug candidates to improve potency, selectivity, and bioavailability [5,6]. Plant alkaloids in particular are traditional starting points for drug development, with many compounds in this class possessing potent biological activity. The bispidine ring system (3,7-diazabicyclo[3.3.1]nonane) is an unusual heterocyclic compound consisting of two piperidine rings fused at a common three carbon junction and is the base structure for the quinolizidine plant alkaloids, including sparteine and related analogues cytisine and anagyrine. Sparteine exhibits antiarrhythmic and antibacterial properties, while cytisine and associated derivatives display analgesic, antihypertensive, antispasmodic, and antidepressant activities [7][8][9][10][11].
The ability of the bispidine ring to adopt different conformational forms in solution (i.e., chair-chair, chair-boat, boat-boat) has been a point of significant research interest, prompting a number of spectroscopic studies [12,13]. This behavior in turn gives rise to the possibility of exo/endo isomeric forms of substituents hosted on the methylene C2, C4, C6, and C8 ring positions leading to further structural diversity. These features, coupled with the high basicity of the ring nitrogen atoms (N3 and N7), make the bispidine system an interesting choice as a privileged scaffold for drug development. A recent review article by Tomassoli and Gundisch provides an excellent overview of the physical, chemical, and pharmacological history of the bispidine system together with an overview of physical, chemical, and spectroscopic properties [14].
Bispidine moieties have been investigated as potential scaffolds for inducing secondary structures in peptides, selected because of their rigidity and lipophilic surface area, potentially enabling interaction with cellular/viral proteins and cell membranes [15]. Bispidine-amino acid conjugates have also been investigated as novel scaffolds for antiviral applications [16].
In contrast to bispidines, few studies investigating the biological properties of bispidinone derivatives and their potential biological effects, in particular regarding anti-cancer activity have to date been undertaken [22,23]. In these cases, considerable variation in substituent attachment and pattern exists making further study of the system essential to develop a more comprehensive understanding of structure-activity relationships. This present study is the first to assess whether selected bispidinone compounds exert cytotoxic effects on pancreatic cancer cell lines. In this initial study we specifically targeted analogues predicted to be blood brain barrier (BBB) impenetrable, readily synthesizable, and allow rapid and subtle modification of analogue physicochemical properties, e.g., topological polar surface area (TPSA) and the fraction of sp3 hybridized carbon atoms (Fsp 3 ) (Figure 1). Molecules 2019, 24, x FOR PEER REVIEW 3 of 15

Synthesis of BisP1-BisP4
The desired symmetrical bispidinones were prepared from readily accessible starting materials. BisP1 was accessed via a "twin double Mannich reaction" from 1,3-diphenylacetone, ethanolamine, and aqueous formaldehyde (Scheme 1) [24]. The bispidinones BisP2-BisP4 were prepared from the easily accessed 1,5-diphenyl-3,7diazabicyclo[3.3.1]nonan-9-one, through an amide bond formation reaction employing HOBt and DCC as coupling reagents (Scheme 2). In all cases, substitution occurred at both aza moieties and products were obtained in high yields (73-93%). The configuration of the amide bonds in these bispidinone species results in the formation of two distinct conformational isomers: a C2 symmetric species wherein the amide moieties are aligned in an anti-parallel orientation and a C1 symmetric isomer with the amide groups in a parallel orientation. The anti-parallel configuration is more energetically favorable due to the lack of steric interaction between the amide substituents. Removal of the Boc protecting group was achieved using 4N HCl in dioxane at room temperature to afford the desired amine salts as solids in high yields.  . 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 (Fsp 3 ) character, increased topological polar surface area (TPSA), and predicted no blood brain barrier (BBB) penetration.

Synthesis of BisP1-BisP4
The desired symmetrical bispidinones were prepared from readily accessible starting materials. BisP1 was accessed via a "twin double Mannich reaction" from 1,3-diphenylacetone, ethanolamine, and aqueous formaldehyde (Scheme 1) [24]. The bispidinones BisP2-BisP4 were prepared from the easily accessed 1,5-diphenyl-3,7diazabicyclo[3.3.1]nonan-9-one, through an amide bond formation reaction employing HOBt and DCC as coupling reagents (Scheme 2). In all cases, substitution occurred at both aza moieties and products were obtained in high yields (73-93%). The configuration of the amide bonds in these bispidinone species results in the formation of two distinct conformational isomers: a C2 symmetric species wherein the amide moieties are aligned in an anti-parallel orientation and a C1 symmetric isomer with the amide groups in a parallel orientation. The anti-parallel configuration is more energetically favorable due to the lack of steric interaction between the amide substituents. Removal of the Boc protecting group was achieved using 4N HCl in dioxane at room temperature to afford the desired amine salts as solids in high yields. The bispidinones BisP2-BisP4 were prepared from the easily accessed 1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one, through an amide bond formation reaction employing HOBt and DCC as coupling reagents (Scheme 2). In all cases, substitution occurred at both aza moieties and products were obtained in high yields (73-93%). The configuration of the amide bonds in these bispidinone species results in the formation of two distinct conformational isomers: a C 2 symmetric species wherein the amide moieties are aligned in an anti-parallel orientation and a C 1 symmetric isomer with the amide groups in a parallel orientation. The anti-parallel configuration is more energetically favorable due to the lack of steric interaction between the amide substituents. Removal of the Boc protecting group was achieved using 4N HCl in dioxane at room temperature to afford the desired amine salts as solids in high yields.

Cytotoxic Effect of Bispidinone Treatment
The cytotoxicity of the bispidinone derivatives was determined using Cell Counting Kit-8 (CCK-8) colorimetric assays after 48 h of treatment. Results from the initial screening of the four bispidinone derivatives (BisP1, BisP2, BisP4, and BisP3) on three pancreatic cancer cell lines (BxPc-3, CFPAC-1, and MiaPaca-2) revealed that derivative BisP4 was the most effective in reducing cell viability. The parent 1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one was not examined in our assays as it failed our preliminary in silico analysis displaying predicted BBB penetration. BisP4 showed significant cytotoxic activity (<50 µM) when compared to the control (Table 1). In contrast, the bispidinone derivative BisP1 demonstrated no significant cytotoxic effects and bispidinone BisP2 was cytotoxic to the pancreatic cancer cell lines at doses of 100 µM and above (Table 1), which was determined to be too high to be considered therapeutically relevant [25] and was therefore excluded from further investigation. IC50 values were then calculated to compare BisP4 treatment between pancreatic cell lines. BisP4 had the lowest IC50 value for MiaPaca-2 cells (16.9 µM), followed by BxPC-3 (23.7 µM) and CFPAC-1 (36.3 µM) ( Figure 2). Consequently, MiaPaca-2 cells were selected for continued investigation into the mechanisms of action of the cytotoxic effects of BisP4. Scheme 2. Synthesis of BisP2, BisP3, and BisP4.

Cytotoxic Effect of Bispidinone Treatment
The cytotoxicity of the bispidinone derivatives was determined using Cell Counting Kit-8 (CCK-8) colorimetric assays after 48 h of treatment. Results from the initial screening of the four bispidinone derivatives (BisP1, BisP2, BisP4, and BisP3) on three pancreatic cancer cell lines (BxPc-3, CFPAC-1, and MiaPaca-2) revealed that derivative BisP4 was the most effective in reducing cell viability. The parent 1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one was not examined in our assays as it failed our preliminary in silico analysis displaying predicted BBB penetration. BisP4 showed significant cytotoxic activity (<50 µM) when compared to the control (Table 1). In contrast, the bispidinone derivative BisP1 demonstrated no significant cytotoxic effects and bispidinone BisP2 was cytotoxic to the pancreatic cancer cell lines at doses of 100 µM and above (Table 1), which was determined to be too high to be considered therapeutically relevant [25] and was therefore excluded from further investigation. IC 50 values were then calculated to compare BisP4 treatment between pancreatic cell lines. BisP4 had the lowest IC 50 value for MiaPaca-2 cells (16.9 µM), followed by BxPC-3 (23.7 µM) and CFPAC-1 (36.3 µM) ( Figure 2). Consequently, MiaPaca-2 cells were selected for continued investigation into the mechanisms of action of the cytotoxic effects of BisP4.

Yes a Yes a Yes b
Cytotoxicity at 10 μM a and 30 μM b . ND = No Data, compound insoluble in testing medium.

Significantly Reduced Cell
Viability at ≤50 µM

Yes a Yes a Yes b
Cytotoxicity at 10 μM a and 30 μM b . ND = No Data, compound insoluble in testing medium.

Yes a Yes a Yes b
Cytotoxicity at 10 μM a and 30 μM b . ND = No Data, compound insoluble in testing medium.

Yes a Yes a Yes b
Cytotoxicity at 10 μM a and 30 μM b . ND = No Data, compound insoluble in testing medium.

Yes a Yes a Yes b
Cytotoxicity at 10 μM a and 30 μM b . ND = No Data, compound insoluble in testing medium.
Yes a Yes a Yes b Cytotoxicity at 10 µM a and 30 µM b . ND = No Data, compound insoluble in testing medium.

BisP4 Induces Apoptosis in Pancreatic Cancer Cells
Morphological changes were observed indicative of apoptosis in a dose-dependent manner on the BisP4 treated MiaPaca-2 cells. MiaPaca-2 cells treated with vehicle ( Figure 3A,D) showed no indication of apoptosis with a homogenous weak blue stain throughout the cell. In contrast, BisP4 treated MiaPaca-2 cells ( Figure 3B,E) displayed strong punctate DAPI indicative of fragmented nuclear bodies. Further, a higher dose treatment of MiaPaca-2 cells with BisP4 resulted in more intense fluorescence consistent with chromatin condensation and fragmentation, synonymous with apoptosis ( Figure 3C,F). In addition, the fluorescence micrographs show a decrease in cell numbers in a dose-dependent manner.
The LIVE/DEAD assay was applied to the BisP4 treated and control cells, which revealed significant compromised cell membrane changes of MiaPaca-2 cells in a dose-dependent manner. No dead cells were identified in the vehicle treatment as determined by the absence of red fluorescence, whereas the proportion of dead cells increased with the dose of BisP4 ( Figure 3G,H,I).
intense fluorescence consistent with chromatin condensation and fragmentation, synonymous with apoptosis ( Figure 3C,F). In addition, the fluorescence micrographs show a decrease in cell numbers in a dose-dependent manner.
The LIVE/DEAD assay was applied to the BisP4 treated and control cells, which revealed significant compromised cell membrane changes of MiaPaca-2 cells in a dose-dependent manner. No dead cells were identified in the vehicle treatment as determined by the absence of red fluorescence, whereas the proportion of dead cells increased with the dose of BisP4 ( Figure 3G,H,I).

Cell Death Analysis by Flow Cytometry
To further evaluate how BisP4 induces apoptosis on MiaPaca-2 cells, a caspase-3/-7 flow cytometry assay was used. BisP4 treatment (17.5 µM and 26 µM) induced a dose-dependent increase in the proportion of apoptotic MiaPaca-2 cells (Figure 4 and Table 2), with negligible effects on necrosis.

Cell Death Analysis by Flow Cytometry
To further evaluate how BisP4 induces apoptosis on MiaPaca-2 cells, a caspase-3/-7 flow cytometry assay was used. BisP4 treatment (17.5 µM and 26 µM) induced a dose-dependent increase in the proportion of apoptotic MiaPaca-2 cells (Figure 4 and Table 2), with negligible effects on necrosis.

Discussion
Treatment options for pancreatic cancer patients remain limited and over the last 30 years there has yet to be any significant advances from clinical drug trials that improve patient survival [26,27]. Due to the known complexity and heterogeneity of pancreatic cancer [28][29][30], there are potentially significant benefits in the continued discovery of novel targeted drug treatments. Recent research has shown that tetraaryl functionalised bispidinone analogues induce apoptosis in HeLa cervical cancer cell lines [22,23]. However, no studies to date have assessed the potential effects of aliphatic and aromatic N,N-disubstituted bispidinone derivatives on PC cell lines.

Discussion
Treatment options for pancreatic cancer patients remain limited and over the last 30 years there has yet to be any significant advances from clinical drug trials that improve patient survival [26,27]. Due to the known complexity and heterogeneity of pancreatic cancer [28][29][30], there are potentially significant benefits in the continued discovery of novel targeted drug treatments. Recent research has shown that tetraaryl functionalised bispidinone analogues induce apoptosis in HeLa cervical cancer cell lines [22,23]. However, no studies to date have assessed the potential effects of aliphatic and aromatic N,N-disubstituted bispidinone derivatives on PC cell lines.
Following the observed cytotoxic activity of derivative BisP4, it was determined that comparison activity should be undertaken against the almost structurally identical Boc-protected derivative, labelled BisP3. The Boc (tert-butyloxycarbonyl) group present on BisP3 serves as a protection group during synthesis, preventing chemical transformation of the α-amino group of tryptophan [31]. The goal of screening BisP3 and comparing cell viability to BisP4 was to examine the structure activity relationship on the cytotoxic effect. These data also highlight the benefit of synthetic drug design, in that structural modification can vary steric and electronic properties to improve drug action and/or efficacy. Derivative BisP3 was screened and cell viability compared to the de-protected BisP4 using the same treatment concentrations, however BisP3 had poor solubility in the culture media, often forming a precipitate. This degree of insolubility is likely to explain the lack of cytotoxic activity shown in the results compared to BisP4 treatment at similar concentrations.
To further support the viability results for BisP4, an additional fluorescent cytotoxicity assay was performed. MiaPaca-2 cells were treated with a range of concentrations of BisP4 with increasing numbers of dead cells, observed in a dose-dependent manner.
Indicators of apoptotic cell death followed BisP4 treatment were observed, such as morphological and biochemical changes like caspase-3/-7 activation. Defects in cell death pathways are one of the defining hallmarks of cancer [32] and many current chemotherapy drugs kill cancer cells by triggering apoptosis [33,34]. Thus, this study investigated the presence of apoptotic markers to determine if BisP4 induced PC cell death was a result of apoptosis. Following a 24 h treatment of BisP4 on MiaPaca-2 cells clear apoptotic characteristics, i.e. nuclear segmentation, apoptotic body formation, and chromatin condensation, occurred in a dose-dependent manner.
Overall, the results revealed apoptotic morphology following BisP4 treatment, which increased in a dose-dependent manner.
These results agree with two recent studies investigating the cytotoxic effect of bispidinone analogues on HeLa cervical cancer cells in vitro [22,23]. Both studies reported significant apoptosis-mediated cytotoxicity from two bispidinone analogues:  [23]. Together, these results emphasize how apoptotic events can have a different time course depending on the concentration of the cytotoxic agent.
Apoptosis is also characterized by activating the caspase cascade that ends in the executioner caspases-3 and -7, which results in cleavage of numerous structural and signaling proteins [33]. The caspase-3/-7 assay revealed a decrease in the percentage of live cells following BisP4 treatment in a dose-dependent manner. These results further support our findings that the cytotoxic effect of derivative BisP4 on MiaPaca-2 cells can be attributed to apoptosis.
While the results presented in this study are preliminary in nature, a number of conclusions can be draw from the findings. All compounds assessed possess a common bispidinone scaffold and are C 2 symmetric in form. Bispidinones possessing short chain aliphatic pendant arms (BisP1 = O, BisP2 = S) exhibit no significant biological activity at concentrations <50 µM. Inclusion of pendant arms containing electron rich character and hydrogen bond donor/acceptor capability induces a dramatic change in cellular toxicity in compound BisP4. All compounds assessed (BisP2-BisP4) contain amide units at the N3 and N7 ring positions that introduce sp2 character to the pendant arm, yet only BisP4 displays significant biological activity suggesting that cytotoxic activity may be derived from the electronic characteristics present in the amino acid residue.

Chemistry General Procedures
All reactions were performed using standard laboratory equipment and laboratory glassware. Organic solids and oils were measured as either thin film anhydrous CHCl 3 solutions between sodium chloride plates or as a cast on a sodium chloride plate.
Concerning compounds BisP1 and BisP3, electron impact mass spectra were determined on a Shimadzu GCMS-QP5050A instrument, using the DI probe and operating conditions of 70 eV and 8000 V accelerating potential. A 20 min temperature program, starting at 100 • C and increasing by 20 • C min −1 up to 400 • C, was followed. Electrospray Ionisation mass spectra were measured on a VG Platform II single quadrupole mass spectrometer coupled to a HPLC binary pump system (Agilent, Santa Clara, CA, USA). The capillary tip was at a potential of ±3.5 kV relative to ground, the source temperature was maintained at 80 • C, and the cone voltage was varied between +20 to +60 for positive-ion and −20 to −50 V for negative ion mode. The electrospray mass spectra of BisP2 and BisP4 were recorded using 10% DMSO/H 2 O or HPLC-grade methanol or acetonitrile as carrier solvents on an Agilent 1260 LCMS system). The mass spectral data is reported according to the following scheme: ion peak (m/z) and intensity. The molecular ion is denoted by (M + ). 1 H NMR spectra were recorded at 300 MHz using a Brüker Avance DPX-300 spectrometer or a Brüker Avance III 400 MHz spectrometer. 13

Preparation of Bispidinone Compounds
Four bispidinone derivatives were synthesized (Table 1)

General Cell Procedures
Stock solutions (200 mM) of the bispidinone compounds (BisP1-BisP4) were prepared in dimethyl sulfoxide (DMSO) and stored at −20 • C. Further dilutions in media were prepared immediately prior to each experiment.

Cell Viability Assays
Cell viability was determined using the Dojindo Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Inc., Rockville, MD, USA). Moreover, 100 µL of suspended cells were seeded into a 96 well plate at 5 × 10 3 cells per well and allowed to adhere for 24 h. The wells were then treated with 100 µL of dissolved bispidinone derivatives at increasing concentrations (1 µM-100 µM) and a vehicle control (0.1% DMSO). Gemcitabine (50 nM) was used as a positive control. After 48 h or 72 h incubation, 10 µL of CCK-8 reagent was added to each well and further incubated at 37 • C for 2 h. The optical density of living cells was read at 450 nm using a microplate spectrophotometer (BIORAD Benchmark Plus, Bio-Rad, Hercules, CA, USA). Wells containing only media and CCK-8 reagent served as the blank. Cell viability was determined as a percentage of the control. All experiments were performed in replicates of 6. Using the mean absorbance of the vehicle control (Ac), the treated groups (A), and the blank (Ab), %viability was determined using the following equation: The 50% inhibitory concentration (IC 50 ) values were calculated by curve fitting the absorbance (viability) vs. log[concentration of treatment], using GraphPad Prism software Version 6.0b (GraphPad Software, Inc., San Diego, CA, USA).

Measurement of Apoptotic Cell Morphology
MiaPaca-2 cells were seeded in 6-well plates at 1 × 10 5 cells per well and allowed to adhere for 24 h. Cells were treated with BisP4 at 17.5 µM and 35 µM or vehicle (0.1% DMSO) and imaged every 30 min for a total of 20 h at 20× magnification using an Axiovert 200M inverted microscope (Carl Zeiss, Inc., Oberkochen, Germany) equipped with an incubation chamber set at 37 • C/5% CO 2 . Images were viewed and analysed using AxioVision LE, Version 4.9.1.0 software (Carl Zeiss, Inc.).

Cell Death Analysis by Fluorescent Staining
To visually observe the effect of bispidinone treatment on the viability of pancreatic cancer cells, two different fluorescent cellular staining techniques were used. In preparation for both cell staining techniques, MiaPaca-2 cells were seeded into 12-well plates containing round coverslips, at a density of 5 × 10 4 cells per well and allowed to adhere for 24 h at 5% CO 2 /37 • C. Cells were then treated with either 0.1% DMSO (control) or BisP4 at concentrations of 17.5 µM, 26 µM, or 35 µM, and further incubated for 24 h before staining.
After treatment, DAPI staining was prepared by gently washing the cells with 3.7% formaldehyde in PBS solution for 5 min. Cells were then permeabilized in 1mL of permeabilization buffer (0.01 M Glycine, 0.1% Triton X-100 diluted in 3.7% formaldehyde/PBS solution), left for 5 min before aspiration, followed by another 5 min wash in 3.7% formaldehyde/PBS solution. The DAPI 4',6-diamino-2-phenylindole stain was prepared immediately prior to use (10 µg/mL DAPI dissolved in 3.7% formaldehyde solution) and protected from light. DAPI stain (1000 µL) was added to each well and left for 5 min at room temperature, protected from light. The stain was then removed, followed by a final wash with the 3.7% formaldehyde/PBS solution. The coverslips were then mounted and viewed (20x objective) with an Axiovert 200M inverted microscope (Carl Zeiss, Inc.). Images were viewed and analyzed using AxioVision LE, Version 4.9.1.0 software. LIVE/DEAD ® staining was prepared by gently washing the cells in 1000 µL of cold PBS. The coverslips were removed and placed face up on parafilm, ready for staining. The two dye reagents were combined and diluted in PBS (0.5 µM of calcein-AM and 2µM of EthD-1) immediately before use and protected from light. The coverslips were covered with 100µL of the prepared reagent, covered, and incubated for 30 min at room temperature. Following incubation, 4 µL of reagent was placed on a clean microscope slide, before carefully inverting and mounting the wet coverslip, then viewed (20× objective) with an Axiovert 200M inverted microscope (Carl Zeiss, Inc.). Images were viewed and analyzed using AxioVision LE, Version 4.9.1.0 software (Carl Zeiss, Inc.).

Cell Death Analysis by Flow Cytometry
MiaPaca-2 cells were seeded into 6-well plates at a concentration of 1 × 10 5 cells per well and incubated for 24 h at 37 • C with 5% CO 2 for cells to adhere. Cells were treated for 24 h at 37 • C with 0.1% DMSO as the control and a range of BisP4 concentrations (17.5 µM, 26 µM, or 35 µM). Following treatment, cells were assessed for apoptosis using the Muse TM Caspase-3/7 Kit (Merck Millipore Corp., Burlington, MA, USA) as per manufacturer's instructions. Briefly, cells were dissociated with trypsin, resuspended in Assay Buffer and incubated for 30 min at 37 • C, protected from light in Muse™ Caspase 3/7 working reagent. After incubation, 150 µL of Muse™ Caspase 7-AAD working reagent was added, mixed thoroughly, and incubated for a further 5min at room temperature, protected from light, and analyzed with the Muse™ Cell Analyser (Merck Millipore Corp.) using Muse™ software Version 1.4.

Statistical Analysis
GraphPad Prism Version 6.0b software (GraphPad Software, Inc.) was used for statistical and graphical evaluations. Results are expressed as the mean ±SD of at least six replicates, unless otherwise stated. Statistical comparisons between the differences in cell viability of treatment groups compared to controls were performed using a one-way ANOVA and a post-hoc Dunnett's Multiple Comparisons test. Statistical significance was set at p < 0.05.

Conclusions
Further work is needed to elucidate the detailed molecular mechanisms of the cytotoxic effect. This will lead to potential opportunities of structural optimization of bispidinone derivative BisP4 to improve potency and targetability. The versatility and relatively straightforward synthesis of bispidinone derivatives provides the potential for further discovery and development of bispidinone derivatives. The necessary demand for novel pancreatic cancer drug treatments and the results of this study of apoptosis-mediated cytotoxicity from in vitro treatment with a bispidinone derivative, makes this an important effort towards the development of potential novel lead compounds to target pancreatic cancer.