3.2. Methods

The hydroxy moiety of lawsone was replaced with a reactive bromine group allowing for reactivity at this moiety. For the purpose of ease, the lawsone derivative 2-Bromo-1,4-naphthoquinone (BrNQ) was purchased and utilized as received. In a fume hood shielded from light, one equivalent of CUR (50 mg, 1.36 mmols) was reacted with two equivalents of BrNQ (64 mg, 2.72 mmols) and two equivalents of potassium carbonate (37.6 mg, 2.72 mmols) in dry DMF (5 mL) as depicted in Figure 1. The reaction was monitored continuously by thin-layer chromatography (TLC) and, after 2 h, the reaction was complete. The resulting reaction was quenched with dilute Hydrochloric acid (HCL) solution and washed three times with ethyl acetate and water. The organic phase was collected, dried over anhydrous magnesium sulphate, filtered, and the organic solvent was removed under reduced pressure to obtain a crude product. The pure product was isolated via silica column chromatography using 30% v/v ethyl acetate:hexane eluent. The combined fractions were subjected to solvent evaporation and the resulting purified product, CurNQ, was dried in vacuo and analyzed.

Multinuclear 1D, ¹H and ¹³C NMR spectroscopy were performed to elucidate the structure of the synthesized molecule. ¹H and ¹³C NMR spectroscopy were performed on the Bruker AVANCE II 500 MHz (Bruker Biospin, Ettlingen, Germany) using deuterated dimethyl sulfoxide (d-DMSO) as a solvent at room temperature. All chemical shift values are reported in parts per million and coupling constants (J-values).

The analysis of CurNQ was performed on a Bruker Compact electrospray ionization (ESI) Q-TOF high resolution compact mass spectrophotometer. 10 μL of the sample was injected into the HPLC and run through the C18 column (5–95% ACN). The solvent system was made up of solvent A, consisting of 0.1% formic acid in H₂O (% v/v) and 50% solvent B, consisting of 0.1% formic acid in acetonitrile (% v/v). The samples were diluted to a concentration of approximately 10 ppm in a mixture of methanol and formic acid (1:1). The mass spectrum analysis was processed using the Bruker Daltonics data analysis program.

FTIR was performed using a single-reflection diamond detector on a PerkinElmer Spectrum 100, (PerkinElmer, Llantrisant, Wales, UK) to analyze the vibrational changes in the chemical structures which occurred during product formation. Through obtaining the spectra of CUR, BrNQ and CurNQ, the chemical structure of the product could be ascertained, as FTIR allows for functional group identification. All samples were scanned from 4000–600 cm⁻¹ at room temperature at a constant pressure of 120 psi.

Differential scanning calorimetry (DSC) was performed on the Mettler Toledo DSC-1 STARe system (Mettler Toledo, DSC1, STARe System, Swchwerzenback, Switzerland) to evaluate the heat flow required for phase transition and thermal properties of the new molecule as well as both starting materials. 6mg of each sample was loaded and sealed into a 40 mL aluminum crucible pan and a small puncture was inserted into the top of the crucible and was heated at a rate of 10 °C/min from 10–350 °C under constant N₂ gas. The instrument was heat- and enthalpically calibrated using indium metal (99.99%).

Powder X-ray diffraction (PXRD), utilizing a variable and fixed slit system, was used to determine the crystallinity and atomic spacing and unit cell dimensions of the new molecule. This characterization technique was performed on the Rigaku MiniFlex 600 Benchtop X-ray Diffractometer (Rigaku Corporation, Tokyo, Japan). A powdered sample was packed onto the sample holder and was analyzed at a scanning rate of 15° per minute at a diffraction angle range of 3°–90° with a degree step of 0.02, a voltage of 40 kV and a current of 15 mA.

The fluorescent and absorbance properties of CurNQ were evaluated to ascertain the fluorescent nature of CurNQ. The fluorescent properties of CurNQ were determined through obtaining the absorption and fluorescence spectra which were measured and recorded on the Shimadzu UV-1800 spectrophotometer and Shimadzu RF-6000 spectrofluorophotometer (Shimadzu Scientific Instruments, Columbia, MD, USA), respectively.

The ovarian carcinoma cancer cell lines OVCAR-5 and SKOV3 were purchased from Fox Chase Cancer Facility, USA, Jerkitown, CA. The healthy fibroblast cell line NIH: 3T3 was utilized as a control. The cells were cultured in DMEM with the media being supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were incubated at 37 °C with 5% CO₂. Upon the cells reaching 70–80% confluency, the cells were washed, and sub-cultured by harvesting using trypsin/EDTA.

Cells were grown until 80% confluency prior to utilization. Upon confluency, media was removed, and cells were washed twice in sterile PBS. Trypsin/EDTA was then added to the flasks and incubated at 37 °C for 5 min, allowing the cells to detach from the bottom of the flask. These cells were then collected and centrifuged. The pellet was resuspended, and the cells were counted using the trypan blue exclusion assay. A predetermined and equal number of cells were then added into each well of a 96-well plate. The cells were incubated overnight at 37 °C and at 5% CO₂, allowing the cells to attach to the plates. The following morning, cells were treated with each sample (CurNQ, CUR and BrNQ) at concentrations of 5 µM, 10 µM, 20 µM, 50 µM and 100 µM. Each treatment was performed in quintuplicate. Treatments occurred for 24 h. Subsequent to this time period, media was removed, and cells were fixed with 5% formaldehyde in PBS for 20 min. Next, cells were incubated for 15 min in a 1 µg/mL solution of DAPI. The plates were then washed several times with PBS to remove all residual DAPI and to reduce background fluorescence. All plates were stored in the dark. Plates were then run on the Logos Biosystem CELENA^® X High Content Imaging System and analyses were performed using their proprietary software. IC₅₀ values were calculated using a Prism Graph. Using the Logos Biosystem software, a pipeline was set up to determine the nuclei count, form factor, compactness and eccentricity. The following definitions were used:

All experiments were performed in triplicate or quadruplet for statistical purchases and standard deviations were calculated. One-way ANOVA, three-way ANOVA and Tukey’s test for post-hoc analysis were performed on acquired data. Statistical analyses were all performed on Stata/IC15.1.