Novel NO-TZDs and trimethoxychalcone-based DHPMs: design, synthesis, and biological evaluation as potential VEGFR-2 inhibitors

Abstract Novel series of nitric oxide-releasing thiazolidine-2,4-diones (NO-TZD-3a-d,5,6) and 3,4,5-trimethoxychalcone-based multifunctional 1,4-dihydropyrimidines (CDHPM-10a-g) have been designed and synthesised as potent broad-spectrum anticancer agents with potential VEGFR-2 inhibition. The designed analogs were evaluated for their anticancer activities towards a full panel of NCI-60 tumour cell lines and CDHPM-10a-g emerged mean %inhibitions ranging from 76.40 to 147.69%. Among them, CDHPM-10e and CDHPM-10f demonstrated the highest MGI% of 147.69 and 140.24%, respectively. Compounds CDHPM-10a,b,d-f showed higher mean %inhibitory activity than the reference drug sorafenib (MGI% = 105.46%). Superiorly, the hybrid CDHPM-10e displayed the highest potencies towards all the herein tested subpanels of nine types of cancer with MGI50 of 1.83 µM. Also, it revealed potent cytostatic single-digit micromolar activity towards the herein examined cancer cell lines. The designed compounds CDHPM-10a-g were exposed as potent non-selective broad-spectrum anticancer agents over all NCI subpanels with an SI range of 0.66–1.97. In addition, the target analog CDHPM-10e revealed potency towards VEGFR-2 kinase comparable to that of sorafenib with a sub-micromolar IC50 value of 0.11 µM. Also, CDHPM-10e could effectively induce Sub-G1-phase arrest and prompt apoptosis via caspase and p53-dependent mechanisms. Furthermore, CDHPM-10e revealed significant anti-metastatic activity as detected by wound healing assay. The modelling study implies that CDHPM-10e overlaid well with sorafenib and formed a strong H-bond in the DFG binding domain. The ADMET studies hinted out that CDHPM-10e met Pfizer’s drug-likeness criteria. The presented novel potent anticancer agent merits further devotion as a new lead product in developing more chalcone-based VEGFR-2 inhibitors.

1 Spectral data

NCI 60 cell one-dose screen
General Description: As of early 2007 all compounds submitted to the NCI 60 Cell screen are tested initially at a single high dose (10 -5 M) in the full NCI 60 cell panel.Only compounds which satisfy predetermined threshold inhibition criteria in a minimum number of cell lines will progress to the full 5-dose assay.The threshold inhibition criteria for progression to the 5-dose screen was selected to efficiently capture compounds with anti-proliferative activity based on careful analysis of historical DTP screening data.The threshold criteria may be updated as additional data becomes available 1, 2 .

NCI 60 cell five-dose screen
Compounds which exhibit significant growth inhibition in the One-Dose Screen are evaluated against the 60-cell panel at five concentration levels 1, 2 .
The human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine.For a typical screening experiment, cells are inoculated into 96 well microtiter plates in 100 μL at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines.After cell inoculation, the microtiter plates are incubated at 37° C, 5 % CO2, 95 % air and 100 % relative humidity for 24 h prior to addition of experimental drugs.
After 24 h, two plates of each cell line are fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz).Experimental drugs are solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use.At the time of drug addition, an aliquot of frozen concentrate is thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 μg/ml gentamicin.Additional four, 10-fold or ½ log serial dilutions are made to provide a total of five drug concentrations plus control.Aliquots of 100 μl of these different drug dilutions are added to the appropriate microtiter wells already containing 100 μl of medium, resulting in the required final drug concentrations.
Following drug addition, the plates are incubated for an additional 48 h at 37°C, 5 % CO2, 95 % air, and 100 % relative humidity.For adherent cells, the assay is terminated by the addition of cold TCA.Cells are fixed in situ by the gentle addition of 50 μl of cold 50 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4°C.The supernatant is discarded, and the plates are washed five times with tap water and air dried.Sulforhodamine B (SRB) solution (100 μl) at 0.4 % (w/v) in 1 % acetic acid is added to each well, and plates are incubated for 10 minutes at room temperature.After staining, unbound dye is removed by washing five times with 1 % acetic acid and the plates are air dried.Bound stain is subsequently solubilized with 10 mM trizma base, and the absorbance is read on an automated plate reader at a wavelength of 515 nm.For suspension cells, the methodology is the same except that the assay is terminated by fixing settled cells at the bottom of the wells by gently adding 50 μl of 80 % TCA (final concentration, 16 % TCA).Using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth is calculated at each of the drug concentrations levels.Percentage growth is calculated as: [(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti>/=Tz [(Ti-Tz)/Tz] x 100 for concentrations for which Ti<Tz.
Three dose response parameters are calculated for each experimental agent.Growth inhibition of 50 % (GI50) is calculated from [(Ti-Tz)/(C-Tz)] x 100 = 50, which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation.The drug concentration resulting in total growth inhibition (TGI) is calculated from Ti = Tz.The LC50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment is calculated from [(Ti-Tz)/Tz] x 100 = -50.Values are calculated for each of these three parameters if the level of activity is reached; however, if the effect is not reached or is exceeded, the value for that parameter is expressed as greater or less than the maximum or minimum concentration tested.