Synthesis and biological evaluation of new derivatives of thieno-thiazole and dihydrothiazolo-thiazole scaffolds integrated with a pyrazoline nucleus as anticancer and multi-targeting kinase inhibitors

Deregulation of various protein kinases is considered as one of the important factors resulting in cancer development and metastasis, thus multi-targeting the kinase family is one of the most important strategies in current cancer therapy. This context represents the design and synthesis of two sets of derivatives bearing a pyrazoline-3-one ring conjugated either with a thieno[3,2-d]thiazole or with a dihydrothiazolo[4,5-d]thiazole scaffold via an NH linker, 3a–d and 5a–d respectively, using the pyrazolinone–thiazolinone derivative 1 as a key precursor. All the newly synthesized compounds were assessed in vitro for their anticancer activity against two cancer cell lines (MCF-7 and HepG-2). The safety profile of the most active cytotoxic candidates 1 and 3c was further examined against the normal cell line WI-38. The compounds 1 and 3c were further evaluated as multi-targeting kinase inhibitors against EGFR, VEGFR-2 and BRAFV600E, exhibiting promising suppression impact. Additionally, the latter compounds were investigated for their impact on cell cycle and apoptosis induction potential in the MCF-7 cell line. Moreover, the antimicrobial activity of all the new analogues was evaluated against a panel of Gram-positive and Gram-negative bacteria, yeast and fungi in comparison to streptomycin and amphotericin-B as reference drugs. Interestingly, both 1 and 3c showed the most promising microbial inhibitory effect. Molecular docking studies showed promising binding patterns of the compounds 1 and 3c with the prospective targets, EGFR, VEGFR-2 and BRAFV600E. Finally, additional toxicity studies were performed for the new derivatives which showed their good drug-like properties and low toxicity risks in humans.

The most active cytotoxic compounds 1 and 3c that showed promising IC 50 values were further examined for their inhibitory activities against EGFR, VEGFR-2 and BRAF V600E . [2][3][4] EGFR assay: The master mixture (6 μL 5X Kinase Buffer + 1 μL ATP (500 μM) + 1 μL 50 X PTK substrate + 17 μL water) was prepared then, 25 μL to every well was added. 5 μL of Inhibitor solution of each well labeled as "Test Inhibitor" was added. However, for the "Positive Control" and "Blank", 5 μL of the same solution without inhibitor (Inhibitor buffer) was added. 3 mL of 1X Kinase Buffer by mixing 600 μL of 5X Kinase Buffer with 2400 μL water was prepared. So, 3 mL of 1X Kinase Buffer became sufficient for 100 reactions. To the wells designated as "Blank", 20 μl of 1X Kinase Buffer was added. EGFR enzyme on ice was thawed.
Upon first thaw, briefly the tube containing enzyme was spun to recover full content of the tube.
The amount of EGFR required for the assay and dilute enzyme to 1 ng/μL with 1X Kinase Buffer was calculated. Moreover, the remaining undiluted enzyme in aliquots was stored at -80°C. The reaction was initiated by adding 20 μL of diluted EGFR enzyme to the wells designated "Positive Control" and "Test Inhibitor Control", after that it was incubated at 30°C for 40 minutes. After the 40 minutes reaction, 50 μL of Kinase-Glo Max reagent was added to each well and the plate was covered with aluminum foil and incubated at room temperature for 15 min. Luminescence was measured using the microplate reader.
VEGFR-2 assay: Also, the effect of the most promising cytotoxic compounds 1 and 3c on the level of VEGFR-2 in human breast cancer cell line MCF-7 was determined. The cells in culture medium were treated with 20 μl of IC50 values of the compounds dissolved in DMSO, then incubated for 24 hours at 37 ºC, in a humidified 5% CO 2 atmosphere. The cells were harvested and the homogenates were prepared in saline using a tight pestle homogenizer until complete cell disruption. The kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the level of human VEGFR-2 in samples. A monoclonal antibody for VEGFR-2 was pre-coated onto 96-well plates. The test samples are added to the wells and a biotinylated detection polyclonal antibody from goat specific for VEGFR-2 was added subsequently followed by washing with PBS buffer. Avidin-Biotin-Peroxidase complex was added and the unbound conjugates were washed away with PBS buffer.
HRP substrate TMB was used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow color is proportional to the human VEGFR-2 amount of the sample captured in the plate. The chroma of color and the concentration of the human VEGFR-2 of the samples were positively correlated and the optical density was determined at 450 nm. The level of human VEGFR-2 in samples was calculated (pg/ml) as duplicate determinations from the standard curve. Percent inhibition was calculated in comparison to control untreated cells. BRAF V600E assay: Reaction Biology Corp. Kinase HotSpotSM service was used for screening of final compounds. Assay protocol: as reported on Reaction Biology Corp. website using 1 μM concentration of ATP. Isolated human BRAF (V599E) was used and MEK1 was used as substrate at 1 uM concentration and 1uM ATP concentration (33P labeled ATP was used to produce 33P-Substrate which was a measure for enzyme activity).

In-vitro DNA-Flow cytometric (cell cycle) analysis
To determine the distribution of cell lines in each phase of cell cycle, the PI was used to stain the DNA content of each cell line. At a density of 1×10 6 -3×10 6 cells/dish, MCF-7 cells were seeded in 30 mm tissue culture plates in 5 ml of complete medium. Cells were incubated and allowed to adhere in CO 2 atmosphere. After 24 h adherence, cells were incubated with compound 13k for 24 h. Then, the cell pellets were collected by trypsinization and washed twice with PBS washing buffer and fixed with 70% ice cold ethanol for a minimum of 24 h at −20 °C.
The cells were stained with PI and RNase Staining Solution according to the manufacturer's instructions. Cell-cycle distribution was evaluated using a BD FACSCalibur flow cytometer.
Data were collected from three individual experiments . 4

Annexin V-FITC apoptosis assay
Annexin V-FITC apoptosis detection kit (BD biosciences) was used to quantify the percentage of cells undergoing apoptosis and to determine the mode of cell death whether by apoptosis or necrosis in the presence or absence of the active compounds 1 and 3c. The experiment was carried out according to the manufacturer's protocol. Briefly, cells were seeded (1×10 6 -3×10 6 ) per dish and allowed to adhere overnight in CO 2 incubator. Following 24 h incubation, the tested compound was added, and plates were incubated for another 24 h in CO 2 atmosphere. Both adherent and nonadherent cells were trypsinized, collected and centrifuged for 5 min at 300g. Cell pellets were washed with 2 ml of cold PBS twice, re-suspended in 100 μl of 1X binding buffer and stained with 5 μl of FITC Annexin V and 5 μl of PI for 15 min in the dark at room temperature. Following incubation, 1 ml of 1X binding buffer was added and the analysis was done using flow cytometer within an hour. Data was collected from three individual Experiments. 4

Antimicrobial activity assay
In vitro microbial activities were carryout at the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. The biological potential of the newly prepared target structures was inspected toward the examined organisms and expressed as the diameter of the inhibition zones due to the agar plate diffusion technique. 5-8 Also, pathological strains (100µl) was outgrowing in 10 mL of fresh media till they reached a count of nearly 108 cells/ml and 105 cells/mL for bacteria and fungi, respectively. Also, each well (10 mm diameter holes cut in the agar gel) included 1mL of each sample (at 0.5 mg/mL). Whoever, incubation of plates was done for 24 h at 37 ˚C for bacteria and72 h at 27˚C for fungi activity.
The plates were done in triplicate and the average inhibition zone diameters were recorded in mm and used as criterion for the microbial activity. Tetracycline (standard drug) was also inspected for antibacterial while amphotericin B for the antifungal activity. DMSO (solvent controls) was used for dissolving the examined compounds and illustrated no inhibition zone, indicating that it has no effect on the growth of the tested biological strains. Furthermore, the proper target compounds were further tested to estimate their antimicrobial activity represented as minimum inhibitory concentration (MIC) using the modified agar well diffusion method.

Minimal Inhibitory Concentration (MIC) Measurement
The bacteriostatic activity of the compounds was then evaluated using the two-fold serial dilution technique. Two-fold serial dilutions of the tested compounds solutions were prepared using the proper nutrient broth. The final concentrations of the solutions were 1000, 500, 250, and 125 μg/mL. The tubes were then inoculated with the test organisms, grown in their suitable broth at 37°C for 24 h for the tested microorganisms (1×10 8 CFU/mL for bacteria and 1 x10 6 CFU/mLof yeast), each 5 mL received 0.1 mL of the above inoculum and incubated at 37°C for 24 h. The lowest concentration showing no growth was taken as the minimum inhibitory concentration (MIC). 5-8

Molecular modeling studies
The 2D structure of the newly synthesized derivatives 1 and 3c was drawn through chem.
Draw. The protonated 3D was employed using standard bond lengths and angles, using Molecular Operating Environment (MOE-Dock) software version 2014.0901. 9,10 Then, the geometry optimization and energy minimization were applied to get the Conf Search module in MOE, followed by saving of the moe file for upcoming docking process. The co-crystallized structures of EGFR, VEGFR-2 and BRAF V600E kinases with their ligands erlotinib, sorafenib and SB-590885 were downloaded (PDB codes: 1M17, 4ASD and 2FB8, respectively) from protein data bank. [11][12][13] All minimizations were performed using MOE until an RMSD gradient of 0.05 kcal•mol −1 Å −1 with MMFF94x force field and the partial charges were automatically calculated.
Preparation of the enzyme structures was done for molecular docking using Protonate 3D protocol with the default options in MOE. London dG scoring function and Triangle Matcher placement method were used in the docking protocol. At the first, validation of the docking processes were established by docking of the native ligands, followed by docking of the derivatives 1 and 3c within the ATP-binding sites after elimination of the co-crystallized ligands.

In silico toxicity potential
Molecular descriptors display the pharmacokinetic, pharmacodynamics and physicochemical effects of all synthesized targets 1-5. The lipophilicity (milogP) and topological polar surface area (tPSA) were calculated using the online software Molinspiration, while the aqueous solubility, drug-likeness, drug score were calculated using the OSIRIS property explorer software. Furthermore, according to Veber et al., good bioavailability is more favorable for targets having TPSA of ≤ 140 A o 2 and ≤ 10 rotatable bonds. Decreased molecular flexibility, as determined by the rotatable bond number, and low polar surface area or total hydrogen bond count, which are vital predictors of good oral bioavailability, independent of molecular weight. 14