Design, synthesis, molecular modeling, and bioactivity evaluation of 1,10-phenanthroline and prodigiosin (Ps) derivatives and their Copper(I) complexes against mTOR and HDAC enzymes as highly potent and effective new anticancer therapeutic drugs

Breast cancer is the second type of cancer with a high probability of brain metastasis and has always been one of the main problems of breast cancer research due to the lack of effective treatment methods. Demand for developing an effective drug against breast cancer brain metastasis and finding molecular mechanisms that play a role in effective treatment are gradually increasing. However, there is no effective anticancer therapeutic drug or treatment method specific to breast cancer, in particular, for patients with a high risk of brain metastases. It is known that mTOR and HDAC enzymes play essential roles in the development of breast cancer brain metastasis. Therefore, it is vital to develop some new drugs and conduct studies toward the inhibition of these enzymes that might be a possible solution to treat breast cancer brain metastasis. In this study, a series of 1,10-phenanthroline and Prodigiosin derivatives consisting of their copper(I) complexes have been synthesized and characterized. Their biological activities were tested in vitro on six different cell lines (including the normal cell line). To obtain additional parallel validations of the experimental data, some in silico modeling studies were carried out with mTOR and HDAC1 enzymes, which are very crucial drug targets, to discover novel and potent drugs for breast cancer and related brain metastases disease.


Synthesis of 4-methoxy-5-(5-ethyl-1H-pyrrol-2-ylmethylidene)-1,5-dihydropyrrol-2-one:
To a solution of 2-formyl-5-ethylpyrrole (0.500 grams, 4.06 mmol) and 4-methoxy-3-pyrrolin-2one (0.920 grams, 8.13 mmol) in 20 mL DMSO was added 2N aq. NaOH (15 mL) and the mixture was stirred at 60 o C for 8 hours. After dilution with 100 mL DI-water, the suspension was extracted with 300 mL dichloromethane (3 x 100 mL). The organic phase was washed (shaken) with saturated brine and DI-water, dried over Na2SO4, and evaporated to dryness. The crude was dissolved in 2-3 mL dichloromethane and then excess hexane (50 mL) was added. The solution was evaporated under vacuum at 45 o C until 15-20 mL hexane was left. The black solid formation occurred. It was filtered and solid was collected over the filter paper. Then it was dissolved in 2-3 mL dichloromethane again and excess hexane (50 mL) was added into it. The solution was started to concentrate by evaporating under vacuum at 45 o C until 15-20 mL hexane was left again.

General procedure for the syntheses of complexes:
A solution of the ligand (L 2 -L 15 ) (0.250 mmol) in DCM (10 mL) and acetonitrile (10 mL) was prepared at room temperature under a nitrogen atmosphere. The light yellow-colored solution was stirred until the ligand was dissolved completely. To this solution, tetrakis(acetonitrile)copper(I) hexafluorophosphate (0.125 mmol) was added and the solution was stirred for 20 minutes at room temperature. The color of the solution turned to a dark brown-red-black. Concentration of the mixture under reduced pressure using a rotary evaporator provided the crude product. Purification on silica gel by flash column chromatography, using DCM-methanol (99:1) as eluent, afforded the corresponding copper(I) complex, [Cu(L n )2]PF6 (C2-C15), where L n = L 2 -L 15 .

Section F. Crystallographic Characterization
All the crystallographic data for the 2-trifluoromethansulfonyloxy-4-methoxy-5-[(5-ethyl-2Hpyrrol-2-ylidene)methyl]-1H-pyrrole (Key Intermediate) structure reported in this paper have been deposited to the Cambridge Crystallographic Data Centre (CCDC) and can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif. CCDC deposition number and all data for the compound can be found in Tables S5-S10. Stemi 305 microscope was used to identify a suitable specimen) and the crystal was mounted on a MῑTiGen holder in Paratone oil on a Bruker Kappa APEX-II CCD diffractometer (operated at 1500 W (50kV, 30 mA) to generate (graphite monochromated) Mo Kα radiation (λ = 0.71073 Å)).
The crystal was kept at 100 K during data collection. Using Olex2 (Dolomanov et al., 2009), the structure was solved with the XT (Sheldrick, 2015) structure solution program using Intrinsic Phasing and refined with the XL (Sheldrick, 2007)

Refinement Details:
After data collection, the unit cell was re-determined using a subset of the full data collection. Intensity data were corrected for Lorentz, polarization, and background effects using the Bruker program APEX 3. A semi-empirical correction for adsorption was applied using the program SADABS (Krause et al., 2014). The SHELXL-2014 (Sheldrick, 2007), series of programs was used for the solution and refinement of the crystal structure. Hydrogen atoms bound to carbon atoms were located in the difference Fourier map and were geometrically constrained using the appropriate AFIX commands. The hydrogen atom bound to N2 (H2A) was last major peak found in the difference Fourier map and was allowed to refine both its position and thermal displacement parameter.   (1) 28 (1)