Design, synthesis, docking study and cytotoxic activity evaluation of some novel letrozole analogs

Background Breast cancer is the most common type of female cancer. One class of hormonal therapy for breast cancer drugs -non steroidal aromatase inhibitors- are triazole analogues. In this work, some derivatives of these drugs was designed and synthesized. All synthesized compounds were evaluated for their cytotoxic activities on breast cancer cell lines (MDA-MB-231, T47D and MCF-7). Methods Our synthetic route for designed compounds started from 4-bromotolunitrile which was reacted with 1H-1,2,4-triazole to afford 4-(4-cyanobenzyl)-1,2,4-triazole. The reaction of later compound with aromatic aldehydes led to formation of the designed compounds. Eleven novel derivatives 1a-k were tested for their cytotoxic activities on three human breast cancer cell lines. Results Among the synthesized compound, 4-[2-(3-chlorophenyl)-1-(1H-1,2,4-triazol-1-yl)ethenyl]benzonitrile (1c) showed the highest activity against MCF-7 and MDA-MB-231 cell lines and 4-[2-(4-methoxyphenyl)-1-(1H-1,2,4-triazol-1-yl)ethenyl]benzonitrile (1 h) exhibited highest activity against T47D cell line. According to cytotoxic activities results, compound 4-[2-(4-dimethylamino)-1-(1H-1,2,4-triazol-1-yl)ethenyl]benzonitrile (1 k) showed comparative activity against T47D and MDA-MB-231 cell lines with compound (1 h) and our reference drug Etoposide. Conclusion In the process of anti-cancer drug discovery, to find new potential anti-breast cancer agents, we designed and synthesized a novel series of letrozole analogs. Cytotoxicity evaluation revealed that compounds (1c) and (1 k) were the most potent compounds with comparative activity with Etoposide. The results revealed that π-π interactions are responsible for the enzyme inhibitions of compounds (1 c) and (1 k).


Background
Breast cancer is the most common female cancer. According to the American cancer society's report about 12% of women in the U.S. will develop some invasive breast cancer during their lifetime. However breast cancer treatment has a complicated process and problems, chemotherapy resistance, surgery and available anti-tumor drugs side effects make it more difficult to gain the appropriate treatment regimen; consequently, there is great demand to introduce new active compounds with more anticancer activity and less unwanted reaction [1,2].
There is some different type of systemic therapy for breast cancer, one kind is hormonal therapy. Hormonal therapy can be given to women whose breast cancers test positive for estrogen to lower estrogen levels. Letrozole is a third generation of non-steroidal aromatase inhibitorone class of hormonal therapy drugs-that was first introduces by Novartis to the market as Femara® for the treatment of local or metastatic breast cancer [3][4][5].
Non-steroidal aromatase inhibitors (as shown in Figure 1) are triazole or imidazole analogues that bind to the active site of enzyme by coordinating the heme iron atom of active site through a heterocyclic nitrogen lone pair [5,6].
As it shown in Figure 1, 1-benzyl-1H-1,2,4-triazole scaffold is a conservative section of aromatase inhibitors which contains various moieties attached to the aliphatic carbon part of this scaffold. In continuation of our research program to find a novel anticancer agent [7][8][9][10][11], and considering the above mentioned data, in the current study, we report the synthesis of a novel series of substituted ethenylbenzene derivatives which linked to1-benzyl-1H-1,2,4triazole (1a-k) and evaluated against three human breast cancer cell lines (Scheme 1).

Chemistry
All raw-materials, solvents and reagents were provided from Aldrich Chemicals and Merck AG. A Kofler hot stage apparatus was used for determination of melting points. The IR and 1 HNMR Spectra were determined on a Shimadzu 470 (potassium bromide disks) and a Bruker 500 spectrophotometer respectively. Tetramethylsilane (TMS) was used as internal standard and chemical shifts are reported in ppm relative to it. The elemental analysis for C, H and N were taken by a Perkin-Elmer 843 spectrometer with using KBr as diluent. Electrospray ionization mass spectra (ESI-MS) were recorded by using Agilent 6410 Triple Quad. LC/MS. Key intermediate 4-(4-cyanobenzyl)-1,2,4-triazole was prepared according to Doiron J. and his collogues report [12].

Physicochemical prediction
Marvin was used for chemical drawing, displaying and characterizing chemical structures, calculator plugins were used for structure property prediction and calculation, (version: Marvin 6.0.3, 2013, ChemAxon scientific package, http://www.chemaxon.com).

Molecular modeling study
Docking studies for selected compounds were performed using Autodock Vina (ver.

In vitro cytotoxicity assay
The in-vitro cytotoxic activity of all synthesized compounds 1a-k was achieved against three human breast cancer cell lines using MTT colorimetric assay according to the method of Mosman [20]. Cells were seeded in 96well plates (Nunc, Denmark) and incubated overnight in a humidified air atmosphere at 37°C with 5% CO 2 to allow cell attachment. The cells were then incubated for another 48 h with various concentrations of compounds 1a-k. The final concentration of DMSO in the highest concentration of the applied compounds was 1%. In each plate, there were three control wells (cells without test compounds) and three blank wells (the medium with 1% DMSO) for cell viability. Etoposide were used as positive controls for cytotoxicity. After 48 h, the culture medium was removed and 200 μl phenol red-free medium containing MTT (final concentration 0.5 mg/mL) was added to wells, followed by 4 h incubation.
After incubation, the culture medium was then replaced with 100 μl of DMSO and the absorbance of each well was measured by using a microplate reader at 492 nm. For each compound, the concentration causing 50% cell growth inhibition (IC 50 ) compared with the control containing 1% DMSO was calculated from concentration response curves by regression analysis.

Physicochemical prediction
In order to investigate the physicochemical properties of products, Vander Waals surface, polar surface and partition-coefficient (Log P) of compounds (1a-k) were predicted by Marvin program and are reported in Table 1.
As it shown primary physicochemical criteria were passed by all designed compounds (1a-k).

Docking study
In order to understand the binding mode of active compounds in the active site pocket of aromatase, docking study was performed using Autodock Vina. To attain this aim, the potent compounds, 1c and 1 k were docked into target enzyme. Docking strongly suggested that the π-π interaction between adjacent phenyl rings and hydrophobic moieties in enzyme residues -Tyrosine 424 and Tyrosine 361-are effective in activity of biologically active synthesized compounds. According to Figure 2, selected compounds fit in the pocket of aromatase enzyme completely, however missing the potentially hydrogen bond between ligands and macromolecule is responsible for moderate activities of compounds (1c) and (1 k).

Conclusion
In the process of anti-cancer drug discovery, to find new potential anti-breast cancer agents, we designed and synthesized a novel series of letrozole analogs. Cytotoxicity evaluation revealed that compounds (1c) and (1 k) were the most potent compounds with comparative activity with Etoposide. Physicochemical properties of products predicted and the binding mode of (1c) and (1 k) were predicted by docking simulation; the results revealed that π-π interactions are responsible for the enzyme inhibitions of compounds (1c) and (1 k).