Design, synthesis, and cytotoxic activity of pyridine-based stilbenes

Abstract In the present study, three series of 35 pyridine-based stilbenes include 10 new compounds prepared by Horner-Wadsworth-Emmons (HWE) reaction were assayed for cytotoxic activities toward two tumoral cell lines (K562 and MDA-MB-231) and one non-tumoral cell line (L-02). The bioassay results indicated that hybrid stilbenes formed at the C-3 position of pyridine displayed stronger antiproliferative activities against K562 cells and C-4 pyridine-based stilbenes showed broad-spectrum cytotoxic effects. Among them, C-3 pyridine-based stilbene PS2g bearing 2,6-dimethoxy possessed extremely potent antiproliferative activity with IC50 values 1.46 µM against K562 cells, along with excellent selectivity towards normal L-02 cells. In summary, the present study contributes to the development of natural stilbene-based derivatives as antitumor agents and PS2g may serve as a promising lead for the treatment of chronic myeloid leukemia (CML) worthy further investigation. Graphical Abstract


Introduction
Chronic myeloid leukemia (CmL) is a malignant myeloproliferative cancer of the hematopoietic system, which constitutes about 15% of diagnosed leukemias in adult (pan et al. 2022).CmL origins from the translocation between the abelson (aBL) gene on chromosome 9 with break point cluster region (BCR) gene on chromosome 22 (Sawyers 1999).the pathogenic and chimeric BCR-aBL protein with high tyrosine kinase activity results in uncontrolled proliferation of myeloid cells through downstream signaling pathways such as RaS, Stat5 and mYC (Jabbour and Kantarjian 2020).the treatment of CmL has achieved striking breakthrough occurred with the development of tyrosine kinase inhibitors (tKis) (Ciftciler and Haznedaroglu 2021).pyridine scaffold is widely applied in drug design due to its significant impact on pharmacological action (Sahu et al. 2022), and pyridine based small molecular drugs occupy a large proportion of tKis used clinically (di Gion et al. 2011), such as Flumatinib, Crizotnib, Sorafenib, axitinib and apatinib.thus, it is a promising and attractive project to develop new highly efficient candidate drugs for the treatment of CmL from pyridine derivatives.
natural products have been the major sources of new drugs due to their novel structures and diverse pharmacological properties (newman and Cragg 2020).natural occurring stilbenes represented by resveratrol are of great interest for antitumor activities (Koh et al. 2021).it is an attractive direction to explore innovative stilbene analogues through molecular hybridization strategy that combines various active fragments to yield hybrid molecules with improved biological profiles (Lin et al. 2021).as a privileged building block in drug designs, the pyridine moiety accounts for 14% of all Fda-approved drugs (Ling et al. 2021).Based on the privileged structural scaffolds of stilbene, a large number of pyridine-based stilbenes have been synthesized by medicinal chemists, of which several compounds were regarding cancer cytotoxic.For example, a resveratrol derivative with amidoxime scaffold was designed as Lysine-specific demethylase 1 (LSd1) inhibitor and demonstrated the development potential for the treatment of gastric cancer (duan et al. 2021).Besides, 2′,6′-dihalopyridine analogues have been identified to exert antitumor activity through regulating methionine adenosyl transferase-2a (mat2a) (Sviripa et al. 2014).Remarkably, methoxy contained pyridine-based stilbenes have been considered to exert antiproliferation activities via mediating the expression of key factors in cancer cell lines (martí-Centelles et al. 2015) (Cushman et al. 1991).
the aforementioned discovery indicated that the rational design of compounds originating from the pyridine and stilbene skeleton may provide candidates for the treatment of cancer.However, the dissimilar research methods and cancer cell lines used by different research groups resulted in an indistinct structure-activity relationships (SaRs) and thus limited the follow-up development of pyridine-based stilbenes.in order to exploit more effective antitumor candidate drugs and supplement the SaRs of pyridine-based stilbenes, in total 35 derivatives including 10 new compounds were synthesized and assayed for cytotoxic activities against CmL cells (K562) and breast cancer cells (mda-mB-231) along with human normal liver cells (L-02).
Commercially available corresponding benzyl alcohol 1 were chlorinated by thionyl chloride in the presence of pyridine to give benzyl chlorides 2. the required diethyl phosphonates (3) were prepared by the classical solvent free michaelis-arbuzov reaction (Kostoudi and pampalakis 2022) of 2 with triethyl phosphite at 150 °C, quantitatively.pyridine-based stilbenes (PS1a-3l) were prepared exclusively in the E conformation by HWE reaction of intermediates 3 with pyridine formaldehyde in dry tetrahydrofuran (tHF) using sodium hydride (naH) as the base at room temperature (rt) with yield 40-70%.

Cytotoxic activity
the cytotoxic effects of all target compounds (PS1a-PS3l) against human tumor cell lines K562 and mda-mB-231 and non-cancer cell line L-02 were evaluated by mtt assay with doxorubicin as the positive control.the results summarized in table 1 are presented as the concentration of the compounds inhibiting 50% cell growth (iC 50 ).
the results showed that pyridine-based stilbenes are of great interest in developing new antitumor agents, especially for the treatment of CmL.thirteen compounds exhibit cytotoxic activity with iC 50 values less than 30 µm, of which eight derivatives possessed extremely potent antiproliferative activity with iC 50 lower than 10 µm. the bioassay results suggested that hybrid stilbenes formed at different position of pyridine had a distinct influence on activity, C-3 pyridine-based stilbenes (series PS2) displayed stronger cytotoxic activities against K562 cells and stilbenes formed at C-4 position of pyridine (series PS3) resulted in significant enhanced antiproliferative effects towards mda-mB-231 cells.
among C-2 pyridine-based stilbenes (PS1a-1j), PS1h and PS1j emerged moderate antiproliferative activities toward K562 cells with iC 50 values 14.75 µm and 12.76 µm, respectively.Favourable impact on potency was observed when stilbenes formed at the C-3 position of pyridine.Four derivatives with dimethoxy and two derivatives with nitrogenous units showed potent cytotoxic effects towards K562 cells, PS2g bearing the 2,6-dimethoxy moiety was the most promising individual with iC 50 values 1.46 µm.interestingly, PS2g and PS2m also emerged suitable cytotoxicity toward mda-mB-231 cells with iC 50 values of 6.13 µm and 14.96 µm, respectively.Hybrid stilbenes formed at the C-4 position of pyridine enhanced the antiproliferative activities toward mda-mB-231 cells while maintaining strong cytotoxicity against K562 cells.the 2,3-dimethoxy substituted PS3d showed the most potent potency against mda-mB-231 cells with iC 50 values 4.38 µm.importantly, synthetic pyridine-based stilbenes except for PS2k exhibited excellent safety with iC 50 values higher than 30 µm against human normal L-02 cells.Given the cytotoxicity and selectivity, PS2g could be selected as a leading molecule for future structural optimization to search for more potential candidate drugs toward CmL.

General experimental procedures
Reagents and solvents were purchased commercially available without future purification.the structures of target compounds were confirmed by characterization with nuclear magnetic resonance ( 1 H nmR and 13 C nmR) and Liquid Chromatography mass Spectrometry (LC-mS).nmR spectra were recorded on the Bruker aVanCE instrument (400 mHz for 1 H nmR and 100 mHz for 13 C nmR) at 25 °C, tmS used as the internal standard.Waters ZQ 2000 (Waters instruments, uK) electrospray ionization (ESi) single quadrupole mass spectrometer gave mass spectra.

Synthesis of pyridine-based stilbenes
to a solution of substituted diethyl benzyl phosphonate ester in dry tHF, naH (2.0 equiv) was added quickly under nitrogen.after the mixture was stirred at −5 °C for 40 min, pyridine formaldehyde (1.0 equiv) in dry tHF was added dropwise to the solution.the reaction mixture was then warmed to room temperature and stirred overnight to afford pyridine-based stilbenes uniquely in the E conformation by recrystallization from ethyl acetate.Characterization data are reported in the Supplemental material.

Cytotoxicity assay
all cell lines were obtained from the Cell Bank of the Chinese academy of Sciences (Shanghai, China).Cells were plated into a 96-well plate at a density of 5 × 10 3 cells per well, after attachment, cells were treated with compounds in different concentrations or 1% dmSo solution as the negative control group.after 72 h, 20 µL of mtt (Sigma-aldrich, St. Louis, mo, uSa) was added to each well.4 h later, the formazan product was dissolved in dmSo and quantitated spectrophotometrically at a wavelength of 570 nm using a microplate reader (Biotek, Winooski, Vt, uSa). the iC 50 value was defined as the concentration that inhibited cell viability by 50% (ma et al. 2023).

Conclusion
inspired by the widespread use of pyridine framework in clinical use, toward the development of natural product-based stilbenes, three series of pyridine-based stilbenes were synthesized and assayed for cytotoxic activities.SaRs studies showed that the cytotoxic activity was closely related to the position of pyridine hybrid with stilbene.Stilbenes formed at the C-3 position of pyridine were more sensitive toward K562 cell and C-4 pyridine-based stilbenes displayed more potent cytotoxic activities against mda-mB-231 cells.among them, compound PS2g could be screened as a high-efficiency (1.46 µm, K562) and low-toxicity (> 30 µm, L-02) lead compound worthy future structural optimization.in conclusion, our study suggest that pyridine-based stilbenes could serve as a bright starting point for the further development of anticancer drugs, especially used in the treatment of CmL.

Table 1 .
structures and cytotoxic activities of pyridine-based stilbenes and doxorubicin.
b * means new compounds.the bold value indicates the inhibitory ability of the most promising compound.