Nicotinonitrile-Based Dual Inhibitors of Tubulin and Topoisomerase II: Design, Synthesis, and Anticancer Evaluation

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Hassan, Abdalla E. A. Hassan, Shaikha S. Al Neyadi, Yasir S. Raouf, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7890915/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract A series of 2,4,6-trisubstituted nicotinonitriles, compounds 10 – 41 , designed as pyridine-bridged analogs of combretastatin A-4 (CA-4), was synthesized to function as dual inhibitors of Topoisomerase II (Topo II) and tubulin polymerization. The anticancer potential of the synthesized compounds was evaluated against three cancer cell lines_MCF-7, HepG2, and HCT-116 using the LDH assay. Notably, several compounds 20, 26, 41, 38, 24, 27, 37, 23, 22, 33, 35, 19, 21 , respectively demonstrated superior cytotoxic activity against MCF-7 cells and compounds 20, 26, 38, 41, 39 , respectively showed moderate activity against HepG2 when compared to Doxorubicin, while maintaining good selectivity towards normal BJ-1 cells. Among these, compounds 26 , 20 , and 37 , respectively exhibited significant tubulin polymerization inhibitory activity ( 26 , 75% inhibition), ( 20 , 74.7% inhibition), ( 37 , 74.3% inhibition), compared to CA-4 (72.1% inhibition). Compound 37 showed strong inhibitory activity against Topo II (82.4% inhibition), while compound 20 showed moderate Topo II inhibitory activity (70.3% inhibition) compared with Doxorubicin (81.6% inhibition), highlighting the dual-target nature of these molecules. Cell cycle analysis further revealed that compounds 20 and 26 induced G2/M phase arrest in MCF-7 cells at rates of 43.30% and 50.69%, respectively, along with evidence of apoptosis induction. Molecular docking studies confirmed the favorable binding interactions of these compounds with both Tubulin and Topo II, aligning well with the in vitro findings. These findings underscore the potential of 2,4,6-trisubstituted nicotinonitriles as promising dual-target anticancer agents and pave the way for more potent derivatives with enhanced therapeutic efficacy. Biological sciences/Biochemistry Biological sciences/Cancer Biological sciences/Chemical biology Physical sciences/Chemistry Biological sciences/Drug discovery 2 4 6-trisubstituted nicotinonitriles azido/tetrazolopyridines. iminophosphorane tubulin polymerization inhibitors Topoisomerase II inhibitors Combretastatin (A-4) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Cancer remains one of the leading causes of mortality worldwide, accounting for nearly 10 million deaths per year (approximately one in six of all deaths) 1 . Global cancer incidence continues to rise, with an estimated 20 million new cases diagnosed in 2022 alone 2 . Beyond the human toll, the economic burden of cancer is enormous – the global cost of cancer is projected to reach about $ 25 trillion (in 2017 international dollars) over 2020–2050 1 . These sobering statistics underscore the urgent need for more effective and innovative anticancer therapies. Current treatment modalities as surgery, radiation, chemotherapy and targeted therapy have improved outcomes for many cancers. Yet treatment failures, resistance, and side effects remain significant challenges. Conventional single-target chemotherapeutic agents often face issues such as dose-limiting toxicity and the emergence of drug resistance, prompting the exploration of new strategies in drug design and discovery. One promising strategy in medicinal chemistry is the development of dual or multi-target inhibitors – single molecules capable of modulating multiple biological targets. By hitting two or more cancer-relevant pathways simultaneously, a multi-target agent can exploit synergistic anticancer effects and potentially overcome resistance mechanisms that would thwart a single-target drug 3 . In this context, dual inhibitors of tubulin polymerization and DNA topoisomerase II (Topo II) are of great therapeutic interest. Both tubulin and Topo-II are well-validated targets in oncology, and their inhibition leads to complementary anticancer effects. Microtubules assembled from tubulin are essential for mitosis and cell division. Agents that disrupt tubulin polymerization, so-called microtubule destabilizers, cause cell cycle arrest and apoptosis of rapidly dividing cells 4 . Topoisomerase-II is an enzyme that relieves DNA supercoiling during replication and transcription; inhibitors of Topo-II induce DNA strand breaks and prevent genome replication, which is also lethal to proliferating cancer cells 5 . Notably, tubulin inhibitors and Topo-II inhibitors are frequently used in combination chemotherapy regimens for synergistic efficacy. This synergism has inspired the design of single molecules capable of concurrently targeting both microtubule assembly and DNA topoisomerase activity 3 . Several examples illustrate the therapeutic value of these target classes and lay the groundwork for dual inhibitors. Combretastatin A-4 (CA-4), a natural cis-stilbene isolated from Combretum Caffrum, is a potent tubulin polymerization inhibitor that binds to the colchicine site on β-tubulin 4 . CA-4 and its analogues cause mitotic arrest and have shown broad anticancer activity, including against multidrug-resistant tumors 6 . However, CA-4’s clinical application is limited by its poor aqueous solubility and the instability of its cis-olefin which can isomerize to the less active trans form (Fig. 1 A) 4 , 6 . Podophyllotoxin, a lignan from the mayapple plant, is another anti-mitotic agent that binds tubulin at a site distinct from the vinca alkaloid site and inhibits microtubule assembly(Fig. 1 A) 7 . Interestingly, semisynthetic derivatives of podophyllotoxin – notably etoposide (VP-16) and teniposide (TM-26) – were developed to forgo tubulin activity and instead act as Topo II inhibitors 8 , 9 . Etoposide, a cornerstone of many chemotherapy regimens, intercalates DNA and stabilizes the Topo-II–DNA cleavable complex, leading to lethal DNA double-strand breaks in cancer cells (Fig. 1 A) 6 , 10 . Another relevant example is Azatoxin (NSC 640737), a synthetic hybrid molecule encompassing the structural elements of etoposide and ellipticine, displays dual mechanism of action; at lower concentrations it behaves predominantly as a microtubule destabilizer, while at higher concentrations it inhibits Topo II, ultimately inducing both mitotic arrest and DNA damage in cancer cells(Fig. 1 B) 5 . compound VI also has dual activity of topo II and tubulin inhibition (Fig. 1 B) 11 , 12 . In this context, diarylnicotinonitriles were selected as a promising scaffold due to their rigid, pyridine-bridged structure that mimics the bioactive conformation of CA-4 while improving metabolic stability. Figure 1 C highlights examples of nicotinonitrile and heterocycle-bridged CA-4 analogues with notable anticancer activity, where the cytotoxic potency is strongly influenced by the substituents on rings A and B and the nature of the central linker 13 , 14 . In this study, we focused on 2,4,6-trisubstituted nicotinonitriles scaffolds that incorporate essential structural elements for dual anti-tubulin and Topoisomerase II inhibition—namely, aryl rings (A and B) connected by a rigid pyridine linker. Modifications at the 2-position, including hydroxy, chloro, tetrazolyl/azido and iminophosphorane moieties on the central pyridine ring, were introduced to enhance DNA intercalation and promote Topo II inhibitory activity (Fig. 2 ). Structure–activity relationship (SAR) studies on CA-4 and its analogues have shown that optimal antiproliferative activity depends on the presence of 3,4,5-trimethoxy and/or 3,5-dimethoxy substitutions on ring A, as well as the cis-configuration of the vinyl bridge. In contrast, ring B tolerates various structural changes; the 3-hydroxy group is not essential for activity, whereas the 4-methoxy group is critical for cytotoxicity (Compounds VII , VIII and IX , Fig. 1 C) 13 – 15 . Compounds exhibiting dual inhibition of tubulin and Topo II often feature per-methoxyphenyl groups, extended cyclic systems, and azole rings within their structures (Compounds IV, V and VI , Fig. 1 B) 16 . Figure 3 presents the design rationale for the hybrid molecules X , which incorporate key structural features aimed at dual inhibition of Topo II and tubulin polymerization. Ring A includes varying numbers of methoxy groups that serve as hydrogen bond acceptors within the colchicine binding site of tubulin, along with a cis-double bond, mimicking the rigid pyridine ring. To enhance TopII binding, the pyridine ring is further functionalized at the 2-position with hydrophobic substituents such as chloro, azido/tetrazolyl, and iminophosphorane groups, while ring B is substituted with different number of methoxy groups (compounds 18–41 , Fig. 3 ). 2. Results and Discussion 2.1. Chemistry The strategy adopted for the synthesis of the target compounds 10–41 is outlined in Scheme 1 and scheme 2. 2-Oxonicotinonitriles (2-ONNs) 10–17 were synthesized by three component-one pot reaction of corresponding aldehydes 1, 2, 3, 7 , corresponding ketones 4, 5, 6, 8, 9 and ethyl cyanoacetate in the presence of ammonium acetate in reflux ethanol 17 – 23 . An alternative pathway for the synthesis of 2-ONNs involves the reaction of chalcones, generated from the corresponding aldehydes and ketone derivatives, with ethyl cyanoacetate in the presence of ammonium acetate 24 – 26 . The structure of the 2-ONNs was confirmed by 1 H-NMR, 13 C-APT-NMR, ATR-IR spectra. For instance, compound 12 displayed a broad singlet at δ 12.69 ppm corresponding to the NH proton and its 13 C-APT-NMR spectrum showed signals at δ 117.0 and δ 162.3 ppm corresponding to the -CN and C = O carbons, respectively (Scheme 1). Treatment of the 2-ONNs derivatives 10–17 with POCl 3 in the presence of N,N -dimethylaniline at reflux temperature provided the corresponding 2-chloronicotinonitriles 18–25 , respectively in good yields. Constructing a tetrazole ring fused at the N-1 and the C-2 position of the pyridine ring envisioned accessible given the fact that nitrogenous six-membered heterocycles with an azido group positioned at the α-position to the ring-nitrogen tend to form a tetrazole ring depending on the structure and the physical state of the molecule 27 , 28 . Treatment of the 2-chloronicotinamide derivatives 18–25 with NaN 3 in DMF at 80 ◦ C provided the corresponding 2-azido/tetrazolo derivatives 26–33 respectively in good yields. ATR-IR spectra of compounds 26–33 showed variable intensities of the signals at 2130 cm − 1 (ν max /cm − 1 ) where is almost absent for compounds 29 and 33 in the solid state. DFT tautomer distribution (Azido/tetrazolo) calculation in polar phase revealed the predominance of the tetrazolo-form for compounds 26 and 27 , while the azido-form predominates for compounds 33 (see Fig. 5 and supplementary information S4.2.6.1). Treatment of the azido/tetrazolo derivatives 26–33 with triphenylphosphine under Staudinger reaction conditions provided the corresponding iminophosphorane derivative 34–41 , respectively in good yields. The 4-furyl derivatives, 16–17, 24–25, 32–33, 40–41 were synthesized using an analogous manner as previously described (Scheme 2). 2.2. Biological evaluations 2.2.1. Cytotoxic activity The cytotoxic activity of the newly synthesized 2-chloro-nicotinonitrile derivatives 18 – 24 , tetrazolo/azido derivatives 26–33 , and their corresponding iminophosphorane analogues 34 – 41 was evaluated in vitro against three human cancer cell lines: HepG2 (liver carcinoma), HCT116 (colorectal carcinoma), and MCF-7 (breast adenocarcinoma), as well as the normal human skin fibroblast line BJ-1(Table 1 ). The assays were performed using the LDH method, with doxorubicin (DOX) serving as the positive control. All tested compounds demonstrated dose-dependent inhibition of the three cancer cell lines, while showing no significant cytotoxicity toward the non-tumor BJ-1 cells, in contrast to doxorubicin. The inhibitory concentrations (IC 50 ) are summarized in Table 1 . Notably, the 2-chloro derivative 20 (4,6-bis(3,4,5-trimethoxyphenyl)) exhibited potent activity against the MCF-7 cell line (IC 50 = 2.4 µM, compared with DOX IC 50 = 2.8 µM) and considerable activity against HepG2 (6.1 µM). In comparison, the 4-(3,5-dimethoxyphenyl)-6-(3,4-dimethoxyphenyl) derivative 21 retained strong activity against MCF-7 (IC 50 = 2.8 µM), but its potency against HepG2 decreased nearly two-fold (IC 50 = 12.3 µM). A similar trend was observed with the tetrazolo derivatives, particularly compound 26 , which showed significant inhibition of MCF-7 (IC 50 = 2.4 µM) and moderate suppression of HepG2 (IC 50 = 6.3 µM). Consistent with these findings, the iminophosphorane series also displayed structure-dependent activity, with the 4-(3,5-dimethoxyphenyl)-6-(4-methoxyphenyl) derivative 38 emerging as one of the most active members against MCF-7 (IC 50 = 2.5 µM) and showing moderate potency against HepG2 (12.2 µM). Importantly, the addition of an extra methoxy group at the 3-position of the 6-phenyl ring diminished activity against HepG2 by 2.7-fold, highlighting the sensitivity of activity to subtle substituent modifications. These results clearly demonstrate that structural modifications within this scaffold strongly influence anticancer potency and selectivity. Compounds bearing trimethoxy-substituted aryl groups at the 4- and 6-positions consistently showed enhanced activity, particularly against the MCF-7 breast cancer cell line. Table 1 In vitro , cytotoxicity of compounds 18–24, 26–41 against MCF-7, HepG-2, and HCT-116 Cell lines. In vitro cytotoxicity IC 50 (µM) ± SD Compd. No. MCF-7 HepG-2 HCT-116 18 2.9 ± 0.2 26.2 ± 2.5 45.6 ± 4.1 19 2.7 ± 0.2 24.1 ± 2.2 44.1 ± 4.7 20 2.4 ± 0.1 6.1 ± 0.5 34.4 ± 3.2 21 2.8 ± 0.2 12.3 ± 1.5 48.1 ± 4.2 22 2.6 ± 0.2 25.3 ± 2.3 36.8 ± 3.5 23 2.6 ± 0.2 29.4 ± 2.3 49.1 ± 5.1 24 2.5 ± 0.2 22.7 ± 2.1 49.3 ± 4.2 26 2.4 ± 0.1 6.3 ± 0.3 63.4 ± 5.3 27 2.5 ± 0.2 25.5 ± 2.4 60.1 ± 6.2 28 3.0 ± 0.2 29.9 ± 3.1 78.6 ± 5.9 29 3.1 ± 0.3 27.1 ± 2.5 55.4 ± 5.1 30 3.2 ± 0.2 27.1 ± 2.2 40.5 ± 3.3 31 3.1 ± 0.3 25.7 ± 2.4 31.0 ± 2.5 32 3.0 ± 0.3 28.2 ± 2.5 98.0 ± 8.6 33 2.6 ± 0.2 25.2 ± 2.5 40.7 ± 3.9 34 5.1 ± 0.2 28.4 ± 2.3 49.9 ± 4.2 35 2.7 ± 0.1 32.4 ± 3.1 57.6 ± 4.1 36 3.0 ± 0.1 25.2 ± 2.1 41.7 ± 4.1 37 2.6 ± 0.1 29.3 ± 2.5 36.0 ± 3.3 38 2.5 ± 0.1 12.2 ± 1.1 31.5 ± 3.2 39 3.0 ± 0.1 12.6 ± 1.1 32.5 ± 3.2 40 5.7 ± 0.2 27.7 ± 2.1 55.7 ± 4.9 41 2.4 ± 0.1 27.3 ± 2.3 38.2 ± 2.8 DOX 2.8 ± 0.3 2.6 ± 0.2 12.5 ± 1.5 a IC 50 values are expressed in µM. b DOX. 2.2.2. Inhibition of Tubulin Polymerization in MCF-7 Cells To assess whether the antiproliferative effects of the most active compounds were associated with tubulin disruption, ten derivatives ( 20, 22, 24, 26, 27, 33, 35, 37, 38 and 41 ) were evaluated for their ability to inhibit tubulin polymerization in MCF-7 cells. Combretastatin A-4 (CA-4) was used as a positive control (Table 2 ). All tested compounds demonstrated significant inhibition of tubulin assembly. Among them, compounds 26, 20, 37 , respectively showed the strongest activity, each surpassing CA-4 in potency. The remaining derivatives exhibited inhibitory activity comparable to that of CA-4. Importantly, there was a clear correlation between the extent of tubulin inhibition and the observed cytotoxicity for most compounds. For instance, 20 and 26 , which were the most potent antiproliferative agents (IC 50 = 2.4 µM), also showed the highest inhibition of tubulin polymerization (74.7% and 75%, respectively). However, this relationship was not universal. For example, compound 41 exhibited stronger antiproliferative activity than 37 (IC 50 = 2.4 µM vs. 2.6 µM), yet its effect on tubulin polymerization was weaker (61.2% vs. 74.3%). This indicates that additional mechanisms may contribute to the cytotoxicity of certain derivatives. These findings strongly support tubulin polymerization inhibition as a major mechanism underlying the cytotoxicity of the most active compounds, particularly 20 and 26 derivatives. Table 2 Inhibition of tubulin polymerization (%) and cytotoxic activity (IC₅₀, µM) of selected compounds in MCF-7 cells Compd. No. tubulin polymerization %inhibition MCF-7 IC 50 (µM) 20 74.7% 2.4 ± 0.1 22 70.9% 2.6 ± 0.2 24 55.4% 2.5 ± 0.2 26 75% 2.4 ± 0.1 27 49.8% 2.5 ± 0.2 33 68.9% 2.6 ± 0.2 35 61.9% 2.7 ± 0.1 37 74.3% 2.6 ± 0.1 38 71.7% 2.5 ± 0.1 41 61.2% 2.4 ± 0.1 CA-4 72.1% ----------- 2.2.3. Inhibition of Topoisomerase II in MCF-7 Cells Following the evaluation of antiproliferative activity and confirmation of tubulin polymerization inhibition, we further examined whether the most active compounds also target topoisomerase II (Topo II), aiming to identify potential dual inhibitors. A selected set of candidates was screened for their ability to inhibit Topo II in vitro in MCF-7 cells, with doxorubicin (DOX) used as a reference standard. Among the tested compounds, the active derivative 37 exhibited the strongest Topo II inhibitory activity, surpassing that of doxorubicin (Table 3 ). In addition, 20 displayed moderate Topo II inhibition. Taken together, these findings indicate that compounds 20 and 37 act as Topo II inhibitors, while compounds 26, 20, 37, 38 and 22 function as potent tubulin inhibitors, respectively. Notably, 20 and 37 emerge as dual-acting agents, capable of interfering with both tubulin polymerization and Topo II function. The combined evidence suggests that the antiproliferative activity of this compound series is primarily mediated through tubulin inhibition, but with certain derivatives, particularly 20 and 37 , also exerting strong Topo II inhibition. This dual-target profile highlights these molecules as promising leads for the development of multitarget anticancer agents with enhanced therapeutic potential. Table 3 Inhibition of Topo II (%) and cytotoxic activity (IC₅₀, µM) of selected compounds in MCF-7 cells. Compd. No. TOPO II % inhibition MCF-7 IC 50 (µM) 20 70.3% 2.4 ± 0.1 22 42% 2.6 ± 0.2 24 55.3% 2.5 ± 0.2 26 43.7% 2.4 ± 0.1 37 82.4% 2.6 ± 0.1 DOX 81.6% 2.8 ± 0.3 2.2.4. In vitro Propidium Iodide Flow Cytometry Cell Cycle Analysis Arrest of cells in the G2/M phase is a hallmark of both tubulin polymerization inhibitors and Topoisomerase II inhibitors. To further elucidate the mechanism of action of the most active derivatives 20 and 26 , we performed cell cycle analysis in MCF-7 cells using propidium iodide. flow cytometry (Table 4 and Fig. 4 a). Compared with untreated MCF-7 cells, which displayed a distribution of 69.01% in G0/G1, 21.73% in S, and 9.26% in G2/M phase, treatment with compounds 20 and 26 caused a dramatic accumulation of cells in the G2/M phase. Specifically, the percentage of cells in G2/M increased to 43.3% for 20 and 50.69% for 26 , accompanied by a marked reduction in both G0/G1 and S populations. Notably, this arrest profile closely resembled that induced by the reference drugs CA-4 (tubulin inhibitor) and DOX (Topo II inhibitor). Taken together, these results confirm that compounds 20 and 26 exert their antiproliferative effects by blocking cell cycle progression at the G2/M phase, consistent with their observed tubulin polymerization inhibition. Compound 20 also exhibited moderate Topo II inhibition, suggesting a dual mechanism of action. The combined evidence indicates that 20 and 26 suppress MCF-7 cell proliferation through induction of G2/M arrest, primarily via disruption of tubulin dynamics, with 20 additionally engaging Topo II. This dual contribution highlights their potential as multitarget anticancer agents with enhanced efficacy. Table 4 Cell cycle analysis of compounds 20 (2.4 µM), and 26 (2.4 µM) in MCF-7 cell line. Compd. No. %G0-G1 %S %G2/M 20 39.69 17.01 43.3 26 35.64 13.67 50.69 cont. MCF-7 69.01 21.73 9.26 2.2.5. Apoptosis and Necrosis Analysis by Annexin V-FITC/PI Staining To further elucidate the mechanism underlying the cytotoxicity of the most active compounds, 20 and 26 , their ability to induce apoptosis in MCF-7 cells was assessed using Annexin V-FITC/PI dual staining followed by flow cytometry quantification and Fluorescence Microscopy detection (Table 5 and Fig. 4 b). Cells were treated at their respective IC₅₀ concentrations, and the distribution of viable, apoptotic, and necrotic populations was analyzed. Compared with untreated control cells, which showed minimal apoptosis (3.04% total apoptotic cells), treatment with compound 20 markedly increased the proportion of apoptotic cells to 38.41% (11.91% early, 22.63% late), while compound 26 induced an even higher apoptotic fraction of 41.26% (7.58% early, 28.16% late). Only minor increases in necrosis were observed (3.87% for 20 and 5.51% for 26), confirming apoptosis as the predominant mechanism of cell death. These findings are consistent with the results of tubulin polymerization and Topo II inhibition studies, as well as cell cycle analysis, where both 20 and 26 induced G2/M arrest in MCF-7 cells. The induction of apoptosis through G2/M blockade strongly supports disruption of microtubule dynamics as the primary mechanism of action. Furthermore, the dual activity of 20 on both tubulin and Topo II may account for its slightly distinct apoptotic profile compared with 26 . Compounds 20 and 26 effectively trigger apoptosis in MCF-7 cells, in line with their strong antiproliferative activity, inhibition of tubulin polymerization, and induction of G2/M arrest. The data firmly establish apoptosis as the main mode of cell death for these derivatives, underscoring their promise as potent anticancer leads with multitarget potential. Table 5 Apoptosis and necrosis analysis of compounds 20 and 26 . Compd. No. Total Apoptosis Early Late Necrosis 20 38.41 11.91 22.63 3.87 26 41.26 7.58 28.16 5.51 cont.MCF-7 3.04 0.46 0.19 2.39 2.3. In silico study 2.3.1. Aqueous-phase Density functional theory supports low energy tetrazole tautomers. As previously mentioned, heteroaryl azides with an azido-group α to a pyridyl ring nitrogen are known to undergo intramolecular cyclization into tetrazole tautomers, especially within the context of six-membered heterocycles. To evaluate this behavior in our ligand series, density functional theory (DFT) calculations were run on relevant azido-compounds 26 , 27 , and 33 . All geometries were first optimized using B3LYP/6-31G** in the aqueous phase using an aqueous continuum solvation model (PCM) before higher-level single point energies were run using B3LYP/6–31 + G**, which incorporated relevant diffuse electronic functions, which better described the delocalized nature of the tetrazole tautomers. From the resulting solution-phase energies (in Hartree), relative Gibbs free energies were obtained for each tautomeric form. Boltzmann distributions (at 298 K) were then used to estimate tautomeric populations. A summary of these results can be found in the figure below (Fig. 5 ). For 26 , the tetrazole tautomer was predicted to be the global minimum, lying approximately ΔG = 1.11 kcal mol − 1 (4.64 kJ mol − 1 ) lower in energy than the corresponding azide form. This relatively large energy difference translated into an estimate equilibrium population of 86.8% (tetrazole) and only 13.2% (azide), supporting a strong thermodynamic preference for ring-closure in aqueous media. In 27 , a similar but less pronounced trend was observed. The tetrazole tautomer was stabilized by ΔG = 0.59 kcal mol − 1 (2.47 kJ mol − 1 ) relative to the azide form, corresponding to a population distribution of 72.9% (tetrazole) compared to 27.1% (azide) under equilibrium conditions. Notably, the smaller energy gap suggests that both species may co-exist in solution, although the tetrazole form remains dominant. Finally, for 33 , the energetics were reversed, revealing the azido-tautomer to be more stable by a value of ΔG = 1.15 kcal mol − 1 (4.81 kJ mol − 1 ). This translated into an estimated population of 12.6% (tetrazole) and 87.4% (azide), suggesting the open-ring form to be more stable in water. Overall, these results confirm that our compounds do exhibit energetically favorable bias towards the cyclized tetrazole form (in water), especially within the larger compounds 26 , and 27 . Overall, this data supports for the hypothesis that heteroaryl azides with proximal nitrogens can preferentially cyclize into tetrazoles. 2.3.2. Glide Docking against TopII, TopII-DNA, and α/β-tubulin To gain insights into the molecular recognition and intermolecular forces that may drive the biochemical activity of our ligands, a series of docking experiments were run against our targets of interest - Topoisomerase II and tubulin protein. Given the inherently complex pharmacology of TopII (due to the presence of multiple binding sites), we elected to use two docking strategies for this target. First, we focused on the catalytic active site, using a human TopII ATPase domain crystal structure co-crystallized with AMP-PNP (1ZXM, r = 1.87 Å), where we probed the orthosteric site. In parallel, we also selected a TopII-DNA crystal complex bound to anti-cancer chemotherapeutic etoposide (3QX3, r = 2.16 Å) where we surveyed the DNA-protein interface as a potential binding site. In addition to Topo II, our ligands do inhibit tubulin polymerization, so we also evaluated protein-ligand docking against a tubulin-RB3-SLD-TTL complex co-crystallized with colchicine analog ABI-274 (6PC4, r = 2.60 Å), where we focused on the colchicine-binding site within the α/β-tubulin heterodimer. These calculations involved the following ligands: 20, 22, 24, 26, 27, 33, 35, 37, 38, 41 and corresponding 26 (tet), 27 (tet), 33 (tet) tautomers. Doxorubicin was also evaluated against all 3 targets as a positive control. All protein targets first underwent a standard protein preparation workflow, including the filling in of missing residues, assignment of bond orders, and the optimization of hydrogen-bond networks. Ligand structures were also prepared using a LigPrep protocol, ensuring correct three-dimensional structures, ionization states (Epik, pH 7.00 ± 2.00), and low energy minimizations at phyioslogical pH. Receptor grids were generated for each protein target (10 x 10 x 10 Å) centered around the cognate co-crystallized ligands, before a glide docking protocol was performed (Glide-XP) enabling flexible sampling of ligand conformations within the prepared grids to deduce relevant binding poses and quantitative docking scores (in kcal mol − 1 ). This calculation was performed against TopII, TopII-DNA, and tubulin proteins, with the most relevant results summarized in Fig. 6 . In the catalytic site within the TopII ATPase domain (1ZXM), several ligands were found to exhibit favorable docking scores. This includes positive control doxorubicin (-7.935 kcal mol − 1 ), as well as the chloropyridyl aryl analog 24 (-7.344 kcal mol − 1 ), furanyl 33 (tet) (-5.786 kcal mol − 1 ), and 22 (-5.187 kcal mol − 1 ). In the active site, 24 was found to form several favorable hydrophobic contacts as well as a hydrogen bond between its aryl methoxy motif and nearby Asn91. The structurally simple 33 (in the tetrazole form) was found to form a stable hydrogen bond network between a tetrazolo-nitrogen and its nearby cyano group with Asn150 in the TopII pocket. Similar to 24 , compound 22 also occupied the TopII active site in a similar pose, exhibiting two hydrogen bonds between the methoxy group and the peptide backbone of Tyr165 and Gly166. Binding poses within the TopII-DNA interface (3QX3) provided important insight into the benefits of the tetrazole tautomer (compared to the azide form). In this docking experiment, the ligands with the most favorable free energies of binding include control doxorubicin (-8.988 kcal mol − 1 ), 26 (tet, -8.560 kcal mol − 1 ), 27 (tet, -8.189 kcal mol − 1 ), 33 (tet, -7.561 kcal mol − 1 ). Interestingly, the highest affinity ligands were all in the tetrazole form, and the ligands returned improved values in the TopII-DNA interface compared to the TopII ATPase active site. As seen in the Fig. 6 , this is primarily driven by the formation of favorable π-π stacking events between the aromatic tetrazole and methoxybenzene motifs and neighboring DNA nucleotides (e.g., DC8, DT9, DG13). π-π stacking is awell-established non-covalent intermolecular force, typically arising from quadrupole-quadrupole or dispersion forces. Depending on its geometry (e.g., face-to-face, edge-to-face, or offset-stacking), a standard π-π stacking event can contribute between ~ 1–3 kcal mol − 1 of energy to the observed binding affinity, often stabilizing protein-ligand interactions through aromatic ring complementarity. Compound 26 (tet) optimally occupies the TopII-DNA interface and engaged the dsDNA strand via two π-π stacking events between the methoxy benzene and DT9 (thymine) as well as the tetrazole ring and DC8 (cytosine). 27 (tet) was able to form similar interactions in the TopII-DNA interfacial pocket, with π-π stacking of its secondary methoxy benzene ring and DG13 (guanine), as well as a similar stacking event between its tetrazole motif and DC8 (cytosine). Finally, while the smaller 33 (tet) lost hydrophobic contacts, it compensated them via three π-π stacking events, two between its furan core and the adenine system of DG13 (guanine), as well as its tetrazole and DC8 (cytosine). These interactions were unique to the tetrazole tautomers of compounds 26 , 27 , and 33 . In the colchicine-binding site within the α/β-tubulin heterodimer (6PC4), the top ligands include 22 (-7.716 kcal mol − 1 ), doxorubicin (-7.218 kcal mol − 1 ), 26 (-7.121 kcal mol − 1 ), and 20 (-7.060 kcal mol − 1 ). First, 22 occupied the colchicine-binding site in a canonical manner, with both methoxy benzene motifs on either end, and uniquely was found to exhibit a powerful π-cation stacking interaction between its central chloro-pyridine ring and a nearby positively charged Lys350. Unlike the standard π-π stacking event, a π–cation interaction involves the electrostatic attraction between an aromatic π-system and a nearby positively charged residue (commonly Lys or Arg) and is generally stronger, contributing ~ 5–10 kcal·mol⁻¹ as a function of distance and orientation. In addition, the pyridyl nitrogen was also found to have a hydrogen bond to nearby Asn256. Remarkably, compound 26 retained the exact same molecular recognition profile, with the π–cation interaction with Lys350 and hydrogen bond to Asn256. Furthermore, 20 also maintained the same interactions with Lys350 and Asn256. This conservation of intermolecular forces strengthens our confidence in these predicted binding poses, given consistent interactions across three separate docking experiments. Overall, our docking analyses demonstrate that the azido-tetrazole scaffolds favorably engage TopII and tubulin targets through a combination of hydrogen bonding, hydrophobic contacts, and aromatic π-π stacking or π-cation interactions. The tetrazole tautomers, in particular, displayed enhanced recognition at the TopoII-DNA interface, where multiple π–π stacking events with nearby nucleotides improved binding affinities relative to the ATPase catalytic site. In tubulin, the identification of a robust π–cation interaction with Lys350, conserved across several ligands, provided further confidence in the reliability of these predicted binding poses. Notably, lead compounds 20 , and 26 emerged among the most favorable binders across all three protein systems, supporting a possible binding mechanism to explain the observed biochemical activities against TopII and tubulin. Taken together, these results support the biological potential of this chemotype and highlight the value of tautomeric control and substituent tuning in optimizing noncovalent target engagement against clinically relevant proteins. 3. Conclusions We have synthesized a series of 2,4,6-trisubstituted nicotinonitriles 10–41 as a dual inhibitors of tubulin polymerization and Topoisomerase II. Compounds 20, 26, 41 showed the highest in vitro cytotoxic activity against MCF-7 cell line, while compounds 20, 26 showed moderate activity against HepG2 cell line maintaining high selectivity toward normal cells. In enzyme-based assays, compounds 26, 20 , and 37 exhibited significant tubulin polymerization inhibitory activity. Compound 37 showed strong inhibitory activity against Top II, while compound 20 showed moderate Top II inhibitory activity. Compounds 20 and 37 exhibited the most pronounced dual-target activity, effectively disrupting tubulin polymerization and inhibiting Topo II. Studies confirmed their ability to induce G2/M phase arrest and trigger apoptosis in MCF-7 cells. Molecular docking analyses support the favorable binding interactions of these compounds with both targets, in line with the biological results. Collectively, these findings establish 2,4,6-trisubstituted nicotinonitriles as a promising structural scaffold for the future development of potent, selective, and dual-target anticancer therapeutics with improved clinical prospects. 4. Experimental section 4.1. Chemistry All chemical reagents were purchased from commercial sources with a high percentage of purity. The melting points (°C) of the synthesized compounds were determined in open capillaries using Stuart melting point apparatus and were uncorrected. The NMR spectra analyses, IR spectra and UV-Vis analyses were carried out at the Applied Nucleic Acid Research Center, Faculty of Sciences, Zagazig University, Zagazig, Egypt. The mass spectra analyses and elemental analyses (C, H, N) were carried out at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Nasr City, Egypt. The 1 H-NMR and 13 C APT NMR spectra were recorded on a Bruker Advance III 400 MHz High Performance Digital FT-NMR spectrometer using dimethyl sulfoxide (DMSO- d 6 ) or deuterated chloroform (CDCl 3 ) as a solvent. Chemical shifts were reported in δ (ppm) using tetramethyl silane (TMS) as an internal standard. The IR spectra (ATR, ν max /cm -1 ) of the compounds were recorded on a Bruker Alpha FT-IR spectrometer. The UV-Visible analyses were carried out by UV-Visible Carry 90 instrument. Mass spectra were obtained using a GC/MS Mat 112 S mass spectrometer under the EI + ionization technique/mode. Elemental analyses were performed using a Vario MICRO cube (Elementar) CHNS analyzer. All reactions were monitored by thin layer chromatography (TLC) (R f ) on silica gel 60 GF245 (E-Merck, Germany) using a UV lamp for visualization at a wavelength (λ) of 254 nm. Compounds 10, 11, 12, 15, 18 were previously reported 29 - 35 . 4.1.1. General Method for Synthesis of 4,6-disubstituted-3-cyano-2-pyridone derivatives ( 10-17 ). A mixture of selected ketone 4, 5, 6, 8, 9 (10 mmol), selected aldehyde 1, 2, 3, 7 (10 mmol), ethyl cyanoacetate (15 mmol, 1.59 ml) and ammonium acetate (80 mmol, 6.1 g) in absolute ethanol (30 ml) was heated to reflux for 12-14 h. The reaction mixture was monitored by TLC till finished then it was left to reach the room temperature. The formed precipitate was filtered off, washed successively with ethanol and dried under vacuum to afford pure compounds 10-17 . 6-(4-Methoxyphenyl)-2-oxo-4-(3,4,5-trimethoxyphenyl)-1,2-dihydropyridine-3-carbonitrile( 10 ) 29 - 31 . Yellow solid; yield: (78%); mp: 270-272 ⁰C(lit. 31 <300 ⁰C); 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 12.63 (s, 1H, NH), 7.89 (d, J = 8.9 Hz, 2H, Ar-H), 7.08 (d, J = 9.0 Hz, 2H, Ar-H), 7.04 (s, 2H, Ar-H), 6.84 (s, 1H, C 5 -H of pyridone), 3.85 (s, 6H, 2OCH 3 ), 3.83 (s, 3H, OCH 3 ), 3.73 (s, 3H, OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 162.20, 161.72, 159.54, 152.85, 150.94, 139.11, 131.36, 129.49, 124.37, 116.98, 114.36, 106.07, 105.16, 97.02, 60.14, 56.16, 55.53; IR (ATR, ν max /cm -1 ): 3446 (NH), 3081 (CH - aromatic), 2830 (CH-aliphatic), 2216 (CN), 1651 (C=O); UV/Vis: λ max 375, 260 nm. 6-(3,4-Dimethoxyphenyl)-2-oxo-4-(3,4,5-trimethoxyphenyl)-1,2-dihydropyridine-3-carbonitrile ( 11 ) 26 . Yellow solid; yield (77%); mp: 275-277 ⁰C (lit. 26 250-252 ⁰C); 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 12.63 (s, 1H, NH), 7.54 (d, J = 8 Hz, 1H,Ar-H), 7.48 (s, 1H, Ar-H ), 7.10 (d, J = 8 Hz, 1H, Ar-H), 7.03 (s, 2H, Ar-H), 6.89 (s, 1H, C 5 -H of pyridone), 3.86 (s, 3H, OCH 3 ), 3.86 (s, 6H, 2OCH 3 ), 3.83 (s, 3H, OCH 3 ), 3.74 (s, 3H, OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 162.12, 159.71, 152.89, 151.47, 148.79, 139.14, 131.43, 124.22, 121.17, 117.01, 111.70, 110.83, 106.10, 60.18, 56.20, 55.76; IR (ATR, ν max /cm -1 ): 3446 (NH), 3013 (CH-aromatic), 2831 (CH-aliphatic), 2215 (CN), 1651 (C=O); UV/Vis: λ max 380 nm. 2-Oxo-4,6-bis(3,4,5-trimethoxyphenyl)-1,2-dihydropyridine-3-carbonitrile (12) 29 . Yellow solid; yield: (75%); mp: 280-282 ⁰C (lit. 29 286-288 ⁰C); 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 12.68 (s, 1H, NH), 7.19 (s, 2H, Ar-H), 7.04 (s, 2H, Ar-H), 6.97 (s, 1H, C 5 -H of pyridone), 3.88 (s, 12H, 4OCH 3 ), 3.74 (s, 6H, 2OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 161.94, 159.80, 153.04, 152.86, 139.96, 139.14, 131.29, 127.22, 116.80, 106.13, 105.40, 60.14, 60.13, 56.21, 56.19; IR (ATR, ν max /cm -1 ): 3502 (NH), 3015 (CH-aromatic), 2837 (CH-aliphatic), 2221 (CN), 1646 (C=O); UV/Vis: λ max 370 nm. 6-(3,4-Dimethoxyphenyl)-4-(3,5-dimethoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile ( 13 ). Yellow solid; yield: (78%); mp: 270-272 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 12.60 (s, 1H, NH), 7.49 (d, J = 8.5 Hz, 1H, Ar-H), 7.42 (s, 1H, Ar-H), 7.03 (d, J = 8.6 Hz, 1H, Ar-H), 6.78 (s, 2H, Ar-H), 6.78 (s, 1H, Ar-H), 6.62 (s, 1H, C 5 -H of pyridone), 3.80 (s, 3H, OCH 3 ), 3.77 (s, 3H, OCH 3 ), 3.76 (s, 6H, 2OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 162.01, 160.48, 159.66, 151.46, 148.76, 138.10, 124.15, 121.14, 116.61, 111.65, 110.79, 106.31, 101.86, 55.72, 55.49; IR (ATR, ν max / cm -1 ): 3502 (NH), 3065 (CH-aromatic), 2834 (CH-aliphatic), 2222 (CN), 1645 (C=O); UV/Vis: λ max 380 nm. 380; MS, m/z : 392.53 (M + ); analysis (calcd., found for C 22 H 20 N 2 O 5 ): C (67.34, 67.48), H (5.14, 5.23), N (7.14, 7.41). 4-(3,5-Dimethoxyphenyl)-6-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile( 14 ). Yellow solid; yield: (76%); mp: 260-262 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 12.68 (s, 1H, NH), 7.90 (d, J = 7.8 Hz, 2H, Ar-H), 7.08 (d, J = 8.8 Hz, 2H, Ar-H), 6.85 (d, J = 2.1 Hz, 2H, Ar-H), 6.77 (s, 1H, Ar-H), 6.68 (s, 1H, C 5 -H of pyridone), 3.84 (s, 3H, OCH 3 ), 3.82 (s, 6H, 2OCH 3 ); 13 C APT-NMR (100 MHz, DMSO- d 6 ) δ ppm: 161.77, 160.50, 138.07, 129.52, 116.59, 114.39, 106.29, 102.00, 55.54, 55.52; IR (ATR, ν max /cm -1 ): 3450 (NH), 3050 (CH-aromatic), 2837 (CH-aliphatic), 2211 (CN), 1646 (C=O); UV/Vis: λ max 375, 265 nm; MS, m/z : 362.67 (M + ); analysis (calcd., found for C 21 H 18 N 2 O 4 ): C (69.60, 69.43), H (5.01, 5.20), N (7.73, 7.96). 6-(3,4-Dimethoxyphenyl)-4-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile( 15 ) 32 - 35 . Yellow solid; yield: (77%); mp: 255-257⁰C(lit. 33 262-264 ⁰C); 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 12.57 (s, 1H, NH), 7.72 (d, J = 8.8 Hz, 2H, Ar-H), 7.53 (d, J = 8.5 Hz, 1H, Ar-H), 7.47 (s, 1H, Ar-H), 7.12 (d, J = 8.8 Hz, 2H, Ar-H), 7.08 (d, J = 8.6 Hz, 1H, Ar-H), 6.80 (s, 1H, C 5 -H of pyridone), 3.86 (s, 3H, OCH 3 ), 3.85 (s, 3H, OCH 3 ), 3.83 (s, 3H, OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 162.21, 161.06, 159.35, 151.40, 148.77, 130.02, 128.26, 124.24, 121.04, 117.08, 114.20, 111.69, 110.76, 55.74, 55.46; IR (ATR, ν max /cm -1 ): 3490 (NH), 3065 (CH-aromatic), 2904 (CH-aliphatic), 2222 (CN), 1651 (C=O); UV/Vis: λ max 380 nm. 4-(Furan-2-yl)-2-oxo-6-(3,4,5-trimethoxyphenyl)-1,2-dihydropyridine-3-carbonitrile ( 16 ). Yellow solid; yield: (78%) ; mp: 254-256 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 12.56 (s, 1H, NH), 8.14 (d, J = 1.3 Hz, 1H, furyl-H), 7.72 (d, J = 3.5 Hz, 1H, furyl-H), 7.18 (s, 2H, Ar-H), 7.08 (s, 1H, C 5 -H of pyridone), 6.87 (dd, J = 3.6, 1.7 Hz, 1H, furyl-H), 3.91 (s, 6H, 2OCH 3 ), 3.74 (s, 3H, OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 153.02, 139.83, 116.79, 116.56, 113.34, 105.23, 60.14, 56.20; IR (ATR, ν max /cm -1 ): 3446 (NH), 3031 (CH-aromatic), 2838 (CH-aliphatic), 2217(CN), 1645 (C=O); UV/Vis: λ max 385, 340 nm; MS, m/z : 352.17 (M + ); analysis (calcd., found for C 19 H 16 N 2 O 5 ): C (64.77, 64.58), H (4.58, 4.72), N (7.95, 8.17). 4-(Furan-2-yl)-6-methyl-2-oxo-1,2-dihydropyridine-3-carbonitrile( 17 ). Yellow solid; yield: (77%); mp: 221-223 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 12.36 (s, 1H, NH), 8.05 (d, J = 1.2 Hz, 1H, C 5 -H of furan), 7.54 (d, J = 3.6 Hz, 1H, C 3 -H of furan), 6.80 (dd, J = 3.6, 1.8 Hz, 1H, C 4 -H of furan), 6.59 (s, 1H, C 5 -H of pyridone), 2.28 (s, 3H, CH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 161.66, 151.93, 147.60, 146.94, 145.30, 116.92, 115.85, 113.31, 101.33, 91.30, 19.20. 4.1.2. General procedure for synthesis of 3-cyano-2-chloro pyridine derivatives ( 18-25 ). A mixture of selected pyridone derivative 10-17 (5 mmol) and POCl 3 (25 mmol, 2.3 ml) in presence of N,N- dimethylaniline (15 mmol, 1.9 ml) was heated to reflux for 12-16 h. The reaction mixture was monitored by TLC till finished. It was cooled to room temperature and poured into crushed ice with stirring till precipitation. The formed precipitate was filtered off, washed successively with water, crystallized from ethanol and dried under vacuum to obtain pure compounds 18-25 . 2-Chloro-6-(4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)nicotinonitrile( 18 ) 30 . Buff solid; yield: (81%); mp: 188-190⁰C; 1 H NMR (400 MHz, CDCl 3 ) δ ppm: 8.06 (d, J = 8.8 Hz, 2H, Ar-H), 7.69 (s, 1H, C 5 -H of pyridine), 7.02 (d, J = 8.8 Hz, 2H, Ar-H), 6.83 (s, 2H, Ar-H), 3.95 (s, 6H, 2OCH 3 ), 3.93 (s, 3H, OCH 3 ), 3.89 (s, 3H, OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 161.93, 158.63, 155.95, 152.97, 152.32, 139.13, 130.42, 129.50, 127.84, 118.40, 115.70, 114.48, 106.63, 105.20, 60.14, 56.21, 55.46; IR (ATR, ν max /cm -1 ): 3001 (CH-aromatic), 2831 (CH-aliphatic), 2224 (CN); UV/Vis: λ max 340 nm; MS, m/z : 410.34 (M + ), 412.07(M +2 ); analysis (calcd., found for C 22 H 19 ClN 2 O 4 ): C (64.32, 64.50), H (4.66, 4.75), N (6.82, 7.09). 2-Chloro-6-(3,4-dimethoxyphenyl)-4-(3,4,5trimethoxyphenyl)nicotinonitrile ( 19 ). Greenish solid; yield: (85%); mp: 212-214 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 8.22 (s, 1H, C 5 -H of pyridine), 7.88 (d, J = 7.4 Hz, 1H, Ar-H), 7.75 (s, 1H, Ar-H), 7.16 (s, 1H, Ar-H), 7.11 (s, 2H, Ar-H), 3.87 (s, 9H, 3OCH 3 ), 3.76 (s, 6H, 2OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 159.22, 156.51, 153.45, 152.64, 152.24, 149.54, 139.57, 130.99, 128.42, 121.89, 119.16, 116.17, 112.24, 111.00, 107.14, 105.81, 60.63, 56.72, 56.24, 56.17; IR (ATR, ν max /cm -1 ): 3010 (CH-aromatic), 2940 (CH-aliphatic), 2220 (CN); UV/Vis: λ max 350 nm; MS, m/z : 440.22 (M + ), 442.34 (M +2 ); analysis (calcd., found for C 23 H 21 ClN 2 O 5 ): C (62.66, 62.89), H (4.80, 4.72), N (6.35, 6.47). 2-Chloro-4,6-bis(3,4,5-trimethoxyphenyl)nicotinonitrile ( 20 ). Green solid; yield: (87%); mp: 244-246 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 8.32 (s, 1H, C 5 -H of pyridine), 7.51 (s, 2H, Ar-H), 7.12 (s, 2H, Ar-H), 3.88 (d, J = 7.5 Hz, 12H, 4OCH 3 ), 3.75 (d, J = 3.0 Hz, 6H, 2OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 158.70, 156.37, 153.39, 153.09, 152.17, 140.48, 139.22, 131.00, 130.54, 119.60, 115.66, 106.80, 106.23, 105.34, 60.32, 60.27, 56.35, 56.32; IR (ATR, ν max /cm -1 ): 3001 (CH-aromatic), 2831 (CH-aliphatic), 2224 (CN); UV/Vis: λ max 345 nm; MS, m/z : 470.84 (M + ), 472.28 (M +2 ); analysis (calcd., found for C 24 H 23 ClN 2 O 6 ): C (61.21, 61.43), H (4.92, 5.06), N (5.95, 6.12). 2-Chloro-6-(3,4-dimethoxyphenyl)-4-(3,5-dimethoxyphenyl)nicotinonitrile( 21 ). Buff solid; yield: (86%); mp: 194-196 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 8.21 (s, 1H, C 5 -H of pyridine), 7.89 (dd, J = 8.5, 1.9 Hz, 1H, Ar-H), 7.76 (d, J = 1.8 Hz, 1H, Ar-H), 7.12 (d, J = 8.6 Hz, 1H, Ar-H), 6.91 (d, J = 2.1 Hz, 2H, Ar-H), 6.72 (s, 1H, Ar-H), 3.87 (s, 3H, OCH 3 ), 3.85 (s, 3H, OCH 3 ), 3.83 (s, 6H, 2OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 161.05, 159.36, 156.50, 152.59, 152.27, 149.55, 137.65, 128.38, 121.92, 119.14, 115.89, 112.26, 111.00, 107.45, 105.94, 102.36, 56.26, 56.17, 56.05; IR (ATR, ν max /cm -1 ): 3011 (CH-aromatic), 2841 (CH-aliphatic), 2233 (CN); UV/Vis: λ max 350, 285 nm; MS, m/z : 410.21 (M + ), 412.38 (M +2 ); analysis (calcd., found for C 22 H 19 ClN 2 O 4 ): C (64.32, 64.57), H (4.66, 4.81), N (6.82, 7.04). 2-Chloro-4-(3,5-dimethoxyphenyl)-6-(4-methoxyphenyl)nicotinonitrile ( 22 ). Buff solid; yield: (84%); mp: 221-223⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 8.22 (d, J = 8.9 Hz, 2H, Ar-H), 8.14 (s, 1H, C 5- H of pyridine), 7.09 (d, J = 8.9 Hz, 2H, Ar-H), 6.92 (d, J = 2.1 Hz, 2H, Ar-H), 6.71 (t, J = 2.1 Hz, 1H, Ar-H), 3.85 (s, 3H, OCH 3 ), 3.83 (s, 6H, 2OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 162.44, 161.05, 159.25, 156.41, 152.75, 137.56, 130.00, 128.27, 118.89, 115.89, 114.98, 107.38, 105.81, 102.48, 56.03, 55.95; IR (ATR, ν max /cm -1 ): 3016 (CH-aromatic), 2841 (CH-aliphatic), 2224 (CN); UV/Vis: λ max 335 nm; MS, m/z : 380.77 (M + ), 382.62 (M +2 ); analysis (calcd., found for C 21 H 17 ClN 2 O 3 ): C (66.23, 66.45), H (4.50, 4.61), N (7.36, 7.63). 2-Chloro-6-(3,4-dimethoxyphenyl)-4-(4-methoxyphenyl)nicotinonitrile ( 23 ). Brownish solid; yield: (88%); mp: 217-219 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 8.16 (s, 1H, C 5 -H of pyridine), 7.88 (s, 1H, Ar-H), 7.76 (s, 2H, Ar-H), 7.17 (s, 2H, Ar-H), 6.71 (s, 1H, Ar-H), 6.63 (s, 1H, Ar-H), 3.86 (s, 9H, 3OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm:160.97, 158.63, 155.71, 152.26, 151.62, 148.97, 130.44, 128.77, 127.93, 127.32, 121.26, 118.32, 116.02, 115.70, 114.27, 112.31, 111.70, 110.38, 104.81, 55.70, 55.62, 55.42; IR (ATR, ν max /cm -1 ): 3079 (CH-aromatic), 2838 (CH-aliphatic), 222 3 (CN); UV/Vis: λ max 345 nm; MS, m/z : 380.25 (M + ), 382.05 (M +2 ); analysis (calcd., found for C 21 H 17 ClN 2 O 3 ): C (66.23, 66.50), H (4.50, 4.62), N (7.36, 7.63). 2-Chloro-4-(furan-2-yl)-6-(3,4,5-trimethoxyphenyl)nicotinonitrile ( 24 ). Green solid; yield: (89%) ; mp: 210-212 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 8.37 (s, 1H, C 5 -H of pyridine), 8.15 (s, 1H, furyl-H), 7.77 (s, 1H, furyl-H), 7.46 (s, 2H, Ar-H), 6.89 (s, 1H, furyl-H), 3.90 (d, J = 5.7 Hz, 6H, 2OCH3), 3.75 (s, 3H, OCH3); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 158.69, 153.35, 153.25, 152.66, 147.25, 142.68, 131.65, 130.85, 115.89, 115.63, 114.47, 114.30, 113.45, 105.11, 104.88, 100.49, 60.27, 56.44, 56.28; IR (ATR, ν max /cm -1 ): 3050 (CH-aromatic), 2835 (CH-aliphatic), 2227 (CN); UV/Vis: λ max 360, 300, 255 nm; MS, m/z : 370.31 (M + ), 372.27(M +2 ); analysis (calcd., found for C 19 H 15 ClN 2 O 4 ): C (61.55, 61.79), H (4.08, 4.21), N (7.56, 7.80). 2-Chloro-4-(furan-2-yl)-6-methylnicotinonitrile ( 25 ). Yellow solid; yield: (77%); mp: 226-228 ⁰C; 1 H NMR (400 MHz, CDCl 3 ) δ ppm: 7.65 (s, 2H, C 5 -H of furan and C 5 -H of pyridine), 7.59 (s, 1H, C 3 -H of furan), 6.64 (d, J = 1.6 Hz, 1H, C 4 -H of furan), 2.63 (s, 3H, CH 3 ); 13 C APT NMR (100 MHz, CDCl 3 ) δ ppm: 163.05, 153.97, 147.23, 145.67, 142.49, 117.10, 115.68, 115.56, 113.31, 100.71, 25.01. 4.1.3. General procedure for synthesis of 2-azido/tetrazolo-nicotinonitrile derivatives ( 26-33 ). To a solution of selected chloro pyridine derivatives 18-25 (3 mmol) in DMF, a solution of sodium azide (12 mmol, 0.7 gm) in least amount of water was dropped into, then the reaction mixture was allowed to be stirred for 12 h at 80⁰C. The reaction mixture was poured into water till precipitation then filtered off, washed successively with water then hexane and dried under vacuum to get compounds 26-33 . 5-(4-methoxyphenyl)-7-(3,4,5-trimethoxyphenyl)tetrazolo[1,5-a]pyridine-8-carbonitrile( 26 ). Green solid; yield: (86%); mp: 184-186 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 8.23 (s, 2H, Ar-H), 7.90 (s, 1H, C 5 -H of pyridine), 7.24 (s, 2H, Ar-H), 7.10 (d, J = 14.2 Hz, 2H, Ar-H), 3.90 (s, 9H, 3OCH 3 ), 3.79 (s, 3H, OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 162.40, 153.44, 152.43, 149.81, 141.65, 139.92, 132.07, 130.23, 121.81, 116.94, 114.64, 107.22, 93.61, 60.62, 56.60, 55.95; IR (ATR, ν max /cm -1 ): 3010 (CH-aromatic), 2841 (CH-aliphatic), 2227 (CN), 2126(N 3 ); UV/Vis: λ max 350, 295 nm; MS, m/z : 417.46 (M + ); analysis (calcd., found for C 22 H 19 N 5 O 4 ): C (63.30, 63.43), H (4.59, 4.78), N (16.78, 17.05). 5-(3,4-dimethoxyphenyl)-7-(3,4,5-trimethoxyphenyl)tetrazolo[1,5-a]pyridine-8-carbonitrile( 27 ). Green solid; yield: (88%); mp: 165-167 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 7.91 (s, 2H, Ar-H), 7.78 (s, 1H, Ar-H), 7.24 (s, 2H, Ar-H), 7.08 (s, 1H, C 5 -H of pyridine), 3.89 (s, 15H, 5OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 153.62, 152.43, 149.96, 149.12, 141.87, 130.42, 124.17, 122.03, 117.34, 114.96, 113.59, 112.10, 107.74, 107.51, 93.93, 60.71, 56.80, 56.39, 56.34; IR (ATR, ν max /cm -1 ): 3022 (CH-aromatic), 2841 (CH-aliphatic), 2225 (CN), 2134 (N 3 ); UV/Vis: λ max 360 nm; MS, m/z : 447.42 (M + ); analysis (calcd., found for C 23 H 21 N 5 O 5 ): C (61.74, 61.85), H (4.73, 4.90), N (15.65, 15.81). 5,7-bis(3,4,5-trimethoxyphenyl)tetrazolo[1,5-a]pyridine-8-carbonitrile( 28 ). Yellow solid; yield: (85%); mp: 212-214 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 8.03 (s, 1H, C 5 -H of pyridine), 7.52 (s, 2H, Ar-H), 7.25 (s, 2H, Ar-H), 3.90 (d, J = 5.2 Hz, 12H, 4OCH 3 ), 3.80 (d, J = 6.8 Hz, 6H, 2OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 153.63, 153.38, 152.57, 149.84, 141.72, 140.96, 140.10, 130.30, 125.11, 118.16, 114.85, 108.24, 107.52, 94.62, 60.77, 60.71, 56.83, 56.79; IR (ATR, ν max /cm -1 ): 3050 (CH- aromatic), 2838 (CH-aliphatic), 2226 (CN), 2135 (N 3 ); UV/Vis: λ max 350 nm; MS, m/z : 477.74 (M + ); analysis (calcd., found for C 24 H 23 N 5 O 6 ): C (60.37, 60.51), H (4.86, 4.92), N (14.67, 14.85). 5-(3,4-dimethoxyphenyl)-7-(3,5-dimethoxyphenyl)tetrazolo[1,5-a]pyridine-8-carbonitrile( 29 ). Yellow solid; yield: (87%); mp: 178-180 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 7.90 (s, 2H, Ar-H), 7.77 (s, 1H, C 5 -H of pyridine), 7.24 (d, J = 7.5 Hz, 1H, Ar-H), 7.02 (s, 2H, Ar-H), 6.77 (s, 1H, Ar-H), 3.90 (s, 3H, OCH 3 ), 3.86 (s, 9H, 3OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 160.77, 152.00, 149.42, 148.67, 141.56, 136.62, 123.74, 121.48, 116.73, 114.17, 113.06, 111.58, 107.29, 102.30, 93.90, 55.92, 55.87, 55.66; IR (ATR, ν max /cm -1 ): 3005 (CH-aromatic), 2838 (CH-aliphatic), 2227 (CN); UV/Vis: λ max 360 nm; MS, m/z : 417.86 (M + ); analysis (calcd., found for C 22 H 19 N 5 O 4 ): C (63.30, 63.47), H (4.59, 4.73), N (16.78, 17.05). 7-(3,5-dimethoxyphenyl)-5-(4-methoxyphenyl)tetrazolo[1,5-a]pyridine-8-carbonitrile (30). Yellow solid; yield: (84%); mp: 180-182 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 8.23 (d, J = 8.0 Hz, 2H, Ar-H), 7.84 (s, 1H, C 5 -H of pyridine), 7.21 (d, J = 8.2 Hz, 2H, Ar-H), 7.03 (s, 2H, Ar-H), 6.76 (s, 1H, Ar-H), 3.90 (s, 3H, OCH 3 ), 3.85 (s, 6H, 2OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 162.14, 160.75, 151.91, 149.41, 141.45, 136.53, 131.79, 121.46, 116.49, 114.31, 114.15, 107.22, 106.74, 102.37, 93.77, 55.64, 55.53; IR (ATR, ν max /cm -1 ): 3004 (CH-aromatic), 2840 (CH-aliphatic), 2218 (CN), 2128 (N 3 ); UV/Vis: λ max 350, 275 nm: 350, 275; MS, m/z : 387.49 (M + ); analysis (calcd., found for C 21 H 17 N 5 O 3 ): C (65.11, 65.37), H (4.42, 4.59), N (18.08, 18.31). 5-(3,4-dimethoxyphenyl)-7-(4-methoxyphenyl)tetrazolo[1,5-a]pyridine-8-carbonitrile( 31 ). Brown solid; yield: (86%); mp: 182-184 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 7.92 (d, J = 6.3 Hz, 2H, Ar-H), 7.87 (s, 1H, C 5 -H of pyridine), 7.78 (s, 1H, Ar-H), 7.74 (d, J = 4.6 Hz, 2H, Ar-H), 7.24 (d, J = 4.8 Hz, 2H, Ar-H), 3.91 (s, 3H, OCH 3 ), 3.90 (s, 6H, 2OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 161.47, 151.83, 141.40, 130.98, 126.86, 123.63, 121.61, 116.72, 114.63, 113.03, 111.58, 92.59, 55.92, 55.84, 55.59; IR (ATR, ν max /cm -1 ): 3011 (CH-aromatic), 2844 (CH-aliphatic), 2223 (CN), 2135 (N 3 ); UV/Vis: λ max 355, 255 nm; MS, m/z : 387.69(M + ); analysis (calcd., found for C 21 H 17 N 5 O 3 ): C (65.11, 64.98), H (4.42, 4.61), N (18.08, 18.27). 7-(furan-2-yl)-8-methyl-5-(3,4,5-trimethoxyphenyl)tetrazolo[1,5-a]pyridine( 32 ). Yellow solid; yield: (88%); mp: 162-164 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 8.25 (s, 1H, C 5 -H of pyridine), 8.02 (s, 1H, furyl-H), 7.93 (s, 1H, furyl-H), 7.44 (s, 2H, Ar-H), 6.95 (s, 1H, furyl-H), 3.90 (s, 6H, 2OCH 3 ), 3.81 (s, 3H, OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 152.86, 149.90, 147.63, 141.57, 140.41, 137.99, 124.58, 117.62, 114.14, 113.83, 113.30, 107.90, 107.62, 87.81, 60.29, 56.33; IR (ATR, ν max /cm -1 ): 3002 (CH-aromatic), 2840 (CH-aliphatic), 2227 (CN), 2136 (N 3 ); UV/Vis: λ max 365, 300, 280 nm; MS, m/z : 377.51 (M + ); analysis (calcd., found for C 19 H 15 N 5 O 4 ): C (60.48, 60.73), H (4.01, 4.20), N (18.56, 18.83). 7-(furan-2-yl)-5-methyltetrazolo[1,5-a]pyridine-8-carbonitrile( 33 ). Green solid; yield: (87%); mp: 228-230 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 8.21 (s, 1H, C 5 -H of pyridine), 7.87 (s, 1H, furyl-H), 7.73 (s, 1H, furyl-H), 6.91 (s, 1H,furyl-H), 2.93 (s, 3H, CH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 149.03, 147.96, 147.50, 141.50, 137.88, 116.95, 114.13, 113.86, 112.96, 87.29, 17.13; IR (ATR, ν max /cm -1 ): 3001 (CH-aromatic), 2840 (CH-aliphatic), 2232 (CN); UV/Vis: λ max 340, 275 nm; MS, m/z : 225.63 (M + ); analysis (calcd., found for C 11 H 7 N 5 O): C (58.67, 58.90), H (3.13, 3.27), N (31.10, 30.98). 4.1.4. General procedure for Synthesis of 2-iminophosphorane-nicotinonitrile derivatives ( 34-41 ). A mixture of selected azido/tetrazolo compounds 26-33 (2 mmol) and triphenylphosphine (2.4 mmol, 629.49 mg) in toluene (15 ml) was heated to reflux for 15 minutes. The reaction mixture was followed by TLC until completion, then evaporated and the precipitate was washed with hexane to remove excess reagent. The pure fluorescent product was dried under vacuum to afford pure compounds 34-41 . 6-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2-((triphenyl-λ 5 -phosphaneylidene)amino)-nicotinonitrile ( 34 ). Buff solid; yield: (89%); mp: 211-213 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 7.86 (s, 6H, Ar-H ), 7.58 (s, 9H, Ar-H), 7.43 (s, 1H, C 5 -H of pyridine), 7.16 (s, 2H, Ar-H), 7.00 (s, 2H, Ar-H), 6.80 (s, 2H, Ar-H), 3.86 (s, 6H, 2OCH 3 ), 3.77 (s, 6H, 2OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 164.89, 164.84, 160.65, 156.65, 154.81, 152.91, 138.31, 132.95, 132.64, 132.54, 131.65, 131.55, 130.48, 129.37, 129.08, 128.96, 128.86, 128.62, 128.37, 119.30, 113.82, 108.39, 106.10, 95.86, 95.62, 60.23, 56.16, 55.35; 31 P NMR (162 MHz, DMSO- d 6 ) δ ppm: 26.31(s), 14.08(s); IR (ATR, ν max /cm -1 ): 3010 (CH-aromatic), 2 8 3 7 (CH-aliphatic), 2 206 (CN); UV/Vis: λ max 355, 295 nm; MS, m/z : 652.32 (M + ); analysis (calcd., found for C 40 H 34 N 3 O 4 P): C (73.72, 73.51), H (5.26, 5.43), N (6.45, 6.72). 6-(3,4-Dimethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2-((triphenyl-λ 5 -phosphaneylidene)-amino)nicotinonitrile ( 35 ). Grey solid; yield: (87%); mp: 212-214 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 7.89 (s, 6H, Ar-H), 7.63 (d, J = 2.9 Hz, 3H, Ar-H), 7.56 (s, 6H, Ar-H), 7.27 (d, J = 6.9 Hz, 1H, Ar-H), 7.21 (s, 1H, Ar-H), 7.12 (s, 1H, C 5 -H of pyridine), 6.99 (s, 2H, Ar-H), 6.87 (d, J = 6.9 Hz, 1H, Ar-H), 3.85 (s, 6H, 2OCH 3 ), 3.75 (d, J = 11.3 Hz, 6H, 2OCH3), 3.40 (s, 3H, OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 165.11, 157.02, 155.11, 153.26, 150.70, 148.98, 138.66, 133.31, 133.06, 132.96, 130.94, 129.70, 129.39, 129.27, 128.70, 120.57, 119.61, 111.75, 110.31, 109.06, 106.52, 96.37, 60.56, 56.53, 55.97, 55.66; 31 P NMR (162 MHz, DMSO- d 6 ) δ ppm: 25.64(s), 13.67(s); IR (ATR, ν max / -1 ): 3005(CH-aromatic), 2 8 3 7 (CH-aliphatic), 2 207 (CN); UV / Vis: λ max 375, 290 nm; MS, m/z : 681.47 (M + ); analysis (calcd., found for C 41 H 36 N 3 O 5 P): C (72.24, 72.43), H (5.32, 5. 39), N (6.16, 6.42). 4,6-Bis(3,4,5-trimethoxyphenyl)-2-((triphenyl-λ 5 -phosphaneylidene)amino)nicotinonitrile( 36 ). Buff solid; yield: (90%); mp: 222-224 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 7.91 (dd, J = 12.0, 7.6 Hz, 6H, Ar-H), 7.64 (d, J = 6.7 Hz, 3H, Ar-H), 7.57 (d, J = 4.8 Hz, 6H, Ar-H), 7.31 (s, 1H, C 5 -H of pyridine), 7.00 (s, 2H, Ar-H), 6.95 (s, 2H, Ar-H), 3.87 (s, 6H, 2OCH 3 ), 3.75 (s, 3H, OCH 3 ), 3.67 (s, 3H, OCH 3 ), 3.54 (s, 6H, 2OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 164.98, 164.92, 156.88, 155.37, 155.33, 153.27, 153.25, 139.36, 138.67, 133.79, 133.21, 133.04, 132.94, 129.55, 129.39, 129.27, 128.55, 119.49, 109.64, 106.57, 104.75, 97.34, 97.09, 60.57, 56.54, 56.27; 31 P NMR (162 MHz, DMSO- d 6 ) δ ppm: 13.28(s); IR (ATR, ν max /cm -1 ): 3003 (CH-aromatic), 2 8 3 8 (CH-aliphatic), 2 214 (CN); UV/Vis: λ max 370, 295 nm; MS, m/z : 711.28 (M + ); analysis (calcd., found for C 42 H 38 N 3 O 6 P): C (70.88, 71.14), H (5.38, 5.47), N (5.90, 6.12). 6-(3,4-Dimethoxyphenyl)-4-(3,5-dimethoxyphenyl)-2-((triphenyl-λ 5 -phosphaneylidene)amino)-nicotinonitrile ( 37 ). Grey solid; yield: (91%); mp: 135-137 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 7.90 (d, J = 6.8 Hz, 6H, Ar-H), 7.63 (d, J = 6.5 Hz, 6H, Ar-H), 7.57 (s, 3H, Ar-H), 7.40 (s, 1H, C 5 -H of pyridine), 7.24 (d, J = 5.9 Hz, 1H, Ar-H), 7.17 (s, 1H, Ar-H), 7.13 (s, 1H, Ar-H), 6.90 – 6.78 (m, 2H, Ar-H), 6.63 (s, 1H, Ar-H), 3.79 (d, J = 18.7 Hz, 6H, 2OCH 3 ), 3.37 (d, J = 30.8 Hz, 6H, 2OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 164.61, 160.42, 156.66, 154.61, 150.28, 148.54, 139.48, 132.61, 132.51, 131.56, 131.46, 130.41, 129.21, 128.96, 128.84, 128.21, 120.13, 118.86, 111.29, 109.83, 108.53, 106.48, 101.04, 95.90, 55.51, 55.42, 55.22; 31 P NMR (162 MHz, DMSO- d 6 ) δ ppm: 27.10(s), 13.82(s); IR (ATR, ν max /cm -1 ): 29 67 (CH-aromatic), 2 8 3 5 (CH-aliphatic), 2 208 (CN); UV/Vis: λ max 370, 275 nm; MS, m/z : 651.55 (M + ); analysis (calcd., found for C 40 H 34 N 3 O 4 P): C (73.72, 73.50), H (5.26, 5.39), N (6.45, 6.67). 4-(3,5-Dimethoxyphenyl)-6-(4-methoxyphenyl)-2-((triphenyl-λ 5 -phosphaneylidene)amino)-nicotinonitrile ( 38 ). Yellow solid; yield: (88%) ; mp: 128-130 ⁰C; 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm: 7.86 (dd, J = 11.5, 7.9 Hz, 6H, Ar-H), 7.64 (d, J = 6.8 Hz, 3H, Ar-H), 7.58 (d, J = 5.9 Hz, 6H, Ar-H), 7.41 (d, J = 8.4 Hz, 2H, Ar-H), 7.11 (s, 1H, C 5 -H of pyridine), 6.81 (d, J = 6.3 Hz, 2H, Ar-H), 6.78 (s, 2H, Ar-H), 6.63 (s, 1H, Ar-H), 3.81 (s, 6H, 2OCH 3 ), 3.76 (s, 3H, OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 164.66, 160.56, 160.42, 156.61, 154.64, 139.48, 132.56, 132.46, 131.55, 131.46, 130.34, 129.27, 128.98, 128.86, 128.51, 128.27, 118.86, 113.72, 108.26, 106.43, 101.11, 95.82, 95.58, 55.41, 55.24; 31 P NMR (162 MHz, DMSO- d 6 ) δ ppm: 26.09(s), 14.09(s); IR (ATR, ν max /cm -1 ): 3012 (CH-aromatic), 2 8 40 (CH-aliphatic) 2 207 (CN); UV/Vis: λ max 370, 265 nm; MS, m/z : 621.40 (M + ); analysis (calcd., found for C 39 H 32 N 3 O 3 P): C (75.35, 75.19), H (5.19, 5.30), N (6.76, 6.94). 6-(3,4-Dimethoxyphenyl)-4-(4-methoxyphenyl)-2-((triphenyl-λ 5 -phosphaneylidene)amino)-nicotinonitrile ( 39 ). brown solid; yield: (86%); mp: 129-131 ⁰C; 1 H NMR (400 MHz, DMSO-d6) δ ppm: 7.90 (s, 6H, Ar-H), 7.61 (d, J = 25.3 Hz, 9H, Ar-H), 7.41 (s, 3H, Ar-H), 7.25 (s, 2H, Ar-H), 7.12 (s, 2H, Ar-H), 6.87 (s, 1H, C 5 -H of pyridine), 3.81 (d, J = 26.1 Hz, 6H, 2OCH 3 ), 3.41 (s, 3H, OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 164.67, 160.12, 156.47, 154.20, 150.16, 148.49, 132.57, 132.47, 132.32, 131.51, 131.41, 130.49, 129.68, 129.25, 128.89, 128.72, 128.68, 128.26, 119.96, 119.11, 114.03, 111.25, 109.79, 108.47, 95.63, 55.46, 55.30, 55.17; 31 P NMR (162 MHz, DMSO- d 6 ) δ ppm: 26.03(s), 13.73(s); IR (ATR, ν max /cm -1 ): 3032 (CH-aromatic), 29 3 1 (CH-aliphatic), 2 2 02 (CN); UV/Vis: λ max 370, 265 nm; MS, m/z : 621.00 (M + ); analysis (calcd., found for C 39 H 32 N 3 O 3 P): C (75.35, 75.12), H (5.19, 5.40), N (6.76, 7.04). 4-(Furan-2-yl)-6-(3,4,5-trimethoxyphenyl)-2-((triphenyl-λ 5 -phosphaneylidene)amino)nicotino-nitrile ( 40 ). Yellow solid; yield: (86%) ; mp: 230-232 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 8.01 (d, J = 1.1 Hz, 1H, furyl-H), 7.89 (dd, J = 12.2, 7.3 Hz, 6H, Ar-H), 7.64 (t, J = 7.3 Hz, 3H, Ar-H), 7.60 – 7.52 (m, 6H, Ar-H), 7.48 (s, 1H, C 5 -H of pyridine), 7.21 (dd, J = 27.4, 7.4 Hz, 1H, furyl-H), 6.93 (s, 2H, Ar-H), 6.80 (dd, J = 3.5, 1.7 Hz, 1H, furyl-H), 3.68 (s, 3H, OCH 3 ), 3.56 (s, 6H, 2OCH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 165.40, 157.21, 153.32, 149.43, 148.33, 141.97, 139.50, 133.76, 133.67, 133.07, 132.97, 129.55, 129.41, 129.29, 128.56, 119.47, 113.25, 104.60, 92.42, 60.57, 56.28; 31 P NMR (162 MHz, DMSO- d 6 ) δ ppm: 28.17(s), 14.19(s); IR (ATR, ν max /cm -1 ): 3012 (CH-aromatic), 29 3 4 (CH-aliphatic), 2 207 (CN); UV/Vis: λ max 385, 300 nm; MS, m/z : 611.45 (M + ); analysis (calcd., found for C 37 H 30 N 3 O 4 P): C (72.66, 72.52), H (4.94, 5.11), N (6.87, 7.05). 4-(Furan-2-yl)-6-methyl-2-((triphenyl-λ 5 -phosphaneylidene)amino)nicotinonitrile ( 41 ) . Cocoa solid; yield: (88%); mp: 222-224 ⁰C; 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm: 7.93 (s, 1H, C 5 -H of pyridine), 7.86 (dd, J = 12.0, 7.3 Hz, 6H, Ar-H), 7.67 – 7.61 (m, 3H, Ar-H), 7.60 – 7.53 (m, 6H, Ar-H), 7.40 (d, J = 3.4 Hz, 1H, furyl-H), 6.80 (s, 1H, furyl-H), 6.73 (d, J = 1.6 Hz, 1H, furyl-H), 2.04 (s, 3H, CH 3 ); 13 C APT NMR (100 MHz, DMSO- d 6 ) δ ppm: 160.04, 144.99, 140.67, 132.84, 132.74, 132.27, 128.93, 128.74, 128.62, 127.93, 119.08, 112.55, 112.29, 106.74, 23.71; 31 P NMR (162 MHz, DMSO- d 6 ) δ ppm: 14.19(s); IR (ATR, ν max /cm -1 ): 30 54 (CH-aromatic), 2840 (CH-aliphatic), 2 20 6 (CN); UV/Vis: λ max 360, 295 nm; MS, m/z : 459.13 (M + ); analysis (calcd., found for C 29 H 22 N 3 OP): C (75.81, 75.68), H (4.83, 4.97), N (9.15, 9.41). About the characterization data of all the final synthesized compounds ( 1 H-NMR, 13 C-APT NMR, IR spectra, UV-Vis analyses, Mass spectra and Elemental analyses), see supplementary information (Fig. S1-S161). 4.2. Biological assay 4.2.1. In vitro anticancer activity. 4.2.1.1. Materials and Methods Roswell Park Memorial Institute (RPMI) 1640 medium was purchased from Sigma Chem. Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) and fetal calf serum (FCS) were purchased from Gibco, UK. Dimethyl sulfoxide (DMSO) and methanol were of HPLC grade, and all other reagents and chemicals were of analytical reagent grade. 4.2.1.2. Cell culture HepG-2 (Human liver carcinoma), HCT116 (human colorectal carcinoma), MCF-7 (human breast adenocarcinoma), and the normal human skin fibroblast (BJ-1) cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in RPMI-1640 medium which was supplemented with 10% heat-inactivated FBS, 100U/ml penicillin and 100U/ml streptomycin. The cells were grown at 37°C in a humidified atmosphere of 5% CO2. All experiments were conducted thrice in triplicate (n = 3). All the values were represented as means ± SD. Significant differences between the means of parameters as well as IC50s were determined by probit analysis using SPSS software program (SPSS Inc., Chicago, IL). 4.2.1.3. Lactate dehydrogenase (LDH) assay To determine the effect of each synthesized compound on membrane permeability in HepG2, MCF-7 and HCT-116 cancer cell lines as well as BJ-1 normal cell line, a lactate dehydrogenase (LDH) release assay was used 36–40 . The cells were seeded in 24-well culture plates at a density of 1 × 104 cells/well in 500 μL volume and allowed to grow for 18h before treatment. After treatment with a series of different concentrations of each compound or Doxorubicin® (positive control), the plates were incubated for 48h. Then, the supernatant (40 μL) was transferred to a new 96 well to determine LDH release and 6% triton X-100 (40 μL) was added to the original plate for determination of total LDH. An aliquot of 0.1 M potassium phosphate buffer (100 μL, pH 7.5) containing 4.6 mM pyruvic acid was mixed to the supernatant using repeated pipetting. Then, 0.1 M potassium phosphate buffer (100 μL, pH 7.5) containing 0.4 mg/mL reduced β-NADH was added to the wells. The kinetic changes were read for 1 min using ELISA microplate reader in absorbance at wavelength 340 nm. This procedure was repeated with 40 μL of the total cell lysate to determine total LDH. The percentage of LDH release was determined by dividing the LDH released into the media by the total LDH following cell lysis in the same well. 4.2.1.4. Statistical analysis All experiments were conducted in triplicate (n = 3). All the values were represented as mean ± SD. Significant differences between the means of parameters as well as IC50s were determined by probit analysis using SPSS software program (SPSS Inc., Chicago, IL). 4.2.2. Inhibition of Tubulin Polymerization in MCF-7 Cells 4.2.2.1. Materials Beta-Tubulin in vitro SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of Beta-Tubulin protein in human cell and tissue homogenate extract samples. This is performed for most active compounds 20, 22, 24, 26, 27, 33, 35, 37, 38, 41 and CA-4 as control against MCF-7 cancer cell line to measure the percentage of β-tubulin polymerization inhibition. 4.2.2.2. Methodolgy The SimpleStep ELISA® employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB Development Solution is added and during incubation is catalyzed by Horseradish Peroxidase (HRP), generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm. The concentration of Beta-Tubulin protein in the samples is determined by comparing the optical density (OD) of the samples to the standard curve. Each experiment was repeated two times (Table 2). 4.2.3. Inhibition of Topoisomerase II in MCF-7 Cells 4.2.3.1. Materials Mouse TOP2B /Topoisomerase II Beta ELISAKit (Sandwich ELISA) is designed for the quantitative measurement of Topo II enzyme. This is performed for most active compounds 20, 22, 24, 26, 37 and doxorubicin as control against MCF-7 cancer cell line to measure the percentage of Topo II enzyme inhibition. 4.2.3.2. Methodology This assay is based on the sandwich ELISA principle. Each well of the supplied microtiter plate has been pre-coated with a target specific capture antibody. Standards or samples are added to the wells and the target antigen binds to the capture antibody. Unbound Standard or sample is washed away. A biotin-conjugateddetection antibody is then added which binds to the captured antigen. Unbound detection antibody is washed away. An Avidin-Horseradish Peroxidase (HRP) conjugate is then added which binds to the biotin. Unbound Avidin-HRP conjugate is washed away. A TMB substrate is then added which reacts with the HRP enzyme resulting in color development. A sulfuric acid stop solution is added to terminate color development reaction and then the optical density (OD) of the well is measured at a wavelength of 450 nm ± 2 nm. An OD standard curve is generated using known antigen concentrations; the OD of an unknown sample can then be compared to the standard curve in order to determine its antigen concentration. Each experiment was repeated two times (Table 3). 4.2.4. In vitro Propidium Iodide Flow Cytometry Cell Cycle Analysis 4.2.4.1. Materials Propidium Iodide Flow Cytometry Kit (ab139418) is designed for quantitative DNA content analysis in tissue culture cells so we performed Propidium Iodide cell cycle analysis for active compounds 20, 26 using MCF-7 cell line. Propidium iodide staining of DNA is the classic means of cell cycle analysis. The staining procedure takes less than 1 hour of total processing time and cells fixed in ethanol are stable for at least several weeks at 4ºC. 4.2.4.2. Methodology Propidium iodide is a fluorescent molecule that binds nucleic acid with little or no sequence preference. Because Propidium iodide binds RNA as well as DNA, RNaseA (ribonuclease A) is included in this kit to digest cellular RNA and thus decrease background RNA staining from the experiment. Since Propidium iodide is membrane impermeant, ethanol is used to both fix and permeabilize cells. A flow cytometer is required for quantitative analysis. First, fix MCF-7 cells in 66% Ethanol, store at +4°C for 2 hours to 4 weeks and rehydrate cells in PBS. Finally, stain cells with Propidium iodide then add Rnase for 30min. Collect Propidium iodide fluorescence intensity on FL2 of a flow cytometer and 488nM laser excitation. A useful way to display Propidium iodide data is on a histogram with the cell count on the y-axis and the propidium iodide fluorescence intensity on the x-axis. 4.2.5. Apoptosis and Necrosis Analysis by Annexin V-FITC/PI Staining 4.2.5.1. Materials Annexin V Apoptosis Detection Kit is used for analyzing apoptosis and necrosis for active compounds 20, 26 using Annexin V-FITC/PI dual staining followed by flow cytometry quantification and Fluorescence Microscopy detection. 4.2.5.2. Methodology Detection is based on the observation that soon after initiating apoptosis, cells translocate the membrane phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface. Once on the cell surface, PS can be easily detected by staining with a fluorescent conjugate of Annexin V, a protein that has a high affinity for PS. The one-step staining procedure takes only 10 minutes. Detection can be analyzed by flow cytometry or by fluorescence microscopy. The kit can differentiate between apoptosis and necrosis when performing both Annexin V-FITC and PI staining. Assay is summarized as first, Induce apoptosis by desired method, Collect 1-5 x 105 cells by centrifugation, Resuspend cells in 500 μl of 1X Binding Buffer, Add 5 μl of Annexin V-FITC and 5 μl of propidium iodide and finally Incubate at room temperature for 5 min in the dark. Analyze Annexin V-FITC binding by flow cytometry (Ex = 488 nm; Em = 530 nm) using FITC signal detector (usually FL1) and PI staining by the phycoerythrin emission signal detector (usually FL2). Place the cell suspension on a glass slide. Cover the cells with a glass coverslip. Observe the cells under a fluorescence microscope using a dual filter set for FITC & Rhodamine (Cells that have bound Annexin V-FITC will show green staining in the plasma membrane. Cells that have lost membrane integrity will show red staining (PI) throughout the nucleus and a halo of green staining (FITC) on the cell surface (plasma membrane). 4.2.6. In silico study You will find general Computational Modelling and Docking in addition to DFT calculations in the supplementary file in S4.2.6 part. Declarations Conflict of interest: There are no conflicts to declare. Funding statement: Yasir S. Raouf is supported by an internal SQU-UAEU grant (Grant #12S244) and Dr. Shaikha Alneyadi is supported by grant number G00004741. Author Contribution Eman S. Hassan: Conceptualization, investigation, methodology, and manuscript writing.Abdalla E. A. Hassan: Conceptualization, data analysis, and manuscript writing.Shaikha Alneyadi: Conceptualization, data analysis, and manuscript writing.Yassir S. Raouf: Conceptualization, methodology, and manuscript writing.Hanem M. Awad: Conceptualization, methodology, and manuscript writing.Zakaria K.M. Abdel-Samii: Conceptualization, data analysis, and manuscript writing.Amany M.M. Al-Mahmoudy: Conceptualization, data analysis, and manuscript writing.Reham A. Abou-Elkhair: Conceptualization, data analysis, and manuscript writing. Acknowledgement We thank Dr. Sara Hosny for recording NMR Data and Ms. Eman Hafez for recording FT-IR and UV-vis data. We thank STDF for the Capacity Building grant # 2698, Dr. Yasir S. Raouf is supported by an internal SQU-UAEU grant (Grant #12S244), Dr. Shaikha Alneyadi is supported by grant number G00004741 Data Availability Data provided within the manuscript or supplementary information files. The raw data for the DFT calculations and docking studies will be shared upon submitting requestto Dr. Yasir S. Raouf. References Chen, S. et al. Estimates and projections of the global economic cost of 29 cancers in 204 countries and territories from 2020 to 2050. JAMA Oncol. 9 , 465–472 (2023). El Hamaky, N. F. M., Hamdi, A., Bayoumi, W. A., Elgazar, A. A. & Nasr, M. N. A. Novel quinazolin-2-yl 1, 2, 3-triazole hybrids as promising multi-target anticancer agents: design, synthesis, and molecular docking study. Bioorg. 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Intermed . 43 , 437–456 (2017). Scheme Scheme 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files supplementaryinformation.docx Scheme12.png Scheme 12 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 25 Nov, 2025 Reviews received at journal 17 Nov, 2025 Reviewers agreed at journal 02 Nov, 2025 Reviewers invited by journal 31 Oct, 2025 Editor assigned by journal 31 Oct, 2025 Editor invited by journal 23 Oct, 2025 Submission checks completed at journal 22 Oct, 2025 First submitted to journal 22 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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08:19:44","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":212658,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7890915/v1/30210c4e00fd524c6d64b063.html"},{"id":95799330,"identity":"fb95254d-0ac7-4332-9a58-89fb3ba224b1","added_by":"auto","created_at":"2025-11-13 08:19:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":59322,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"AEHESHoctober162025Figutres1.png","url":"https://assets-eu.researchsquare.com/files/rs-7890915/v1/3e60d0643f8dfde850f309dc.png"},{"id":95676078,"identity":"df080b37-bd92-431b-ac79-f92eac76156a","added_by":"auto","created_at":"2025-11-11 18:38:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":43002,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"AEHESHoctober162025Figutres2.png","url":"https://assets-eu.researchsquare.com/files/rs-7890915/v1/1b510bae3a481a44bc9d1aed.png"},{"id":95675954,"identity":"43e9df8f-dfe5-440c-9888-1e53d2f52071","added_by":"auto","created_at":"2025-11-11 18:38:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":62485,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"AEHESHoctober162025Figutres3.png","url":"https://assets-eu.researchsquare.com/files/rs-7890915/v1/65b5f2832cbd9abe33a59c74.png"},{"id":95676072,"identity":"13d30713-2ad0-44d6-8e98-dc12f7e9a9f1","added_by":"auto","created_at":"2025-11-11 18:38:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":565375,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"AEHESHoctober162025Figutres5.png","url":"https://assets-eu.researchsquare.com/files/rs-7890915/v1/aa8f0c1e026a563fb46ab1c4.png"},{"id":95675958,"identity":"e57c1288-57a4-4a27-a704-101d1b135a54","added_by":"auto","created_at":"2025-11-11 18:38:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":62566,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"AEHESHoctober162025Figutres6.png","url":"https://assets-eu.researchsquare.com/files/rs-7890915/v1/743d600eb3dd1792c42aab76.png"},{"id":95676149,"identity":"757a4a77-183d-4885-9006-4e39c15a6de2","added_by":"auto","created_at":"2025-11-11 18:38:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1281888,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"AEHESHoctober162025Figutres7.png","url":"https://assets-eu.researchsquare.com/files/rs-7890915/v1/80094d420fd65d553a813565.png"},{"id":95804714,"identity":"8ad2ee3f-020e-416d-8243-3391eaabe946","added_by":"auto","created_at":"2025-11-13 08:39:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3681435,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7890915/v1/f63de18e-65fa-49f8-984e-7c202464ce0a.pdf"},{"id":95676089,"identity":"3386734f-37fc-4378-824a-ecd42106e5a9","added_by":"auto","created_at":"2025-11-11 18:38:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11164566,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7890915/v1/2a047ca5f9f903b002f47f05.docx"},{"id":95798747,"identity":"625ab7af-b236-436e-bfde-4609e78734c1","added_by":"auto","created_at":"2025-11-13 08:17:44","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":58700,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 12\u003c/p\u003e","description":"","filename":"Scheme12.png","url":"https://assets-eu.researchsquare.com/files/rs-7890915/v1/457722eb40ec06b29f000a42.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nicotinonitrile-Based Dual Inhibitors of Tubulin and Topoisomerase II: Design, Synthesis, and Anticancer Evaluation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCancer remains one of the leading causes of mortality worldwide, accounting for nearly 10\u0026nbsp;million deaths per year (approximately one in six of all deaths)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Global cancer incidence continues to rise, with an estimated 20\u0026nbsp;million new cases diagnosed in 2022 alone\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Beyond the human toll, the economic burden of cancer is enormous \u0026ndash; the global cost of cancer is projected to reach about \u003cspan\u003e$\u003c/span\u003e25 trillion (in 2017 international dollars) over 2020\u0026ndash;2050\u003csup\u003e1\u003c/sup\u003e. These sobering statistics underscore the urgent need for more effective and innovative anticancer therapies. Current treatment modalities as surgery, radiation, chemotherapy and targeted therapy have improved outcomes for many cancers. Yet treatment failures, resistance, and side effects remain significant challenges. Conventional single-target chemotherapeutic agents often face issues such as dose-limiting toxicity and the emergence of drug resistance, prompting the exploration of new strategies in drug design and discovery. One promising strategy in medicinal chemistry is the development of dual or multi-target inhibitors \u0026ndash; single molecules capable of modulating multiple biological targets. By hitting two or more cancer-relevant pathways simultaneously, a multi-target agent can exploit synergistic anticancer effects and potentially overcome resistance mechanisms that would thwart a single-target drug \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In this context, dual inhibitors of tubulin polymerization and DNA topoisomerase II (Topo II) are of great therapeutic interest. Both tubulin and Topo-II are well-validated targets in oncology, and their inhibition leads to complementary anticancer effects. Microtubules assembled from tubulin are essential for mitosis and cell division. Agents that disrupt tubulin polymerization, so-called microtubule destabilizers, cause cell cycle arrest and apoptosis of rapidly dividing cells\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Topoisomerase-II is an enzyme that relieves DNA supercoiling during replication and transcription; inhibitors of Topo-II induce DNA strand breaks and prevent genome replication, which is also lethal to proliferating cancer cells \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Notably, tubulin inhibitors and Topo-II inhibitors are frequently used in combination chemotherapy regimens for synergistic efficacy. This synergism has inspired the design of single molecules capable of concurrently targeting both microtubule assembly and DNA topoisomerase activity\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Several examples illustrate the therapeutic value of these target classes and lay the groundwork for dual inhibitors. Combretastatin A-4 (CA-4), a natural cis-stilbene isolated from Combretum Caffrum, is a potent tubulin polymerization inhibitor that binds to the colchicine site on β-tubulin \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. CA-4 and its analogues cause mitotic arrest and have shown broad anticancer activity, including against multidrug-resistant tumors \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However, CA-4\u0026rsquo;s clinical application is limited by its poor aqueous solubility and the instability of its cis-olefin which can isomerize to the less active trans form (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Podophyllotoxin, a lignan from the mayapple plant, is another anti-mitotic agent that binds tubulin at a site distinct from the vinca alkaloid site and inhibits microtubule assembly(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Interestingly, semisynthetic derivatives of podophyllotoxin \u0026ndash; notably etoposide (VP-16) and teniposide (TM-26) \u0026ndash; were developed to forgo tubulin activity and instead act as Topo II inhibitors \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Etoposide, a cornerstone of many chemotherapy regimens, intercalates DNA and stabilizes the Topo-II\u0026ndash;DNA cleavable complex, leading to lethal DNA double-strand breaks in cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Another relevant example is Azatoxin (NSC 640737), a synthetic hybrid molecule encompassing the structural elements of etoposide and ellipticine, displays dual mechanism of action; at lower concentrations it behaves predominantly as a microtubule destabilizer, while at higher concentrations it inhibits Topo II, ultimately inducing both mitotic arrest and DNA damage in cancer cells(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. compound VI also has dual activity of topo II and tubulin inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In this context, diarylnicotinonitriles were selected as a promising scaffold due to their rigid, pyridine-bridged structure that mimics the bioactive conformation of CA-4 while improving metabolic stability. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC highlights examples of nicotinonitrile and heterocycle-bridged CA-4 analogues with notable anticancer activity, where the cytotoxic potency is strongly influenced by the substituents on rings A and B and the nature of the central linker \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn this study, we focused on 2,4,6-trisubstituted nicotinonitriles scaffolds that incorporate essential structural elements for dual anti-tubulin and Topoisomerase II inhibition\u0026mdash;namely, aryl rings (A and B) connected by a rigid pyridine linker. Modifications at the 2-position, including hydroxy, chloro, tetrazolyl/azido and iminophosphorane moieties on the central pyridine ring, were introduced to enhance DNA intercalation and promote Topo II inhibitory activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eStructure\u0026ndash;activity relationship (SAR) studies on CA-4 and its analogues have shown that optimal antiproliferative activity depends on the presence of 3,4,5-trimethoxy and/or 3,5-dimethoxy substitutions on ring A, as well as the cis-configuration of the vinyl bridge. In contrast, ring B tolerates various structural changes; the 3-hydroxy group is not essential for activity, whereas the 4-methoxy group is critical for cytotoxicity (Compounds \u003cb\u003eVII\u003c/b\u003e, \u003cb\u003eVIII\u003c/b\u003e and \u003cb\u003eIX\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC)\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Compounds exhibiting dual inhibition of tubulin and Topo II often feature per-methoxyphenyl groups, extended cyclic systems, and azole rings within their structures (Compounds \u003cb\u003eIV, V\u003c/b\u003e and \u003cb\u003eVI\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents the design rationale for the hybrid molecules \u003cb\u003eX\u003c/b\u003e, which incorporate key structural features aimed at dual inhibition of Topo II and tubulin polymerization.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRing A includes varying numbers of methoxy groups that serve as hydrogen bond acceptors within the colchicine binding site of tubulin, along with a cis-double bond, mimicking the rigid pyridine ring. To enhance TopII binding, the pyridine ring is further functionalized at the 2-position with hydrophobic substituents such as chloro, azido/tetrazolyl, and iminophosphorane groups, while ring B is substituted with different number of methoxy groups (compounds \u003cb\u003e18\u0026ndash;41\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e"},{"header":"2. Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Chemistry\u003c/h2\u003e\u003cp\u003eThe strategy adopted for the synthesis of the target compounds \u003cb\u003e10\u0026ndash;41\u003c/b\u003e is outlined in Scheme 1 and scheme 2. 2-Oxonicotinonitriles (2-ONNs) \u003cb\u003e10\u0026ndash;17\u003c/b\u003e were synthesized by three component-one pot reaction of corresponding aldehydes \u003cb\u003e1, 2, 3, 7\u003c/b\u003e, corresponding ketones \u003cb\u003e4, 5, 6, 8, 9\u003c/b\u003e and ethyl cyanoacetate in the presence of ammonium acetate in reflux ethanol\u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAn alternative pathway for the synthesis of 2-ONNs involves the reaction of chalcones, generated from the corresponding aldehydes and ketone derivatives, with ethyl cyanoacetate in the presence of ammonium acetate\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The structure of the 2-ONNs was confirmed by \u003csup\u003e1\u003c/sup\u003eH-NMR, \u003csup\u003e13\u003c/sup\u003eC-APT-NMR, ATR-IR spectra. For instance, compound \u003cb\u003e12\u003c/b\u003e displayed a broad singlet at δ 12.69 ppm corresponding to the NH proton and its \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC-APT-NMR spectrum showed signals at \u003cem\u003eδ\u003c/em\u003e 117.0 and \u003cem\u003eδ\u003c/em\u003e 162.3 ppm corresponding to the -CN and C\u0026thinsp;=\u0026thinsp;O carbons, respectively (Scheme 1). Treatment of the 2-ONNs derivatives \u003cb\u003e10\u0026ndash;17\u003c/b\u003e with POCl\u003csub\u003e3\u003c/sub\u003e in the presence of \u003cem\u003eN,N\u003c/em\u003e-dimethylaniline at reflux temperature provided the corresponding 2-chloronicotinonitriles \u003cb\u003e18\u0026ndash;25\u003c/b\u003e, respectively in good yields.\u003c/p\u003e\u003cp\u003eConstructing a tetrazole ring fused at the N-1 and the C-2 position of the pyridine ring envisioned accessible given the fact that nitrogenous six-membered heterocycles with an azido group positioned at the α-position to the ring-nitrogen tend to form a tetrazole ring depending on the structure and the physical state of the molecule\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Treatment of the 2-chloronicotinamide derivatives \u003cb\u003e18\u0026ndash;25\u003c/b\u003e with NaN\u003csub\u003e3\u003c/sub\u003e in DMF at 80\u003csup\u003e◦\u003c/sup\u003eC provided the corresponding 2-azido/tetrazolo derivatives \u003cb\u003e26\u0026ndash;33\u003c/b\u003e respectively in good yields. ATR-IR spectra of compounds \u003cb\u003e26\u0026ndash;33\u003c/b\u003e showed variable intensities of the signals at 2130 cm\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u0026thinsp;1\u003c/sup\u003e (ν\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) where is almost absent for compounds \u003cb\u003e29\u003c/b\u003e and \u003cb\u003e33\u003c/b\u003e in the solid state. DFT tautomer distribution (Azido/tetrazolo) calculation in polar phase revealed the predominance of the tetrazolo-form for compounds \u003cb\u003e26\u003c/b\u003e and \u003cb\u003e27\u003c/b\u003e, while the azido-form predominates for compounds \u003cb\u003e33\u003c/b\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e and supplementary information S4.2.6.1). Treatment of the azido/tetrazolo derivatives \u003cb\u003e26\u0026ndash;33\u003c/b\u003e with triphenylphosphine under Staudinger reaction conditions provided the corresponding iminophosphorane derivative \u003cb\u003e34\u0026ndash;41\u003c/b\u003e, respectively in good yields. The 4-furyl derivatives, \u003cb\u003e16\u0026ndash;17, 24\u0026ndash;25, 32\u0026ndash;33, 40\u0026ndash;41\u003c/b\u003e were synthesized using an analogous manner as previously described (Scheme 2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Biological evaluations\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1. Cytotoxic activity\u003c/h2\u003e\u003cp\u003eThe cytotoxic activity of the newly synthesized 2-chloro-nicotinonitrile derivatives \u003cb\u003e18\u003c/b\u003e\u0026ndash;\u003cb\u003e24\u003c/b\u003e, tetrazolo/azido derivatives \u003cb\u003e26\u0026ndash;33\u003c/b\u003e, and their corresponding iminophosphorane analogues \u003cb\u003e34\u003c/b\u003e\u0026ndash;\u003cb\u003e41\u003c/b\u003e was evaluated in vitro against three human cancer cell lines: HepG2 (liver carcinoma), HCT116 (colorectal carcinoma), and MCF-7 (breast adenocarcinoma), as well as the normal human skin fibroblast line BJ-1(Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The assays were performed using the LDH method, with doxorubicin (DOX) serving as the positive control. All tested compounds demonstrated dose-dependent inhibition of the three cancer cell lines, while showing no significant cytotoxicity toward the non-tumor BJ-1 cells, in contrast to doxorubicin. The inhibitory concentrations (IC\u003csub\u003e50\u003c/sub\u003e) are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Notably, the 2-chloro derivative \u003cb\u003e20\u003c/b\u003e (4,6-bis(3,4,5-trimethoxyphenyl)) exhibited potent activity against the MCF-7 cell line (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.4 \u0026micro;M, compared with DOX IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.8 \u0026micro;M) and considerable activity against HepG2 (6.1 \u0026micro;M). In comparison, the 4-(3,5-dimethoxyphenyl)-6-(3,4-dimethoxyphenyl) derivative \u003cb\u003e21\u003c/b\u003e retained strong activity against MCF-7 (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.8 \u0026micro;M), but its potency against HepG2 decreased nearly two-fold (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;12.3 \u0026micro;M). A similar trend was observed with the tetrazolo derivatives, particularly compound \u003cb\u003e26\u003c/b\u003e, which showed significant inhibition of MCF-7 (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.4 \u0026micro;M) and moderate suppression of HepG2 (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.3 \u0026micro;M). Consistent with these findings, the iminophosphorane series also displayed structure-dependent activity, with the 4-(3,5-dimethoxyphenyl)-6-(4-methoxyphenyl) derivative \u003cb\u003e38\u003c/b\u003e emerging as one of the most active members against MCF-7 (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.5 \u0026micro;M) and showing moderate potency against HepG2 (12.2 \u0026micro;M). Importantly, the addition of an extra methoxy group at the 3-position of the 6-phenyl ring diminished activity against HepG2 by 2.7-fold, highlighting the sensitivity of activity to subtle substituent modifications. These results clearly demonstrate that structural modifications within this scaffold strongly influence anticancer potency and selectivity. Compounds bearing trimethoxy-substituted aryl groups at the 4- and 6-positions consistently showed enhanced activity, particularly against the MCF-7 breast cancer cell line.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e, cytotoxicity of compounds \u003cb\u003e18\u0026ndash;24, 26\u0026ndash;41\u003c/b\u003e against MCF-7, HepG-2, and HCT-116 Cell lines.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIn vitro cytotoxicity IC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;M)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompd. No.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMCF-7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHepG-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHCT-116\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e45.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e44.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e34.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e48.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e36.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e49.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e49.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e63.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e78.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e98.0\u0026thinsp;\u0026plusmn;\u0026thinsp;8.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e49.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e32.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e57.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e41.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e36.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDOX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003eIC\u003csub\u003e50\u003c/sub\u003e values are expressed in \u0026micro;M. \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003eDOX.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2. Inhibition of Tubulin Polymerization in MCF-7 Cells\u003c/h2\u003e\u003cp\u003eTo assess whether the antiproliferative effects of the most active compounds were associated with tubulin disruption, ten derivatives (\u003cb\u003e20, 22, 24, 26, 27, 33, 35, 37, 38 and 41\u003c/b\u003e) were evaluated for their ability to inhibit tubulin polymerization in MCF-7 cells. Combretastatin A-4 (CA-4) was used as a positive control (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All tested compounds demonstrated significant inhibition of tubulin assembly. Among them, compounds \u003cb\u003e26, 20, 37\u003c/b\u003e, respectively showed the strongest activity, each surpassing CA-4 in potency. The remaining derivatives exhibited inhibitory activity comparable to that of CA-4. Importantly, there was a clear correlation between the extent of tubulin inhibition and the observed cytotoxicity for most compounds. For instance, \u003cb\u003e20\u003c/b\u003e and \u003cb\u003e26\u003c/b\u003e, which were the most potent antiproliferative agents (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.4 \u0026micro;M), also showed the highest inhibition of tubulin polymerization (74.7% and 75%, respectively). However, this relationship was not universal. For example, compound \u003cb\u003e41\u003c/b\u003e exhibited stronger antiproliferative activity than \u003cb\u003e37\u003c/b\u003e (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.4 \u0026micro;M vs. 2.6 \u0026micro;M), yet its effect on tubulin polymerization was weaker (61.2% vs. 74.3%). This indicates that additional mechanisms may contribute to the cytotoxicity of certain derivatives. These findings strongly support tubulin polymerization inhibition as a major mechanism underlying the cytotoxicity of the most active compounds, particularly \u003cb\u003e20\u003c/b\u003e and \u003cb\u003e26\u003c/b\u003e derivatives.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInhibition of tubulin polymerization (%) and cytotoxic activity (IC₅₀, \u0026micro;M) of selected compounds in MCF-7 cells\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompd.\u003c/p\u003e\u003cp\u003eNo.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003etubulin polymerization\u003c/p\u003e\u003cp\u003e%inhibition\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMCF-7\u003c/p\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;M)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e74.7%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e70.9%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e55.4%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e75%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e49.8%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e68.9%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e61.9%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e74.3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e71.7%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e61.2%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCA-4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e72.1%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e-----------\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3. Inhibition of Topoisomerase II in MCF-7 Cells\u003c/h2\u003e\u003cp\u003eFollowing the evaluation of antiproliferative activity and confirmation of tubulin polymerization inhibition, we further examined whether the most active compounds also target topoisomerase II (Topo II), aiming to identify potential dual inhibitors. A selected set of candidates was screened for their ability to inhibit Topo II in vitro in MCF-7 cells, with doxorubicin (DOX) used as a reference standard. Among the tested compounds, the active derivative \u003cb\u003e37\u003c/b\u003e exhibited the strongest Topo II inhibitory activity, surpassing that of doxorubicin (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In addition, \u003cb\u003e20\u003c/b\u003e displayed moderate Topo II inhibition. Taken together, these findings indicate that compounds \u003cb\u003e20\u003c/b\u003e and \u003cb\u003e37\u003c/b\u003e act as Topo II inhibitors, while compounds \u003cb\u003e26, 20, 37, 38 and 22\u003c/b\u003e function as potent tubulin inhibitors, respectively. Notably, \u003cb\u003e20\u003c/b\u003e and \u003cb\u003e37\u003c/b\u003e emerge as dual-acting agents, capable of interfering with both tubulin polymerization and Topo II function. The combined evidence suggests that the antiproliferative activity of this compound series is primarily mediated through tubulin inhibition, but with certain derivatives, particularly \u003cb\u003e20\u003c/b\u003e and \u003cb\u003e37\u003c/b\u003e, also exerting strong Topo II inhibition. This dual-target profile highlights these molecules as promising leads for the development of multitarget anticancer agents with enhanced therapeutic potential.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInhibition of Topo II (%) and cytotoxic activity (IC₅₀, \u0026micro;M) of selected compounds in MCF-7 cells.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompd.\u003c/p\u003e\u003cp\u003eNo.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTOPO II\u003c/p\u003e\u003cp\u003e% inhibition\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMCF-7\u003c/p\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e(\u0026micro;M)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"1\" nameend=\"c4\" namest=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e70.3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e42%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e55.3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e43.7%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e82.4%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDOX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e81.6%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.2.4. In vitro Propidium Iodide Flow Cytometry Cell Cycle Analysis\u003c/h2\u003e\u003cp\u003eArrest of cells in the G2/M phase is a hallmark of both tubulin polymerization inhibitors and Topoisomerase II inhibitors. To further elucidate the mechanism of action of the most active derivatives \u003cb\u003e20\u003c/b\u003e and \u003cb\u003e26\u003c/b\u003e, we performed cell cycle analysis in MCF-7 cells using propidium iodide. flow cytometry (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Compared with untreated MCF-7 cells, which displayed a distribution of 69.01% in G0/G1, 21.73% in S, and 9.26% in G2/M phase, treatment with compounds \u003cb\u003e20\u003c/b\u003e and \u003cb\u003e26\u003c/b\u003e caused a dramatic accumulation of cells in the G2/M phase. Specifically, the percentage of cells in G2/M increased to 43.3% for \u003cb\u003e20\u003c/b\u003e and 50.69% for \u003cb\u003e26\u003c/b\u003e, accompanied by a marked reduction in both G0/G1 and S populations. Notably, this arrest profile closely resembled that induced by the reference drugs CA-4 (tubulin inhibitor) and DOX (Topo II inhibitor). Taken together, these results confirm that compounds \u003cb\u003e20\u003c/b\u003e and \u003cb\u003e26\u003c/b\u003e exert their antiproliferative effects by blocking cell cycle progression at the G2/M phase, consistent with their observed tubulin polymerization inhibition. Compound \u003cb\u003e20\u003c/b\u003e also exhibited moderate Topo II inhibition, suggesting a dual mechanism of action. The combined evidence indicates that \u003cb\u003e20\u003c/b\u003e and \u003cb\u003e26\u003c/b\u003e suppress MCF-7 cell proliferation through induction of G2/M arrest, primarily \u003cem\u003evia\u003c/em\u003e disruption of tubulin dynamics, with \u003cb\u003e20\u003c/b\u003e additionally engaging Topo II. This dual contribution highlights their potential as multitarget anticancer agents with enhanced efficacy.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCell cycle analysis of compounds \u003cb\u003e20\u003c/b\u003e (2.4 \u0026micro;M), and \u003cb\u003e26\u003c/b\u003e (2.4 \u0026micro;M) in MCF-7 cell line.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompd. No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e%G0-G1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e%S\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e%G2/M\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e39.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e43.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e35.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50.69\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003econt. MCF-7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e69.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.2.5. Apoptosis and Necrosis Analysis by Annexin V-FITC/PI Staining\u003c/h2\u003e\u003cp\u003eTo further elucidate the mechanism underlying the cytotoxicity of the most active compounds, \u003cb\u003e20\u003c/b\u003e and \u003cb\u003e26\u003c/b\u003e, their ability to induce apoptosis in MCF-7 cells was assessed using Annexin V-FITC/PI dual staining followed by flow cytometry quantification and Fluorescence Microscopy detection (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Cells were treated at their respective IC₅₀ concentrations, and the distribution of viable, apoptotic, and necrotic populations was analyzed. Compared with untreated control cells, which showed minimal apoptosis (3.04% total apoptotic cells), treatment with compound \u003cb\u003e20\u003c/b\u003e markedly increased the proportion of apoptotic cells to 38.41% (11.91% early, 22.63% late), while compound \u003cb\u003e26\u003c/b\u003e induced an even higher apoptotic fraction of 41.26% (7.58% early, 28.16% late). Only minor increases in necrosis were observed (3.87% for 20 and 5.51% for 26), confirming apoptosis as the predominant mechanism of cell death. These findings are consistent with the results of tubulin polymerization and Topo II inhibition studies, as well as cell cycle analysis, where both \u003cb\u003e20\u003c/b\u003e and \u003cb\u003e26\u003c/b\u003e induced G2/M arrest in MCF-7 cells. The induction of apoptosis through G2/M blockade strongly supports disruption of microtubule dynamics as the primary mechanism of action. Furthermore, the dual activity of \u003cb\u003e20\u003c/b\u003e on both tubulin and Topo II may account for its slightly distinct apoptotic profile compared with \u003cb\u003e26\u003c/b\u003e. Compounds \u003cb\u003e20\u003c/b\u003e and \u003cb\u003e26\u003c/b\u003e effectively trigger apoptosis in MCF-7 cells, in line with their strong antiproliferative activity, inhibition of tubulin polymerization, and induction of G2/M arrest. The data firmly establish apoptosis as the main mode of cell death for these derivatives, underscoring their promise as potent anticancer leads with multitarget potential.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cem\u003eApoptosis and necrosis analysis of compounds\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e \u003cem\u003eand\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompd. No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eApoptosis\u003c/p\u003e\u003cp\u003eEarly\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNecrosis\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e38.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e22.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e41.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003econt.MCF-7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.3. In silico study\u003c/h2\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1. Aqueous-phase Density functional theory supports low energy tetrazole tautomers.\u003c/h2\u003e\u003cp\u003eAs previously mentioned, heteroaryl azides with an azido-group α to a pyridyl ring nitrogen are known to undergo intramolecular cyclization into tetrazole tautomers, especially within the context of six-membered heterocycles. To evaluate this behavior in our ligand series, density functional theory (DFT) calculations were run on relevant azido-compounds \u003cb\u003e26\u003c/b\u003e, \u003cb\u003e27\u003c/b\u003e, and \u003cb\u003e33\u003c/b\u003e. All geometries were first optimized using B3LYP/6-31G** in the aqueous phase using an aqueous continuum solvation model (PCM) before higher-level single point energies were run using B3LYP/6\u0026ndash;31\u0026thinsp;+\u0026thinsp;G**, which incorporated relevant diffuse electronic functions, which better described the delocalized nature of the tetrazole tautomers. From the resulting solution-phase energies (in Hartree), relative Gibbs free energies were obtained for each tautomeric form. Boltzmann distributions (at 298 K) were then used to estimate tautomeric populations. A summary of these results can be found in the figure below (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor \u003cb\u003e26\u003c/b\u003e, the tetrazole tautomer was predicted to be the global minimum, lying approximately ΔG\u0026thinsp;=\u0026thinsp;1.11 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (4.64 kJ mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) lower in energy than the corresponding azide form. This relatively large energy difference translated into an estimate equilibrium population of 86.8% (tetrazole) and only 13.2% (azide), supporting a strong thermodynamic preference for ring-closure in aqueous media. In \u003cb\u003e27\u003c/b\u003e, a similar but less pronounced trend was observed. The tetrazole tautomer was stabilized by ΔG\u0026thinsp;=\u0026thinsp;0.59 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (2.47 kJ mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) relative to the azide form, corresponding to a population distribution of 72.9% (tetrazole) compared to 27.1% (azide) under equilibrium conditions. Notably, the smaller energy gap suggests that both species may co-exist in solution, although the tetrazole form remains dominant. Finally, for \u003cb\u003e33\u003c/b\u003e, the energetics were reversed, revealing the azido-tautomer to be more stable by a value of ΔG\u0026thinsp;=\u0026thinsp;1.15 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (4.81 kJ mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). This translated into an estimated population of 12.6% (tetrazole) and 87.4% (azide), suggesting the open-ring form to be more stable in water. Overall, these results confirm that our compounds do exhibit energetically favorable bias towards the cyclized tetrazole form (in water), especially within the larger compounds \u003cb\u003e26\u003c/b\u003e, and \u003cb\u003e27\u003c/b\u003e. Overall, this data supports for the hypothesis that heteroaryl azides with proximal nitrogens can preferentially cyclize into tetrazoles.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2. Glide Docking against TopII, TopII-DNA, and α/β-tubulin\u003c/h2\u003e\u003cp\u003eTo gain insights into the molecular recognition and intermolecular forces that may drive the biochemical activity of our ligands, a series of docking experiments were run against our targets of interest - Topoisomerase II and tubulin protein. Given the inherently complex pharmacology of TopII (due to the presence of multiple binding sites), we elected to use two docking strategies for this target. First, we focused on the catalytic active site, using a human TopII ATPase domain crystal structure co-crystallized with AMP-PNP (1ZXM, r\u0026thinsp;=\u0026thinsp;1.87 \u0026Aring;), where we probed the orthosteric site. In parallel, we also selected a TopII-DNA crystal complex bound to anti-cancer chemotherapeutic etoposide (3QX3, r\u0026thinsp;=\u0026thinsp;2.16 \u0026Aring;) where we surveyed the DNA-protein interface as a potential binding site. In addition to Topo II, our ligands do inhibit tubulin polymerization, so we also evaluated protein-ligand docking against a tubulin-RB3-SLD-TTL complex co-crystallized with colchicine analog ABI-274 (6PC4, r\u0026thinsp;=\u0026thinsp;2.60 \u0026Aring;), where we focused on the colchicine-binding site within the α/β-tubulin heterodimer. These calculations involved the following ligands: \u003cb\u003e20, 22, 24, 26, 27, 33, 35, 37, 38, 41\u003c/b\u003e and corresponding \u003cb\u003e26\u003c/b\u003e (tet), \u003cb\u003e27\u003c/b\u003e (tet), \u003cb\u003e33\u003c/b\u003e (tet) tautomers. Doxorubicin was also evaluated against all 3 targets as a positive control. All protein targets first underwent a standard protein preparation workflow, including the filling in of missing residues, assignment of bond orders, and the optimization of hydrogen-bond networks. Ligand structures were also prepared using a LigPrep protocol, ensuring correct three-dimensional structures, ionization states (Epik, pH 7.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00), and low energy minimizations at phyioslogical pH. Receptor grids were generated for each protein target (10 x 10 x 10 \u0026Aring;) centered around the cognate co-crystallized ligands, before a glide docking protocol was performed (Glide-XP) enabling flexible sampling of ligand conformations within the prepared grids to deduce relevant binding poses and quantitative docking scores (in kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). This calculation was performed against TopII, TopII-DNA, and tubulin proteins, with the most relevant results summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. In the catalytic site within the TopII ATPase domain (1ZXM), several ligands were found to exhibit favorable docking scores. This includes positive control doxorubicin (-7.935 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), as well as the chloropyridyl aryl analog \u003cb\u003e24\u003c/b\u003e (-7.344 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), furanyl \u003cb\u003e33\u003c/b\u003e (tet) (-5.786 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and \u003cb\u003e22\u003c/b\u003e (-5.187 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). In the active site, \u003cb\u003e24\u003c/b\u003e was found to form several favorable hydrophobic contacts as well as a hydrogen bond between its aryl methoxy motif and nearby Asn91. The structurally simple \u003cb\u003e33\u003c/b\u003e (in the tetrazole form) was found to form a stable hydrogen bond network between a tetrazolo-nitrogen and its nearby cyano group with Asn150 in the TopII pocket. Similar to \u003cb\u003e24\u003c/b\u003e, compound \u003cb\u003e22\u003c/b\u003e also occupied the TopII active site in a similar pose, exhibiting two hydrogen bonds between the methoxy group and the peptide backbone of Tyr165 and Gly166. Binding poses within the TopII-DNA interface (3QX3) provided important insight into the benefits of the tetrazole tautomer (compared to the azide form). In this docking experiment, the ligands with the most favorable free energies of binding include control doxorubicin (-8.988 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), \u003cb\u003e26\u003c/b\u003e (tet, -8.560 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), \u003cb\u003e27\u003c/b\u003e (tet, -8.189 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), \u003cb\u003e33\u003c/b\u003e (tet, -7.561 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Interestingly, the highest affinity ligands were all in the tetrazole form, and the ligands returned improved values in the TopII-DNA interface compared to the TopII ATPase active site. As seen in the Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, this is primarily driven by the formation of favorable π-π stacking events between the aromatic tetrazole and methoxybenzene motifs and neighboring DNA nucleotides (e.g., DC8, DT9, DG13). π-π stacking is awell-established non-covalent intermolecular force, typically arising from quadrupole-quadrupole or dispersion forces. Depending on its geometry (e.g., face-to-face, edge-to-face, or offset-stacking), a standard π-π stacking event can contribute between ~\u0026thinsp;1\u0026ndash;3 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of energy to the observed binding affinity, often stabilizing protein-ligand interactions through aromatic ring complementarity. Compound \u003cb\u003e26\u003c/b\u003e (tet) optimally occupies the TopII-DNA interface and engaged the dsDNA strand via two π-π stacking events between the methoxy benzene and DT9 (thymine) as well as the tetrazole ring and DC8 (cytosine). \u003cb\u003e27\u003c/b\u003e (tet) was able to form similar interactions in the TopII-DNA interfacial pocket, with π-π stacking of its secondary methoxy benzene ring and DG13 (guanine), as well as a similar stacking event between its tetrazole motif and DC8 (cytosine). Finally, while the smaller \u003cb\u003e33\u003c/b\u003e (tet) lost hydrophobic contacts, it compensated them via three π-π stacking events, two between its furan core and the adenine system of DG13 (guanine), as well as its tetrazole and DC8 (cytosine). These interactions were unique to the tetrazole tautomers of compounds \u003cb\u003e26\u003c/b\u003e, \u003cb\u003e27\u003c/b\u003e, and \u003cb\u003e33\u003c/b\u003e. In the colchicine-binding site within the α/β-tubulin heterodimer (6PC4), the top ligands include \u003cb\u003e22\u003c/b\u003e (-7.716 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), doxorubicin (-7.218 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), \u003cb\u003e26\u003c/b\u003e (-7.121 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and \u003cb\u003e20\u003c/b\u003e (-7.060 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). First, \u003cb\u003e22\u003c/b\u003e occupied the colchicine-binding site in a canonical manner, with both methoxy benzene motifs on either end, and uniquely was found to exhibit a powerful π-cation stacking interaction between its central chloro-pyridine ring and a nearby positively charged Lys350. Unlike the standard π-π stacking event, a π\u0026ndash;cation interaction involves the electrostatic attraction between an aromatic π-system and a nearby positively charged residue (commonly Lys or Arg) and is generally stronger, contributing\u0026thinsp;~\u0026thinsp;5\u0026ndash;10 kcal\u0026middot;mol⁻\u0026sup1; as a function of distance and orientation. In addition, the pyridyl nitrogen was also found to have a hydrogen bond to nearby Asn256. Remarkably, compound \u003cb\u003e26\u003c/b\u003e retained the exact same molecular recognition profile, with the π\u0026ndash;cation interaction with Lys350 and hydrogen bond to Asn256.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, \u003cb\u003e20\u003c/b\u003e also maintained the same interactions with Lys350 and Asn256. This conservation of intermolecular forces strengthens our confidence in these predicted binding poses, given consistent interactions across three separate docking experiments.\u003c/p\u003e\u003cp\u003eOverall, our docking analyses demonstrate that the azido-tetrazole scaffolds favorably engage TopII and tubulin targets through a combination of hydrogen bonding, hydrophobic contacts, and aromatic π-π stacking or π-cation interactions. The tetrazole tautomers, in particular, displayed enhanced recognition at the TopoII-DNA interface, where multiple π\u0026ndash;π stacking events with nearby nucleotides improved binding affinities relative to the ATPase catalytic site. In tubulin, the identification of a robust π\u0026ndash;cation interaction with Lys350, conserved across several ligands, provided further confidence in the reliability of these predicted binding poses. Notably, lead compounds \u003cb\u003e20\u003c/b\u003e, and \u003cb\u003e26\u003c/b\u003e emerged among the most favorable binders across all three protein systems, supporting a possible binding mechanism to explain the observed biochemical activities against TopII and tubulin. Taken together, these results support the biological potential of this chemotype and highlight the value of tautomeric control and substituent tuning in optimizing noncovalent target engagement against clinically relevant proteins.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Conclusions","content":"\u003cp\u003eWe have synthesized a series of 2,4,6-trisubstituted nicotinonitriles \u003cb\u003e10\u0026ndash;41\u003c/b\u003e as a dual inhibitors of tubulin polymerization and Topoisomerase II. Compounds \u003cb\u003e20, 26, 41\u003c/b\u003e showed the highest \u003cem\u003ein vitro\u003c/em\u003e cytotoxic activity against MCF-7 cell line, while compounds \u003cb\u003e20, 26\u003c/b\u003e showed moderate activity against HepG2 cell line maintaining high selectivity toward normal cells. In enzyme-based assays, compounds \u003cb\u003e26, 20\u003c/b\u003e, and \u003cb\u003e37\u003c/b\u003e exhibited significant tubulin polymerization inhibitory activity. Compound \u003cb\u003e37\u003c/b\u003e showed strong inhibitory activity against Top II, while compound \u003cb\u003e20\u003c/b\u003e showed moderate Top II inhibitory activity. Compounds \u003cb\u003e20\u003c/b\u003e and \u003cb\u003e37\u003c/b\u003e exhibited the most pronounced dual-target activity, effectively disrupting tubulin polymerization and inhibiting Topo II. Studies confirmed their ability to induce G2/M phase arrest and trigger apoptosis in MCF-7 cells. Molecular docking analyses support the favorable binding interactions of these compounds with both targets, in line with the biological results. Collectively, these findings establish 2,4,6-trisubstituted nicotinonitriles as a promising structural scaffold for the future development of potent, selective, and dual-target anticancer therapeutics with improved clinical prospects.\u003c/p\u003e"},{"header":"4. Experimental section","content":"\u003cp\u003e\u003cem\u003e4.1. Chemistry\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll chemical reagents were purchased from commercial sources with a high percentage of purity. The melting points (\u0026deg;C) of the synthesized compounds were determined in open capillaries using Stuart melting point apparatus and were uncorrected. The NMR spectra analyses, IR spectra and UV-Vis analyses were carried out at the Applied Nucleic Acid Research Center, Faculty of Sciences, Zagazig University, Zagazig, Egypt. The mass spectra analyses and elemental analyses (C, H, N) were carried out at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Nasr City, Egypt. The \u003csup\u003e1\u003c/sup\u003eH-NMR and \u003csup\u003e13\u003c/sup\u003eC APT NMR spectra were recorded on a Bruker Advance III 400 MHz High Performance Digital FT-NMR spectrometer using dimethyl sulfoxide (DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) or deuterated chloroform (CDCl\u003csub\u003e3\u003c/sub\u003e) as a solvent. Chemical shifts were reported in \u0026delta; (ppm) using tetramethyl silane (TMS) as an internal standard. The IR spectra (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e) of the compounds were recorded on a Bruker Alpha FT-IR spectrometer. The UV-Visible analyses were carried out by UV-Visible Carry 90 instrument. Mass spectra were obtained using a GC/MS Mat 112 S mass spectrometer under the EI\u003csup\u003e+\u003c/sup\u003e ionization technique/mode. Elemental analyses were performed using a Vario MICRO cube (Elementar) CHNS analyzer. All reactions were monitored by thin layer chromatography (TLC) (R\u003csub\u003ef\u003c/sub\u003e) on silica gel 60 GF245 (E-Merck, Germany) using a UV lamp for visualization at a wavelength (\u0026lambda;) of 254 nm. Compounds \u003cstrong\u003e10, 11, 12, 15, 18\u003c/strong\u003e were previously reported\u003csup\u003e29\u003c/sup\u003e\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e35\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e4.1.1. \u003cem\u003eGeneral Method for Synthesis of\u0026nbsp;\u003c/em\u003e\u003cem\u003e4,6-disubstituted-3-cyano-2-pyridone derivatives\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003e10-17\u003c/strong\u003e). A mixture of selected ketone \u003cstrong\u003e4, 5, 6, 8, 9\u003c/strong\u003e (10 mmol), selected aldehyde \u003cstrong\u003e1, 2, 3, 7\u003c/strong\u003e (10 mmol), ethyl cyanoacetate (15 mmol, 1.59 ml) and ammonium acetate (80 mmol, 6.1 g) in absolute ethanol (30 ml) was heated to reflux for 12-14 h. The reaction mixture was monitored by TLC till finished then it was left to reach the room temperature. The formed precipitate was filtered off, washed successively with ethanol and dried under vacuum to afford pure compounds \u003cstrong\u003e10-17\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e6-(4-Methoxyphenyl)-2-oxo-4-(3,4,5-trimethoxyphenyl)-1,2-dihydropyridine-3-carbonitrile(\u003cstrong\u003e10\u003c/strong\u003e)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003csup\u003e29\u003c/sup\u003e\u003cstrong\u003e\u003csup\u003e-\u003c/sup\u003e\u003c/strong\u003e\u003csup\u003e31\u003c/sup\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eYellow solid; yield: (78%); mp: 270-272 ⁰C(lit.\u003csup\u003e31\u003c/sup\u003e \u0026lt;300 ⁰C); \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 12.63 (s, 1H, NH), 7.89 (d, \u003cem\u003eJ\u003c/em\u003e = 8.9 Hz, 2H, Ar-H), 7.08 (d, \u003cem\u003eJ\u003c/em\u003e = 9.0 Hz, 2H, Ar-H), 7.04 (s, 2H, Ar-H), 6.84 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridone), 3.85 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e), 3.83 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.73 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003csub\u003e6\u003c/sub\u003e\u003c/em\u003e) \u0026delta; ppm: 162.20, 161.72, 159.54, 152.85, 150.94, 139.11, 131.36, 129.49, 124.37, 116.98, 114.36, 106.07, 105.16, 97.02, 60.14, 56.16, 55.53; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3446 (NH), 3081 (CH\u003cspan dir=\"RTL\"\u003e-\u003c/span\u003earomatic),\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e2830 (CH-aliphatic), 2216 (CN), 1651 (C=O); UV/Vis: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e 375, 260 nm.\u003c/p\u003e\n\u003cp\u003e6-(3,4-Dimethoxyphenyl)-2-oxo-4-(3,4,5-trimethoxyphenyl)-1,2-dihydropyridine-3-carbonitrile\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003e11\u003c/strong\u003e)\u003csup\u003e26\u003c/sup\u003e. Yellow solid; yield (77%); mp: 275-277 ⁰C (lit.\u003csup\u003e26\u003c/sup\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003e250-252 ⁰C); \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 12.63 (s, 1H, NH), 7.54 (d, \u003cem\u003eJ\u003c/em\u003e = 8 Hz, 1H,Ar-H), 7.48 (s, 1H, Ar-H ), 7.10 (d, \u003cem\u003eJ\u003c/em\u003e = 8 Hz, 1H, Ar-H), 7.03 (s, 2H, Ar-H), 6.89 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridone), 3.86 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.86 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e), 3.83 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.74 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 162.12, 159.71, 152.89, 151.47, 148.79, 139.14, 131.43, 124.22, 121.17, 117.01, 111.70, 110.83, 106.10, 60.18, 56.20, 55.76; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3446 (NH), 3013 (CH-aromatic), 2831 (CH-aliphatic), 2215 (CN), 1651 (C=O); UV/Vis: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e 380 nm.\u003c/p\u003e\n\u003cp\u003e2-Oxo-4,6-bis(3,4,5-trimethoxyphenyl)-1,2-dihydropyridine-3-carbonitrile\u003cstrong\u003e(12)\u003c/strong\u003e\u003csup\u003e29\u003c/sup\u003e. Yellow solid; yield: (75%); mp: 280-282 ⁰C (lit.\u003csup\u003e29\u003c/sup\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003e286-288 ⁰C); \u003csup\u003e1\u003c/sup\u003eH NMR \u0026nbsp;(400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 12.68 (s, 1H, NH), 7.19 (s, 2H, Ar-H), 7.04 (s, 2H, Ar-H), 6.97 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridone), 3.88 (s, 12H, 4OCH\u003csub\u003e3\u003c/sub\u003e), 3.74 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 161.94, 159.80, 153.04, 152.86, 139.96, 139.14, 131.29, 127.22, 116.80, 106.13, 105.40, 60.14, 60.13, 56.21, 56.19; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3502 (NH), 3015 (CH-aromatic), 2837 (CH-aliphatic), 2221 (CN), 1646 (C=O); UV/Vis: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e 370 nm.\u003c/p\u003e\n\u003cp\u003e6-(3,4-Dimethoxyphenyl)-4-(3,5-dimethoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003e13\u003c/strong\u003e). \u0026nbsp;Yellow solid; yield: (78%); mp: 270-272 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 12.60 (s, 1H, NH), 7.49 (d, \u003cem\u003eJ\u003c/em\u003e = 8.5 Hz, 1H, Ar-H), 7.42 (s, 1H, Ar-H), 7.03 (d, \u003cem\u003eJ\u003c/em\u003e = 8.6 Hz, 1H, Ar-H), 6.78 (s, 2H, Ar-H), 6.78 (s, 1H, Ar-H), 6.62 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridone), 3.80 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.77 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.76 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 162.01, 160.48, 159.66, 151.46, 148.76, 138.10, 124.15, 121.14, 116.61, 111.65, 110.79, 106.31, 101.86, 55.72, 55.49; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/ cm\u003csup\u003e-1\u003c/sup\u003e): 3502 (NH), 3065 (CH-aromatic), 2834 (CH-aliphatic), 2222 (CN), 1645 (C=O); UV/Vis: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e 380 nm. 380; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;392.53\u0026nbsp;(M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e): C (67.34, 67.48), H (5.14, 5.23), N (7.14, 7.41).\u003c/p\u003e\n\u003cp\u003e4-(3,5-Dimethoxyphenyl)-6-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile(\u003cstrong\u003e14\u003c/strong\u003e). Yellow solid; yield: (76%); mp: 260-262 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 12.68 (s, 1H, NH), 7.90 (d, \u003cem\u003eJ\u003c/em\u003e = 7.8 Hz, 2H, Ar-H), 7.08 (d, \u003cem\u003eJ\u003c/em\u003e = 8.8 Hz, 2H, Ar-H), 6.85 (d, \u003cem\u003eJ\u003c/em\u003e = 2.1 Hz, 2H, Ar-H), 6.77 (s, 1H, Ar-H), 6.68 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridone), 3.84 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.82 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT-NMR (100 MHz, DMSO-\u003cem\u003ed\u003csub\u003e6\u003c/sub\u003e\u003c/em\u003e) \u0026delta; ppm: 161.77, 160.50, 138.07, 129.52, 116.59, 114.39, 106.29, 102.00, 55.54, 55.52; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3450 (NH), 3050\u003cu\u003e\u0026nbsp;\u003c/u\u003e(CH-aromatic), 2837 (CH-aliphatic), 2211 (CN), 1646 (C=O); UV/Vis: \u0026lambda;\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e375, 265 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;362.67 (M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e): C (69.60, 69.43), H (5.01, 5.20), N (7.73, 7.96).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e6-(3,4-Dimethoxyphenyl)-4-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile(\u003cstrong\u003e15\u003c/strong\u003e)\u003csup\u003e32\u003c/sup\u003e\u003cstrong\u003e\u003csup\u003e-\u003c/sup\u003e\u003c/strong\u003e\u003csup\u003e35\u003c/sup\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eYellow solid; yield: (77%); mp: 255-257⁰C(lit.\u003csup\u003e33\u003c/sup\u003e 262-264 ⁰C); \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 12.57 (s, 1H, NH), 7.72 (d, \u003cem\u003eJ\u003c/em\u003e = 8.8 Hz, 2H, Ar-H), 7.53 (d, \u003cem\u003eJ\u003c/em\u003e = 8.5 Hz, 1H, Ar-H), 7.47 (s, 1H, Ar-H), 7.12 (d, \u003cem\u003eJ\u003c/em\u003e = 8.8 Hz, 2H, Ar-H), 7.08 (d, \u003cem\u003eJ\u003c/em\u003e = 8.6 Hz, 1H, Ar-H), 6.80 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridone), 3.86 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.85 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.83 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e);\u003csup\u003e\u0026nbsp;13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 162.21, 161.06, 159.35, 151.40, 148.77, 130.02, 128.26, 124.24, 121.04, 117.08, 114.20, 111.69, 110.76, 55.74, 55.46; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3490 (NH), 3065 (CH-aromatic), 2904 (CH-aliphatic), 2222 (CN), 1651 (C=O); UV/Vis: \u0026lambda;\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e380\u003csub\u003e\u0026nbsp;\u003c/sub\u003enm.\u003c/p\u003e\n\u003cp\u003e4-(Furan-2-yl)-2-oxo-6-(3,4,5-trimethoxyphenyl)-1,2-dihydropyridine-3-carbonitrile (\u003cstrong\u003e16\u003c/strong\u003e). Yellow solid; yield: (78%) ; mp: 254-256 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 12.56 (s, 1H, NH), 8.14 (d, \u003cem\u003eJ\u003c/em\u003e = 1.3 Hz, 1H, furyl-H), 7.72 (d, \u003cem\u003eJ\u003c/em\u003e = 3.5 Hz, 1H, furyl-H), 7.18 (s, 2H, Ar-H), 7.08 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridone), 6.87 (dd, \u003cem\u003eJ\u003c/em\u003e = 3.6, 1.7 Hz, 1H, furyl-H), 3.91 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e), 3.74 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 153.02, 139.83, 116.79, 116.56, 113.34, 105.23, 60.14, 56.20; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3446 (NH), 3031 (CH-aromatic), 2838 (CH-aliphatic), 2217(CN), 1645 (C=O); UV/Vis: \u0026lambda;\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e385, 340 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;352.17 (M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e): C (64.77, 64.58), H (4.58, 4.72), N (7.95, 8.17).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4-(Furan-2-yl)-6-methyl-2-oxo-1,2-dihydropyridine-3-carbonitrile(\u003cstrong\u003e17\u003c/strong\u003e). Yellow solid; yield: (77%); mp: 221-223 ⁰C;\u003csup\u003e\u0026nbsp;1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 12.36 (s, 1H, NH), 8.05 (d, \u003cem\u003eJ\u003c/em\u003e = 1.2 Hz, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of furan), 7.54 (d, J = 3.6 Hz, 1H, C\u003csub\u003e3\u003c/sub\u003e-H of furan), 6.80 (dd, \u003cem\u003eJ\u003c/em\u003e = 3.6, 1.8 Hz, 1H, C\u003csub\u003e4\u003c/sub\u003e-H of furan), 6.59 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridone), 2.28 (s, 3H, CH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 161.66, 151.93, 147.60, 146.94, 145.30, 116.92, 115.85, 113.31, 101.33, 91.30, 19.20.\u003c/p\u003e\n\u003cp\u003e4.1.2. General procedure for synthesis of 3-cyano-2-chloro pyridine derivatives (\u003cstrong\u003e18-25\u003c/strong\u003e). A mixture of selected pyridone derivative \u003cstrong\u003e10-17\u003c/strong\u003e (5 mmol) and POCl\u003csub\u003e3\u003c/sub\u003e (25 mmol, 2.3 ml) in presence of \u003cem\u003eN,N-\u003c/em\u003edimethylaniline (15 mmol, 1.9 ml) was heated to reflux for 12-16 h. The reaction mixture was monitored by TLC till finished. It was cooled to room temperature and poured into crushed ice with stirring till precipitation. The formed precipitate was filtered off, washed successively with water, crystallized from ethanol and dried under vacuum to obtain pure compounds \u003cstrong\u003e18-25\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e2-Chloro-6-(4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)nicotinonitrile(\u003cstrong\u003e18\u003c/strong\u003e)\u003csup\u003e30\u003c/sup\u003e. Buff solid; yield: (81%); mp: 188-190⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta; ppm: 8.06 (d, \u003cem\u003eJ\u003c/em\u003e = 8.8 Hz, 2H, Ar-H), 7.69 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 7.02 (d, \u003cem\u003eJ\u003c/em\u003e = 8.8 Hz, 2H, Ar-H), 6.83 (s, 2H, Ar-H), 3.95 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e), 3.93 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.89 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 161.93, 158.63, 155.95, 152.97, 152.32, 139.13, 130.42, 129.50, 127.84, 118.40, 115.70, 114.48, 106.63, 105.20, 60.14, 56.21, 55.46; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3001 (CH-aromatic), 2831 (CH-aliphatic), 2224 (CN); UV/Vis: \u0026lambda;\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e340\u003csub\u003e\u0026nbsp;\u003c/sub\u003enm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;410.34\u0026nbsp;(M\u003csup\u003e+\u003c/sup\u003e), 412.07(M\u003csup\u003e+2\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eClN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e): C (64.32, 64.50), H (4.66, 4.75), N (6.82, 7.09).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2-Chloro-6-(3,4-dimethoxyphenyl)-4-(3,4,5trimethoxyphenyl)nicotinonitrile (\u003cstrong\u003e19\u003c/strong\u003e). Greenish solid; yield: (85%); mp: 212-214 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 8.22 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 7.88 (d, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 1H, Ar-H), 7.75 (s, 1H, Ar-H), 7.16 (s, 1H, Ar-H), 7.11 (s, 2H, Ar-H), 3.87 (s, 9H, 3OCH\u003csub\u003e3\u003c/sub\u003e), 3.76 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 159.22, 156.51, 153.45, 152.64, 152.24, 149.54, 139.57, 130.99, 128.42, 121.89, 119.16, 116.17, 112.24, 111.00, 107.14, 105.81, 60.63, 56.72, 56.24, 56.17; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3010 (CH-aromatic), 2940 (CH-aliphatic), 2220 (CN); UV/Vis: \u0026lambda;\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e350 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;440.22 (M\u003csup\u003e+\u003c/sup\u003e), 442.34 (M\u003csup\u003e+2\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e23\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eClN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e): C (62.66, \u0026nbsp;62.89), H (4.80, \u0026nbsp;4.72), N (6.35, 6.47).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2-Chloro-4,6-bis(3,4,5-trimethoxyphenyl)nicotinonitrile (\u003cstrong\u003e20\u003c/strong\u003e). Green solid; yield: (87%); mp: 244-246 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 8.32 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 7.51 (s, 2H, Ar-H), 7.12 (s, 2H, Ar-H), 3.88 (d, \u003cem\u003eJ\u003c/em\u003e = 7.5 Hz, 12H, 4OCH\u003csub\u003e3\u003c/sub\u003e), 3.75 (d, \u003cem\u003eJ\u003c/em\u003e = 3.0 Hz, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 158.70, 156.37, 153.39, 153.09, 152.17, 140.48, 139.22, 131.00, 130.54, 119.60, 115.66, 106.80, 106.23, 105.34, 60.32, 60.27, 56.35, 56.32; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3001 (CH-aromatic), 2831 (CH-aliphatic), 2224 (CN); UV/Vis: \u0026lambda;\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e345\u003csub\u003e\u0026nbsp;\u003c/sub\u003enm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;470.84 (M\u003csup\u003e+\u003c/sup\u003e), 472.28 (M\u003csup\u003e+2\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e24\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eClN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e): C (61.21, 61.43), H (4.92, 5.06), N (5.95, 6.12).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2-Chloro-6-(3,4-dimethoxyphenyl)-4-(3,5-dimethoxyphenyl)nicotinonitrile(\u003cstrong\u003e21\u003c/strong\u003e). Buff solid; yield: (86%); mp: 194-196 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 8.21 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 7.89 (dd, \u003cem\u003eJ\u003c/em\u003e = 8.5, 1.9 Hz, 1H, Ar-H), 7.76 (d, \u003cem\u003eJ\u003c/em\u003e = 1.8 Hz, 1H, Ar-H), 7.12 (d, \u003cem\u003eJ\u003c/em\u003e = 8.6 Hz, 1H, Ar-H), 6.91 (d, \u003cem\u003eJ\u003c/em\u003e = 2.1 Hz, 2H, Ar-H), 6.72 (s, 1H, Ar-H), 3.87 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.85 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.83 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 161.05, 159.36, 156.50, 152.59, 152.27, 149.55, 137.65, 128.38, 121.92, 119.14, 115.89, 112.26, 111.00, 107.45, 105.94, 102.36, 56.26, 56.17, 56.05; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3011 (CH-aromatic), 2841 (CH-aliphatic), 2233 (CN); UV/Vis: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e 350, 285 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;410.21\u0026nbsp;(M\u003csup\u003e+\u003c/sup\u003e), 412.38 (M\u003csup\u003e+2\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eClN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e): C (64.32, 64.57), H (4.66, 4.81), N (6.82, 7.04).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2-Chloro-4-(3,5-dimethoxyphenyl)-6-(4-methoxyphenyl)nicotinonitrile (\u003cstrong\u003e22\u003c/strong\u003e). Buff solid; yield: (84%); mp: 221-223⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 8.22 (d, \u003cem\u003eJ\u003c/em\u003e = 8.9 Hz, 2H, Ar-H), 8.14 (s, 1H, C\u003csub\u003e5-\u003c/sub\u003eH of pyridine), 7.09 (d, \u003cem\u003eJ\u003c/em\u003e = 8.9 Hz, 2H, Ar-H), 6.92 (d, \u003cem\u003eJ\u003c/em\u003e = 2.1 Hz, 2H, Ar-H), 6.71 (t, \u003cem\u003eJ\u003c/em\u003e = 2.1 Hz, 1H, Ar-H), 3.85 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.83 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR \u0026nbsp;(100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 162.44, 161.05, 159.25, 156.41, 152.75, 137.56, 130.00, 128.27, 118.89, 115.89, 114.98, 107.38, 105.81, 102.48, 56.03, 55.95; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3016 (CH-aromatic), 2841 (CH-aliphatic), 2224 (CN); UV/Vis: \u0026lambda;\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e335 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;380.77 (M\u003csup\u003e+\u003c/sup\u003e), 382.62 (M\u003csup\u003e+2\u003c/sup\u003e); \u0026nbsp;analysis (calcd., found for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eClN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e): C (66.23, 66.45), H (4.50, 4.61), N (7.36, 7.63).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2-Chloro-6-(3,4-dimethoxyphenyl)-4-(4-methoxyphenyl)nicotinonitrile (\u003cstrong\u003e23\u003c/strong\u003e). Brownish solid; yield: (88%); mp: 217-219 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR \u0026nbsp;(400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 8.16 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 7.88 (s, 1H, Ar-H), 7.76 (s, 2H, Ar-H), 7.17 (s, 2H, Ar-H), 6.71 (s, 1H, Ar-H), 6.63 (s, 1H, Ar-H), 3.86 (s, 9H, 3OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR \u0026nbsp; (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm:160.97, 158.63, 155.71, 152.26, 151.62, 148.97, 130.44, 128.77, 127.93, 127.32, 121.26, 118.32, 116.02, 115.70, 114.27, 112.31, 111.70, 110.38, 104.81, 55.70, 55.62, 55.42; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3079 (CH-aromatic), 2838 (CH-aliphatic), 222\u003cspan dir=\"RTL\"\u003e3\u0026nbsp;\u003c/span\u003e(CN); UV/Vis: \u0026lambda;\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e345\u003csub\u003e\u0026nbsp;\u003c/sub\u003enm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;380.25 (M\u003csup\u003e+\u003c/sup\u003e), 382.05 (M\u003csup\u003e+2\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eClN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e): C (66.23, 66.50), H (4.50, 4.62), N (7.36, 7.63). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2-Chloro-4-(furan-2-yl)-6-(3,4,5-trimethoxyphenyl)nicotinonitrile (\u003cstrong\u003e24\u003c/strong\u003e).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eGreen solid; yield: (89%) ; mp: 210-212 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 8.37 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 8.15 (s, 1H, furyl-H), 7.77 (s, 1H, furyl-H), 7.46 (s, 2H, Ar-H), 6.89 (s, 1H, furyl-H), 3.90 (d, \u003cem\u003eJ\u003c/em\u003e = 5.7 Hz, 6H, 2OCH3), 3.75 (s, 3H, OCH3); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 158.69, 153.35, 153.25, 152.66, 147.25, 142.68, 131.65, 130.85, 115.89, 115.63, 114.47, 114.30, 113.45, 105.11, 104.88, 100.49, 60.27, 56.44, 56.28; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3050 (CH-aromatic), 2835 (CH-aliphatic), 2227 (CN); UV/Vis: \u0026lambda;\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e360, 300, 255 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;370.31 (M\u003csup\u003e+\u003c/sup\u003e), 372.27(M\u003csup\u003e+2\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eClN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e): C (61.55, 61.79), H (4.08, 4.21), N (7.56, 7.80).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2-Chloro-4-(furan-2-yl)-6-methylnicotinonitrile (\u003cstrong\u003e25\u003c/strong\u003e). Yellow solid; yield: (77%); mp: 226-228 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta; ppm: 7.65 (s, 2H, C\u003csub\u003e5\u003c/sub\u003e-H of furan and C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 7.59 (s, 1H, C\u003csub\u003e3\u003c/sub\u003e-H of furan), 6.64 (d, \u003cem\u003eJ\u003c/em\u003e = 1.6 Hz, 1H, C\u003csub\u003e4\u003c/sub\u003e-H of furan), 2.63 (s, 3H, CH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta; ppm: 163.05, 153.97, 147.23, 145.67, 142.49, 117.10, 115.68, 115.56, 113.31, 100.71, 25.01.\u003c/p\u003e\n\u003cp\u003e4.1.3. General procedure for synthesis of 2-azido/tetrazolo-nicotinonitrile derivatives (\u003cstrong\u003e26-33\u003c/strong\u003e). To a solution of selected chloro pyridine derivatives \u003cstrong\u003e18-25\u003c/strong\u003e (3 mmol) in DMF, a solution of sodium azide (12 mmol, 0.7 gm) in least amount of water was dropped into, then the reaction mixture was allowed to be stirred for 12 h at 80⁰C. The reaction mixture was poured into water till precipitation then filtered off, washed successively with water then hexane and dried under vacuum to get compounds \u003cstrong\u003e26-33\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e5-(4-methoxyphenyl)-7-(3,4,5-trimethoxyphenyl)tetrazolo[1,5-a]pyridine-8-carbonitrile(\u003cstrong\u003e26\u003c/strong\u003e). Green solid; yield: (86%); mp: 184-186 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 8.23 (s, 2H, Ar-H), 7.90 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 7.24 (s, 2H, Ar-H), 7.10 (d, \u003cem\u003eJ\u003c/em\u003e = 14.2 Hz, 2H, Ar-H), 3.90 (s, 9H, 3OCH\u003csub\u003e3\u003c/sub\u003e), 3.79 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 162.40, 153.44, 152.43, 149.81, 141.65, 139.92, 132.07, 130.23, 121.81, 116.94, 114.64, 107.22, 93.61, 60.62, 56.60, 55.95; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3010 (CH-aromatic), 2841 (CH-aliphatic), 2227 (CN), 2126(N\u003csub\u003e3\u003c/sub\u003e); UV/Vis: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e 350, 295 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;417.46 (M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e): C (63.30, 63.43), H (4.59, 4.78), N (16.78, 17.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e5-(3,4-dimethoxyphenyl)-7-(3,4,5-trimethoxyphenyl)tetrazolo[1,5-a]pyridine-8-carbonitrile(\u003cstrong\u003e27\u003c/strong\u003e). Green solid; yield: (88%); mp: 165-167 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 7.91 (s, 2H, Ar-H), 7.78 (s, 1H, Ar-H), 7.24 (s, 2H, Ar-H), 7.08 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 3.89 (s, 15H, 5OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 153.62, 152.43, 149.96, 149.12, 141.87, 130.42, 124.17, 122.03, 117.34, 114.96, 113.59, 112.10, 107.74, 107.51, 93.93, 60.71, 56.80, 56.39, 56.34; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3022 (CH-aromatic), 2841 (CH-aliphatic), 2225 (CN), 2134 (N\u003csub\u003e3\u003c/sub\u003e); UV/Vis: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e 360 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;447.42 (M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e23\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e): C (61.74, 61.85), H (4.73, 4.90), N (15.65, 15.81).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e5,7-bis(3,4,5-trimethoxyphenyl)tetrazolo[1,5-a]pyridine-8-carbonitrile(\u003cstrong\u003e28\u003c/strong\u003e). Yellow solid; yield: (85%); mp: 212-214 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 8.03 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 7.52 (s, 2H, Ar-H), 7.25 (s, 2H, Ar-H), 3.90 (d, \u003cem\u003eJ\u003c/em\u003e = 5.2 Hz, 12H, 4OCH\u003csub\u003e3\u003c/sub\u003e), 3.80 (d, \u003cem\u003eJ\u003c/em\u003e = 6.8 Hz, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 153.63, 153.38, 152.57, 149.84, 141.72, 140.96, 140.10, 130.30, 125.11, 118.16, 114.85, 108.24, 107.52, 94.62, 60.77, 60.71, 56.83, 56.79; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3050 (CH- aromatic), 2838 (CH-aliphatic), 2226 (CN), 2135 (N\u003csub\u003e3\u003c/sub\u003e); UV/Vis: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e 350 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;477.74 (M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e24\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e): C (60.37, 60.51), H (4.86, 4.92), N (14.67, 14.85).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e5-(3,4-dimethoxyphenyl)-7-(3,5-dimethoxyphenyl)tetrazolo[1,5-a]pyridine-8-carbonitrile(\u003cstrong\u003e29\u003c/strong\u003e). Yellow solid; yield: (87%); mp: 178-180 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 7.90 (s, 2H, Ar-H), 7.77 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 7.24 (d, \u003cem\u003eJ\u003c/em\u003e = 7.5 Hz, 1H, Ar-H), 7.02 (s, 2H, Ar-H), 6.77 (s, 1H, Ar-H), 3.90 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.86 (s, 9H, 3OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 160.77, 152.00, 149.42, 148.67, 141.56, 136.62, 123.74, 121.48, 116.73, 114.17, 113.06, 111.58, 107.29, 102.30, 93.90, 55.92, 55.87, 55.66; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3005 (CH-aromatic), 2838 (CH-aliphatic), 2227 (CN); UV/Vis: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e 360 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;417.86 (M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e): C (63.30, 63.47), H (4.59, 4.73), N (16.78, 17.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e7-(3,5-dimethoxyphenyl)-5-(4-methoxyphenyl)tetrazolo[1,5-a]pyridine-8-carbonitrile\u003cstrong\u003e(30).\u003c/strong\u003e Yellow solid; yield: (84%); mp: 180-182 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 8.23 (d, \u003cem\u003eJ\u003c/em\u003e = 8.0 Hz, 2H, Ar-H), 7.84 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 7.21 (d, \u003cem\u003eJ\u003c/em\u003e = 8.2 Hz, 2H, Ar-H), 7.03 (s, 2H, Ar-H), 6.76 (s, 1H, Ar-H), 3.90 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.85 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 162.14, 160.75, 151.91, 149.41, 141.45, 136.53, 131.79, 121.46, 116.49, 114.31, 114.15, 107.22, 106.74, 102.37, 93.77, 55.64, 55.53; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3004 (CH-aromatic), 2840 (CH-aliphatic), 2218 (CN), 2128 (N\u003csub\u003e3\u003c/sub\u003e); UV/Vis: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e 350, 275 nm: 350, 275; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;387.49 (M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e): C (65.11, 65.37), H (4.42, 4.59), N (18.08, 18.31).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e5-(3,4-dimethoxyphenyl)-7-(4-methoxyphenyl)tetrazolo[1,5-a]pyridine-8-carbonitrile(\u003cstrong\u003e31\u003c/strong\u003e).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eBrown solid; yield: (86%); mp: 182-184 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 7.92 (d, \u003cem\u003eJ\u003c/em\u003e = 6.3 Hz, 2H, Ar-H), 7.87 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 7.78 (s, 1H, Ar-H), 7.74 (d, \u003cem\u003eJ\u003c/em\u003e = 4.6 Hz, 2H, Ar-H), 7.24 (d, \u003cem\u003eJ\u003c/em\u003e = 4.8 Hz, 2H, Ar-H), 3.91 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.90 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 161.47, 151.83, 141.40, 130.98, 126.86, 123.63, 121.61, 116.72, 114.63, 113.03, 111.58, 92.59, 55.92, 55.84, 55.59; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3011 (CH-aromatic), 2844 (CH-aliphatic), 2223 (CN), 2135 (N\u003csub\u003e3\u003c/sub\u003e); UV/Vis: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e 355, 255 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;387.69(M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e): C (65.11, 64.98), H (4.42, 4.61), N (18.08, 18.27).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e7-(furan-2-yl)-8-methyl-5-(3,4,5-trimethoxyphenyl)tetrazolo[1,5-a]pyridine(\u003cstrong\u003e32\u003c/strong\u003e). Yellow solid; yield: (88%); mp: 162-164 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 8.25 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 8.02 (s, 1H, furyl-H), 7.93 (s, 1H, furyl-H), 7.44 (s, 2H, Ar-H), 6.95 (s, 1H, furyl-H), 3.90 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e), 3.81 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 152.86, 149.90, 147.63, 141.57, 140.41, 137.99, 124.58, 117.62, 114.14, 113.83, 113.30, 107.90, 107.62, 87.81, 60.29, 56.33; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3002 (CH-aromatic), 2840 (CH-aliphatic), 2227 (CN), 2136 (N\u003csub\u003e3\u003c/sub\u003e); UV/Vis: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e 365, 300, 280 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;377.51 (M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e): C (60.48, 60.73), H (4.01, 4.20), N (18.56, 18.83).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e7-(furan-2-yl)-5-methyltetrazolo[1,5-a]pyridine-8-carbonitrile(\u003cstrong\u003e33\u003c/strong\u003e). Green solid; yield: (87%); mp: 228-230 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 8.21 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 7.87 (s, 1H, furyl-H), 7.73 (s, 1H, furyl-H), 6.91 (s, 1H,furyl-H), 2.93 (s, 3H, CH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 149.03, 147.96, 147.50, 141.50, 137.88, 116.95, 114.13, 113.86, 112.96, 87.29, 17.13; IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3001 (CH-aromatic), 2840 (CH-aliphatic), 2232 (CN); UV/Vis: \u0026lambda;\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e340, 275 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;225.63\u0026nbsp;(M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e11\u003c/sub\u003eH\u003csub\u003e7\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO): C (58.67, 58.90), H (3.13, 3.27), N (31.10, 30.98).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4.1.4. General procedure for Synthesis of 2-iminophosphorane-nicotinonitrile derivatives (\u003cstrong\u003e34-41\u003c/strong\u003e). A mixture of selected azido/tetrazolo compounds \u003cstrong\u003e26-33\u003c/strong\u003e (2 mmol) and triphenylphosphine (2.4 mmol, 629.49 mg) in toluene (15 ml) was heated to reflux for 15 minutes. The reaction mixture was followed by TLC until completion, then evaporated and the precipitate was washed with hexane to remove excess reagent. The pure fluorescent product was dried under vacuum to afford pure compounds \u003cstrong\u003e34-41\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e6-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2-((triphenyl-\u0026lambda;\u003csup\u003e5\u003c/sup\u003e-phosphaneylidene)amino)-nicotinonitrile (\u003cstrong\u003e34\u003c/strong\u003e). Buff solid; yield: (89%); mp: 211-213 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 7.86 (s, 6H, Ar-H ), 7.58 (s, 9H, Ar-H), 7.43 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 7.16 (s, 2H, Ar-H), 7.00 (s, 2H, Ar-H), 6.80 (s, 2H, Ar-H), 3.86 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e), 3.77 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003csub\u003e6\u003c/sub\u003e\u003c/em\u003e) \u0026delta; ppm: 164.89, 164.84, 160.65, 156.65, 154.81, 152.91, 138.31, 132.95, 132.64, 132.54, 131.65, 131.55, 130.48, 129.37, 129.08, 128.96, 128.86, 128.62, 128.37, 119.30, 113.82, 108.39, 106.10, 95.86, 95.62, 60.23, 56.16, 55.35;\u003csup\u003e\u0026nbsp;31\u003c/sup\u003eP NMR (162 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 26.31(s), 14.08(s); IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3010 (CH-aromatic), 2\u003cspan dir=\"RTL\"\u003e8\u003c/span\u003e3\u003cspan dir=\"RTL\"\u003e7\u0026nbsp;\u003c/span\u003e(CH-aliphatic), 2\u003cspan dir=\"RTL\"\u003e206\u003c/span\u003e (CN); UV/Vis: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e 355, 295 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;652.32 (M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e40\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eP): C (73.72, 73.51), H (5.26, 5.43), N (6.45, 6.72).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e6-(3,4-Dimethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2-((triphenyl-\u0026lambda;\u003csup\u003e5\u003c/sup\u003e-phosphaneylidene)-amino)nicotinonitrile (\u003cstrong\u003e35\u003c/strong\u003e). Grey solid; yield: (87%); mp: 212-214 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 7.89 (s, 6H, Ar-H), 7.63 (d, \u003cem\u003eJ\u003c/em\u003e = 2.9 Hz, 3H, Ar-H), 7.56 (s, 6H, Ar-H), 7.27 (d, \u003cem\u003eJ\u003c/em\u003e = 6.9 Hz, 1H, Ar-H), 7.21 (s, 1H, Ar-H), 7.12 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 6.99 (s, 2H, Ar-H), 6.87 (d, \u003cem\u003eJ\u003c/em\u003e = 6.9 Hz, 1H, Ar-H), 3.85 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e), 3.75 (d, \u003cem\u003eJ\u003c/em\u003e = 11.3 Hz, 6H, 2OCH3), 3.40 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e);\u003csup\u003e\u0026nbsp;13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 165.11, 157.02, 155.11, 153.26, 150.70, 148.98, 138.66, 133.31, 133.06, 132.96, 130.94, 129.70, 129.39, 129.27, 128.70, 120.57, 119.61, 111.75, 110.31, 109.06, 106.52, 96.37, 60.56, 56.53, 55.97, 55.66; \u003csup\u003e31\u003c/sup\u003eP NMR (162 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 25.64(s), 13.67(s); IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/\u003csup\u003e-1\u003c/sup\u003e): 3005(CH-aromatic), 2\u003cspan dir=\"RTL\"\u003e8\u003c/span\u003e3\u003cspan dir=\"RTL\"\u003e7\u003c/span\u003e(CH-aliphatic), 2\u003cspan dir=\"RTL\"\u003e207\u003c/span\u003e(CN); UV\u003cspan dir=\"RTL\"\u003e/\u003c/span\u003eVis: \u0026lambda;\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e375, 290 nm; MS, \u003cem\u003em/z\u003c/em\u003e: 681.47 (M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e41\u003c/sub\u003eH\u003csub\u003e36\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003eP): C (72.24, 72.43), H (5.32, 5. 39), N (6.16, 6.42).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4,6-Bis(3,4,5-trimethoxyphenyl)-2-((triphenyl-\u0026lambda;\u003csup\u003e5\u003c/sup\u003e-phosphaneylidene)amino)nicotinonitrile(\u003cstrong\u003e36\u003c/strong\u003e). Buff solid; yield: (90%); mp: 222-224 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 7.91 (dd, \u003cem\u003eJ\u003c/em\u003e = 12.0, 7.6 Hz, 6H, Ar-H), 7.64 (d, \u003cem\u003eJ\u003c/em\u003e = 6.7 Hz, 3H, Ar-H), 7.57 (d, \u003cem\u003eJ\u003c/em\u003e = 4.8 Hz, 6H, Ar-H), 7.31 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 7.00 (s, 2H, Ar-H), 6.95 (s, 2H, Ar-H), 3.87 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e), 3.75 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.67 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.54 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 164.98, 164.92, 156.88, 155.37, 155.33, 153.27, 153.25, 139.36, 138.67, 133.79, 133.21, 133.04, 132.94, 129.55, 129.39, 129.27, 128.55, 119.49, 109.64, 106.57, 104.75, 97.34, 97.09, 60.57, 56.54, 56.27; \u003csup\u003e31\u003c/sup\u003eP NMR (162 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 13.28(s); IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3003 (CH-aromatic), 2\u003cspan dir=\"RTL\"\u003e8\u003c/span\u003e3\u003cspan dir=\"RTL\"\u003e8\u0026nbsp;\u003c/span\u003e(CH-aliphatic), 2\u003cspan dir=\"RTL\"\u003e214\u003c/span\u003e (CN); UV/Vis: \u0026lambda;\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e370, 295 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;711.28 (M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e42\u003c/sub\u003eH\u003csub\u003e38\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003eP): C (70.88, 71.14), H (5.38, 5.47), N (5.90, 6.12).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e6-(3,4-Dimethoxyphenyl)-4-(3,5-dimethoxyphenyl)-2-((triphenyl-\u0026lambda;\u003csup\u003e5\u003c/sup\u003e-phosphaneylidene)amino)-nicotinonitrile\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003e37\u003c/strong\u003e).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eGrey solid; yield: (91%); mp: 135-137 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 7.90 (d, \u003cem\u003eJ\u003c/em\u003e = 6.8 Hz, 6H, Ar-H), 7.63 (d, \u003cem\u003eJ\u003c/em\u003e = 6.5 Hz, 6H, Ar-H), 7.57 (s, 3H, Ar-H), 7.40 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 7.24 (d, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 1H, Ar-H), 7.17 (s, 1H, Ar-H), 7.13 (s, 1H, Ar-H), 6.90 \u0026ndash; 6.78 (m, 2H, Ar-H), 6.63 (s, 1H, Ar-H), 3.79 (d, \u003cem\u003eJ\u003c/em\u003e = 18.7 Hz, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e), 3.37 (d, \u003cem\u003eJ\u003c/em\u003e = 30.8 Hz, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e);\u003csup\u003e\u0026nbsp;13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 164.61, 160.42, 156.66, 154.61, 150.28, 148.54, 139.48, 132.61, 132.51, 131.56, 131.46, 130.41, 129.21, 128.96, 128.84, 128.21, 120.13, 118.86, 111.29, 109.83, 108.53, 106.48, 101.04, 95.90, 55.51, 55.42, 55.22; \u003csup\u003e31\u003c/sup\u003eP NMR (162 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 27.10(s), 13.82(s); IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 29\u003cspan dir=\"RTL\"\u003e67\u0026nbsp;\u003c/span\u003e(CH-aromatic), 2\u003cspan dir=\"RTL\"\u003e8\u003c/span\u003e3\u003cspan dir=\"RTL\"\u003e5\u003c/span\u003e (CH-aliphatic), 2\u003cspan dir=\"RTL\"\u003e208\u003c/span\u003e (CN); UV/Vis: \u0026lambda;\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e370, 275 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;651.55 (M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e40\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eP): C (73.72, 73.50), H (5.26, 5.39), N (6.45, 6.67).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4-(3,5-Dimethoxyphenyl)-6-(4-methoxyphenyl)-2-((triphenyl-\u0026lambda;\u003csup\u003e5\u003c/sup\u003e-phosphaneylidene)amino)-nicotinonitrile\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003e38\u003c/strong\u003e).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eYellow solid; yield: (88%) ; mp: 128-130 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 7.86 (dd, \u003cem\u003eJ\u003c/em\u003e = 11.5, 7.9 Hz, 6H, Ar-H), 7.64 (d, \u003cem\u003eJ\u003c/em\u003e = 6.8 Hz, 3H, Ar-H), 7.58 (d, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 6H, Ar-H), 7.41 (d, \u003cem\u003eJ\u003c/em\u003e = 8.4 Hz, 2H, Ar-H), 7.11 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 6.81 (d, \u003cem\u003eJ\u003c/em\u003e = 6.3 Hz, 2H, Ar-H), 6.78 (s, 2H, Ar-H), 6.63 (s, 1H, Ar-H), 3.81 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e), 3.76 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 164.66, 160.56, 160.42, 156.61, 154.64, 139.48, 132.56, 132.46, 131.55, 131.46, 130.34, 129.27, 128.98, 128.86, 128.51, 128.27, 118.86, 113.72, 108.26, 106.43, 101.11, 95.82, 95.58, 55.41, 55.24; \u003csup\u003e31\u003c/sup\u003eP NMR (162 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 26.09(s), 14.09(s); IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): \u003cspan dir=\"RTL\"\u003e3012\u0026nbsp;\u003c/span\u003e(CH-aromatic), 2\u003cspan dir=\"RTL\"\u003e8\u003c/span\u003e40 (CH-aliphatic) 2\u003cspan dir=\"RTL\"\u003e207\u003c/span\u003e (CN); UV/Vis: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e 370, 265 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;621.40 (M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e39\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eP): C (75.35, 75.19), H (5.19, 5.30), N (6.76, 6.94).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e6-(3,4-Dimethoxyphenyl)-4-(4-methoxyphenyl)-2-((triphenyl-\u0026lambda;\u003csup\u003e5\u003c/sup\u003e-phosphaneylidene)amino)-nicotinonitrile\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003e39\u003c/strong\u003e).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ebrown solid; yield: (86%); mp: 129-131 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-d6) \u0026delta; ppm: 7.90 (s, 6H, Ar-H), 7.61 (d, \u003cem\u003eJ\u003c/em\u003e = 25.3 Hz, 9H, Ar-H), 7.41 (s, 3H, Ar-H), 7.25 (s, 2H, Ar-H), 7.12 (s, 2H, Ar-H), 6.87 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 3.81 (d, \u003cem\u003eJ\u003c/em\u003e = 26.1 Hz, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e), 3.41 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 164.67, 160.12, 156.47, 154.20, 150.16, 148.49, 132.57, 132.47, 132.32, 131.51, 131.41, 130.49, 129.68, 129.25, 128.89, 128.72, 128.68, 128.26, 119.96, 119.11, 114.03, 111.25, 109.79, 108.47, 95.63, 55.46, 55.30, 55.17; \u003csup\u003e31\u003c/sup\u003eP NMR (162 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 26.03(s), 13.73(s); IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): 3032 (CH-aromatic), 29\u003cspan dir=\"RTL\"\u003e3\u003c/span\u003e1 (CH-aliphatic), 2\u003cspan dir=\"RTL\"\u003e2\u003c/span\u003e02 (CN); UV/Vis: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e 370, 265 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;621.00 (M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e39\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eP): C (75.35, 75.12), H (5.19, 5.40), N (6.76, 7.04).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4-(Furan-2-yl)-6-(3,4,5-trimethoxyphenyl)-2-((triphenyl-\u0026lambda;\u003csup\u003e5\u003c/sup\u003e-phosphaneylidene)amino)nicotino-nitrile\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003e40\u003c/strong\u003e).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e Yellow solid; yield: (86%) ; mp: 230-232 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 8.01 (d, \u003cem\u003eJ\u003c/em\u003e = 1.1 Hz, 1H, furyl-H), 7.89 (dd, \u003cem\u003eJ\u003c/em\u003e = 12.2, 7.3 Hz, 6H, Ar-H), 7.64 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 3H, Ar-H), 7.60 \u0026ndash; 7.52 (m, 6H, Ar-H), 7.48 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 7.21 (dd, \u003cem\u003eJ\u003c/em\u003e = 27.4, 7.4 Hz, 1H, furyl-H), 6.93 (s, 2H, Ar-H), 6.80 (dd, \u003cem\u003eJ\u003c/em\u003e = 3.5, 1.7 Hz, 1H, furyl-H), 3.68 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.56 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 165.40, 157.21, 153.32, 149.43, 148.33, 141.97, 139.50, 133.76, 133.67, 133.07, 132.97, 129.55, 129.41, 129.29, 128.56, 119.47, 113.25, 104.60, 92.42, 60.57, 56.28; \u003csup\u003e31\u003c/sup\u003eP NMR (162 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 28.17(s), 14.19(s); IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): \u003cspan dir=\"RTL\"\u003e3012\u0026nbsp;\u003c/span\u003e(CH-aromatic), 29\u003cspan dir=\"RTL\"\u003e3\u003c/span\u003e4 (CH-aliphatic), 2\u003cspan dir=\"RTL\"\u003e207\u003c/span\u003e (CN); UV/Vis: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e 385, 300 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;611.45 (M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e37\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eP): C (72.66, 72.52), H (4.94, 5.11), N (6.87, 7.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4-(Furan-2-yl)-6-methyl-2-((triphenyl-\u0026lambda;\u003csup\u003e5\u003c/sup\u003e-phosphaneylidene)amino)nicotinonitrile (\u003cstrong\u003e41\u003c/strong\u003e)\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eCocoa solid; yield: (88%); mp: 222-224 ⁰C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 7.93 (s, 1H, C\u003csub\u003e5\u003c/sub\u003e-H of pyridine), 7.86 (dd, \u003cem\u003eJ\u003c/em\u003e = 12.0, 7.3 Hz, 6H, Ar-H), 7.67 \u0026ndash; 7.61 (m, 3H, Ar-H), 7.60 \u0026ndash; 7.53 (m, 6H, Ar-H), 7.40 (d, \u003cem\u003eJ\u003c/em\u003e = 3.4 Hz, 1H, furyl-H), 6.80 (s, 1H, furyl-H), 6.73 (d, \u003cem\u003eJ\u003c/em\u003e = 1.6 Hz, 1H, furyl-H), 2.04 (s, 3H, CH\u003csub\u003e3\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC APT NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 160.04, 144.99, 140.67, 132.84, 132.74, 132.27, 128.93, 128.74, 128.62, 127.93, 119.08, 112.55, 112.29, 106.74, 23.71; \u003csup\u003e31\u003c/sup\u003eP NMR (162 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; ppm: 14.19(s); IR (ATR, \u0026nu;\u003csub\u003emax\u003c/sub\u003e/cm\u003csup\u003e-1\u003c/sup\u003e): \u003cspan dir=\"RTL\"\u003e30\u003c/span\u003e54 (CH-aromatic), 2840 (CH-aliphatic), 2\u003cspan dir=\"RTL\"\u003e20\u003c/span\u003e6 (CN); UV/Vis: \u0026lambda;\u003csub\u003emax\u003c/sub\u003e 360, 295 nm; MS, \u003cem\u003em/z\u003c/em\u003e:\u0026nbsp;459.13 (M\u003csup\u003e+\u003c/sup\u003e); analysis (calcd., found for C\u003csub\u003e29\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eOP): C (75.81, 75.68), H (4.83, 4.97), N (9.15, 9.41).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAbout the characterization data of all the final synthesized compounds (\u003csup\u003e1\u003c/sup\u003eH-NMR, \u003csup\u003e13\u003c/sup\u003eC-APT NMR, IR spectra, UV-Vis analyses, Mass spectra and Elemental analyses), see supplementary information (Fig. S1-S161).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2. Biological assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.1. In vitro anticancer activity.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.1.1. Materials and Methods\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRoswell Park Memorial Institute (RPMI) 1640 medium was purchased from Sigma Chem. Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) and fetal calf serum (FCS) were purchased from Gibco, UK. Dimethyl sulfoxide (DMSO) and methanol were of HPLC grade, and all other reagents and chemicals were of analytical reagent grade.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.1.2. Cell culture\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHepG-2 (Human liver carcinoma), HCT116 (human colorectal carcinoma), MCF-7 (human breast adenocarcinoma), and the normal human skin fibroblast (BJ-1) cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in RPMI-1640 medium which was supplemented with 10% heat-inactivated FBS, 100U/ml penicillin and 100U/ml streptomycin. The cells were grown at 37\u0026deg;C in a humidified atmosphere of 5% CO2. All experiments were conducted thrice in triplicate (n = 3). All the values were represented as means \u0026plusmn; SD. Significant differences between the means of parameters as well as IC50s were determined by probit analysis using SPSS software program (SPSS Inc., Chicago, IL).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.1.3. Lactate dehydrogenase (LDH) assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the effect of each synthesized compound on membrane permeability in HepG2, MCF-7 and HCT-116 cancer cell lines as well as BJ-1 normal cell line, a lactate dehydrogenase (LDH) release assay was used\u003csup\u003e36\u0026ndash;40\u003c/sup\u003e. The cells were seeded in 24-well culture plates at a density of 1 \u0026times; 104 cells/well in 500 \u0026mu;L volume and allowed to grow for 18h before treatment. After treatment with a series of different concentrations of each compound or Doxorubicin\u0026reg; (positive control), the plates were incubated for 48h. Then, the supernatant (40 \u0026mu;L) was transferred to a new 96 well to determine LDH release and 6% triton X-100 (40 \u0026mu;L) was added to the original plate for determination of total LDH. An aliquot of 0.1 M potassium phosphate buffer (100 \u0026mu;L, pH 7.5) containing 4.6 mM pyruvic acid was mixed to the supernatant using repeated pipetting. Then, 0.1 M potassium phosphate buffer (100 \u0026mu;L, pH 7.5) containing 0.4 mg/mL reduced \u0026beta;-NADH was added to the wells. The kinetic changes were read for 1 min using ELISA microplate reader in absorbance at wavelength 340 nm. This procedure was repeated with 40 \u0026mu;L of the total cell lysate to determine total LDH. The percentage of LDH release was determined by dividing the LDH released into the media by the total LDH following cell lysis in the same well.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.1.4. Statistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were conducted in triplicate (n = 3). All the values were represented as mean \u0026plusmn; SD. Significant differences between the means of parameters as well as IC50s were determined by probit analysis using SPSS software program (SPSS Inc., Chicago, IL).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.2.\u0026nbsp; \u0026nbsp;Inhibition of Tubulin Polymerization in MCF-7 Cells\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.2.1. Materials\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBeta-Tubulin in vitro SimpleStep ELISA\u0026reg; (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of Beta-Tubulin protein in human cell and tissue homogenate extract samples. This is performed for most active compounds \u003cstrong\u003e20, 22, 24, 26, 27, 33, 35, 37, 38, 41\u003c/strong\u003e and CA-4 as control against MCF-7 cancer cell line to measure the percentage of \u0026beta;-tubulin polymerization inhibition.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.2.2. Methodolgy\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe SimpleStep ELISA\u0026reg; employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB Development Solution is added and during incubation is catalyzed by Horseradish Peroxidase (HRP), generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm. The concentration of Beta-Tubulin protein in the samples is determined by comparing the optical density (OD) of the samples to the standard curve. Each experiment was repeated two times (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.3. Inhibition of Topoisomerase II in MCF-7 Cells\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.3.1. Materials\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMouse TOP2B /Topoisomerase II Beta ELISAKit (Sandwich ELISA) is designed for the quantitative measurement of Topo II enzyme. This is performed for most active compounds \u003cstrong\u003e20, 22, 24, 26, 37\u003c/strong\u003e and doxorubicin as control against MCF-7 cancer cell line to measure the percentage of Topo II enzyme inhibition.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.3.2. Methodology\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis assay is based on the sandwich ELISA principle. Each well of the supplied microtiter plate has been pre-coated with a target specific capture antibody. Standards or samples are added to the wells and the target antigen binds to the capture antibody. Unbound Standard or sample is washed away. A biotin-conjugateddetection antibody is then added which binds to the captured antigen. Unbound detection antibody is washed away. An Avidin-Horseradish Peroxidase (HRP) conjugate is then added which binds to the biotin. Unbound Avidin-HRP conjugate is washed away. A TMB substrate is then added which reacts with the HRP enzyme resulting in color development. A sulfuric acid stop solution is added to terminate color development reaction and then the optical density (OD) of the well is measured at a wavelength of 450 nm \u0026plusmn; 2 nm. An OD standard curve is generated using known antigen concentrations; the OD of an unknown sample can then be compared to the standard curve in order to determine its antigen concentration. Each experiment was repeated two times (Table 3).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.4. In vitro Propidium Iodide Flow Cytometry Cell Cycle Analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.4.1. Materials\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePropidium Iodide Flow Cytometry Kit (ab139418) is designed for quantitative DNA content analysis in tissue culture cells so we performed Propidium Iodide cell cycle analysis for active compounds \u003cstrong\u003e20, 26\u003c/strong\u003e using MCF-7 cell line. Propidium iodide staining of DNA is the classic means of cell cycle analysis. The staining procedure takes less than 1 hour of total processing time and cells fixed in ethanol are stable for at least several weeks at 4\u0026ordm;C.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.4.2. Methodology\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePropidium iodide is a fluorescent molecule that binds nucleic acid with little or no sequence preference. Because Propidium iodide binds RNA as well as DNA, RNaseA (ribonuclease A) is included in this kit to digest cellular RNA and thus decrease background RNA staining from the experiment. Since Propidium iodide is membrane impermeant, ethanol is used to both fix and permeabilize cells. A flow cytometer is required for quantitative analysis. First, fix MCF-7 cells in 66% Ethanol, store at +4\u0026deg;C for 2 hours to 4 weeks and rehydrate cells in PBS. Finally, stain cells with Propidium iodide then add Rnase for 30min. Collect Propidium iodide fluorescence intensity on FL2 of a flow cytometer and 488nM laser excitation. A useful way to display Propidium iodide data is on a histogram with the cell count on the y-axis and the propidium iodide fluorescence intensity on the x-axis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.5. Apoptosis and Necrosis Analysis by Annexin V-FITC/PI Staining\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.5.1. Materials\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAnnexin V Apoptosis Detection Kit is used for analyzing apoptosis and necrosis for active compounds \u003cstrong\u003e20, 26\u003c/strong\u003e using Annexin V-FITC/PI dual staining followed by flow cytometry quantification and Fluorescence Microscopy detection.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.5.2. Methodology\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDetection is based on the observation that soon after initiating apoptosis, cells translocate the membrane phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface. Once on the cell surface, PS can be easily detected by staining with a fluorescent conjugate of Annexin V, a protein that has a high affinity for PS. The one-step staining procedure takes only 10 minutes. Detection can be analyzed by flow cytometry or by fluorescence microscopy. The kit can differentiate between apoptosis and necrosis when performing both Annexin V-FITC and PI staining. Assay is summarized as first, Induce apoptosis by desired method, Collect 1-5 x 105 cells by centrifugation, Resuspend cells in 500 \u0026mu;l of 1X Binding Buffer, Add 5 \u0026mu;l of Annexin V-FITC and 5 \u0026mu;l of propidium iodide and finally Incubate at room temperature for 5 min in the dark. Analyze Annexin V-FITC binding by flow cytometry (Ex = 488 nm; Em = 530 nm) using FITC signal detector (usually FL1) and PI staining by the phycoerythrin emission signal detector (usually FL2). Place the cell suspension on a glass slide. Cover the cells with a glass coverslip. Observe the cells under a fluorescence microscope using a dual filter set for FITC \u0026amp; Rhodamine (Cells that have bound Annexin V-FITC will show green staining in the plasma membrane. Cells that have lost membrane integrity will show red staining (PI) throughout the nucleus and a halo of green staining (FITC) on the cell surface (plasma membrane).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2.6. In silico study\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eYou will find general Computational Modelling and Docking in addition to DFT calculations in the supplementary file in S4.2.6 part.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interest:\u003c/h2\u003e\u003cp\u003eThere are no conflicts to declare.\u003c/p\u003e\u003ch2\u003eFunding statement:\u003c/h2\u003e\u003cp\u003eYasir S. Raouf is supported by an internal SQU-UAEU grant (Grant #12S244) and Dr. Shaikha Alneyadi is supported by grant number G00004741.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eEman S. Hassan: Conceptualization, investigation, methodology, and manuscript writing.Abdalla E. A. Hassan: Conceptualization, data analysis, and manuscript writing.Shaikha Alneyadi: Conceptualization, data analysis, and manuscript writing.Yassir S. Raouf: Conceptualization, methodology, and manuscript writing.Hanem M. Awad: Conceptualization, methodology, and manuscript writing.Zakaria K.M. Abdel-Samii: Conceptualization, data analysis, and manuscript writing.Amany M.M. Al-Mahmoudy: Conceptualization, data analysis, and manuscript writing.Reham A. Abou-Elkhair: Conceptualization, data analysis, and manuscript writing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Dr. Sara Hosny for recording NMR Data and Ms. Eman Hafez for recording FT-IR and UV-vis data. We thank STDF for the Capacity Building grant # 2698, Dr. Yasir S. Raouf is supported by an internal SQU-UAEU grant (Grant #12S244), Dr. Shaikha Alneyadi is supported by grant number G00004741\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData provided within the manuscript or supplementary information files. The raw data for the DFT calculations and docking studies will be shared upon submitting requestto Dr. Yasir S. Raouf.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChen, S. et al. 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Intermed\u003c/em\u003e. \u003cb\u003e43\u003c/b\u003e, 437\u0026ndash;456 (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"2,4,6-trisubstituted nicotinonitriles, azido/tetrazolopyridines. iminophosphorane, tubulin polymerization inhibitors, Topoisomerase II inhibitors, Combretastatin (A-4)","lastPublishedDoi":"10.21203/rs.3.rs-7890915/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7890915/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA series of 2,4,6-trisubstituted nicotinonitriles, compounds \u003cb\u003e10\u003c/b\u003e\u0026ndash;\u003cb\u003e41\u003c/b\u003e, designed as pyridine-bridged analogs of combretastatin A-4 (CA-4), was synthesized to function as dual inhibitors of Topoisomerase II (Topo II) and tubulin polymerization. The anticancer potential of the synthesized compounds was evaluated against three cancer cell lines_MCF-7, HepG2, and HCT-116 using the LDH assay. Notably, several compounds \u003cb\u003e20, 26, 41, 38, 24, 27, 37, 23, 22, 33, 35, 19, 21\u003c/b\u003e, respectively demonstrated superior cytotoxic activity against MCF-7 cells and compounds \u003cb\u003e20, 26, 38, 41, 39\u003c/b\u003e, respectively showed moderate activity against HepG2 when compared to Doxorubicin, while maintaining good selectivity towards normal BJ-1 cells. Among these, compounds \u003cb\u003e26\u003c/b\u003e, \u003cb\u003e20\u003c/b\u003e, and \u003cb\u003e37\u003c/b\u003e, respectively exhibited significant tubulin polymerization inhibitory activity (\u003cb\u003e26\u003c/b\u003e, 75% inhibition), (\u003cb\u003e20\u003c/b\u003e, 74.7% inhibition), (\u003cb\u003e37\u003c/b\u003e, 74.3% inhibition), compared to CA-4 (72.1% inhibition). Compound \u003cb\u003e37\u003c/b\u003e showed strong inhibitory activity against Topo II (82.4% inhibition), while compound \u003cb\u003e20\u003c/b\u003e showed moderate Topo II inhibitory activity (70.3% inhibition) compared with Doxorubicin (81.6% inhibition), highlighting the dual-target nature of these molecules. Cell cycle analysis further revealed that compounds \u003cb\u003e20\u003c/b\u003e and \u003cb\u003e26\u003c/b\u003e induced G2/M phase arrest in MCF-7 cells at rates of 43.30% and 50.69%, respectively, along with evidence of apoptosis induction. Molecular docking studies confirmed the favorable binding interactions of these compounds with both Tubulin and Topo II, aligning well with the in vitro findings. These findings underscore the potential of 2,4,6-trisubstituted nicotinonitriles as promising dual-target anticancer agents and pave the way for more potent derivatives with enhanced therapeutic efficacy.\u003c/p\u003e","manuscriptTitle":"Nicotinonitrile-Based Dual Inhibitors of Tubulin and Topoisomerase II: Design, Synthesis, and Anticancer Evaluation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-11 18:38:33","doi":"10.21203/rs.3.rs-7890915/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-25T12:07:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-17T06:47:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158920538163145452152860703676578028667","date":"2025-11-02T05:06:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-31T04:48:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-31T04:42:35+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-24T03:40:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-22T19:31:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-22T19:25:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7fa4ca7b-4be0-42bf-849b-32abdc7f6646","owner":[],"postedDate":"November 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":57695648,"name":"Biological sciences/Biochemistry"},{"id":57695649,"name":"Biological sciences/Cancer"},{"id":57695650,"name":"Biological sciences/Chemical biology"},{"id":57695651,"name":"Physical sciences/Chemistry"},{"id":57695652,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2026-04-06T05:09:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-11 18:38:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7890915","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7890915","identity":"rs-7890915","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}