Synthesis of novel 4-substituted isatin Schiff base derivatives as potential autophagy inducers and evaluation of their antitumour activity

Synthesis of novel 4-substituted isatin Schiff base derivatives as potential autophagy inducers and evaluation of their antitumour activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis of novel 4-substituted isatin Schiff base derivatives as potential autophagy inducers and evaluation of their antitumour activity Huayuan Tan, Guanglong Zhang, Chenlu Xu, Xue Lei, Jiayi Chen, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4633192/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Aug, 2024 Read the published version in Molecular Diversity → Version 1 posted 9 You are reading this latest preprint version Abstract Autophagy is a potential target in cancer therapy. In the present study, we designed and synthesized a series of isatin Schiff base derivatives containing thioether structures. After discovering the highly active target compound H13 (IC50 = 4.83 μM) based on in vitro antiproliferation, we also found it had a high safety against normal cells HEK293 with CC50 of 69.01 μM. In addition, to provide reference for subsequent studies, a model was successfully constructed by Sybyl software. Preliminary mechanistic studies suggested that H13-induced apoptosis may be closely related to ROS accumulation and mitochondrial dysfunction. Subsequent studies revealed that H13 inhibited cell proliferation by inducing cellular autophagy mainly through blocking signal of the PI3K/AKT/mTOR pathway. Altogether, these results suggested that H13 was potentially valuable as a lead compound. Isatin Schiff bases antitumor activity 3D-QSAR autophagy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction To date, tumors remain a major threat to human health[ 1 ]. The World Health Organization (WHO) predicts an increase of 29.5 million cancer cases by 2040[ 2 ]. According to the WHO, only 12 countries will be able to reduce cancer mortality by more than 30% by 2030[ 3 ]. Chemotherapy has long been an important drug therapy approach in oncology[ 4 ], and the greatest drawback of chemotherapy is its inability to distinguish between cancer and normal cells, leading to significant toxic side effects[ 5 – 7 ]. This also highlights the need for a continuous search for an efficient and less toxic small molecule inhibitor. Much evidence suggests the therapeutic role of autophagy modulators in cancer treatment. Therefore, pharmacological intervention of the autophagy pathway may be a promising strategy for the treatment of a wide range of pathological conditions. Isatin (1H-indole-2,3-dione) has been found to be a common structural motif in a variety of dyes, pesticides and pharmacologically active compounds due to its unique size and specific electronic properties[ 8 – 10 ]. Modern pharmacological studies have found that compounds containing these structures have a wide spectrum of biological activity[ 11 ], such as antitumor[ 12 – 14 ], antiviral[ 15 – 17 ], bactericidal[ 18 , 19 ], anti-inflammatory[ 20 , 21 ], anti-hypertensive[ 22 ], anti-cardiovascular disease[ 23 ] and so on. Chemicals and drug candidates based on isatin have been applied in the pharmaceutical industry[ 24 ]. For example, sunitinib, an oral tyrosine kinase inhibitor, was approved in 2006 for the treatment of metastatic renal cell carcinoma and imatinib-resistant gastrointestinal mesenchymal tumors[ 25 ]. In addition, isatin scaffolds are present in nintedanib, Intedanib, Semaxanib and TSU-68. Structurally, these marketed drugs are all close analogs of sunitinib, as they all share the isatin backbone. In recent years, compounds containing hydrazide or hydrazone fractions have been attractive for new drug development because of their potential multifunctional biological activity, including antiproliferative activity[ 26 , 27 ]. It has been reported that the antiproliferative activity of these hydrazones may be attributed to kinase inhibition[ 28 – 30 ], through free radical production[ 31 ] or involved in the regulation of p53[ 32 ]. Inspired by such interesting and potentially valuable research of isatin and hydrazone fractions, in this study, we designed and synthesized 28 thioether-containing derivatives of isatin Schiff base. Subsequently, growth inhibitory activity of the synthesized target compounds against four tumor cell lines was determined. In addition, the preliminary mechanism of this type of isatin Schiff base derivatives against human non-small cell lung cancer cell line (A549) was examined. 2 Results 2.1 Chemistry The synthesis route of compounds H1 - H28 is shown in Scheme 1 . The commercially available 4-chloroindigo was converted into intermediate 2 in N,N -dimethylformamide (DMF) by reacting with thiophenol substituted at different positions in the presence of caesium carbonate. The target compounds H1-H28 were obtained by the condensation reaction of intermediate 2 with various hydrazides in methanol. All the target compounds were confirmed by 1 H NMR, 13 C NMR, and HRMS. In addition, H23 , H24 and H25 were also characterized by 19 F NMR. Satisfactorily, the yields of all the target compounds were between 76%-89%. 2.2 In vitro antiproliferative activity In the present study, in vitro antiproliferative activity of all the target compounds was evaluated by MTT assay against A549, prostate (PC-3), chronic myeloid leukemia (K562) and liver (HepG2) cell lines using 5-fluorouracil ( 5-Fu ) as a positive control. After treatments with compounds H1 - H28 for 48 h, most of the target compounds showed good inhibitory activity against A549, PC-3, HepG2 and K562 cancer cells, respectively. H3 and H13 showed significant inhibitory activity against A549 cells, which was superior to that of 5-Fu (Table 1 ). Meanwhile, we evaluated the cytotoxicity of H6 , H8 , H10 , H12 , H13 , H14 and H18 on normal cells HEK293 using 5-Fu as a control (Table 2 ). Compared with 5-Fu , most of the target compounds in vitro cytotoxicity tests have satisfactory selectivity index. Only the cytotoxicity of H8 against A549 cells was comparable to that of 5-Fu . Table 1 Antiproliferative activity of target compounds against cancer cell lines after 48 hours of treatment. Compounds IC 50 ± SD 1 (µM) R 1 R 2 A549 PC-3 HepG2 K562 H1 3-F 3-pyridylformyl 15.78 ± 1.11 10.44 ± 0.97 17.44 ± 3.95 − H2 4-OCH 3 2-pyridylformyl 15.43 ± 1.60 10.47 ± 0.86 18.13 ± 3.39 − H3 4OCH 3 3-pyridylformyl 6.22 ± 0.10 27.07 ± 2.20 > 50 − H4 3-Cl 3-Cyanophenyl 21.15 ± 3.55 > 50 28.92 ± 3.38 − H5 4-Br Trifluoromethylphenyl 33.76 ± 10.93 7.43 ± 0.67 > 50 − H6 3-Cl 3-Fluorophenyl 11.02 ± 0.65 38.73 ± 3.51 > 50 14.59 ± 2.60 H7 4-Br 2-pyridylformyl 22.30 ± 6.42 8.26 ± 1.02 33.84 ± 6.06 − H8 4-OCH 3 4-pyridylformyl 7.32 ± 0.66 12.70 ± 4.33 11.09 ± 3.45 12.64 ± 1.94 H9 3-Cl 2-Nitrophenyl 34.90 ± 7.58 31.13 ± 7.86 17.72 ± 6.01 26.30 ± 3.26 H10 4-Br Thioformamide 16.67 ± 2.65 41.90 ± 7.27 11.68 ± 1.94 7.45 ± 1.30 H11 3-F 4-pyridylformyl 16.52 ± 3.34 30.81 ± 1.67 15.73 ± 1.01 − H12 4-Br 3-Fluorophenyl 11.07 ± 1.27 40.20 ± 6.17 30.78 ± 4.79 16.43 ± 2.05 H13 4-Br Thiohydrazide 4.83 ± 0.16 > 50 7.56 ± 1.16 9.33 ± 1.34 H14 4-OCH 3 Trifluoromethylphenyl 8.22 ± 1.11 29.42 ± 7.07 > 50 − H15 4-OCH 3 Thioformamide 16.83 ± 6.17 17.73 ± 5.75 10.27 ± 0.65 > 50 H16 4-Br 3-pyridylformyl 12.44 ± 0.64 8.15 ± 0.33 34.75 ± 3.79 > 50 H17 4-OCH 3 Thiohydrazide > 50 > 50 23.56 ± 4.19 30.55 ± 11.97 H18 3-Cl 4-bromo-2-fluorophenyl 17.24 ± 2.91 > 50 > 50 15.87 ± 2.06 H19 4-Cl Phenyl 19.75 ± 0.95 5.84 ± 1.26 24.20 ± 3.10 13.42 ± 0.20 H20 2-F Phenyl > 50 8.12 ± 0.95 > 50 17.97 ± 0.29 H21 4-Br Phenyl 19.13 ± 5.80 8.01 ± 0.39 > 50 16.89 ± 0.50 H22 4-F Phenyl > 50 > 50 > 50 10.37 ± 1.87 H23 4-F Trifluoromethylphenyl 22.27 ± 6.30 9.95 ± 2.38 > 50 16.43 ± 0.50 H24 4-Cl Trifluoromethylphenyl > 50 9.35 ± 0.74 > 50 12.66 ± 0.22 H25 2-F Trifluoromethylphenyl > 50 7.20 ± 0.84 17.48 ± 6.60 17.49 ± 0.51 H26 4-F 3-Cyanophenyl 15.25 ± 2.45 8.06 ± 0.50 11.61 ± 4.57 12.46 ± 0.84 H27 4-Cl 3-Cyanophenyl 11.91 ± 2.50 6.23 ± 0.43 10.91 ± 2.61 12.44 ± 0.34 H28 2-F 3-Cyanophenyl > 50 9.03 ± 0.30 > 50 > 50 5-Fu 2 − − 7.30 ± 1.81 6.11 ± 0.40 12.94 ± 2.84 10.78 ± 0.99 1 Data represent the mean values ± standard deviation of three independent experiments performed in triplicate. 2 5-Fu (5-Fluorouracil) was used as control. Table 2 In vitro cytotoxicity testing of normal cells HEK293 after 48 hours of treatment with the target compounds. Compounds IC 50 ± SD 1 (µM) R 1 R 2 A549 HEK293 SI 2 H6 3Cl 3-Fluorophenyl 11.02 ± 0.65 32.44 ± 7.65 2.94 H8 4OCH 3 4-pyridylformyl 7.32 ± 0.66 6.15 ± 0.16 − H10 4Br Thioformamide 16.67 ± 2.65 94.19 ± 7.855 5.65 H12 4Br 3-Fluorophenyl 11.07 ± 1.27 71.73 ± 3.741 6.48 H13 4Br Thiohydrazide 4.83 ± 0.16 69.01 ± 16.37 14.29 H14 4OCH 3 Trifluoromethylphenyl 8.22 ± 1.11 69.17 ± 2.16 8.41 H18 3Cl 4-bromo-2-fluorophenyl 17.24 ± 2.91 31.97 ± 3.57 1.85 5-Fu 3 − − 7.30 ± 1.81 4.88 ± 1.34 − 1 Data represent the mean values ± standard deviation of three independent experiments performed in triplicate. 2 Selectivity ratio = (IC 50 A549) / (IC 50 HEK293). 3 5-Fu (5-Fluorouracil) was used as control. Based on the low toxicity to HEK293 normal cells of compound H13 , it suggested that H13 may have a higher safety profile. Due to its high in vitro inhibitory activity against A549 and low toxicity to HEK293, H13 was selected as a candidate for further mechanistic study. 2.3 3D-QSAR Analysis The CoMFA model was constructed based on the IC 50 values of compounds H1 – H28 against A549 cancer cells. The values of q 2 and r 2 were 0.541 and 0.973, respectively ( Table S1 ). Experimental values, predicted values and residuals of the training and test sets are listed in Table 3 . pIC 50 values of most compounds were clustered around the trend line (Fig. 2 a), suggesting that the CoMFA model may be reliable. As shown in Fig. 2 b, in the stereo field diagram, the green isopotential lines were concentrated in the para position of the benzene ring, suggesting that the bulky groups were unfavorable for the anti-A549 cell line activity, while the small ones might be more favorable for the anti-A549 cell line activity. Also, in the electrostatic field diagram, the presence of blue isobars on the 3-position of the benzene ring side indicated that negatively charged groups were more favorable for antitumor activity. As H1 (R 1 = 3F, IC 50 = 15.78 µM) > H20 (R 1 = 2F, IC 50 > 50 µM), H22 (R 1 = 4F, IC 50 > 50 µM), the introduction of electronegative group fluorine atom at position 3 was more active than the introduction of electronegative group at other positions. The presence of a red isobaric line in the para position on the benzene ring side suggested that groups with electron-bearing groups were more favorable for antitumor activity. Further, the presence of a red isobaric line in the para position on the benzene ring side suggested that groups with electron-bearing groups were more favorable for antitumor activity. For example, H3 (R 1 = 4OCH 3 , IC 50 = 6.22 µM) > H16 (R 1 = 4Br, IC 50 = 12.44 µM) > H1 (R 1 = 3F, IC 50 = 15.78 µM). Interestingly, when the substituent of R 2 was 3-pyridylformyl, H3 (R 1 = 4OCH 3 , IC 50 = 6.22 µM) > H16 (R 1 = 4Br, IC 50 = 12.44 µM) > H1 (R 1 = 3F, IC 50 = 15.78 µM), and when nitrogen atoms on the pyridine were in different positions, H3 (R 1 = 4OCH 3 , R 2 = 3-pyridylformyl, IC 50 = 6.22 µM) > H8 (R 1 = 4OCH 3 , R 2 = 4-pyridylformyl, IC 50 = 7.32 µM) > H1 (R 1 = 4OCH 3 , R 2 = 2-pyridylformyl, IC 50 = 15.43 µM). All the results showed that the CoMFA model had good predictive ability for the anti-A549 cell line. Table 3 Experimental and predicted results of pIC 50 values for CoMFA. Compounds CoMFA R 1 R 2 A549 Exptl 1 Predicted 2 Relative error 3 H1 3F 3-pyridylformyl 15.78 ± 1.11 4.801 4.809 −0.008 H2 4OCH 3 2-pyridylformyl 15.43 ± 1.60 4.812 5.153 −0.341 H3 4OCH 3 3-pyridylformyl 6.22 ± 0.10 5.206 5.219 −0.013 H4 3Cl 3-Cyanophenyl 21.15 ± 3.55 4.675 4.711 −0.036 H5 4Br Trifluoromethylphenyl 33.76 ± 10.93 4.472 5.165 −0.693 H6 3Cl 3-Fluorophenyl 11.02 ± 0.65 4.958 4.579 0.379 H7 4Br 2-pyridylformyl 22.30 ± 6.42 4.652 5.158 −0.506 H8 4OCH 3 4-pyridylformyl 7.32 ± 0.66 5.135 5.117 0.018 H9 3Cl 2-Nitrophenyl 34.90 ± 7.58 4.457 4.450 0.007 H10 4Br Thioformamide 16.67 ± 2.65 4.778 5.259 −0.481 H11 3F 4-pyridylformyl 16.52 ± 3.34 4.782 4.768 0.014 H12 4Br 3-Fluorophenyl 11.07 ± 1.27 4.956 4.950 0.006 H13 4Br Thiohydrazide 4.83 ± 0.16 5.316 5.306 0.010 H14 4OCH 3 Trifluoromethylphenyl 8.22 ± 1.11 5.085 5.105 −0.020 H15 4OCH 3 Thioformamide 16.83 ± 6.17 4.774 4.768 0.006 H16 4Br 3-pyridylformyl 12.44 ± 0.64 4.905 5.198 −0.293 H17 4OCH 3 Thiohydrazide > 50 4.301 4.654 −0.353 H18 3Cl 4-bromo-2-fluorophenyl 17.24 ± 2.91 4.763 4.776 −0.013 H19 4Cl phenyl 19.75 ± 0.95 4.704 4.701 0.003 H20 2F phenyl > 50 4.301 4.196 0.105 H21 4Br phenyl 19.13 ± 5.80 4.718 4.635 0.083 H22 4F phenyl > 50 4.301 4.489 −0.188 H23 4F Trifluoromethylphenyl 22.27 ± 6.30 4.652 4.423 0.229 H24 4Cl Trifluoromethylphenyl > 50 4.301 4.482 −0.181 H25 2F Trifluoromethylphenyl > 50 4.301 4.356 −0.055 H26 4F 3-Cyanophenyl 15.25 ± 2.45 4.817 4.803 0.014 H27 4Cl 3-Cyanophenyl 11.91 ± 2.50 4.924 4.863 0.061 H28 2F 3-Cyanophenyl > 50 4.301 4.342 −0.041 1 Experimental pIC 50 . 2 Predicted pIC 50 . 3 Residual error (Exp. – Pred.). 2.4 Colony formation assay and Wound-healing assay To further verify whether the target compounds could reduce the in vitro growth ability of A549 cells, we completed colony formation experiments of the cells. The experimental results are shown in Fig. 3 a. After 14 days of co-incubation, compound H13 had a strong inhibitory effect on A549 cell colony formation at 5 µM and was close to the semi-inhibitory concentration value of compound H13 . Thus, compound H13 did inhibit A549 colony formation. On top of that, the ability of cancer cells to migrate is a high-risk factor that can lead to metastasis of the tumor, making it more difficult for the patient to be cured. Wound healing assay is considered a convenient method to study directed migration in vitro [ 33 ]. The inhibitory effect of compound H13 on the A549 cell line was assessed through a wound healing assay. As shown in Fig. 3 c, treatment of compound H13 (5, 10 µM) on A549 cell line showed a dose-dependent antimigration effect compared to the negative control, and compound H13 significantly inhibited the motility of A549 cell line, which was superior to the positive control 5-Fu . 2.5 RNA sequencing analysis To further understand the anti-tumor mechanism of H13 in NSCLC, we first performed RNAseq on DMSO- and H13 -treated A549 cells. Analysis of the RNAseq data revealed that there were 6410 differentially expressed genes (DEGs) in H13 -treated A549 cells, of which 2948 genes were up-regulated and 3462 genes were down-regulated (adjusted P |1|; Fig. 4 a, b). Subsequently, we significantly enriched Pathways in differentially expressed genes by KEGG database. Interestingly, among the top 20 pathways, the following pathways were associated with cell death, (i) reactive oxygen species production; (ii) autophagy; (iii) mTOR signaling pathway; and (iv) cell cycle. Subsequently, we also explored the anti-tumor mechanism of H13 in non-small cell lung cancer based on RNAseq data. 2.6 Apoptosis induction assay To detect apoptosis, in this study, Annexin V-FITC/PI kit was used to detect apoptosis. As can be seen in Fig. 5 A, the total apoptosis increased to 21.33% and 21.25% after 24 h of treatment of cells with compound H13 (5,10 µM) compared with the control group (6.2%), respectively. Unexpectedly, a large number of cells appeared in the Q1 region after 24 h treatment of both cells and compound H13 . We hypothesized that compound H13 may have altered the permeability of the cell membrane, resulting in a large amount of PI dye entering the cells. It is worth mentioning that the membrane of a large number of cells could be observed in the support material S1 after 24 h of compound H13 action. These results suggest that compound H13 strongly induced apoptosis. In order to visualize the apoptotic effects of compounds on the A549 cell line in a more intuitive manner, Hoechst 33258 staining is often considered to be an economical and convenient method. In this work, we treated the A549 cell line with different concentrations of H13 , as well as the positive control 5-Fu . As shown in Fig. 5 B, the normal nucleus of the control A549 cells were small and round. In contrast, early signs of apoptosis were characterized by chromatin condensation and blistering of the cell membrane after treatment with 5 µM and 10 µM of H13 , respectively. Also, as the concentration increased, the cells exhibited shrinkage of chromatin and aggregation towards the nucleus membrane. The nucleus disintegrated to form fragments. The fluorescence which emitted due to Hoechst 33258 bound to DNA enhanced. The result indicated that compound H13 induced the A549 cell line apoptosis in dose-dependent. 2.7 Cell cycle analysis To evaluate the effect of the targeting compounds on the tumor cell cycle, A549 cells were treated with H13 at concentrations of 5 µM and 10 µM, respectively, for 24 hours. Surprisingly, compound H13 was not able to significantly block the cell cycle, and this result was further verified by Western blot assay of P21 protein expression, which showed that compound H13 was not involved in cycle block as shown in Fig. 6 . 2.8 Effect of the compound H13 on reactive oxygen species levels in A549 cells The level of reactive oxygen species (ROS) is relatively higher in most cancer cells compared to normal cells[ 34 ], and when ROS increases to a certain level, it may suppress the antioxidant capacity of the cell and trigger cell death; therefore, the difference in the intracellular ROS level can be used as a potential strategy for the development of anticancer drugs with high therapeutic indexes[ 35 , 36 ]. Therefore, we used the fluorescent probe DCFH-DA to detect ROS in A549 cells treated with the compound H13 for 24 h. We observed and counted the fluorescence intensity of ROS using an inverted fluorescence microscope. As shown in Fig. 7 a, the result showed that H13 significantly elevated the ROS in the cells compared to the negative control, whereas the positive control 5-Fu hardly affected the ROS level in the cells. Accordingly, we also detected changes in ROS more precisely by flow cytometry. The results are shown in Fig. 7 c, where the ROS level was elevated about 2.7-fold after 10 Μm of H13 treatment compared with the control group. Compound H13 significantly increased the intracellular level of ROS. 2.9 Mitochondrial membrane potential assay Mitochondria involved in apoptosis of cells, and mitochondrial membrane potential (MMP) reflects the function of mitochondria[ 37 ]. To determine whether H13 induced apoptosis through the mitochondrial pathway, we assessed changes in MMP in A549 cells using fluorescence microscopy. The results showed that MMP decreased when A549 cells exposed to H13 (5 µM and 10 µM) for 24 h, which was a classical feature of early markers of apoptosis (Fig. 8 ). 2.10 Western blot assay The PI3K/AKT/mTOR pathway has an important role in cellular value-adding and metabolism[ 38 , 39 ], and is also a very important signaling pathway in tumor research. Therefore, we first detected the effects of the target compounds on p-AKT and p-mTOR after 5 µM and 10 µM treatments. The results showed that H13 could significantly reduce the expression of p-AKT and p-mTOR. In addition to this, we found that compound H13 not only blocked the conduction of the PI3K/AKT/mTOR pathway, but also significantly increased the expression of the autophagy marker LC3II protein. Interestingly, tumor autophagy supports tumorigenesis through different mechanisms, including the inhibition of p53 activation[ 40 ]. The two exist in opposition. This result was also characterized in a subsequent validation as shown in Fig. 9 c. The MDM2 oncogene is a major cellular regulator and repressor of p53. Accordingly, we also examined the expression level of MDM2 and surprisingly, compound H13 reduced the expression level of MDM2 protein, but did not depend on p53. In summary, H13 inhibits cell proliferation by inducing cellular autophagy mainly by blocking signaling of the PI3K/AKT/mTOR pathway. 3. Conclusion In this study, a series of isatin Schiff base derivatives containing thioether structures was obtained by active splicing with isatin. 28 target compounds showed good antitumor activity against A549, PC-3, HepG2 and K562 cells, respectively, with the strongest antitumor activity in A549 cells. Based on the activity data, a model with good predictive ability was constructed. Meanwhile, the mechanism of action of compound H13 in inhibiting the proliferation of A549 cells was analyzed and verified based on the results of RNAseq. It was found that compound H13 induced cellular autophagy mainly through negative regulation of PI3K/AKT/mTOR signaling pathway. In conclusion, compound H13 is a potential class of autophagy inducers. 4. Materials and Methods 4.1 Chemistry All chemical reagents were purchased from BiDe PharmaTech (Shanghai, China). The melting points of the compounds were tested on an X-4D melting point apparatus. NMR spectra were tested on a Bruker (Avance) 400 MHz instrument, chemically reported as δ and DMSO- d6 , and tetramethylsilane (TMS) was used as an internal standard, courtesy of the School of Pharmacy of Guizhou University. 4.1.1 General procedure for the synthesis of intermediate 2 . Intermediate 2 was synthesized as follows: 4-chloroindigo red was dissolved in DMF solution, Cs 2 CO 3 (3.0–5.0 eq) was added, thiophenol (1.0 eq) was added, and after adding the reaction materials, heating was carried out at a reaction temperature of 80 ℃ for 4–5 hours, and the reaction was monitored by TLC until the end of the reaction. After the reaction was completed, the appropriate amount of water was added to the reaction solution, left for half an hour, then filtered, the filter cake was washed with water and vacuum dried to obtain intermediates 2 . 4.1.2 General procedure for the synthesis of compounds H1 - H28 . After the intermediate 2 was dissolved in an appropriate amount of methanol, 2–3 drops of acetic acid were added to the reaction solution as a catalyst, and various substituted hydrazines (3.0 eq) were added, and the mixture was heated to 65℃, then refluxed for 3–4 h, and the whole reaction was monitored by TLC. After the reaction was complete, the reaction solution was filtrated, and the filtrated slag was washed with methanol 2–3 times and dried under vacuum to obtain the target compounds H1 - H28. H1 (Z)-N'-(4-((3-fluorophenyl)thio)-2-oxoindolin-3-ylidene)nicotinohydrazide . A yellow solid, yield of 86%, m.p 271.6–272.7 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 9.15–9.08 (m, 1H), 8.81 (s, 1H), 8.30 (d, J = 7.9 Hz, 1H), 7.67–7.52 (m, 2H), 7.51–7.32 (m, 3H), 7.25 (t, J = 8.0 Hz, 1H), 6.78 (d, J = 7.7 Hz, 1H), 6.40 (d, J = 8.1 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 164.06, 162.94, 161.60, 143.50, 132.70, 132.63, 132.44, 132.25, 132.17, 120.42, 117.15, 116.94, 115.68, 108.58. HRMS (AP-ESI) m/z calcd for C 20 H 13 FN 4 O 2 S [M-H] − 391.0663; found 391.066. H2 (Z)-N'-(4-((4-methoxyphenyl)thio)-2-oxoindolin-3-ylidene)picolinohydrazide. A yellow solid, yield of 76%, m.p > 300 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 14.72 (s, 1H), 11.36 (s, 1H), 8.78 (ddd, J = 4.8, 1.7, 0.9 Hz, 1H), 8.23 (dt, J = 7.8, 1.1 Hz, 1H), 8.12 (td, J = 7.7, 1.7 Hz, 1H), 7.77–7.73 (m, 1H), 7.60–7.54 (m, 2H), 7.21–7.10 (m, 3H), 6.67 (dd, J = 7.7, 0.8 Hz, 1H), 6.21 (dd, J = 8.2, 0.8 Hz, 1H), 3.85 (s, 3H). 13 C NMR (101 MHz, DMSO- d6 ) δ 162.57, 161.19, 161.07, 149.52, 148.98, 143.42, 139.67, 138.85, 138.51, 137.99, 132.08, 128.29, 123.49, 119.95, 118.76, 116.19, 114.93, 107.29. HRMS (AP-ESI) m/z calcd for C 21 H 16 N 4 O 3 S [M-H] − 403.0863; found 403.0859. H3 : (Z)-N'-(4-((4-methoxyphenyl)thio)-2-oxoindolin-3-ylidene)nicotinohydrazide. A yellow solid, yield of 78%, m.p 286.1–286.5 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 9.13 (s, 1H), 8.81 (s, 1H), 8.31 (s, 1H), 7.62 (s, 1H), 7.52 (d, J = 8.1 Hz, 2H), 7.18 (t, J = 8.0 Hz, 1H), 7.11 (d, J = 8.6 Hz, 2H), 6.68 (d, J = 7.6 Hz, 1H), 6.20 (d, J = 8.2 Hz, 1H), 3.84 (s, 3H). 13 C NMR (101 MHz, DMSO- d6 ) δ 161.10, 143.31, 137.92, 132.20, 119.75, 118.90, 116.19, 114.59, 107.51. HRMS (AP-ESI) m/z calcd for C 21 H 16 N 4 O 3 S [M-H] − 403.0863: found 403.0859. H4 (Z)-3-(2-(4-((3-chlorophenyl)thio)-2-oxoindolin-3-ylidene)hydrazinyl)benzonitrile . A yellow solid, yield of 80%, m.p 296.2–296.8 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 12.77 (s, 1H), 11.25 (s, 1H), 7.77–7.69 (m, 2H), 7.58–7.53 (m, 2H), 7.50–7.46 (m, 3H), 7.21 (t, J = 7.9 Hz, 1H), 6.84 (dd, J = 7.8, 0.8 Hz, 1H), 6.56 (dd, J = 8.1, 0.8 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 162.89, 144.00, 141.50, 134.53, 134.49, 132.29, 131.89, 131.78, 131.17, 130.01, 129.96, 129.77, 128.93, 126.41, 122.00, 119.21, 119.19, 118.03, 117.68, 112.56, 109.14. HRMS (AP-ESI) m/z calcd for C 21 H 13 ClN 4 OS [M-H] − 403.0420; found 403.0415. H5 (Z)-4-((4-bromophenyl)thio)-3-(2-(3-(trifluoromethyl)phenyl)hydrazono)indolin-2-one . A yellow solid, yield of 78%, m.p 286.0–286.2 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 12.74 (s, 1H), 11.21 (s, 1H), 7.71–7.64 (m, 2H), 7.52–7.46 (m, 2H), 7.38 (td, J = 8.4, 6.5 Hz, 1H), 7.26–7.19 (m, 2H), 7.16 (t, J = 7.9 Hz, 1H), 6.87–6.75 (m, 2H), 6.45 (dd, J = 8.1, 0.8 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 162.90, 144.02, 141.32, 135.67, 133.22, 131.30, 131.00, 130.78, 130.70, 130.46, 129.82, 129.58, 125.93, 123.22, 122.64, 121.20, 119.24, 118.39, 117.72, 111.12, 108.71. HRMS (AP-ESI) m/z calcd for C 21 H 13 BrF 3 N 3 OS [M-H] − 489.9832; found 489.9831. H6 (Z)-4-((3-chlorophenyl)thio)-3-(2-(3-fluorophenyl)hydrazono)indolin-2-one . A yellow solid, yield of 82%, m.p 237.8–238.2 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 12.76 (s, 1H), 11.24 (s, 1H), 7.59 (dt, J = 2.6, 1.2 Hz, 1H), 7.51–7.43 (m, 3H), 7.43–7.35 (m, 1H), 7.25–7.15 (m, 3H), 6.87–6.79 (m, 2H), 6.54 (dd, J = 8.1, 0.8 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 164.70, 163.00, 162.30, 145.16, 145.05, 141.23, 134.51, 132.43, 131.86, 131.62, 131.52, 129.83, 129.71, 129.05, 128.93, 121.82, 118.10, 110.91, 110.88, 109.73, 109.51, 109.03, 101.71, 101.44. HRMS (AP-ESI) m/z calcd for C 20 H 13 ClFN 3 OS [M-H] − 396.0370; found 396.0368. H7 (Z)-N'-(4-((4-bromophenyl)thio)-2-oxoindolin-3-ylidene)picolinohydrazide . A yellow solid, yield of 79%, m.p > 300 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 14.71 (s, 1H), 11.40 (s, 1H), 8.77 (d, J = 4.8 Hz, 1H), 8.18 (dd, J = 36.2, 8.0 Hz, 2H), 7.86–7.65 (m, 3H), 7.56 (dd, J = 14.8, 8.1 Hz, 2H), 7.30 (dt, J = 56.1, 8.0 Hz, 1H), 6.69 (dd, J = 39.4, 7.7 Hz, 1H), 6.28 (dd, J = 46.3, 8.3 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 162.51, 161.21, 149.53, 148.92, 143.58, 139.42, 138.85, 137.75, 137.65, 135.88, 133.63, 133.46, 132.34, 129.91, 128.32, 123.81, 123.51, 119.83, 115.74, 108.11. HRMS (AP-ESI) m/z calcd for C 20 H 13 BrN 4 O 2 S [M-H] − 450.9864; found 456.9859. H8 (Z)-N'-(4-((4-methoxyphenyl)thio)-2-oxoindolin-3-ylidene)isonicotinohydrazide . A yellow solid, yield of 89%, m.p 286.1–286.5 ℃; 1 H NMR (400 MHz, DMSO-d6) δ 14.11 (s, 1H), 11.49 (s, 1H), 8.85 (s, 2H), 7.87–7.78 (m, 2H), 7.59–7.46 (m, 2H), 7.18 (t, J = 8.0 Hz, 1H), 7.11 (d, J = 8.3 Hz, 2H), 6.68 (d, J = 7.6 Hz, 1H), 6.21 (d, J = 8.2 Hz, 1H), 3.84 (s, 3H). 13 C NMR (101 MHz, DMSO- d6 ) δ 161.10, 143.44, 139.83, 137.90, 132.35, 119.72, 118.92, 116.19, 114.52, 107.53, 55.86. HRMS (AP-ESI) m/z calcd for C 21 H 16 N 4 O 3 S [M-H] − 403.0861; found 403.0859. H9 (Z)-4-((3-chlorophenyl)thio)-3-(2-(2-nitrophenyl)hydrazono)indolin-2-one . A yellow solid, yield of 78%, m.p 297.2–298.4 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 14.34 (s, 1H), 11.36 (s, 1H), 8.30–8.20 (m, 1H), 8.08–8.01 (m, 1H), 7.83 (t, J = 7.7 Hz, 1H), 7.72–7.64 (m, 1H), 7.54 (p, J = 3.0 Hz, 3H), 7.29–7.14 (m, 2H), 6.84 (d, J = 7.7 Hz, 1H), 6.50 (d, J = 8.1 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 162.54, 142.46, 139.76, 137.10, 134.58, 133.65, 133.40, 133.29, 132.73, 132.11, 132.01, 131.16, 129.46, 126.31, 122.15, 121.19, 116.97, 116.32, 108.90. HRMS (AP-ESI) m/z calcd for C 20 H 13 ClN 4 O 3 S [M-H] − 423.0320; found 423.0313. H10 (Z)-2-(4-((4-bromophenyl)thio)-2-oxoindolin-3-ylidene)hydrazine-1-carbothioamide . A yellow solid, yield of 76%, m.p 288.9–290.2 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 12.76–12.55 (m, 1H), 11.41 (s, 1H), 9.25 (s, 1H), 7.74–7.65 (m, 2H), 7.52–7.48 (m, 3H), 7.22 (t, J = 8.0 Hz, 1H), 6.75 (d, J = 7.7 Hz, 1H), 6.38 (d, J = 8.1 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 - d6 ) δ 178.87, 162.83, 143.45, 136.51, 134.48, 133.37, 133.08, 132.06, 130.24, 123.30, 120.63, 115.74, 108.66. HRMS (AP-ESI) m/z calcd for C 15 H 11 BrN 4 OS 2 [M-H] − 404.9479; found 404.9474. H11 (Z)-N'-(4-((3-fluorophenyl)thio)-2-oxoindolin-3-ylidene)isonicotinohydrazide . A yellow solid, yield of 78%, m.p 281.1–282.3 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 14.08 (s, 1H), 11.54 (s, 1H), 8.85 (s, 2H), 7.85–7.78 (m, 2H), 7.61–7.53 (m, 1H), 7.52–7.32 (m, 3H), 7.26 (t, J = 8.0 Hz, 1H), 6.77 (dd, J = 7.7, 0.8 Hz, 1H), 6.40 (dd, J = 8.2, 0.7 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 164.07, 162.97, 161.61, 143.63, 139.73, 132.61, 132.57, 132.27, 132.18, 120.39, 117.19, 116.98, 115.58, 108.57. HRMS (AP-ESI) m/z calcd for C 20 H 13 FN 4 O 2 S [M-H] − 391.0667; found 391.0660. H12 (Z)-4-((4-bromophenyl)thio)-3-(2-(3-fluorophenyl)hydrazono)indolin-2-one . A yellow solid, yield of 77%, m.p 266.0–267.2 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 12.74 (s, 1H), 11.21 (s, 1H), 7.71–7.64 (m, 2H), 7.52–7.46 (m, 2H), 7.38 (td, J = 8.4, 6.5 Hz, 1H), 7.26–7.19 (m, 2H), 7.16 (t, J = 7.9 Hz, 1H), 6.87–6.75 (m, 2H), 6.45 (dd, J = 8.1, 0.8 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 163.01, 145.19, 145.09, 141.14, 135.93, 133.22, 131.64, 131.54, 131.23, 130.82, 129.65, 129.23, 122.73, 120.95, 117.61, 110.86, 110.83, 109.67, 109.46, 108.57, 101.66, 101.40. HRMS (AP-ESI) m/z calcd for C 20 H 13 BrFN 3 OS [M-H] − 439.9867; found 439.9863. H13 (Z)-N'-(4-((4-bromophenyl)thio)-2-oxoindolin-3-ylidene)hydrazinecarbothiohydrazide . A yellow solid, yield of 85%, m.p 241.5–242.4 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 7.75–7.68 (m, 2H), 7.59–7.48 (m, 2H), 7.19 (dt, J = 17.6, 8.0 Hz, 1H), 6.73 (dd, J = 20.0, 7.7 Hz, 1H), 6.34 (dd, J = 39.5, 8.1 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 161.10, 143.44, 139.83, 137.90, 132.35, 119.72, 118.92, 116.19, 114.51, 107.53, 55.86. HRMS (AP-ESI) m/z calcd for C 15 H 12 BrN 5 OS 2 [M-H] − 419.9585; found 419.9583. H14 (Z)-4-((4-methoxyphenyl)thio)-3-(2-(3-(trifluoromethyl)phenyl)hydrazono)indolin-2-one . A yellow solid, yield of 76%, m.p > 300 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 12.75 (s, 1H), 11.17 (s, 1H), 7.90 (d, J = 2.0 Hz, 1H), 7.80 (dd, J = 8.1, 2.2 Hz, 1H), 7.64–7.54 (m, 3H), 7.37–7.32 (m, 1H), 7.14–7.07 (m, 3H), 6.69 (dd, J = 7.7, 0.8 Hz, 1H), 6.22 (dd, J = 8.2, 0.8 Hz, 1H), 3.84 (s, 3H). 13 C NMR (101 MHz, DMSO- d6 ) δ 162.92, 160.96, 144.19, 141.05, 137.79, 134.93, 131.06, 130.08, 129.57, 125.97, 123.26, 120.35, 119.02, 118.50, 118.22, 116.12, 111.04, 111.00, 107.17, 55.86. HRMS (AP-ESI) m/z calcd for C 22 H 16 F 3 N 3 O 2 S [M-H] − 442.0832; found 442.0832. H15 (Z)-2-(4-((4-methoxyphenyl)thio)-2-oxoindolin-3-ylidene)hydrazine-1-carbothioamide . A yellow solid, yield of 79%, m.p 283.9–284.5 ℃; 1H NMR (400 MHz, DMSO- d6 ) δ 12.67 (d, J = 1.4 Hz, 1H), 11.37 (s, 1H), 9.27 (s, 1H), 7.60 (d, J = 2.4 Hz, 1H), 7.57–7.50 (m, 2H), 7.20–7.08 (m, 3H), 6.67 (dd, J = 7.7, 0.8 Hz, 1H), 6.19 (dd, J = 8.3, 0.8 Hz, 1H), 3.83 (s, 3H). 13 C NMR (101 MHz, DMSO- d6 ) δ 178.88, 162.87, 161.04, 143.24, 137.88, 137.78, 133.46, 131.83, 119.68, 118.88, 116.14, 114.52, 107.56, 55.86. HRMS (AP-ESI) m/z calcd for C 16 H 14 N 4 O 2 S 2 [M-H] − 357.0479; found 357.0474. H16 (Z)-N'-(4-((4-bromophenyl)thio)-2-oxoindolin-3-ylidene)nicotinohydrazide . A yellow solid, yield of 80%,m.p 280.4–281.5 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 13.98 (s, 1H), 11.51 (s, 1H), 9.11 (s, 1H), 8.82 (s, 1H), 8.30 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.0 Hz, 2H), 7.63 (s, 1H), 7.53 (d, J = 14.9 Hz, 2H), 7.24 (t, J = 8.0 Hz, 1H), 6.76 (d, J = 7.7 Hz, 1H), 6.36 (d, J = 8.1 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 162.97, 143.49, 133.45, 132.48, 129.79, 123.75, 120.14, 115.51, 108.41. HRMS (AP-ESI) m/z calcd for C 20 H 13 BrN 4 O 2 S [M-H] − 450.9863; found 450.9859. H17 (Z)-N'-(4-((4-methoxyphenyl)thio)-2-oxoindolin-3-ylidene)hydrazinecarbothiohydrazide . A yellow solid, yield of 81%, m.p 244.2–244.8 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 14.86–13.60 (m, 1H), 12.72 (s, 1H), 11.31 (d, J = 38.8 Hz, 1H), 7.62–7.50 (m, 2H), 7.18–7.05 (m, 3H), 6.72–6.60 (m, 1H), 6.21–6.13 (m, 1H), 3.83 (s, 3H). 13 C NMR (101 MHz, DMSO- d6 ) δ 180.64, 162.94, 162.24, 161.04, 142.40, 137.81, 136.32, 131.01, 120.54, 119.83, 118.81, 116.16, 107.48, 55.86. HRMS (AP-ESI) m/z calcd for C 16 H 15 N 5 O 2 S2 [M-K] − 372.0587; found 372.0583. H18 (Z)-3-(2-(4-chloro-2-fluorophenyl)hydrazono)-4-((3-chlorophenyl)thio)indolin-2-one . A yellow solid, yield of 78%, m.p 251.7–252.2 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 12.93 (d, J = 2.4 Hz, 1H), 11.35 (s, 1H), 7.68–7.58 (m, 2H), 7.54–7.41 (m, 5H), 7.21 (t, J = 7.9 Hz, 1H), 6.84 (d, J = 7.7 Hz, 1H), 6.54 (d, J = 8.1 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 141.49, 134.52, 134.24, 132.66, 132.12, 131.94, 130.29, 129.12, 109.19. HRMS (AP-ESI) m/z calcd for C 20 H 12 BrClFN 3 OS [M-H] − 473.9480; found 473.9473. H19 (Z)-4-((4-chlorophenyl)thio)-3-(2-phenylhydrazono)indolin-2-one . A yellow solid, yield of 85%, m.p 243.9–244.0 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 12.80 (s, 1H), 11.19 (s, 1H), 7.60–7.50 (m, 4H), 7.42–7.33 (m, 4H), 7.14 (t, J = 7.9 Hz, 1H), 7.04 (tt, J = 6.6, 1.8 Hz, 1H), 6.79 (dd, J = 7.8, 0.8 Hz, 1H), 6.43 (dd, J = 8.1, 0.8 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 163.21, 142.98, 140.67, 135.82, 134.13, 130.81, 130.67, 130.28, 129.92, 129.15, 128.19, 123.43, 120.74, 117.78, 114.62, 108.44. HRMS (AP-ESI) m/z calcd for C 20 H 14 ClN 3 OS [M-H] − 378.0469; found 378.0462. H20 (Z)-4-((2-fluorophenyl)thio)-3-(2-phenylhydrazono)indolin-2-one . A yellow solid, yield of 81%, m.p 239.4–240.2 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 12.78 (s, 1H), 11.19 (s, 1H), 7.65–7.56 (m, 2H), 7.48–7.42 (m, 3H), 7.41–7.30 (m, 3H), 7.12 (t, J = 7.9 Hz, 1H), 7.09–6.98 (m, 1H), 6.78 (d, J = 7.7 Hz, 1H), 6.31 (d, J = 8.1 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 163.73, 163.16, 161.28, 143.01, 140.69, 137.01, 132.71, 129.94, 129.18, 128.30, 126.34, 123.42, 119.45, 118.02, 117.84, 117.37, 117.04, 116.82, 114.60, 108.19. HRMS (AP-ESI) m/z calcd for C 20 H 14 FN 3 OS [M-H] − 362.0773; found 362.0758. H21 (Z)-4-((4-bromophenyl)thio)-3-(2-phenylhydrazono)indolin-2-one . A yellow solid, yield of 82%, m.p 299.6–299.8 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 12.81 (s, 1H), 11.19 (s, 1H), 7.72–7.63 (m, 2H), 7.54–7.45 (m, 2H), 7.43–7.33 (m, 4H), 7.14 (t, J = 7.9 Hz, 1H), 7.04 (tt, J = 6.3, 2.1 Hz, 1H), 6.80 (d, J = 7.7 Hz, 1H), 6.46 (d, J = 8.1 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 163.22, 142.98, 140.69, 135.87, 133.17, 131.48, 130.36, 129.91, 129.16, 128.17, 123.44, 122.62, 120.97, 117.91, 114.64, 108.54. HRMS (AP-ESI) m/z calcd for C 20 H 14 BrN 3 OS [M-H] − 421.9965; found 421.9957. H22 (Z)-4-((4-fluorophenyl)thio)-3-(2-phenylhydrazono)indolin-2-one . A yellow solid, yield of 83%, m.p 236.5–237.3 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 12.78 (s, 1H), 11.29–10.99 (m, 1H), 7.73–7.61 (m, 2H), 7.54–7.44 (m, 2H), 7.38 (dtd, J = 8.8, 6.7, 5.9, 2.1 Hz, 4H), 7.15–6.98 (m, 2H), 6.74 (d, J = 7.7 Hz, 1H), 6.29 (d, J = 8.1 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 164.45, 163.21, 162.00, 143.05, 140.55, 137.81, 137.73, 132.50, 129.95, 129.09, 128.43, 126.55, 126.52, 123.35, 119.29, 117.66, 117.45, 116.90, 114.56, 107.74. HRMS (AP-ESI) m/z calcd for C 20 H 14 FN 3 OS [M-H] − 362.0764; found 362.0758. H23 (Z)-4-((4-fluorophenyl)thio)-3-(2-(3-(trifluoromethyl)phenyl)hydrazono)indolin-2-one . A yellow solid, yield of 82%, m.p 276.2–276.4 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 12.78 (s, 1H), 11.20 (s, 1H), 7.86 (t, J = 2.0 Hz, 1H), 7.77 (dd, J = 8.2, 2.2 Hz, 1H), 7.71–7.63 (m, 2H), 7.60 (t, J = 8.0 Hz, 1H), 7.41–7.30 (m, 3H), 7.13 (t, J = 7.9 Hz, 1H), 6.74 (d, J = 7.7 Hz, 1H), 6.30 (d, J = 8.1 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 164.48, 162.89, 162.02, 144.11, 141.18, 137.68, 132.90, 131.03, 129.72, 126.40, 125.95, 123.24, 119.47, 119.15, 119.11, 118.28, 117.72, 116.70, 111.10, 107.86. 19 F NMR (376 MHz, DMSO- d6 ) δ − 61.43, − 111.74. HRMS (AP-ESI) m/z calcd for C 21 H 13 F 4 N 3 OS [M-H] − 430.0634; found 430.0632. H24 (Z)-4-((4-chlorophenyl)thio)-3-(2-(3-(trifluoromethyl)phenyl)hydrazono)indolin-2-one . A yellow solid, yield of 77%, m.p 288.2–288.6 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 12.80 (s, 1H), 11.22 (s, 1H), 7.81 (d, J = 2.1 Hz, 1H), 7.71 (dd, J = 8.2, 2.2 Hz, 1H), 7.62–7.51 (m, 5H), 7.41–7.26 (m, 1H), 7.17 (t, J = 7.9 Hz, 1H), 6.87–6.71 (m, 1H), 6.46 (dd, J = 8.1, 0.7 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 162.90, 144.04, 141.31, 135.64, 134.17, 131.02, 131.00, 130.77, 130.64, 130.46, 130.33, 129.80, 129.61, 125.93, 123.22, 120.96, 119.19, 118.37, 117.59, 111.12, 111.08, 108.59. 19 F NMR (376 MHz, DMSO-d6) δ − 61.35. HRMS (AP-ESI) m/z calcd for C 21 H 13 ClF 3 N 3 OS [M-H] − 446.0342; found 446.0336. H25 (Z)-4-((2-fluorophenyl)thio)-3-(2-(3-(trifluoromethyl)phenyl)hydrazono)indolin-2-one . A yellow solid, yield of 80%, m.p 253.0–253.2 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 12.79 (s, 1H), 11.23 (s, 1H), 7.84 (d, J = 2.0 Hz, 1H), 7.75 (dd, J = 8.1, 2.1 Hz, 1H), 7.65–7.53 (m, 3H), 7.43 (ddd, J = 9.3, 8.1, 1.2 Hz, 1H), 7.34 (td, J = 7.5, 1.3 Hz, 2H), 7.16 (t, J = 7.9 Hz, 1H), 6.79 (d, J = 7.6 Hz, 1H), 6.35 (d, J = 8.1 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 163.60, 162.86, 161.15, 144.05, 141.33, 136.71, 132.67, 131.02, 130.81, 130.49, 130.18, 129.82, 126.36, 123.22, 119.77, 119.22, 118.32, 117.77, 117.26, 111.12, 108.38. 19 F NMR (376 MHz, DMSO-d6) δ − 61.44, − 107.54. HRMS (AP-ESI) m/z calcd for C 21 H 13 F 4 N 3 OS [M-H] − 430.06; found 430.06. H26 (Z)-3-(2-(4-((4-fluorophenyl)thio)-2-oxoindolin-3-ylidene)hydrazinyl)benzonitrile . A yellow solid, yield of 83%, m.p 274.1–274.5 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 7.84–7.76 (m, 2H), 7.75–7.63 (m, 2H), 7.62–7.50 (m, 1H), 7.44 (dt, J = 7.6, 1.3 Hz, 1H), 7.40–7.31 (m, 2H), 7.13 (t, J = 7.9 Hz, 1H), 6.78–6.68 (m, 1H), 6.35–6.25 (m, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 164.46, 162.90, 162.00, 144.11, 141.28, 137.67, 132.99, 131.23, 130.20, 129.85, 126.40, 126.36, 126.26, 119.56, 119.25, 119.08, 117.71, 117.50, 116.61, 112.57, 107.90. HRMS (AP-ESI) m/z calcd for C 21 H 13 FN 4 OS [M-H] − 387.0725; found 387.0710. H27 (Z)-3-(2-(4-((4-chlorophenyl)thio)-2-oxoindolin-3-ylidene)hydrazinyl)benzonitrile . A yellow solid, yield of 89%, m.p 278.5–279.3 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 12.76 (s, 1H), 11.23 (s, 1H), 7.74 (dtd, J = 5.1, 2.4, 1.1 Hz, 2H), 7.55 (s, 5H), 7.44 (dt, J = 7.6, 1.3 Hz, 1H), 7.17 (t, J = 7.9 Hz, 1H), 6.80 (dd, J = 7.8, 0.8 Hz, 1H), 6.46 (dd, J = 8.1, 0.8 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 162.91, 144.04, 141.41, 135.56, 134.11, 131.18, 131.04, 130.70, 130.33, 129.95, 129.93, 126.34, 121.11, 119.20, 119.18, 117.58, 117.55, 112.56, 108.67. HRMS (AP-ESI) m/z calcd for C 21 H 13 ClN 4 OS [M-H] − 403.0420; found 403.0415. H28 (Z)-3-(2-(4-((2-fluorophenyl)thio)-2-oxoindolin-3-ylidene)hydrazinyl)benzonitrile . A yellow solid, yield of 81%, m.p 299.6–300.2 ℃; 1 H NMR (400 MHz, DMSO- d6 ) δ 12.75 (s, 1H), 11.24 (s, 1H), 7.88–7.70 (m, 2H), 7.59 (ddd, J = 8.9, 7.6, 5.4 Hz, 3H), 7.49–7.40 (m, 2H), 7.34 (td, J = 7.5, 1.3 Hz, 1H), 7.17 (t, J = 7.9 Hz, 1H), 6.83–6.73 (m, 1H), 6.36 (d, J = 8.1 Hz, 1H). 13 C NMR (101 MHz, DMSO- d6 ) δ 162.86, 144.09, 141.46, 136.61, 131.25, 130.19, 129.99, 126.38, 120.01, 119.17, 117.61, 117.22, 112.59, 108.50. HRMS (AP-ESI) m/z calcd for C 21 H 13 FN 4 OS [M-H] − 387.0716; found 387.0710. 4.2 MTT assay A549, K562, PC-3, and HepG2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Kunming, China). A549, K562, and PC-3 cells were cultured in RPMI-1640 medium and HepG2 cells were cultured in MEM medium. All mediums were supplemented with 10% fetal bovine serum (FBS), 100 µg/mL penicillin and 100 µg/mL streptomycin. All the cells were cultivated in 5% CO 2 incubator at 37°C. According to the MTT method, the inhibitory activity of target compounds against PC-3, HepG2, K562 and A549 cells were detected after treatment in different concentration ranges (1–30 µM) for 48 h. 4.3 3D-QSAR Analysis All the molecules (compounds H1 – H28 ) were submitted in Mol2 format and divided into a test set (7 compounds) and a training set (21 compounds). IC 50 values were converted to pIC 50 and analysed by Sybyl 2.0 software. CoMFA analyses were performed using partial least squares (PLS) regression, cross-validated correlation coefficients (q 2 ), non-cross-validated correlation coefficients (r 2 ) and predicted correlation coefficients (r 2 pred). The force field model was visualized using Sybyl 2.0 software and the compound H13 with the highest antitumor activity in the training set was selected as a template molecule. 4.4 Colony formation assay and Wound healing assay Colony formation assay: The cells were inoculated into 6-well plates according to 1500 cells per well and treated with different concentrations of compounds according to groups for 15 days, then stained with 1% crystal violet and photographed. Wound healing assay: Cells in logarithmic growth phase were inoculated into 6-well plates at a density of 1 × 10 6 cells/mL and incubated overnight at 37°C with 5% CO 2 . The compounds were dissolved in 1% FBS to obtain different concentrations (5, 10 µM). DMSO and 5-Fu (10 µM) were used as negative and positive controls, respectively, and a line was drawn vertically downwards. The scratches were observed, the liquid in the plate was discarded, and pictures were taken at 0, 6, 12 and 24 h after dosing to observe the migration changes of the cells. The pictures were processed by Image J software. 4.5 RNA sequencing analysis About 1×10 7 cells were counted, treated with compounds, and washed with PBS 2–3 times before adding TRIzol reagent. The cells were fully lysed and the samples were preserved in liquid nitrogen and finally analyzed by Illumina Novaseq 6000 sequencing platform. 4.6 Apoptosis analysis Cells in logarithmic growth phase were inoculated into 6-well plates at a density of 1×10 6 cells/mL and incubated overnight at 37°C with 5% CO 2 . An appropriate amount of Hoechst 33258 Working Solution was added to cover the samples. Then the cells were stained and incubated for 20–30 minutes at a temperature suitable for cell culture. The staining solution was discarded, and the cells were washed 2–3 times with PBS or culture medium for fluorescence detection. Annexin-V/FITC assay: each group of adherent cells was reacted with different concentrations of compound solutions for 48 h. Cells were collected and rinsed twice with PBS, then apoptosis was detected by Annexin V-FITC/PI Apoptosis Detection Kit (Solarbio, Beijing, China) according to the instructions of the kit, and flow cytometry detection was performed with a BD FACSCalibur instrument (BD Corporation Shanghai, China, USA). 4.7 Cell cycle analysis The adherent cells were reacted with different concentrations of compound solutions in each group for 48 h. The cells were collected, rinsed twice with PBS, fixed with ice-water ethanol (70%), and then stored at 4°C overnight. Flow cytometry detection was performed with a BD FACSCalibur instrument (BD Shanghai Co., Ltd., USA). 4.8 Western blot assay Cells (1×10 5 )/well were inoculated into 6-well plates and incubated for 24 h. Cells were treated with different concentrations of compounds for the indicated time and lysed with lysis buffer (Beyotime) containing protease and phosphatase inhibitors if necessary. The suspension was centrifuged at 12000 rpm for 20 minutes and the supernatant was collected. Proteins (25 µg) were separated by 8% SDS-PAGE and transferred to a PVDF membrane (Millipore). After incubation with primary and secondary antibodies, the membranes were imaged and collated using Image Lab software. All antibodies for the experiments were purchased from Cell Signaling. The colour was developed in the gel imager (VilberLourmat), and the data was analyzed by ImageJ software. 4.9 Statistical analysis Statistical processing of results was performed using GraphPad Prism 8.0 software (GraphPad Inc, San Diego, CA). All data were expressed as mean ± SD and all the data provided had been verified by at least three independent experiments. Differences among groups were considered significant at P ≤ 0.05. Declarations Supplementary data Photographs about the cell morphology after 24 h of H13 action are in Fig. S1 . 1 H NMR, 13 C NMR, 19 F NMR and HR-MS spectra for the target compounds ( Fig. S2 − S88 ). The authors declare that there are no competing financial interests. Author Contribution Zhenchao wang and zhurui Li made substantial contributions to the conception and revised the manuscript. Huayuan Tan and Guanglong Zhang designed and synthesized the compounds, and wrote the main manuscript. Chenlu Xu, Xue Lei, Jiayi Chen, Haitao Long, Xuemei Qiu, and Wenhang Wang carried out the biological experiments. Yue Zhou, Danping Chen, and Chengpeng Li analyzed the data. All authors reviewed the manuscript. Acknowledgements This study was supported by the National Natural Science Foundation of China (22007022, 32360689, 22364008, 32260694, 21867004), Guizhou Provincial Natural Science Foundation (ZZK[2021]034, ZK[2022]073), Guizhou Provincial Young Science and Technology Talents Development Project (KY[2022]146), and Top Science and Technology Talent Program of Guizhou Education Department (2022075). References Ferlay J, Colombet M, Soerjomataram I, Parkin DM, Piñeros M, Znaor A, Bray F (2021) Cancer statistics for the year 2020: an overview. Int J Cancer 149:778–789. https://doi.org/10.1002/ijc.33588 Yin LJ, Bin A, Kamar AKD,Liang CT, Avupati VR (2022) Review of anticancer potentials and structure-activity relationships (SAR) of rhodanine derivatives. 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Supplementary Files Supplementarydata.docx floatimage1.jpg Graphical abstract scheme1.jpg Scheme 1 Synthetic route of target molecules. Reagents and conditions: (i) Different substituted thiophenols, Cs 2 CO 3 , DMF, room temperature; (ii) Various hydrazides, catalytic amount of hydrochloric acid, methanol, 65 ℃. 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University","correspondingAuthor":false,"prefix":"","firstName":"Zhenchao","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-06-25 03:21:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4633192/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4633192/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11030-024-10954-1","type":"published","date":"2024-08-07T15:56:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60482376,"identity":"47823405-002f-4d41-97e1-ad47e9150f3c","added_by":"auto","created_at":"2024-07-17 08:56:07","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":132151,"visible":true,"origin":"","legend":"\u003cp\u003eDesign of target compounds \u003cstrong\u003eH1\u003c/strong\u003e-\u003cstrong\u003eH28\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4633192/v1/dc42a028319775c401a2b6d5.jpg"},{"id":60482379,"identity":"63e0089c-d56f-48c3-a263-5fe392ff27ee","added_by":"auto","created_at":"2024-07-17 08:56:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":169739,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the CoMFA analysis. \u003cstrong\u003e(a)\u003c/strong\u003e Plots of experimental and predicted pIC\u003csub\u003e50\u003c/sub\u003e values for the CoMFA model. The blue squares represent the test set, and the red circles represent the training set. \u003cstrong\u003e(b) \u003c/strong\u003eThe field contour maps of the CoMFA model based on the antibacterial activity against A549 cell line. \u003cstrong\u003e(c)\u003c/strong\u003e Structure−activity relationships based on the CoMFA model.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4633192/v1/5bf62b68d22daf707acdee51.jpg"},{"id":60482377,"identity":"560bae9b-46ae-44d7-b7f9-d30b27806c34","added_by":"auto","created_at":"2024-07-17 08:56:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":290782,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) A549 cells were treated with different concentrations of compound \u003cstrong\u003eH13 \u003c/strong\u003efor 14 d to form colonies. (\u003cstrong\u003eb\u003c/strong\u003e) Compound \u003cstrong\u003eH13\u003c/strong\u003e affects clone formation in A549 cells at concentrations of 2.5 μM, 5 μM. (\u003cstrong\u003eb\u003c/strong\u003e) Wound healing assay. Images were taken at 0 h, 6 h, 12 h and 24 h, respectively. The scale bar was 100 μm. (\u003cstrong\u003ed\u003c/strong\u003e) Compound \u003cstrong\u003eH13\u003c/strong\u003eaffects the migration of A549 cells at concentrations of 5 μM, 10 μM. The values are expressed as mean SD from three individual experiments. \u003cstrong\u003e*\u003c/strong\u003eStatistically significant (\u003cstrong\u003e**\u003c/strong\u003ep \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4633192/v1/ab582c4577595957a23cdabd.jpg"},{"id":60483192,"identity":"243b5476-c738-4cdc-90bc-5147578f57be","added_by":"auto","created_at":"2024-07-17 09:04:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":296591,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic analysis of A549 cells inhibited by compound \u003cstrong\u003eH13\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) and (\u003cstrong\u003eB\u003c/strong\u003e), histograms and Volcano plots of differentially expressed genes in cells after treatment with 10 μM \u003cstrong\u003eH13\u003c/strong\u003e for 24 h. (\u003cstrong\u003eC\u003c/strong\u003e) Gene set enrichment analysis showing the top 20 most significant pathways.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4633192/v1/357ae5e9118f86512090afd4.jpg"},{"id":60482380,"identity":"5f75b1d4-5268-4870-8805-40a909e20ed1","added_by":"auto","created_at":"2024-07-17 08:56:07","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":318226,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Apoptosis of \u003cstrong\u003eH13 \u003c/strong\u003ewas analyzed by flow cytometry. (\u003cstrong\u003eb\u003c/strong\u003e) Drug treated cells were stained with Hoechest 33258 to show apoptotic effects. Cell morphology was observed under fluorescence microscope (20x). Scale bar is 100 μm.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4633192/v1/c83916ac5d6c5fee874c5a7c.jpg"},{"id":60482382,"identity":"3aafcacd-6b95-4b29-89e1-97f3a15a92b5","added_by":"auto","created_at":"2024-07-17 08:56:07","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":193512,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Cell cycle of \u003cstrong\u003eH13 \u003c/strong\u003ewas analyzed by flow cytometry. (\u003cstrong\u003eb\u003c/strong\u003e) Cycle distribution statistics of A549 cells. (\u003cstrong\u003ec\u003c/strong\u003e) Expression of cyclin P21 after treatment with compound \u003cstrong\u003eH13\u003c/strong\u003efor 24 h.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4633192/v1/d5168513d0e20cf7bae30734.jpg"},{"id":60482384,"identity":"45db402d-aeda-4a5d-99b6-540fdd311486","added_by":"auto","created_at":"2024-07-17 08:56:07","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":209167,"visible":true,"origin":"","legend":"\u003cp\u003eEffect on ROS levels following the action of compound \u003cstrong\u003eH13\u003c/strong\u003e. (a) A fluorescent probe DCFH-DA was used to detect the photo of ROS in A549 cells after 24 h treatment with the compound \u003cstrong\u003eH13\u003c/strong\u003e. (b) Reactive oxygen fluorescence intensity statistics of A549 cells. (c) The ROS levels of the cells after the action of compound H13 were detected by flow cytometry. (d)Statistical analysis of changes in ROS levels in A549 cells. The values are expressed as mean SD from three individual experiments. * Statistically significant (* p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4633192/v1/91232c76a1703f7e532c4f92.jpg"},{"id":60482383,"identity":"0adea5e3-196d-49f9-bd15-1d8eca653943","added_by":"auto","created_at":"2024-07-17 08:56:07","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":493827,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of the compound \u003cstrong\u003eH13\u003c/strong\u003e on the MMP was observed by fluorescence microscopy.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4633192/v1/d60354a0dc9da850b0a70347.jpg"},{"id":60482386,"identity":"ccbed342-1b41-4dfc-bfce-9dd499dd9943","added_by":"auto","created_at":"2024-07-17 08:56:07","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":299256,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot analysis of phosphorylated AKT (p-AKT), phosphorylated mTOR (p-mTOR), P53, MDM2, and LC3II was performed at 5 μM and 10 μM using \u003cstrong\u003e5F\u003c/strong\u003e and compound \u003cstrong\u003eH13\u003c/strong\u003e.\u003cstrong\u003e*\u003c/strong\u003eStatistically significant (* p \u0026lt; 0.05,****p \u0026lt; 0.0001 ).\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4633192/v1/d1752f0cff0b9211696c3c02.jpg"},{"id":62298150,"identity":"7e42afa5-24ad-49d8-b0c0-cb3f4be71d1d","added_by":"auto","created_at":"2024-08-12 16:09:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3850398,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4633192/v1/cd79c9c7-6872-4ab8-9cf4-e07c528e5dbb.pdf"},{"id":60482385,"identity":"24d46379-6e19-400d-95c4-afc93b5083a1","added_by":"auto","created_at":"2024-07-17 08:56:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7875904,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-4633192/v1/62a8c0300fe521a767ff281b.docx"},{"id":60484181,"identity":"ac4f7e68-1216-4511-98b7-474811cfdfef","added_by":"auto","created_at":"2024-07-17 09:12:07","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":217856,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4633192/v1/4492c2a0b2e9cd267e331d4c.jpg"},{"id":60483190,"identity":"bb175225-2d57-4f89-aed4-77a6c8082b1f","added_by":"auto","created_at":"2024-07-17 09:04:07","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":285143,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e Synthetic route of target molecules. Reagents and conditions: (\u003cstrong\u003ei\u003c/strong\u003e) Different substituted thiophenols, Cs\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e, DMF, room temperature; (\u003cstrong\u003eii\u003c/strong\u003e) Various hydrazides, catalytic amount of hydrochloric acid, methanol, 65 ℃.\u003c/p\u003e","description":"","filename":"scheme1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4633192/v1/54222ab28313996aef095832.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis of novel 4-substituted isatin Schiff base derivatives as potential autophagy inducers and evaluation of their antitumour activity","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTo date, tumors remain a major threat to human health[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The World Health Organization (WHO) predicts an increase of 29.5\u0026nbsp;million cancer cases by 2040[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. According to the WHO, only 12 countries will be able to reduce cancer mortality by more than 30% by 2030[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Chemotherapy has long been an important drug therapy approach in oncology[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and the greatest drawback of chemotherapy is its inability to distinguish between cancer and normal cells, leading to significant toxic side effects[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This also highlights the need for a continuous search for an efficient and less toxic small molecule inhibitor. Much evidence suggests the therapeutic role of autophagy modulators in cancer treatment. Therefore, pharmacological intervention of the autophagy pathway may be a promising strategy for the treatment of a wide range of pathological conditions.\u003c/p\u003e \u003cp\u003eIsatin (1H-indole-2,3-dione) has been found to be a common structural motif in a variety of dyes, pesticides and pharmacologically active compounds due to its unique size and specific electronic properties[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Modern pharmacological studies have found that compounds containing these structures have a wide spectrum of biological activity[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], such as antitumor[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], antiviral[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], bactericidal[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], anti-inflammatory[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], anti-hypertensive[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], anti-cardiovascular disease[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and so on. Chemicals and drug candidates based on isatin have been applied in the pharmaceutical industry[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. For example, sunitinib, an oral tyrosine kinase inhibitor, was approved in 2006 for the treatment of metastatic renal cell carcinoma and imatinib-resistant gastrointestinal mesenchymal tumors[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In addition, isatin scaffolds are present in nintedanib, Intedanib, Semaxanib and TSU-68. Structurally, these marketed drugs are all close analogs of sunitinib, as they all share the isatin backbone.\u003c/p\u003e \u003cp\u003eIn recent years, compounds containing hydrazide or hydrazone fractions have been attractive for new drug development because of their potential multifunctional biological activity, including antiproliferative activity[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It has been reported that the antiproliferative activity of these hydrazones may be attributed to kinase inhibition[\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], through free radical production[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] or involved in the regulation of p53[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInspired by such interesting and potentially valuable research of isatin and hydrazone fractions, in this study, we designed and synthesized 28 thioether-containing derivatives of isatin Schiff base. Subsequently, growth inhibitory activity of the synthesized target compounds against four tumor cell lines was determined. In addition, the preliminary mechanism of this type of isatin Schiff base derivatives against human non-small cell lung cancer cell line (A549) was examined.\u003c/p\u003e"},{"header":"2 Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemistry\u003c/h2\u003e \u003cp\u003eThe synthesis route of compounds \u003cb\u003eH1\u003c/b\u003e-\u003cb\u003eH28\u003c/b\u003e is shown in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The commercially available 4-chloroindigo was converted into intermediate \u003cb\u003e2\u003c/b\u003e in \u003cem\u003eN,N\u003c/em\u003e-dimethylformamide (DMF) by reacting with thiophenol substituted at different positions in the presence of caesium carbonate. The target compounds \u003cb\u003eH1-H28\u003c/b\u003e were obtained by the condensation reaction of intermediate \u003cb\u003e2\u003c/b\u003e with various hydrazides in methanol. All the target compounds were confirmed by \u003csup\u003e1\u003c/sup\u003eH NMR, \u003csup\u003e13\u003c/sup\u003eC NMR, and HRMS. In addition, \u003cb\u003eH23\u003c/b\u003e, \u003cb\u003eH24\u003c/b\u003e and \u003cb\u003eH25\u003c/b\u003e were also characterized by \u003csup\u003e19\u003c/sup\u003eF NMR. Satisfactorily, the yields of all the target compounds were between 76%-89%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 \u003cem\u003eIn vitro\u003c/em\u003e antiproliferative activity\u003c/h2\u003e \u003cp\u003eIn the present study, \u003cem\u003ein vitro\u003c/em\u003e antiproliferative activity of all the target compounds was evaluated by MTT assay against A549, prostate (PC-3), chronic myeloid leukemia (K562) and liver (HepG2) cell lines using 5-fluorouracil (\u003cb\u003e5-Fu\u003c/b\u003e) as a positive control. After treatments with compounds \u003cb\u003eH1\u003c/b\u003e-\u003cb\u003eH28\u003c/b\u003e for 48 h, most of the target compounds showed good inhibitory activity against A549, PC-3, HepG2 and K562 cancer cells, respectively. \u003cb\u003eH3\u003c/b\u003e and \u003cb\u003eH13\u003c/b\u003e showed significant inhibitory activity against A549 cells, which was superior to that of \u003cb\u003e5-Fu\u003c/b\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Meanwhile, we evaluated the cytotoxicity of \u003cb\u003eH6\u003c/b\u003e, \u003cb\u003eH8\u003c/b\u003e, \u003cb\u003eH10\u003c/b\u003e, \u003cb\u003eH12\u003c/b\u003e, \u003cb\u003eH13\u003c/b\u003e, \u003cb\u003eH14\u003c/b\u003e and \u003cb\u003eH18\u003c/b\u003e on normal cells HEK293 using \u003cb\u003e5-Fu\u003c/b\u003e as a control (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Compared with \u003cb\u003e5-Fu\u003c/b\u003e, most of the target compounds \u003cem\u003ein vitro\u003c/em\u003e cytotoxicity tests have satisfactory selectivity index. Only the cytotoxicity of \u003cb\u003eH8\u003c/b\u003e against A549 cells was comparable to that of \u003cb\u003e5-Fu\u003c/b\u003e.\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\u003eAntiproliferative activity of target compounds against cancer cell lines after 48 hours of treatment.\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\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompounds\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;SD \u003csup\u003e1\u003c/sup\u003e (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA549\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePC-3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHepG2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK562\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-pyridylformyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17.44\u0026thinsp;\u0026plusmn;\u0026thinsp;3.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2-pyridylformyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e 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align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3-Cl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-Fluorophenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38.73\u0026thinsp;\u0026plusmn;\u0026thinsp;3.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14.59\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4-Br\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e 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align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31.13\u0026thinsp;\u0026plusmn;\u0026thinsp;7.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17.72\u0026thinsp;\u0026plusmn;\u0026thinsp;6.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e26.30\u0026thinsp;\u0026plusmn;\u0026thinsp;3.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4-Br\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThioformamide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e 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align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4-Br\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-Fluorophenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40.20\u0026thinsp;\u0026plusmn;\u0026thinsp;6.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30.78\u0026thinsp;\u0026plusmn;\u0026thinsp;4.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16.43\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4-Br\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eThiohydrazide\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e 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\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThioformamide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.83\u0026thinsp;\u0026plusmn;\u0026thinsp;6.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17.73\u0026thinsp;\u0026plusmn;\u0026thinsp;5.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4-Br\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-pyridylformyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e34.75\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThiohydrazide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e23.56\u0026thinsp;\u0026plusmn;\u0026thinsp;4.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e30.55\u0026thinsp;\u0026plusmn;\u0026thinsp;11.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH18\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3-Cl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4-bromo-2-fluorophenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.24\u0026thinsp;\u0026plusmn;\u0026thinsp;2.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH19\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4-Cl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24.20\u0026thinsp;\u0026plusmn;\u0026thinsp;3.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH21\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4-Br\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.13\u0026thinsp;\u0026plusmn;\u0026thinsp;5.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH22\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH23\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrifluoromethylphenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.27\u0026thinsp;\u0026plusmn;\u0026thinsp;6.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.95\u0026thinsp;\u0026plusmn;\u0026thinsp;2.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH24\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4-Cl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrifluoromethylphenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH25\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrifluoromethylphenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17.48\u0026thinsp;\u0026plusmn;\u0026thinsp;6.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH26\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-Cyanophenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.61\u0026thinsp;\u0026plusmn;\u0026thinsp;4.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH27\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4-Cl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-Cyanophenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH28\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-Cyanophenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5-Fu \u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003e1\u003c/sup\u003eData represent the mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of three independent experiments performed in triplicate.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003e2\u003c/sup\u003e \u003cb\u003e5-Fu\u003c/b\u003e (5-Fluorouracil) was used as control.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \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\u003e\u003cem\u003eIn vitro\u003c/em\u003e cytotoxicity testing of normal cells HEK293 after 48 hours of treatment with the target compounds.\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=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" 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\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompounds\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;SD \u003csup\u003e1\u003c/sup\u003e (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA549\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHEK293\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSI \u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3Cl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-Fluorophenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e11.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e32.44\u0026thinsp;\u0026plusmn;\u0026thinsp;7.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4-pyridylformyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e7.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e6.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4Br\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThioformamide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e16.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e94.19\u0026thinsp;\u0026plusmn;\u0026thinsp;7.855\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4Br\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-Fluorophenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e11.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e71.73\u0026thinsp;\u0026plusmn;\u0026thinsp;3.741\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4Br\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eThiohydrazide\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e69.01\u0026thinsp;\u0026plusmn;\u0026thinsp;16.37\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e14.29\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrifluoromethylphenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e8.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e69.17\u0026thinsp;\u0026plusmn;\u0026thinsp;2.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH18\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3Cl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4-bromo-2-fluorophenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e17.24\u0026thinsp;\u0026plusmn;\u0026thinsp;2.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e31.97\u0026thinsp;\u0026plusmn;\u0026thinsp;3.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5-Fu \u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e7.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e1\u003c/sup\u003e Data represent the mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of three independent experiments performed in triplicate.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e2\u003c/sup\u003e Selectivity ratio = (IC\u003csub\u003e50\u003c/sub\u003e A549) / (IC\u003csub\u003e50\u003c/sub\u003e HEK293).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e3\u003c/sup\u003e \u003cb\u003e5-Fu\u003c/b\u003e (5-Fluorouracil) was used as control.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBased on the low toxicity to HEK293 normal cells of compound \u003cb\u003eH13\u003c/b\u003e, it suggested that \u003cb\u003eH13\u003c/b\u003e may have a higher safety profile. Due to its high \u003cem\u003ein vitro\u003c/em\u003e inhibitory activity against A549 and low toxicity to HEK293, \u003cb\u003eH13\u003c/b\u003e was selected as a candidate for further mechanistic study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 3D-QSAR Analysis\u003c/h2\u003e \u003cp\u003eThe CoMFA model was constructed based on the IC\u003csub\u003e50\u003c/sub\u003e values of compounds \u003cb\u003eH1\u003c/b\u003e\u0026ndash;\u003cb\u003eH28\u003c/b\u003e against A549 cancer cells. The values of q\u003csup\u003e2\u003c/sup\u003e and r\u003csup\u003e2\u003c/sup\u003e were 0.541 and 0.973, respectively (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Experimental values, predicted values and residuals of the training and test sets are listed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. pIC\u003csub\u003e50\u003c/sub\u003e values of most compounds were clustered around the trend line (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), suggesting that the CoMFA model may be reliable. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, in the stereo field diagram, the green isopotential lines were concentrated in the para position of the benzene ring, suggesting that the bulky groups were unfavorable for the anti-A549 cell line activity, while the small ones might be more favorable for the anti-A549 cell line activity. Also, in the electrostatic field diagram, the presence of blue isobars on the 3-position of the benzene ring side indicated that negatively charged groups were more favorable for antitumor activity. As \u003cb\u003eH1\u003c/b\u003e (R\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3F, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;15.78 \u0026micro;M)\u0026thinsp;\u0026gt;\u0026thinsp;\u003cb\u003eH20\u003c/b\u003e (R\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2F, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;50 \u0026micro;M), \u003cb\u003eH22\u003c/b\u003e (R\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4F, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;50 \u0026micro;M), the introduction of electronegative group fluorine atom at position 3 was more active than the introduction of electronegative group at other positions. The presence of a red isobaric line in the para position on the benzene ring side suggested that groups with electron-bearing groups were more favorable for antitumor activity. Further, the presence of a red isobaric line in the para position on the benzene ring side suggested that groups with electron-bearing groups were more favorable for antitumor activity. For example, \u003cb\u003eH3\u003c/b\u003e (R\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4OCH\u003csub\u003e3\u003c/sub\u003e, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.22 \u0026micro;M)\u0026thinsp;\u0026gt;\u0026thinsp;\u003cb\u003eH16\u003c/b\u003e (R\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4Br, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;12.44 \u0026micro;M)\u0026thinsp;\u0026gt;\u0026thinsp;\u003cb\u003eH1\u003c/b\u003e (R\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3F, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;15.78 \u0026micro;M). Interestingly, when the substituent of R\u003csub\u003e2\u003c/sub\u003e was 3-pyridylformyl, \u003cb\u003eH3\u003c/b\u003e (R\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4OCH\u003csub\u003e3\u003c/sub\u003e, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.22 \u0026micro;M)\u0026thinsp;\u0026gt;\u0026thinsp;\u003cb\u003eH16\u003c/b\u003e (R\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4Br, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;12.44 \u0026micro;M)\u0026thinsp;\u0026gt;\u0026thinsp;\u003cb\u003eH1\u003c/b\u003e (R\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3F, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;15.78 \u0026micro;M), and when nitrogen atoms on the pyridine were in different positions, \u003cb\u003eH3\u003c/b\u003e (R\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4OCH\u003csub\u003e3\u003c/sub\u003e, R\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3-pyridylformyl, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.22 \u0026micro;M)\u0026thinsp;\u0026gt;\u0026thinsp;\u003cb\u003eH8\u003c/b\u003e (R\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4OCH\u003csub\u003e3\u003c/sub\u003e, R\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4-pyridylformyl, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.32 \u0026micro;M)\u0026thinsp;\u0026gt;\u0026thinsp;\u003cb\u003eH1\u003c/b\u003e (R\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4OCH\u003csub\u003e3\u003c/sub\u003e, R\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2-pyridylformyl, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;15.43 \u0026micro;M). All the results showed that the CoMFA model had good predictive ability for the anti-A549 cell line.\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\u003eExperimental and predicted results of pIC\u003csub\u003e50\u003c/sub\u003e values for CoMFA.\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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompounds\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eCoMFA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA549\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExptl \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePredicted \u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRelative error \u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-pyridylformyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.801\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;0.008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2-pyridylformyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.812\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.153\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;0.341\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-pyridylformyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.206\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.219\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;0.013\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3Cl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-Cyanophenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.15\u0026thinsp;\u0026plusmn;\u0026thinsp;3.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.675\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.711\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;0.036\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4Br\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrifluoromethylphenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.76\u0026thinsp;\u0026plusmn;\u0026thinsp;10.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.472\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;0.693\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3Cl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-Fluorophenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.958\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.579\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.379\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4Br\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2-pyridylformyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.30\u0026thinsp;\u0026plusmn;\u0026thinsp;6.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.652\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;0.506\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4-pyridylformyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.135\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.018\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3Cl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2-Nitrophenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.90\u0026thinsp;\u0026plusmn;\u0026thinsp;7.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.457\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.450\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4Br\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThioformamide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.778\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.259\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;0.481\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4-pyridylformyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.52\u0026thinsp;\u0026plusmn;\u0026thinsp;3.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.768\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4Br\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-Fluorophenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.956\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.950\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4Br\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThiohydrazide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.316\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.306\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrifluoromethylphenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.085\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;0.020\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThioformamide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.83\u0026thinsp;\u0026plusmn;\u0026thinsp;6.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.774\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.768\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4Br\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-pyridylformyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.905\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.198\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;0.293\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThiohydrazide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.654\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;0.353\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH18\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3Cl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4-bromo-2-fluorophenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.24\u0026thinsp;\u0026plusmn;\u0026thinsp;2.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.763\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.776\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;0.013\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH19\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4Cl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ephenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.704\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.701\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ephenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.196\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.105\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH21\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4Br\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ephenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.13\u0026thinsp;\u0026plusmn;\u0026thinsp;5.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.718\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.635\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.083\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH22\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ephenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.489\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;0.188\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH23\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrifluoromethylphenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.27\u0026thinsp;\u0026plusmn;\u0026thinsp;6.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.652\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.423\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.229\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH24\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4Cl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrifluoromethylphenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.482\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;0.181\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH25\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrifluoromethylphenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.356\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;0.055\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH26\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-Cyanophenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.817\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.803\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH27\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4Cl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-Cyanophenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.924\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.863\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.061\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH28\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3-Cyanophenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.342\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;0.041\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003e1\u003c/sup\u003e Experimental pIC\u003csub\u003e50\u003c/sub\u003e.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003e2\u003c/sup\u003e Predicted pIC\u003csub\u003e50\u003c/sub\u003e.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003e3\u003c/sup\u003e Residual error (Exp. \u0026ndash; Pred.).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Colony formation assay and Wound-healing assay\u003c/h2\u003e \u003cp\u003eTo further verify whether the target compounds could reduce the \u003cem\u003ein vitro\u003c/em\u003e growth ability of A549 cells, we completed colony formation experiments of the cells. The experimental results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. After 14 days of co-incubation, compound \u003cb\u003eH13\u003c/b\u003e had a strong inhibitory effect on A549 cell colony formation at 5 \u0026micro;M and was close to the semi-inhibitory concentration value of compound \u003cb\u003eH13\u003c/b\u003e. Thus, compound \u003cb\u003eH13\u003c/b\u003e did inhibit A549 colony formation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn top of that, the ability of cancer cells to migrate is a high-risk factor that can lead to metastasis of the tumor, making it more difficult for the patient to be cured. Wound healing assay is considered a convenient method to study directed migration \u003cem\u003ein vitro\u003c/em\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The inhibitory effect of compound \u003cb\u003eH13\u003c/b\u003e on the A549 cell line was assessed through a wound healing assay. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, treatment of compound \u003cb\u003eH13\u003c/b\u003e (5, 10 \u0026micro;M) on A549 cell line showed a dose-dependent antimigration effect compared to the negative control, and compound \u003cb\u003eH13\u003c/b\u003e significantly inhibited the motility of A549 cell line, which was superior to the positive control \u003cb\u003e5-Fu\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 RNA sequencing analysis\u003c/h2\u003e \u003cp\u003eTo further understand the anti-tumor mechanism of \u003cb\u003eH13\u003c/b\u003e in NSCLC, we first performed RNAseq on DMSO- and \u003cb\u003eH13\u003c/b\u003e-treated A549 cells. Analysis of the RNAseq data revealed that there were 6410 differentially expressed genes (DEGs) in \u003cb\u003eH13\u003c/b\u003e-treated A549 cells, of which 2948 genes were up-regulated and 3462 genes were down-regulated (adjusted P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; log2 fold change (FC) \u0026gt; |1|; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Subsequently, we significantly enriched Pathways in differentially expressed genes by KEGG database. Interestingly, among the top 20 pathways, the following pathways were associated with cell death, (i) reactive oxygen species production; (ii) autophagy; (iii) mTOR signaling pathway; and (iv) cell cycle. Subsequently, we also explored the anti-tumor mechanism of \u003cb\u003eH13\u003c/b\u003e in non-small cell lung cancer based on RNAseq data.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Apoptosis induction assay\u003c/h2\u003e \u003cp\u003eTo detect apoptosis, in this study, Annexin V-FITC/PI kit was used to detect apoptosis. As can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, the total apoptosis increased to 21.33% and 21.25% after 24 h of treatment of cells with compound \u003cb\u003eH13\u003c/b\u003e (5,10 \u0026micro;M) compared with the control group (6.2%), respectively. Unexpectedly, a large number of cells appeared in the Q1 region after 24 h treatment of both cells and compound \u003cb\u003eH13\u003c/b\u003e. We hypothesized that compound \u003cb\u003eH13\u003c/b\u003e may have altered the permeability of the cell membrane, resulting in a large amount of PI dye entering the cells. It is worth mentioning that the membrane of a large number of cells could be observed in the support material \u003cb\u003eS1\u003c/b\u003e after 24 h of compound \u003cb\u003eH13\u003c/b\u003e action. These results suggest that compound \u003cb\u003eH13\u003c/b\u003e strongly induced apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to visualize the apoptotic effects of compounds on the A549 cell line in a more intuitive manner, Hoechst 33258 staining is often considered to be an economical and convenient method. In this work, we treated the A549 cell line with different concentrations of \u003cb\u003eH13\u003c/b\u003e, as well as the positive control \u003cb\u003e5-Fu\u003c/b\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, the normal nucleus of the control A549 cells were small and round. In contrast, early signs of apoptosis were characterized by chromatin condensation and blistering of the cell membrane after treatment with 5 \u0026micro;M and 10 \u0026micro;M of \u003cb\u003eH13\u003c/b\u003e, respectively. Also, as the concentration increased, the cells exhibited shrinkage of chromatin and aggregation towards the nucleus membrane. The nucleus disintegrated to form fragments. The fluorescence which emitted due to Hoechst 33258 bound to DNA enhanced. The result indicated that compound \u003cb\u003eH13\u003c/b\u003e induced the A549 cell line apoptosis in dose-dependent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cell cycle analysis\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of the targeting compounds on the tumor cell cycle, A549 cells were treated with \u003cb\u003eH13\u003c/b\u003e at concentrations of 5 \u0026micro;M and 10 \u0026micro;M, respectively, for 24 hours. Surprisingly, compound \u003cb\u003eH13\u003c/b\u003e was not able to significantly block the cell cycle, and this result was further verified by Western blot assay of P21 protein expression, which showed that compound \u003cb\u003eH13\u003c/b\u003e was not involved in cycle block as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Effect of the compound H13 on reactive oxygen species levels in A549 cells\u003c/h2\u003e \u003cp\u003eThe level of reactive oxygen species (ROS) is relatively higher in most cancer cells compared to normal cells[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and when ROS increases to a certain level, it may suppress the antioxidant capacity of the cell and trigger cell death; therefore, the difference in the intracellular ROS level can be used as a potential strategy for the development of anticancer drugs with high therapeutic indexes[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Therefore, we used the fluorescent probe DCFH-DA to detect ROS in A549 cells treated with the compound \u003cb\u003eH13\u003c/b\u003e for 24 h. We observed and counted the fluorescence intensity of ROS using an inverted fluorescence microscope. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, the result showed that \u003cb\u003eH13\u003c/b\u003e significantly elevated the ROS in the cells compared to the negative control, whereas the positive control \u003cb\u003e5-Fu\u003c/b\u003e hardly affected the ROS level in the cells. Accordingly, we also detected changes in ROS more precisely by flow cytometry. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, where the ROS level was elevated about 2.7-fold after 10 Μm of \u003cb\u003eH13\u003c/b\u003e treatment compared with the control group. Compound \u003cb\u003eH13\u003c/b\u003e significantly increased the intracellular level of ROS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Mitochondrial membrane potential assay\u003c/h2\u003e \u003cp\u003eMitochondria involved in apoptosis of cells, and mitochondrial membrane potential (MMP) reflects the function of mitochondria[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. To determine whether \u003cb\u003eH13\u003c/b\u003e induced apoptosis through the mitochondrial pathway, we assessed changes in MMP in A549 cells using fluorescence microscopy. The results showed that MMP decreased when A549 cells exposed to \u003cb\u003eH13\u003c/b\u003e (5 \u0026micro;M and 10 \u0026micro;M) for 24 h, which was a classical feature of early markers of apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Western blot assay\u003c/h2\u003e \u003cp\u003eThe PI3K/AKT/mTOR pathway has an important role in cellular value-adding and metabolism[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and is also a very important signaling pathway in tumor research. Therefore, we first detected the effects of the target compounds on p-AKT and p-mTOR after 5 \u0026micro;M and 10 \u0026micro;M treatments. The results showed that \u003cb\u003eH13\u003c/b\u003e could significantly reduce the expression of p-AKT and p-mTOR. In addition to this, we found that compound \u003cb\u003eH13\u003c/b\u003e not only blocked the conduction of the PI3K/AKT/mTOR pathway, but also significantly increased the expression of the autophagy marker LC3II protein. Interestingly, tumor autophagy supports tumorigenesis through different mechanisms, including the inhibition of p53 activation[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The two exist in opposition. This result was also characterized in a subsequent validation as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec. The MDM2 oncogene is a major cellular regulator and repressor of p53. Accordingly, we also examined the expression level of MDM2 and surprisingly, compound \u003cb\u003eH13\u003c/b\u003e reduced the expression level of MDM2 protein, but did not depend on p53. In summary, \u003cb\u003eH13\u003c/b\u003e inhibits cell proliferation by inducing cellular autophagy mainly by blocking signaling of the PI3K/AKT/mTOR pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003eIn this study, a series of isatin Schiff base derivatives containing thioether structures was obtained by active splicing with isatin. 28 target compounds showed good antitumor activity against A549, PC-3, HepG2 and K562 cells, respectively, with the strongest antitumor activity in A549 cells. Based on the activity data, a model with good predictive ability was constructed. Meanwhile, the mechanism of action of compound \u003cb\u003eH13\u003c/b\u003e in inhibiting the proliferation of A549 cells was analyzed and verified based on the results of RNAseq.\u0026nbsp;It was found that compound \u003cb\u003eH13\u003c/b\u003e induced cellular autophagy mainly through negative regulation of PI3K/AKT/mTOR signaling pathway. In conclusion, compound \u003cb\u003eH13\u003c/b\u003e is a potential class of autophagy inducers.\u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Chemistry\u003c/h2\u003e \u003cp\u003eAll chemical reagents were purchased from BiDe PharmaTech (Shanghai, China). The melting points of the compounds were tested on an X-4D melting point apparatus. NMR spectra were tested on a Bruker (Avance) 400 MHz instrument, chemically reported as \u003cem\u003eδ\u003c/em\u003e and DMSO-\u003cem\u003ed6\u003c/em\u003e, and tetramethylsilane (TMS) was used as an internal standard, courtesy of the School of Pharmacy of Guizhou University.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e4.1.1 General procedure for the synthesis of intermediate \u003cb\u003e2\u003c/b\u003e.\u003c/h2\u003e \u003cp\u003eIntermediate \u003cb\u003e2\u003c/b\u003e was synthesized as follows: 4-chloroindigo red was dissolved in DMF solution, Cs\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (3.0\u0026ndash;5.0 eq) was added, thiophenol (1.0 eq) was added, and after adding the reaction materials, heating was carried out at a reaction temperature of 80 ℃ for 4\u0026ndash;5 hours, and the reaction was monitored by TLC until the end of the reaction. After the reaction was completed, the appropriate amount of water was added to the reaction solution, left for half an hour, then filtered, the filter cake was washed with water and vacuum dried to obtain intermediates \u003cb\u003e2\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e4.1.2 General procedure for the synthesis of compounds \u003cb\u003eH1\u003c/b\u003e-\u003cb\u003eH28\u003c/b\u003e.\u003c/h2\u003e \u003cp\u003eAfter the intermediate \u003cb\u003e2\u003c/b\u003e was dissolved in an appropriate amount of methanol, 2\u0026ndash;3 drops of acetic acid were added to the reaction solution as a catalyst, and various substituted hydrazines (3.0 eq) were added, and the mixture was heated to 65℃, then refluxed for 3\u0026ndash;4 h, and the whole reaction was monitored by TLC. After the reaction was complete, the reaction solution was filtrated, and the filtrated slag was washed with methanol 2\u0026ndash;3 times and dried under vacuum to obtain the target compounds \u003cb\u003eH1\u003c/b\u003e-\u003cb\u003eH28.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH1\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-N'-(4-((3-fluorophenyl)thio)-2-oxoindolin-3-ylidene)nicotinohydrazide\u003c/em\u003e. A yellow solid, yield of 86%, m.p 271.6\u0026ndash;272.7 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 9.15\u0026ndash;9.08 (m, 1H), 8.81 (s, 1H), 8.30 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.67\u0026ndash;7.52 (m, 2H), 7.51\u0026ndash;7.32 (m, 3H), 7.25 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 6.78 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 1H), 6.40 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 1H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 164.06, 162.94, 161.60, 143.50, 132.70, 132.63, 132.44, 132.25, 132.17, 120.42, 117.15, 116.94, 115.68, 108.58. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eFN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 391.0663; found 391.066.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH2\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-N'-(4-((4-methoxyphenyl)thio)-2-oxoindolin-3-ylidene)picolinohydrazide.\u003c/em\u003e A yellow solid, yield of 76%, m.p\u0026thinsp;\u0026gt;\u0026thinsp;300 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 14.72 (s, 1H), 11.36 (s, 1H), 8.78 (ddd, J\u0026thinsp;=\u0026thinsp;4.8, 1.7, 0.9 Hz, 1H), 8.23 (dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1.1 Hz, 1H), 8.12 (td, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7, 1.7 Hz, 1H), 7.77\u0026ndash;7.73 (m, 1H), 7.60\u0026ndash;7.54 (m, 2H), 7.21\u0026ndash;7.10 (m, 3H), 6.67 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7, 0.8 Hz, 1H), 6.21 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2, 0.8 Hz, 1H), 3.85 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 162.57, 161.19, 161.07, 149.52, 148.98, 143.42, 139.67, 138.85, 138.51, 137.99, 132.08, 128.29, 123.49, 119.95, 118.76, 116.19, 114.93, 107.29. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 403.0863; found 403.0859.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eH3\u003c/b\u003e: \u003cem\u003e(Z)-N'-(4-((4-methoxyphenyl)thio)-2-oxoindolin-3-ylidene)nicotinohydrazide.\u003c/em\u003e A yellow solid, yield of 78%, m.p 286.1\u0026ndash;286.5 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 9.13 (s, 1H), 8.81 (s, 1H), 8.31 (s, 1H), 7.62 (s, 1H), 7.52 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 2H), 7.18 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 7.11 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.6 Hz, 2H), 6.68 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 6.20 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2 Hz, 1H), 3.84 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 161.10, 143.31, 137.92, 132.20, 119.75, 118.90, 116.19, 114.59, 107.51. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 403.0863: found 403.0859.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH4\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-3-(2-(4-((3-chlorophenyl)thio)-2-oxoindolin-3-ylidene)hydrazinyl)benzonitrile\u003c/em\u003e. A yellow solid, yield of 80%, m.p 296.2\u0026ndash;296.8 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 12.77 (s, 1H), 11.25 (s, 1H), 7.77\u0026ndash;7.69 (m, 2H), 7.58\u0026ndash;7.53 (m, 2H), 7.50\u0026ndash;7.46 (m, 3H), 7.21 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 6.84 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 0.8 Hz, 1H), 6.56 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1, 0.8 Hz, 1H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 162.89, 144.00, 141.50, 134.53, 134.49, 132.29, 131.89, 131.78, 131.17, 130.01, 129.96, 129.77, 128.93, 126.41, 122.00, 119.21, 119.19, 118.03, 117.68, 112.56, 109.14. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eClN\u003csub\u003e4\u003c/sub\u003eOS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 403.0420; found 403.0415.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH5\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-4-((4-bromophenyl)thio)-3-(2-(3-(trifluoromethyl)phenyl)hydrazono)indolin-2-one\u003c/em\u003e. A yellow solid, yield of 78%, m.p 286.0\u0026ndash;286.2 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 12.74 (s, 1H), 11.21 (s, 1H), 7.71\u0026ndash;7.64 (m, 2H), 7.52\u0026ndash;7.46 (m, 2H), 7.38 (td, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4, 6.5 Hz, 1H), 7.26\u0026ndash;7.19 (m, 2H), 7.16 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 6.87\u0026ndash;6.75 (m, 2H), 6.45 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1, 0.8 Hz, 1H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 162.90, 144.02, 141.32, 135.67, 133.22, 131.30, 131.00, 130.78, 130.70, 130.46, 129.82, 129.58, 125.93, 123.22, 122.64, 121.20, 119.24, 118.39, 117.72, 111.12, 108.71. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eBrF\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eOS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 489.9832; found 489.9831.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH6\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-4-((3-chlorophenyl)thio)-3-(2-(3-fluorophenyl)hydrazono)indolin-2-one\u003c/em\u003e. A yellow solid, yield of 82%, m.p 237.8\u0026ndash;238.2 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 12.76 (s, 1H), 11.24 (s, 1H), 7.59 (dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.6, 1.2 Hz, 1H), 7.51\u0026ndash;7.43 (m, 3H), 7.43\u0026ndash;7.35 (m, 1H), 7.25\u0026ndash;7.15 (m, 3H), 6.87\u0026ndash;6.79 (m, 2H), 6.54 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1, 0.8 Hz, 1H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 164.70, 163.00, 162.30, 145.16, 145.05, 141.23, 134.51, 132.43, 131.86, 131.62, 131.52, 129.83, 129.71, 129.05, 128.93, 121.82, 118.10, 110.91, 110.88, 109.73, 109.51, 109.03, 101.71, 101.44. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eClFN\u003csub\u003e3\u003c/sub\u003eOS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 396.0370; found 396.0368.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH7\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-N'-(4-((4-bromophenyl)thio)-2-oxoindolin-3-ylidene)picolinohydrazide\u003c/em\u003e. A yellow solid, yield of 79%, m.p\u0026thinsp;\u0026gt;\u0026thinsp;300 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 14.71 (s, 1H), 11.40 (s, 1H), 8.77 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.8 Hz, 1H), 8.18 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;36.2, 8.0 Hz, 2H), 7.86\u0026ndash;7.65 (m, 3H), 7.56 (dd, J\u0026thinsp;=\u0026thinsp;14.8, 8.1 Hz, 2H), 7.30 (dt, J\u0026thinsp;=\u0026thinsp;56.1, 8.0 Hz, 1H), 6.69 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;39.4, 7.7 Hz, 1H), 6.28 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;46.3, 8.3 Hz, 1H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 162.51, 161.21, 149.53, 148.92, 143.58, 139.42, 138.85, 137.75, 137.65, 135.88, 133.63, 133.46, 132.34, 129.91, 128.32, 123.81, 123.51, 119.83, 115.74, 108.11. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eBrN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 450.9864; found 456.9859.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH8\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-N'-(4-((4-methoxyphenyl)thio)-2-oxoindolin-3-ylidene)isonicotinohydrazide\u003c/em\u003e. A yellow solid, yield of 89%, m.p 286.1\u0026ndash;286.5 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-d6) \u003cem\u003eδ\u003c/em\u003e 14.11 (s, 1H), 11.49 (s, 1H), 8.85 (s, 2H), 7.87\u0026ndash;7.78 (m, 2H), 7.59\u0026ndash;7.46 (m, 2H), 7.18 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 7.11 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 2H), 6.68 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 6.21 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2 Hz, 1H), 3.84 (s, 3H).\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 161.10, 143.44, 139.83, 137.90, 132.35, 119.72, 118.92, 116.19, 114.52, 107.53, 55.86. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 403.0861; found 403.0859.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH9\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-4-((3-chlorophenyl)thio)-3-(2-(2-nitrophenyl)hydrazono)indolin-2-one\u003c/em\u003e. A yellow solid, yield of 78%, m.p 297.2\u0026ndash;298.4 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 14.34 (s, 1H), 11.36 (s, 1H), 8.30\u0026ndash;8.20 (m, 1H), 8.08\u0026ndash;8.01 (m, 1H), 7.83 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 1H), 7.72\u0026ndash;7.64 (m, 1H), 7.54 (p, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.0 Hz, 3H), 7.29\u0026ndash;7.14 (m, 2H), 6.84 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 1H), 6.50 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 1H).\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 162.54, 142.46, 139.76, 137.10, 134.58, 133.65, 133.40, 133.29, 132.73, 132.11, 132.01, 131.16, 129.46, 126.31, 122.15, 121.19, 116.97, 116.32, 108.90. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eClN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 423.0320; found 423.0313.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH10\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-2-(4-((4-bromophenyl)thio)-2-oxoindolin-3-ylidene)hydrazine-1-carbothioamide\u003c/em\u003e. A yellow solid, yield of 76%, m.p 288.9\u0026ndash;290.2 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 12.76\u0026ndash;12.55 (m, 1H), 11.41 (s, 1H), 9.25 (s, 1H), 7.74\u0026ndash;7.65 (m, 2H), 7.52\u0026ndash;7.48 (m, 3H), 7.22 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 6.75 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 1H), 6.38 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 1H).\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 178.87, 162.83, 143.45, 136.51, 134.48, 133.37, 133.08, 132.06, 130.24, 123.30, 120.63, 115.74, 108.66. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e11\u003c/sub\u003eBrN\u003csub\u003e4\u003c/sub\u003eOS\u003csub\u003e2\u003c/sub\u003e [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 404.9479; found 404.9474.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH11\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-N'-(4-((3-fluorophenyl)thio)-2-oxoindolin-3-ylidene)isonicotinohydrazide\u003c/em\u003e. A yellow solid, yield of 78%, m.p 281.1\u0026ndash;282.3 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 14.08 (s, 1H), 11.54 (s, 1H), 8.85 (s, 2H), 7.85\u0026ndash;7.78 (m, 2H), 7.61\u0026ndash;7.53 (m, 1H), 7.52\u0026ndash;7.32 (m, 3H), 7.26 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 6.77 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7, 0.8 Hz, 1H), 6.40 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2, 0.7 Hz, 1H).\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 164.07, 162.97, 161.61, 143.63, 139.73, 132.61, 132.57, 132.27, 132.18, 120.39, 117.19, 116.98, 115.58, 108.57. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eFN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 391.0667; found 391.0660.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH12\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-4-((4-bromophenyl)thio)-3-(2-(3-fluorophenyl)hydrazono)indolin-2-one\u003c/em\u003e. A yellow solid, yield of 77%, m.p 266.0\u0026ndash;267.2 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 12.74 (s, 1H), 11.21 (s, 1H), 7.71\u0026ndash;7.64 (m, 2H), 7.52\u0026ndash;7.46 (m, 2H), 7.38 (td, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4, 6.5 Hz, 1H), 7.26\u0026ndash;7.19 (m, 2H), 7.16 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 6.87\u0026ndash;6.75 (m, 2H), 6.45 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1, 0.8 Hz, 1H).\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 163.01, 145.19, 145.09, 141.14, 135.93, 133.22, 131.64, 131.54, 131.23, 130.82, 129.65, 129.23, 122.73, 120.95, 117.61, 110.86, 110.83, 109.67, 109.46, 108.57, 101.66, 101.40. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eBrFN\u003csub\u003e3\u003c/sub\u003eOS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 439.9867; found 439.9863.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH13\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-N'-(4-((4-bromophenyl)thio)-2-oxoindolin-3-ylidene)hydrazinecarbothiohydrazide\u003c/em\u003e. A yellow solid, yield of 85%, m.p 241.5\u0026ndash;242.4 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 7.75\u0026ndash;7.68 (m, 2H), 7.59\u0026ndash;7.48 (m, 2H), 7.19 (dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.6, 8.0 Hz, 1H), 6.73 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20.0, 7.7 Hz, 1H), 6.34 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;39.5, 8.1 Hz, 1H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 161.10, 143.44, 139.83, 137.90, 132.35, 119.72, 118.92, 116.19, 114.51, 107.53, 55.86. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eBrN\u003csub\u003e5\u003c/sub\u003eOS\u003csub\u003e2\u003c/sub\u003e [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 419.9585; found 419.9583.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH14\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-4-((4-methoxyphenyl)thio)-3-(2-(3-(trifluoromethyl)phenyl)hydrazono)indolin-2-one\u003c/em\u003e. A yellow solid, yield of 76%, m.p\u0026thinsp;\u0026gt;\u0026thinsp;300 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 12.75 (s, 1H), 11.17 (s, 1H), 7.90 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H), 7.80 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1, 2.2 Hz, 1H), 7.64\u0026ndash;7.54 (m, 3H), 7.37\u0026ndash;7.32 (m, 1H), 7.14\u0026ndash;7.07 (m, 3H), 6.69 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7, 0.8 Hz, 1H), 6.22 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2, 0.8 Hz, 1H), 3.84 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 162.92, 160.96, 144.19, 141.05, 137.79, 134.93, 131.06, 130.08, 129.57, 125.97, 123.26, 120.35, 119.02, 118.50, 118.22, 116.12, 111.04, 111.00, 107.17, 55.86. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 442.0832; found 442.0832.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH15\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-2-(4-((4-methoxyphenyl)thio)-2-oxoindolin-3-ylidene)hydrazine-1-carbothioamide\u003c/em\u003e. A yellow solid, yield of 79%, m.p 283.9\u0026ndash;284.5 ℃; 1H NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 12.67 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.4 Hz, 1H), 11.37 (s, 1H), 9.27 (s, 1H), 7.60 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4 Hz, 1H), 7.57\u0026ndash;7.50 (m, 2H), 7.20\u0026ndash;7.08 (m, 3H), 6.67 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7, 0.8 Hz, 1H), 6.19 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3, 0.8 Hz, 1H), 3.83 (s, 3H).\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 178.88, 162.87, 161.04, 143.24, 137.88, 137.78, 133.46, 131.83, 119.68, 118.88, 116.14, 114.52, 107.56, 55.86. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 357.0479; found 357.0474.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH16\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-N'-(4-((4-bromophenyl)thio)-2-oxoindolin-3-ylidene)nicotinohydrazide\u003c/em\u003e. A yellow solid, yield of 80%,m.p 280.4\u0026ndash;281.5 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 13.98 (s, 1H), 11.51 (s, 1H), 9.11 (s, 1H), 8.82 (s, 1H), 8.30 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 7.72 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 7.63 (s, 1H), 7.53 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14.9 Hz, 2H), 7.24 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 6.76 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 1H), 6.36 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 1H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 162.97, 143.49, 133.45, 132.48, 129.79, 123.75, 120.14, 115.51, 108.41. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eBrN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 450.9863; found 450.9859.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH17\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-N'-(4-((4-methoxyphenyl)thio)-2-oxoindolin-3-ylidene)hydrazinecarbothiohydrazide\u003c/em\u003e. A yellow solid, yield of 81%, m.p 244.2\u0026ndash;244.8 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 14.86\u0026ndash;13.60 (m, 1H), 12.72 (s, 1H), 11.31 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;38.8 Hz, 1H), 7.62\u0026ndash;7.50 (m, 2H), 7.18\u0026ndash;7.05 (m, 3H), 6.72\u0026ndash;6.60 (m, 1H), 6.21\u0026ndash;6.13 (m, 1H), 3.83 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 180.64, 162.94, 162.24, 161.04, 142.40, 137.81, 136.32, 131.01, 120.54, 119.83, 118.81, 116.16, 107.48, 55.86. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eS2 [M-K]\u003csup\u003e\u0026minus;\u003c/sup\u003e 372.0587; found 372.0583.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH18\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-3-(2-(4-chloro-2-fluorophenyl)hydrazono)-4-((3-chlorophenyl)thio)indolin-2-one\u003c/em\u003e. A yellow solid, yield of 78%, m.p 251.7\u0026ndash;252.2 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 12.93 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4 Hz, 1H), 11.35 (s, 1H), 7.68\u0026ndash;7.58 (m, 2H), 7.54\u0026ndash;7.41 (m, 5H), 7.21 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 6.84 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 1H), 6.54 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 1H).\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 141.49, 134.52, 134.24, 132.66, 132.12, 131.94, 130.29, 129.12, 109.19. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eBrClFN\u003csub\u003e3\u003c/sub\u003eOS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 473.9480; found 473.9473.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH19\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-4-((4-chlorophenyl)thio)-3-(2-phenylhydrazono)indolin-2-one\u003c/em\u003e. A yellow solid, yield of 85%, m.p 243.9\u0026ndash;244.0 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 12.80 (s, 1H), 11.19 (s, 1H), 7.60\u0026ndash;7.50 (m, 4H), 7.42\u0026ndash;7.33 (m, 4H), 7.14 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.04 (tt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.6, 1.8 Hz, 1H), 6.79 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 0.8 Hz, 1H), 6.43 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1, 0.8 Hz, 1H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 163.21, 142.98, 140.67, 135.82, 134.13, 130.81, 130.67, 130.28, 129.92, 129.15, 128.19, 123.43, 120.74, 117.78, 114.62, 108.44. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eClN\u003csub\u003e3\u003c/sub\u003eOS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 378.0469; found 378.0462.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH20\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-4-((2-fluorophenyl)thio)-3-(2-phenylhydrazono)indolin-2-one\u003c/em\u003e. A yellow solid, yield of 81%, m.p 239.4\u0026ndash;240.2 ℃;\u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 12.78 (s, 1H), 11.19 (s, 1H), 7.65\u0026ndash;7.56 (m, 2H), 7.48\u0026ndash;7.42 (m, 3H), 7.41\u0026ndash;7.30 (m, 3H), 7.12 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.09\u0026ndash;6.98 (m, 1H), 6.78 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 1H), 6.31 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 1H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 163.73, 163.16, 161.28, 143.01, 140.69, 137.01, 132.71, 129.94, 129.18, 128.30, 126.34, 123.42, 119.45, 118.02, 117.84, 117.37, 117.04, 116.82, 114.60, 108.19. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eFN\u003csub\u003e3\u003c/sub\u003eOS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 362.0773; found 362.0758.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH21\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-4-((4-bromophenyl)thio)-3-(2-phenylhydrazono)indolin-2-one\u003c/em\u003e. A yellow solid, yield of 82%, m.p 299.6\u0026ndash;299.8 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 12.81 (s, 1H), 11.19 (s, 1H), 7.72\u0026ndash;7.63 (m, 2H), 7.54\u0026ndash;7.45 (m, 2H), 7.43\u0026ndash;7.33 (m, 4H), 7.14 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.04 (tt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.3, 2.1 Hz, 1H), 6.80 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 1H), 6.46 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 1H).\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 163.22, 142.98, 140.69, 135.87, 133.17, 131.48, 130.36, 129.91, 129.16, 128.17, 123.44, 122.62, 120.97, 117.91, 114.64, 108.54. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eBrN\u003csub\u003e3\u003c/sub\u003eOS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 421.9965; found 421.9957.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH22\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-4-((4-fluorophenyl)thio)-3-(2-phenylhydrazono)indolin-2-one\u003c/em\u003e. A yellow solid, yield of 83%, m.p 236.5\u0026ndash;237.3 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 12.78 (s, 1H), 11.29\u0026ndash;10.99 (m, 1H), 7.73\u0026ndash;7.61 (m, 2H), 7.54\u0026ndash;7.44 (m, 2H), 7.38 (dtd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8, 6.7, 5.9, 2.1 Hz, 4H), 7.15\u0026ndash;6.98 (m, 2H), 6.74 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 1H), 6.29 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 1H).\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 164.45, 163.21, 162.00, 143.05, 140.55, 137.81, 137.73, 132.50, 129.95, 129.09, 128.43, 126.55, 126.52, 123.35, 119.29, 117.66, 117.45, 116.90, 114.56, 107.74. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eFN\u003csub\u003e3\u003c/sub\u003eOS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 362.0764; found 362.0758.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH23\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-4-((4-fluorophenyl)thio)-3-(2-(3-(trifluoromethyl)phenyl)hydrazono)indolin-2-one\u003c/em\u003e. A yellow solid, yield of 82%, m.p 276.2\u0026ndash;276.4 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 12.78 (s, 1H), 11.20 (s, 1H), 7.86 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H), 7.77 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2, 2.2 Hz, 1H), 7.71\u0026ndash;7.63 (m, 2H), 7.60 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 7.41\u0026ndash;7.30 (m, 3H), 7.13 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 6.74 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 1H), 6.30 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 1H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 164.48, 162.89, 162.02, 144.11, 141.18, 137.68, 132.90, 131.03, 129.72, 126.40, 125.95, 123.24, 119.47, 119.15, 119.11, 118.28, 117.72, 116.70, 111.10, 107.86. \u003csup\u003e19\u003c/sup\u003eF NMR (376 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e\u0026thinsp;\u0026minus;\u0026thinsp;61.43, \u0026minus;\u0026thinsp;111.74. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eF\u003csub\u003e4\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eOS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 430.0634; found 430.0632.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH24\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-4-((4-chlorophenyl)thio)-3-(2-(3-(trifluoromethyl)phenyl)hydrazono)indolin-2-one\u003c/em\u003e. A yellow solid, yield of 77%, m.p 288.2\u0026ndash;288.6 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 12.80 (s, 1H), 11.22 (s, 1H), 7.81 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.1 Hz, 1H), 7.71 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2, 2.2 Hz, 1H), 7.62\u0026ndash;7.51 (m, 5H), 7.41\u0026ndash;7.26 (m, 1H), 7.17 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 6.87\u0026ndash;6.71 (m, 1H), 6.46 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1, 0.7 Hz, 1H).\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 162.90, 144.04, 141.31, 135.64, 134.17, 131.02, 131.00, 130.77, 130.64, 130.46, 130.33, 129.80, 129.61, 125.93, 123.22, 120.96, 119.19, 118.37, 117.59, 111.12, 111.08, 108.59. \u003csup\u003e19\u003c/sup\u003eF NMR (376 MHz, DMSO-d6) \u003cem\u003eδ\u003c/em\u003e\u0026thinsp;\u0026minus;\u0026thinsp;61.35. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eClF\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eOS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 446.0342; found 446.0336.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH25\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-4-((2-fluorophenyl)thio)-3-(2-(3-(trifluoromethyl)phenyl)hydrazono)indolin-2-one\u003c/em\u003e. A yellow solid, yield of 80%, m.p 253.0\u0026ndash;253.2 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 12.79 (s, 1H), 11.23 (s, 1H), 7.84 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H), 7.75 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1, 2.1 Hz, 1H), 7.65\u0026ndash;7.53 (m, 3H), 7.43 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.3, 8.1, 1.2 Hz, 1H), 7.34 (td, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5, 1.3 Hz, 2H), 7.16 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 6.79 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 6.35 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 1H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 163.60, 162.86, 161.15, 144.05, 141.33, 136.71, 132.67, 131.02, 130.81, 130.49, 130.18, 129.82, 126.36, 123.22, 119.77, 119.22, 118.32, 117.77, 117.26, 111.12, 108.38. \u003csup\u003e19\u003c/sup\u003eF NMR (376 MHz, DMSO-d6) \u003cem\u003eδ\u003c/em\u003e\u0026thinsp;\u0026minus;\u0026thinsp;61.44, \u0026minus;\u0026thinsp;107.54. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eF\u003csub\u003e4\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eOS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 430.06; found 430.06.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH26\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-3-(2-(4-((4-fluorophenyl)thio)-2-oxoindolin-3-ylidene)hydrazinyl)benzonitrile\u003c/em\u003e. A yellow solid, yield of 83%, m.p 274.1\u0026ndash;274.5 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 7.84\u0026ndash;7.76 (m, 2H), 7.75\u0026ndash;7.63 (m, 2H), 7.62\u0026ndash;7.50 (m, 1H), 7.44 (dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6, 1.3 Hz, 1H), 7.40\u0026ndash;7.31 (m, 2H), 7.13 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 6.78\u0026ndash;6.68 (m, 1H), 6.35\u0026ndash;6.25 (m, 1H).\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 164.46, 162.90, 162.00, 144.11, 141.28, 137.67, 132.99, 131.23, 130.20, 129.85, 126.40, 126.36, 126.26, 119.56, 119.25, 119.08, 117.71, 117.50, 116.61, 112.57, 107.90. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eFN\u003csub\u003e4\u003c/sub\u003eOS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 387.0725; found 387.0710.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH27\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-3-(2-(4-((4-chlorophenyl)thio)-2-oxoindolin-3-ylidene)hydrazinyl)benzonitrile\u003c/em\u003e. A yellow solid, yield of 89%, m.p 278.5\u0026ndash;279.3 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 12.76 (s, 1H), 11.23 (s, 1H), 7.74 (dtd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.1, 2.4, 1.1 Hz, 2H), 7.55 (s, 5H), 7.44 (dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6, 1.3 Hz, 1H), 7.17 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 6.80 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 0.8 Hz, 1H), 6.46 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1, 0.8 Hz, 1H).\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 162.91, 144.04, 141.41, 135.56, 134.11, 131.18, 131.04, 130.70, 130.33, 129.95, 129.93, 126.34, 121.11, 119.20, 119.18, 117.58, 117.55, 112.56, 108.67. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eClN\u003csub\u003e4\u003c/sub\u003eOS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 403.0420; found 403.0415.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eH28\u003c/strong\u003e \u003cp\u003e \u003cem\u003e(Z)-3-(2-(4-((2-fluorophenyl)thio)-2-oxoindolin-3-ylidene)hydrazinyl)benzonitrile\u003c/em\u003e. A yellow solid, yield of 81%, m.p 299.6\u0026ndash;300.2 ℃; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e 12.75 (s, 1H), 11.24 (s, 1H), 7.88\u0026ndash;7.70 (m, 2H), 7.59 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.9, 7.6, 5.4 Hz, 3H), 7.49\u0026ndash;7.40 (m, 2H), 7.34 (td, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5, 1.3 Hz, 1H), 7.17 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 6.83\u0026ndash;6.73 (m, 1H), 6.36 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 1H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u003cem\u003eδ\u003c/em\u003e162.86, 144.09, 141.46, 136.61, 131.25, 130.19, 129.99, 126.38, 120.01, 119.17, 117.61, 117.22, 112.59, 108.50. HRMS (AP-ESI) m/z calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eFN\u003csub\u003e4\u003c/sub\u003eOS [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 387.0716; found 387.0710.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.2 MTT assay\u003c/h2\u003e \u003cp\u003eA549, K562, PC-3, and HepG2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Kunming, China). A549, K562, and PC-3 cells were cultured in RPMI-1640 medium and HepG2 cells were cultured in MEM medium. All mediums were supplemented with 10% fetal bovine serum (FBS), 100 \u0026micro;g/mL penicillin and 100 \u0026micro;g/mL streptomycin. All the cells were cultivated in 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C. According to the MTT method, the inhibitory activity of target compounds against PC-3, HepG2, K562 and A549 cells were detected after treatment in different concentration ranges (1\u0026ndash;30 \u0026micro;M) for 48 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.3 3D-QSAR Analysis\u003c/h2\u003e \u003cp\u003eAll the molecules (compounds \u003cb\u003eH1\u003c/b\u003e\u0026ndash;\u003cb\u003eH28\u003c/b\u003e) were submitted in Mol2 format and divided into a test set (7 compounds) and a training set (21 compounds). IC\u003csub\u003e50\u003c/sub\u003e values were converted to pIC\u003csub\u003e50\u003c/sub\u003e and analysed by Sybyl 2.0 software. CoMFA analyses were performed using partial least squares (PLS) regression, cross-validated correlation coefficients (q\u003csup\u003e2\u003c/sup\u003e), non-cross-validated correlation coefficients (r\u003csup\u003e2\u003c/sup\u003e) and predicted correlation coefficients (r\u003csup\u003e2\u003c/sup\u003e pred). The force field model was visualized using Sybyl 2.0 software and the compound \u003cb\u003eH13\u003c/b\u003e with the highest antitumor activity in the training set was selected as a template molecule.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Colony formation assay and Wound healing assay\u003c/h2\u003e \u003cp\u003eColony formation assay: The cells were inoculated into 6-well plates according to 1500 cells per well and treated with different concentrations of compounds according to groups for 15 days, then stained with 1% crystal violet and photographed.\u003c/p\u003e \u003cp\u003eWound healing assay: Cells in logarithmic growth phase were inoculated into 6-well plates at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL and incubated overnight at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The compounds were dissolved in 1% FBS to obtain different concentrations (5, 10 \u0026micro;M). DMSO and \u003cb\u003e5-Fu\u003c/b\u003e (10 \u0026micro;M) were used as negative and positive controls, respectively, and a line was drawn vertically downwards. The scratches were observed, the liquid in the plate was discarded, and pictures were taken at 0, 6, 12 and 24 h after dosing to observe the migration changes of the cells. The pictures were processed by Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.5 RNA sequencing analysis\u003c/h2\u003e \u003cp\u003eAbout 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells were counted, treated with compounds, and washed with PBS 2\u0026ndash;3 times before adding TRIzol reagent. The cells were fully lysed and the samples were preserved in liquid nitrogen and finally analyzed by Illumina Novaseq 6000 sequencing platform.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Apoptosis analysis\u003c/h2\u003e \u003cp\u003eCells in logarithmic growth phase were inoculated into 6-well plates at a density of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL and incubated overnight at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. An appropriate amount of Hoechst 33258 Working Solution was added to cover the samples. Then the cells were stained and incubated for 20\u0026ndash;30 minutes at a temperature suitable for cell culture. The staining solution was discarded, and the cells were washed 2\u0026ndash;3 times with PBS or culture medium for fluorescence detection.\u003c/p\u003e \u003cp\u003eAnnexin-V/FITC assay: each group of adherent cells was reacted with different concentrations of compound solutions for 48 h. Cells were collected and rinsed twice with PBS, then apoptosis was detected by Annexin V-FITC/PI Apoptosis Detection Kit (Solarbio, Beijing, China) according to the instructions of the kit, and flow cytometry detection was performed with a BD FACSCalibur instrument (BD Corporation Shanghai, China, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.7 Cell cycle analysis\u003c/h2\u003e \u003cp\u003eThe adherent cells were reacted with different concentrations of compound solutions in each group for 48 h. The cells were collected, rinsed twice with PBS, fixed with ice-water ethanol (70%), and then stored at 4\u0026deg;C overnight. Flow cytometry detection was performed with a BD FACSCalibur instrument (BD Shanghai Co., Ltd., USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e4.8 Western blot assay\u003c/h2\u003e \u003cp\u003eCells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e)/well were inoculated into 6-well plates and incubated for 24 h. Cells were treated with different concentrations of compounds for the indicated time and lysed with lysis buffer (Beyotime) containing protease and phosphatase inhibitors if necessary. The suspension was centrifuged at 12000 rpm for 20 minutes and the supernatant was collected. Proteins (25 \u0026micro;g) were separated by 8% SDS-PAGE and transferred to a PVDF membrane (Millipore). After incubation with primary and secondary antibodies, the membranes were imaged and collated using Image Lab software. All antibodies for the experiments were purchased from Cell Signaling. The colour was developed in the gel imager (VilberLourmat), and the data was analyzed by ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e4.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical processing of results was performed using GraphPad Prism 8.0 software (GraphPad Inc, San Diego, CA). All data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and all the data provided had been verified by at least three independent experiments. Differences among groups were considered significant at P\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhotographs about the cell morphology after 24 h of \u003cstrong\u003eH13\u003c/strong\u003e action are in \u003cstrong\u003eFig.\u003c/strong\u003e \u003cstrong\u003eS1\u003c/strong\u003e. \u003csup\u003e1\u003c/sup\u003eH NMR, \u003csup\u003e13\u003c/sup\u003eC NMR, \u003csup\u003e19\u003c/sup\u003eF NMR and HR-MS spectra for the target compounds\u0026nbsp;(\u003cstrong\u003eFig. S2\u003c/strong\u003e\u0026minus;\u003cstrong\u003eS88\u003c/strong\u003e).\u003c/p\u003e\u003cp\u003eThe authors declare that there are no competing financial interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZhenchao wang and zhurui Li made substantial contributions to the conception and revised the manuscript. Huayuan Tan and Guanglong Zhang designed and synthesized the compounds, and wrote the main manuscript. Chenlu Xu, Xue Lei, Jiayi Chen, Haitao Long, Xuemei Qiu, and Wenhang Wang carried out the biological experiments. Yue Zhou, Danping Chen, and Chengpeng Li analyzed the data. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis study was supported by the National Natural Science Foundation of China (22007022, 32360689, 22364008, 32260694, 21867004), Guizhou Provincial Natural Science Foundation (ZZK[2021]034, ZK[2022]073), Guizhou Provincial Young Science and Technology Talents Development Project (KY[2022]146), and Top Science and Technology Talent Program of Guizhou Education Department (2022075).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFerlay J, Colombet M, Soerjomataram I, Parkin DM, Pi\u0026ntilde;eros M, Znaor A, Bray F (2021) Cancer statistics for the year 2020: an overview. 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Csh Perspect Med 6:a026120. https://doi.org/10.1101/cshperspect.a026120\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is 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":"molecular-diversity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"modi","sideBox":"Learn more about [Molecular Diversity](http://link.springer.com/journal/11030)","snPcode":"11030","submissionUrl":"https://submission.nature.com/new-submission/11030/3","title":"Molecular Diversity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Isatin Schiff bases, antitumor activity, 3D-QSAR, autophagy","lastPublishedDoi":"10.21203/rs.3.rs-4633192/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4633192/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Autophagy is a potential target in cancer therapy. In the present study, we designed and synthesized a series of isatin Schiff base derivatives containing thioether structures. After discovering the highly active target compound H13 (IC50 = 4.83 μM) based on in vitro antiproliferation, we also found it had a high safety against normal cells HEK293 with CC50 of 69.01 μM. In addition, to provide reference for subsequent studies, a model was successfully constructed by Sybyl software. Preliminary mechanistic studies suggested that H13-induced apoptosis may be closely related to ROS accumulation and mitochondrial dysfunction. Subsequent studies revealed that H13 inhibited cell proliferation by inducing cellular autophagy mainly through blocking signal of the PI3K/AKT/mTOR pathway. 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