Synthesis and antiinflammatory, antiproliferative activities of new coxib–combretastatin hybrids

Through the determination of apoptosis induction through three methods of

detecting apoptosis of the active substance by staining the cell nucleus with

Hoechst 33342, caspase - 3 and cytometry can be summarized:

 Sample 82 at test concentrations slightly increased the proportion of cells

in early apoptosis and late apoptosis compared with negative control,

reaching 22.57-26.21%.

 Sample 102, at a concentration of 20 µg / ml increased the incidence of

early and late apoptosis cells from 18.3% to 42.2% and 1.90% to 6.7% in

the negative control, respectively. At lower concentrations there was a

slight increase (10 µg / ml and 5 /g / ml) in the incidence of apoptosis

compared to the negative control.

 Camptothecin - positive control increased apoptosis rate, reaching from

78.79%.

 Sample 82 has not clearly shown the ability to induce apoptosis to

research concentrations in the trials performed;

 Sample at a concentration of 10 M was able to induce apoptosis in MCF7

cells through caspase 3 induction with an increase of 1.21 times compared

with negative control (P <0.05);

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tastatin A-4 phosphate are currently is clinically tested to treat Alzheimer's disease and cancer. The recently isolated stilbene has been shown to have a diverse range of biological activities, including antioxidant, antibacterial, anti-malarial, cytotoxic, liver protective and anti-inflammatory properties. Combretastatin A-4 (CA4) is also considered to be a potential cytotoxic agent by strongly inhibiting microtubule polymerization by binding to the binding point of colchicine on tubulin. CA-4 is highly toxic on many cancer cell models, making it a very interesting target structure. Overview of Pyrazole are pentagonal heterocyclics that form a group of compounds that are particularly useful in organic synthesis. They are one of the most studied groups of compounds in the azol family. Pyrazole is reported through the available literature and SAR shows that it is necessary for the design of selective COX2 inhibitors. One of the most important and commonly used compounds in commercially applied pyrazole compounds is celecoxib, a 5 substance known for its potent anti-inflammatory activity, which selectively inhibits COX2 through its action. Prostaglandins induce inflammation and pain without effects on prostaglandins COX1 that have a protective effect on the gastrointestinal tract. Furthermore, Celecoxib inhibits the proliferation of human breast cancer in vitro models such as MCF7 and MDAMB-231. Some studies indicate that celecoxib and related compounds can induce cell cycle arrest at G0 /G1 stage leading to apotosis cyclic apoptosis, inhibition of tumor growth and prevents tumor angiogenesis in the absence of COX2 Thereby, it can be seen that combretastatin celecoxib hybrid compounds are promising classes that are still new in terms of structural development as well as bioactivity, contributing to the construction of new research projects looking for different types anticancer drugs in the pharmaceutical industry. 6 CHAPTER 2. EXPERIMENTS 2.1. Materials and equipments 2.1.1. Materials 2.1.2. Equipments 2.2. Methods 2.2.1. Organic synthesis method 2.2.3. Biological activity test method 2.3. Synthesis of coxib- combrestatin hybrid compounds 2.3.1. Synthesis of ester derivatives of coxib - combretastatin hybrid ester hybrid Figure 2.2. Ester derivative synthesis of coxib - combrestatin hybrids compound (i) Alkaline: t-BuOLi (3 mmol, 3 eq), refluxe, (ii) Ethyl chlorooxoacetate (1 mmol, 1 eq) (77), 5 ml THF; (iii) HCl (4 mmol); refluxe, 5 ml C2H5OH dry; phenylhydrazin (1 mmol, 1 eq) (78). Synthesized 20 hybrids esters of coxib - combretastatin ester form substances from 79 to 98. 2.3.2. Synthesis of coxib - combrestastatin hybrids compounds containing groups CF3 Figure 2.5. Synthesis of coxib - combretastatin hybridization containing groups CF3 7 (a) 100 (1.0 mmol), 99 etyl trifluoroacetate (1,2 eq) and NaH (2,5 eq) in THF (5 mL), 6 h. (b), EtOH (5 mL), axit (1.0 eq); arylhydrazin hydrochloride 78 (1.0 mmol) is added consecutively to the residue and restored for 6 hours. Substance 102 was isolated by column chromatography. 2.3.3. Synthesis of acid derivatives of coxib - combretastatin hybrid Figure 2.8. Synthesis of coxib - combretastatin hybrids (i) Alkaline: t-BuOLi (3 mmol, 3 eq), refluxe, (ii) Ethyl chlorooxoacetate (1 mmol, 1 eq) (77), 5 ml THF; (iii) HCl (4 mmol); refluxe, 5 ml C2H5OH dry; phenylhydrazin (1 mmol, 1 eq) (78). The product obtained after isolation through column chromatography was dissolved in the solvent system THF / MeOH / H2O = 3: 1: 1, then NaH (1,2 eq) was added to the mixture. Carry out the reaction in 3 hours to obtain compounds acid hybrids 103-122. successful synthesis of 20 acid hybridization of coxib – combretastatin hybrids 103-122 2.5. Biologically active testing of research compounds The synthetic compounds were screened for anti-breast cancer activity MCF7, colon cancer HT-29, hepatic carcinoma Hep-G2 and inhibited NO production. 8 CHAPTER 3. RESULTS AND DISCUSSIONS The advantages of using a hybrid molecule over co-combination of multiple drugs at the same time may improve the limitations of adverse effects and resistance [107]. Despite its outstanding activity, combretastatin still has many undesirable effects. This is why the team aims to combine combretastatin, an anticancer compound, and celecoxib, a COX2-engineered anti-inflammatory agent, as derivatives for new hybrid compounds in hopes of finding new It has interesting biologically active properties such as the anticancer and anti- inflammatory properties of the parent substance and has less side effects. 3.1. Design of the structure and biological activity of the hybrids 3.1.1. Design of hybrid molecular structure In this study, we adopted the hybridization strategy to incorporate the important pharmacophoric groups of two original compounds celecoxib and CA-4 in a single molecule. We utilized 1,2-diphenyl substituted pyrazole ring of celecoxib as a scaffold to mimic the cis-1,2-diphenylethylene motif in CA-4. Replacement of the double bond with heterocyclic five membered rings was demonstrated to retain both cytotoxic and antitubulin activities of the compounds [108]. Indeed, these cis-locked analogues provide several advantages: preventing of isomerization from cis to trans; increasing specificity of these drugs to cellular targets; and improving the therapeutic potential of these drugs. The presence of the trimethoxybenzene moiety of CA-4 is also crucial to obtain relevant cytotoxic and antitubulin responses. [76]. We were particularly interested in maintaining the sulfonamid group or related bioisosteres of celecoxib which seems to result in its COX-2 selectivity [109]. The COX-2 inhibitory effect, if any, will contribute to the overall anti-tumor activity of new molecules. 9 Figure 3.1. C Structure of celecoxib, combretastatin A4 and the coxib– combretastatin hybrids 3.1.2. Design of hybrid molecular biology activities The cytotoxic effects of these compounds against three human cancer cell lines HT-29, Hep-G2, MCF-7 as well as the inhibition of NO production using lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells were studied. The role of NO in tumour biology is complex, because it has both facilitatory and inhibitory roles in cellular processes depending on the conditions, so NO inhibition is directly unrelated to the cytotoxicity. But NO displays a variety of the same useful pharmacological properties as prostaglandins in the cardiovascular system including vasodilation, inhibition of platelet aggregation, modulation of platelet and leukocytes adherence to vessels [110]. The use of selective COX-2 inhibitor celecoxib in chemoprevention of breast cancer and other malignancies has been limited by its adverse effects, in particular the risk of cardiovascular events principally connected to their ability to reduce the production of prostacyclin PGI2 [111]. Based on the observation that maintaining of NO production together with coxib-induced activities thus might help avoiding risk of cardiovascular toxicity, a strategy to design a multi- 10 target drug by combining COX-2-selective inhibition with nitric oxide (NO)- dependent activities has been initiated [112-114]. In infammation, the activated immune cells such as macrophages secrete everal infammatory mediators such as proinfammatory cytokines, nitric oxide (NO) and prostaglandin E2 (PGE2). In our previous study, tested compounds and celecoxib were examined for their efects on nitric oxide (NO) production in LPS-activated murine macrophage RAW 264.7 cells 264.7 cells [115]. Prostaglandin E2 is present in high concentrations in breast tumors and in metastatic cancers, in the absence of estrogen and progesterone receptors [116- 117]. Hence, the ability to inhibit production of PGE2 can be considered a good strategy in the design of anticancer agents. However, the MCF7 breast cancer cells produced very low levels of PGE2, according to another report [109]. Therefore, the active efficacy of the hybrid compounds was tested for the inflammatory response induced by LPS instead of by inhibiting PGE2 on MCF7 [118].. Therefore, cancer cell lines HT-29, Hep-G2, MCF-7 and inhibition of NO production were selected as initial tools to screen for anti-inflammatory activity of the study agent class. And to determine the anti-inflammatory and anti- cancer mechanisms of the hybrid substances, studies of PGE2 production inhibition activity, cell cycle analysis methods, apoptosis methods by nuclear staining. cells with Hoechst 33342, study of apotosis-inducing activity by caspase-3 indicator, study of apoptosis induction by FITC-anexin V and PI stained cell photometric method. Finally, substances selected and studied on biological mechanisms will be tested to interact with tubulin and COX2 targets by molecular docking method to confirm the biologic accuracy of the model in vitro. 3.2. Synthesis of coxib - combretastatin hybrids The single-reaction method has been applied to prepare the 1,4,5-triaryl- 1H pyrazole-3-carboxylate ethyl oxalyl chlorid, 1,2-diarylethanon and ethyl 11 oxalyl chloride arylhydrazine hydrochloride derivatives (Figure 2.1 , Figure 2.2, Figure 2.7, Figure 2.8) [109]. Reaction of Claisen condensation between two components 1,2-diarylethanon and ethyl oxalyl chloride with alkaline agent using tert-BuOLi, obtained the lithium salt intermediate product of ethyl 2,4- dioxo-3,4-diarylbutanoate , the reaction is continued with arylhydrazine hydrochloride through the Knorr reaction catalyzed by hydrochloric acid to produce triarylpyrazole-3-carboxylate (hybrids compound 79 to 98) and (hybrids compound 103 to 122). A cycle of celecoxib derivatization was also applied (Figure 2.4, Figure 2.5) 3,4,5-trimethoxyphenyl - 1,1,1-trifluoro-2,4- butanedion (65, 102) obtained from Claisen condensation between acetophenone and ethyl trifluoroacetate derivatives then reacts with 4- sulfamidophenyl hydrazine halide salt to form 3,4,5-trimethoxyphenyl, a substance with structure similar to celecoxib. According to the reaction procedures described in Figure 2.1, Figure 2.2, 20 coxib-combretastatin hybrid compounds were successfully synthesized with efficiency from 64 to 83% (Table 3.1) 02 compounds containing CF3 group with difference rates are 95% respectively. 78% (table 3.2). 20 acidic compounds of coxib - combretastatin hybrid with efficiency from 54 - 74% (Table 3.3); The substances are structurally determined by the methods of purification and nuclear magnetic resonance spectroscopy method NMR 1D, 2D. Of the hybrid compounds obtained, substances 79 to 98 and 102 to 122 were synthesized for the first time and never previously published. And 01 compound has been published and also used as a comparator is celecoxib (65) which is known for many studies [78-79]. 3.3. Biologically active coxib - combretastatin hybrids 12 3.3.1. Screening for biological activity of coxib - combretastatin hybrids Screening activity of 20 ester-based coxib-combretastatin hybrid compounds and 02 hybrid compounds containing groups CF3 The cytotoxicity of 20 coxib ester hybrid compounds and 02 CF3- containing compounds were evaluated for inhibition of cell growth on three lines HT-29, Hep-G2 and MCF-7 by method developed by Monks et al. [120]. The results are summarized in Table 3.4 according to descending activity. The anti-proliferative activity of celecoxib expressed on human cancer cells such as breast cancer (MCF-7), liver cancer (Hep-G2) rectal cancer (HT-29) has been published [ 121-123]. In the scope of this study, 12 new hybrid hybrid compounds 102, 96, 81, 82, 94, 93, 91, 92, 89, 90, 97, 85 exhibit cytotoxicity on strong MCF-7 strains. than celecoxib and five compounds 102, 96, 81, 82, 94 have IC50 <10 µM. In which, some compounds such as 102, 96, 94, 93 and 97 also have Hep-G2 toxi ity equiva ent to e e oxi ase su stan e . Furthermore five hy rid ompounds 0 0 showed - times stronger resistan e to H -2 e s than e e oxi radi a and ompound 0 with IC 0 0 . here y it an e onfirmed that the presence of nitrogen and halogen substituents at the para- position of both adjacent phenyl rings 96, 93, 91, 92, 97 contributes to increased cytotoxicity. Compound 102 with full structural features of celecoxib and CA4 frames showed outstanding cytotoxic activity with MCF-7 line. 21 compounds and celecoxib (65) were also tested for inhibitory effects of nitric oxide (NO) upon activation of lipopolysaccharide (LPS) on RAW 264.7 macrophages [124]. Interestingly, with the exception of compounds 102 and 98 with an exceptionally strong double activity for inhibiting NO production as well as cytotoxic activity on the cancer cell line MCF-7, the other compounds. in this group exhibited weaker NO production 13 inhibitory activity, emphasizing that they may have a better safety in the cardiovascular system than celecoxib [110]. Table 3.4. The cytotoxic effect of ester-type hybrid compounds on three cancer cell lines HT-29, Hep-G2, MCF7 and inhibition of NO production Entry Comp. R R’ R’’ IC50 (µM) Inhibits NO production HT-29 Hep-G2 MCF-7 1 102 3,4,5- triOMe 4-NH2SO2 1.95±0.11 43.0 ± 6.0 39.96± 3.36 4.63± 0.59 2 96 H 4-F 4-NH2SO2 81.6 ± 10.6 18.7 ± 1.7 29.37± 1.90 7.97±0.51 3 81 H H 4-CN 116.83±7.7 37.6±3.5 >254 8.20± 0.93 4 82 H H 4-NO2 129.88±15.7 21.2±2.1 >242 9.67± 0.92 5 94 H 4-OH 4-NH2SO2 92.3 ± 7.2 18.8 ± 1.5 14.51±1.36 8.90± 0.79 6 93 H 4-Br 4-NH2SO2 73.7 ± 2.6 23.5 ± 1.8 35.48±2.85 19.27±0.27 7 91 H 4-Br 4-CN 116.01±13.6 32.6± 3.0 >211 20.16± 1.83 8 92 H 4-Br 4-NO2 91.58±7.8 153.2± 10 >203 21.30± 1.40 9 89 H 4-Br 4-OMe 21.01±4.3 39.6±2.9 >209 21.51± 1.48 10 90 H 4-Br 4-CF3 > 233 10.0±0.7 75.87±4.41 21.65± 1.86 11 97 H 4-Cl 4-NH2SO2 122.2 ± 9.7 14.1 ± 1 37.04±4.95 23.79± 3.11 12 85 4- OMe 4-OMe 4-CF3 134.10±15.7 107± 10 >201 29.80± 2.71 13 43 4-Me 4-NH2SO2 83.59 ± 5.0 58.4 ± 3.4 37.11±2.14 30.67±3.79 14 98 H 4,6- diOMe 4-NH2SO2 4.41±0.17 136.0 ± 12 >197 39.93± 2.64 15 83 H H 4-NH2SO2 > 233 100 ± 10 54.35±4.57 40.54±2.56 14 Table 3.5. Comparison of cytotoxic effects of acid and ester hybrid compounds on MCF-7 cancer cells and inhibition of NO production 16 79 H H 4-OMe 30.0±13.4 >251 21.43±1.93 43.19±4.86 17 87 4- OMe 4-OMe 4-NO2 148.85±8.4 >211 >211 48.81± 2.62 18 86 4- OMe 4-OMe 4-CN 76.42±9.4 110.3±7.0 >220 48.86± 3.13 19 84 4- OMe 4-OMe 4-OMe 67.06±3.7 160±15 >218 49.32± 1.72 20 80 H H 4-CF3 59.52±5.5 17.3±2.0 63.17± 3.01 50.66± 4.46 21 88 4- OMe 4-OMe 4-NH2SO2 103.4 ±10.5 99.0±6.3 58.57± 4.70 68.43± 7.05 22 98 H 4,6-diOH 4-NH2SO2 > 208.5 173.9 ± 12 64.03± 5.75 83.60± 3.44 L-NMMA 8.39±0.31 Ellipticine 0.18± 0.04 0.44± 0.05 0.36± 0.04 STT R R’ R’’ IC50 (µM) Este (NO) Acid (NO) Este (MCF-7) Acid (MCF7) 1 3,4,5-triOMe 4-NH2SO2 1.95±0.11 43.0 ± 6.0 39.96± 3.36 4.63± 0.59 2 H 4-F 4-NH2SO2 81.6 ± 10.6 194.5±7.6 7.97±0.51 187.7±9.5 3 H H 4-CN 116.83±7.7 256.7±22.1 8.20± 0.93 154.5±15.1 4 H H 4-NO2 129.88±15.7 >261.1 9.67± 0.92 187.6±10.4 5 H 4-OH 4-NH2SO2 92.3 ± 7.2 75.8±2.6 8.90± 0.79 34.6±2.6 6 H 4-Br 4-NH2SO2 73.7 ± 2.6 >202.0 19.27±0.27 149.5±7.3 7 H 4-Br 4-CN 116.01±13.6 97.9±9.4 20.16± 1.83 70.1± 4.6 15 Note: *: Statistics column of ester activity **: Statistics column of acidic activity The super-active hybrid compounds 102, 96, 81, 82, 94 and celecoxib continued to be transferred to the PGE2 production test to further screen for the anti-inflammatory and anticancer activities of the active ingredient. 3.3.2. Study on anti-inflammatory and anti-cancer mechanisms 8 H 4-Br 4-NO2 91.58±7.8 111.2±8.7 21.30± 1.40 34.0±6.4 9 H 4-Br 4-OMe 21.01±4.3 >223.7 21.51± 1.48 >223.7 10 H 4-Br 4-CF3 > 233 80.0±6.3 21.65± 1.86 129.5±9.7 11 H 4-Cl 4-NH2SO2 122.2 ± 9.7 >220.7 23.79± 3.11 191.1±20.6 12 4-OMe 4-OMe 4-CF3 134.10±15.7 140.3±16.7 29.80± 2.71 136.0±8.4 13 4-Me 4-NH2SO2 83.59 ± 5.0 30.67±3.79 30.0±3.3 14 H 4,6- diOMe 4-NH2SO2 4.41±0.17 >208.7 39.93± 2.64 94.0±6.4 15 H H 4-NH2SO2 > 233 >239.8 40.54±2.56 178.9±12.9 16 H H 4-OMe 30.0±13.4 >271.7 43.19±4.86 209.3 ± 20.0 17 4-OMe 4-OMe 4-NO2 148.85±8.4 185.9±9.8 48.81± 2.62 31.8±3.7 18 4-OMe 4-OMe 4-CN 76.42±9.4 157.0±12.2 48.86± 3.13 83.0±10.3 19 4-OMe 4-OMe 4-OMe 67.06±3.7 145.7±15.2 49.32± 1.72 75.7±6.9 20 H H 4-CF3 59.52±5.5 109.4±5.8 50.66± 4.46 203.8 ± 5.1 21 4-OMe 4-OMe 4-NH2SO2 103.4 ±10.5 >209 68.43± 7.05 150.0±14.7 22 H 4,6-diOH 4-NH2SO2 > 208.5 >222.0 83.60± 3.44 >222.0 L-NMMA 8.39±0.31 Ellipticine 0.36± 0.04 16 3.3.2.1. Study on PGE2 production inhibition activity Typical compounds 102, 96, 81, 82, 94, celecoxib continued to be tested for the ability to inhibit PGE2 production. Table 3.7. Effect of test compounds on PEG2 production in LPS-stimulated RAW 264.7 macrophages PGE2 (pg/ml) Concentration (µM) 102 96 81 82 94 Concentration (µM) Celecoxib (43) 20 65.31 223.82 253.60 155.07 246.88 100 41.45 4 133.59 326.20 277.23 383.49 488.82 20 129.83 0.8 225.56 399.56 490.73 478.52 560.34 4 278.64 LPS 329.60 3.3.2.2. Cell cycle analysis Compounds 96, 81, 82 and 94 increased the number of cells in G0/G1 phase and decreased them in S and G2/M phases as celecoxib did. Celecoxib and related compounds ere also known to be able to inhibit tumor growth and to induce apoptosis Some research also resulted in the stable G0 /G1 block efect of celecoxib with unclear involvement of COX-2 as well as of PGE2 [104]. On other hand, compound 102 caused G2/M phase arrest as evidenced by the increase in cell number with a concomitant decrease in cells in phase S. G2/M phase arrest prevents cells from exiting mitosis, a feature shared by microtubule inhibiting agents such as colchicine or combretastatins [78]. In case of compound 102, replacement of the double bond of CA4 with pyrazole ring of celecoxib and maintaining trimethoxybenzene moiety of CA-4 were demonstrated to be crucial to obtain relevant cytotoxic and antimitotic efects [98, 115]. Table 3.8. Percentage of cell by phases of tested compounds in MCF7cells entrie Entries Compound Percentage of cell by phases (%) % G0/G1 % S % G2/M 1 Negative control (DMSO 0,5%) 42.46 40.47 13.15 2 102 (10 µM) 40.72 35.09 21.04 3 96 (10 µM) 49.00 34.17 14.17 4 81 (10 µM) 45.22 34.46 17.45 5 82 (10 µM) 50.96 30.75 14.80 6 94 (10 µM) 49.55 34.50 14.34 7 Celecoxib (30 µM) 46.80 36.69 11.45 17 (-) control 102 96 81 82 94 Celecoxib (43) Figure 3.3. Cell cycle analysis of tested compounds including 102, 96, 81, 82, 94 and celecoxib at the concentration of 10 µM on MCF-7 human breast cancer cells using Novocyte fow cytometry system (the experiment has been done one time) 3.3.2.3. Research results of apoptosis-causing activity of compounds 82 and 102 Since compounds 102 and 82 are potential inhibitors of antiproliferative activity, inhibiting PGE2 production more potent than celecoxib and selectively inhibiting cell cycle, it was selected for further evaluation of potential for apoptosis. Study on apoptosis activity of active substance through nuclear staining with Hoechst 33342 18 Table 3. 9. Percent of condensation or fragmentation in the cell nucleus caused by compounds 102 and 82 Tế bào Apoptosis (%) 82 (20 µM) 82 (10 µM) 82 (5 µM) 102 (20 µM) 102 (10 µM) 102 (5 µM) Camptotheci n (5µM) (-) Control 5.33 ± 0.34 2.69± 0.22 2.42± 0.27 12.38± 0.96 5.18± 0.33 3.55± 0.39 22.71± 2.01 2.71± 0.29 The study samples obtained MCF7 cell image after being stained with Hoechst 33342 at different concentrations: Negative control Camptothecin (5 µM) 102 (20 µM) 102 (10 µM) 82 (20 µM) 82 (10 µM) Figure 3.4. Image of MCF7 cells under the influence of research samples stained with Hoechst 33342 at different concentrations Study of apotosis-inducing activity by caspase-3 indicator Table 3.10. Activation of caspase 3 by compounds 102 and 82 % Tế bào Apoptosis Comp. 102 (20 µM) 102 (10 µM) 102 (5 µM) 82 (20µM) 82 (10 µM) 82 (5 µM) Camptothecin (5µM) (-) control Mean 1.36* 1.21* 1.01 1.08 1.12 1.11 1.67** 1.00 (SD) 0.033 0.013 0.037 0.023 0.056 0.040 0.020 0.070 - * P<0.05 and ** P<0.01 19 Study on apoptosis induction by FITC-anexin V and PI stained cell photometric method Table 3.11. Percentage of apoptosis cells Samples % Necrotic cells % Early apoptosis cell % % late apoptosis cell % Apoptosis cells Negative control 0.22 18.32 1.90 20.22 82 (20 µM) 1.39 18.49 4.08 22.57 82 (10 µM) 0.27 21.62 4.59 26.21 82 (5 µM) 0.16 22.74 3.27 26.01 102 (20 µM) 0.42 42.24 6.73 48.97* 102 (10 µM) 0.49 19.90 3.45 23.35 102 (5 µM) 0.27 15.84 2.17 18.01 Camptothecin (5µM) 0.08 73.83 4.96 78.79** Ghi chú: * là P<0.05; ** là P<0.01 Cell incubated with compound 82 (20 µg/ml) in 48h Cell incubated with compound 82 (10 µg/ml) in 48h Cell incubated with compound 82 (5 µg/ml) in 48h Cell incubated with compound 102 (20 µg/ml) in 48h Cell incubated with compound 102 (10 µg/ml) in 48h Cell incubated with compound 102 (5 µg/ml) in 48h 20 Cell incubated with 0.5% DMSO trong 48h Cell incubated with5 µM camptothecin in 48 h Fig 3.5. Cell apoptosis of tested compounds via Dead Cell Apoptosis Kit with Annexin V FITC and PI for fowcytometry. The x-axis represents the level of FITC-Annexin V staining; the y-axis represents the level of PI staining in log units. The conclusions determine the apoptosis induction potential of two study samples Through the determination of apoptosis induction through three methods of detecting apoptosis of the active substance by staining the cell nucleus with Hoechst 33342, caspase - 3 and cytometry can be summarized:  Sample 82 at test concentrations slightly increased the proportion of cells in early apoptosis and late apoptosis compared with negative control, reaching 22.57-26.21%.  Sample 102, at a concentration of 20 µg / ml increased the incidence of early and late apoptosis cells from 18.3% to 42.2% and 1.90% to 6.7% in the negative control, respectively. At lower concentrations there was a slight increase (10 µg / ml and 5 /g / ml) in the incidence of apoptosis compared to the negative control.  Camptothecin - positive control increased apoptosis rate, reaching from 78.79%.  Sample 82 has not clearly shown the ability to induce apoptosis to research concentrations in the trials performed;  Sample at a concentration of 10 M was able to induce apoptosis in MCF7 cells through caspase 3 induction with an increase of 1.21 times compared with negative control (P <0.05); 21  Sample 102 at a concentration of 20 M induces apoptosis on MCF7 cells through: inducing concentration or fragmentation of the cell nucleus at a rate of 12,386%; induction of caspase -3 generation with concentration increased 1.36 times compared with negative control (P <0.01); Apoptosis increased and especially early apoptosis increased 42.24% through cell photometric technique (P <0.05). Thereby, based on the results of cytotoxic activity, NO, PGE2 anti- inflammatory and apoptosis induction, we confirmed the mechanism and activity characteristics of the obtained hybrid compounds. The results showed that the hybrid molecule in terms of both molecular structure and biological activity according to the design target model of the original hybrid molecule. We have continued to opt for molecular docking analysis to simulate the interaction between engineered hybrid with COX2 and tubulin targets. 4. Molecular docking study According to AutoDock's rating criteria 4.2.6, the negative the value of energy, the better the ability of the compound to bind to the targeted receptor. Compounds 102 and 82 were shown to be two potential candidates with good binding energies for both targets and can be considered as potential COX2 / tubulin dual inhibitors, their interactions analyzed. added using Model Studio Visualizer. The resulting computation can be used to better predict a protein mode inhi itor ased on the differen e Δ etween the COX2 intera tion value and tubulin (Equation 1–2). ΔG=Do k s oreCOX2−Dockscroretubulin (1) ΔLE=LECOX2−Letubulin (2) Dock pose of compound 102 in the active site of COX2 showed three hydrog

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