Dissertation summary Organic chemistry: Synthesis and antiinflammatory, antiproliferative activities of new Coxib–Combretastatin hybrids

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Objectives of the dissertation: Structured design of coxib - combretastatin hybrid compounds; synthesize coxib - combretastatin hybrid compounds; screening for anti-cancer and anti-inflammatory activities of hybrid compounds; icreening for anti-cancer and anti-inflammatory activities of hybrid compounds.

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Nội dung Text: Dissertation summary Organic chemistry: Synthesis and antiinflammatory, antiproliferative activities of new Coxib–Combretastatin hybrids

MINISTERY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY ------------- NGUYEN THI THUY HANG “SYNTHESIS AND ANTIINFLAMMATORY, ANTIPROLIFERATIVE ACTIVITIES OF NEW COXIB–COMBRETASTATIN HYBRIDS’’ Scientific Field: Organic Chemistry Classification Code: 9.44.01.14 DISSERTATION SUMMARY HA NOI - 2021
The dissertation was completed at: Institute of Chemistry Vietnam Academy of Science and Technology Scientific Supervisors: 1. Assoc. Prof. Dr. Ngo Quoc Anh 2. Assoc. Prof. Dr. Vu Dinh Hoang 1st Reviewer: ........................................................................... ................................................................................. 2nd Reviewer: .......................................................................... ................................................................................. 3rd Reviewer:........................................................................... ................................................................................. The dissertation will be defended at Graduate University of Science And Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay District, Ha Noi City. At ….. hour….. date….. month …..2021. The dissertation can be found in National Library of Vietnam and the library of Graduate University of Science And Technology, Vietnam Academy of Science and Technology.
1 INSTRODUCTION 1. The urgency of the thesis Cancer is a group of diseases that involve disorganized cell division and cells that have the ability to invade other tissues by either growing directly into nearby tissue or moving to multiple locations different (metastatic). According to the Global Cancer Organization GLOBOCAN 2018, there are currently more than 300,000 people living with cancer nationwide, there are 164,671 new cases, 114,871 people die from this disease. Globally, there are about 23 million people infected, of which more than 14 million people are newly infected and 8 million 2 hundred thousand people die. The World Health Organization (WHO) ranked Vietnam in the top 50 countries in the top 2 of the cancer map. Researching to find cancer treatment drugs with few side effects is one of the directions that is always interested in the scientific community. Among current treatments, chemotherapy is a cancer treatment that uses one or more anticancer drugs - cytotoxic. One of the anti-cancer drugs, used today in chemotherapy, influences the cell cycle to inhibit cancer cell growth and subsequently induce apoptosis (apoptosis). Prospective treatment guidelines often recommend a combination of anticancer drugs acting on a variety of mechanisms. Nowadays, research discovering drugs targeting multiple targets is gaining attention, with a desire to combine different molecular targets on a single chemical agent. The advantages of a hybrid molecule over a combination of many drugs can improve the technical limitations, side effects and resistance of a single target [1]. One of the most important groups of anticancer agents are tubulin-binding molecules. Although some drugs that target microtubule have been used in clinical
2 practice, it is still necessary to look for new agents that can overcome the limitations of resistance and the undesirable side effects of these therapies. current method [3]. 2. Objectives of the dissertation 1. Structured design of coxib - combretastatin hybrid compounds 2. Synthesize coxib - combretastatin hybrid compounds 3. Screening for anti-cancer and anti-inflammatory activities of hybrid compounds 4. Identifying anti-inflammatory and anti-cancer mechanisms of hybrid compounds 5. Docking of a hybrid coxib - combretastatin compound with two purpose effect COX2 and tubulin 3. The main research contents of the thesis - Research on coxib - combretastatin hybrid compounds synthesis - Determination of the structure of coxib-combretastatin hybrid compounds - Screening the activity of coxib - combretastatin hybrid substances - Study on mechanism of action of some coxib - combretastatin hybrid substances - Research docking of typical hybrid compounds with target effects of colchicine and COX2
3 Figure 31: Simulation of coxib - combretastatin hybrid compound according to set target
4 DISSERTATION CONTENTS CHAPTER 1. LITERATURE REVIEW General presentation of anticancer compounds by tubulin inhibitory mechanism, profile of tubulin mechanism. The group of tubulin-based compounds has always been a topic of concern in the field of anti-cancer drug research. Combretastatin compounds with rich biological activity have been used in the treatment of a number of cancers. They are known for their cytotoxic activity by inhibiting tubulin polymerization at the colchicine site [ 2]. Up to now, these cancer treatment compounds with this mechanism are still being widely used and always a research direction that receives a lot of attention. Overview of combretastatin compounds, which belong to the class of cis- stilbene, a rich source of pathogens in the search for new drugs, typical compounds such as resveratrol and combretastatin 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
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 HT-29 Hep-G2 MCF-7 production 1 102 3,4,5- 4-NH2SO2 1.95±0.11 43.0 ± 6.0 39.96± 3.36 4.63± 0.59 triOMe 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- 4-OMe 4-CF3 134.10±15.7 107± 10 >201 29.80± 2.71 OMe 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- 4-NH2SO2 4.41±0.17 136.0 ± 12 >197 39.93± 2.64 diOMe 15 83 H H 4-NH2SO2 > 233 100 ± 10 54.35±4.57 40.54±2.56
14 16 79 H H 4-OMe 30.0±13.4 >251 21.43±1.93 43.19±4.86 17 87 4- 4-OMe 4-NO2 148.85±8.4 >211 >211 48.81± 2.62 OMe 18 86 4- 4-OMe 4-CN 76.42±9.4 110.3±7.0 >220 48.86± 3.13 OMe 19 84 4- 4-OMe 4-OMe 67.06±3.7 160±15 >218 49.32± 1.72 OMe 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- 4-OMe 4-NH2SO2 103.4 ±10.5 99.0±6.3 58.57± 4.70 68.43± 7.05 OMe 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 Table 3.5. Comparison of cytotoxic effects of acid and ester hybrid compounds on MCF-7 cancer cells and inhibition of NO production IC50 (µM) STT R R’ R’’ Este (NO) Acid (NO) Este Acid (MCF7) (MCF-7) 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 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 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
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 Concentration 102 96 81 82 94 Celecoxib (43) (µM) (µM) 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 Percentage of cell by phases (%) Entries Compound % G0/G1 %S % G2/M Negative control 1 42.46 40.47 13.15 (DMSO 0,5%) 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 (%) Camptotheci (-) 82 82 82 102 102 102 n Control (20 µM) (10 µM) (5 µM) (20 µM) (10 µM) (5 µM) (5µM) 2.42± 5.18± 3.55± 2.71± 5.33 ± 0.34 2.69± 0.22 0.27 12.38± 0.96 0.33 0.39 22.71± 2.01 0.29 The study samples obtained MCF7 cell image after being stained with Hoechst 33342 at different concentrations: Negative control Camptothecin (5 102 (20 µM) 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. 102 102 (10 102 82 82 82 Camptothecin (-) (20 µM) µM) (5 µM) (20µM) (10 µM) (5 µM) (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