THE DERIVATIZATION OF SOME INDONESIAN NATURAL COMPOUNDS AS UROKINASE PLASMINOGEN ACTIVATOR ENZYME INHIBITOR AS ANTICANCER CANDIDATE

• Main Author: Lohita Sari, Bina
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/62963
• Institution: Institut Teknologi Bandung

Cancer is a disease characterized by uncontrolled cell division. Cancer cells have the ability to invade other biological tissues, either by direct growth in adjacent tissues (invasion) or by migration of cells to distant sites (metastasis). Urokinase-type plasminogen activator (uPA) is a serine protease enzyme group that plays an important role in physiological and pathological regulatory processes. The binding of uPA to the membrane-specific uPA receptor (uPAR) can degrade the extracellular matrix (ECM) and cause tumor cell progression, invasion, and metastasis. Research on inhibition of uPA activity is considered to increase the efficiency of cancer therapy. Treatment of cancer cells begins with chemotherapy, immunotherapy, hormone therapy and radiation. In vitro tests on two synthetic uPA inhibitors, namely p-aminobenzamide, and amiloride, have been shown to reduce prostate tumors in humans. Doxorubicin, an anthracycline antibiotic, is used as a cancer treatment drug. Although many other synthetic drugs are used for cancer therapy, they can induce a secondary cancer. Natural compounds and its derivative can be alternative. Quercetin (3,3',4',5'7-pentahydroxyflavone) in vegetables and fruits such as broccoli and apples, may play a role in the prevention or treatment of cancer because they have been consumed traditionally in cancer patients as supporting food. The discovery of anticancer drug can be done through the search for active compound or random screening of candidate drug compound from a database based on cancer macromolecular targets. Structure-Based Virtual Screening (SBVS) from the Indonesian Herbal Database (HerbalDB) using a structure-based pharmacophore model, was used to identify and design potent inhibitors of uPA enzyme target. Verification of the pharmacophore model using the Demanding Evaluating Kits for Objective In Silico Screening database (Dekois 2.0). The validated pharmacophore model and 1412 ligands from HerbalDB were inputted online into Pharmit (http://pharmit.csb.pitt.edu) and Lipinski's rule was used to perform virtual screening. The hits compound result was determined by docking score (S score) using the Molecular Operating Environment (MOE 2009.10) as molecular docking protocol. Validation of the molecular docking program using decoys from A Directory of Useful Decoys (DUD) consist of 162 active compounds and 9840 decoys. Compounds with more negative docking scores than others, and the bond interactions with amino acid residues were determined. Pharmacokinetics prediction of hit compounds using the pkCSM website. The stability of the uPA enzyme complex with the compound because of molecular bonding was determined by molecular dynamics (MD) simulations. Simulations were carried out until the system was in a stable state (based on the analysis of energy, pressure, temperature, Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF). The binding free energy was determined by calculating Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA). One of the developments of natural material compounds through the formation of analogues or their derivatives. Acetylation of the hits compound of quercetin, is one of the analogues formations that can increase the bioavailability. Acetylation using acetic anhydride reagent and sodium acetate as a catalyst for 2 hours at 0°C to synthesize 3,3',4',7-tetraacetylquercetin (Compound 1) and and acetic anhydride reagent and pyridine catalyst for 6 hours at room temperature to synthesize 3,3',4',5,7- pentaacetylquercetin (Compound 2). The characterization of the synthesis product was identified by FT-IR, ESI-MS, and 1H- and 13C-NMR spectroscopy. Molecular docking, visualization of the docking result, pharmacokinetic prediction, molecular dynamics simulation, and binding free energy based on MM-PBSA were done to the analog of compounds 1 and 2. One of the developments of natural material compounds is through the formation of analogues or their derivatives. Acetylation of compounds hits quercetin is one of the formations of analogs that can increase bioavailability. Acetylation using acetic anhydride reagent and pyridine catalyst for 10 minutes and 6 hours at room temperature to synthesize 3,3',4',7-tetraacetylquercetin (compound 1) and 3,3',4',5,7- pentaacetylquercetin (compound 1). 2). The characterization of the synthesis product was identified by FT-IR, ESI-MS, and 1H- and 13C-NMR spectroscopy. Molecular anchoring was carried out, visualization of tethering results, pharmacokinetic predictions, molecular dynamics simulation, and binding energy based on MM-PBSA to analog compounds 1 and 2. In vitro assay of uPA inhibitor on the anchored compound and the two analogs used a spectrofluorometric method based on the cleavage of 7-amino-4- trifluoromethylcoumarin (AFC) with a substrate base to form a fluorescent AFC (?ex = 350 nm/?em = 450 nm). The value of % Relative Inhibition is the result of the fluorescence intensity concerning to time within a certain time range (every 5 minutes). Calculation of IC50 value is the concentration of compounds that can inhibit 50% of uPA enzyme activity calculated using a sigmoid curve with a slope value based on the Hill coefficient. The result of the pharmacophore verification is that Model _3 of the PCH scheme consists of four pharmacophore features, namely two hydrophobic groups (benzene and thiophene), one atom as a hydrogen bond donor, cationic, metal ligator (NH2 + ), and one metal ligator (S atom) meeting the modeling requirements. pharmacophore with a hit score of Enrichment Factor (EF, Goodness of Hit Score (GH), sensitivity, and specificity namely 2.48 0.723, 0.6, and 0.94 respectively. So that Model_3 can be used to determine bioactive molecules using virtual screening. The virtual screening yielded 72 compound hits. Molecular anchoring validation by redocking resulted in an RMSD 0.5174, Area Under the Curve (AUC) 0.709 and EF 1% 61.74. Four hits compounds with more negative S score, namely Isorhamnetin, Rhamnetin, Quercetin, and Kaempferol (-129.64; -128.37; -126.03 and -116.83 kcal/mol. The four hits compounds are the flavonol group. Isorhamnetin and Rhamnetin are O-methyl flavonol (3'-methoxy quercetin and 7-methoxy quercetin) and Kaempferol a 3,4',5,7- tetrahydroxyflavone. The pharmacokinetic prediction of isorhamnetin and rhamnetin is better than the other two hit compounds, namely easy absorption, as inhibitors of metabolism CYP1A2, easy to excrete, toxicity LD50 value < 5000 mg/kg (harmful if swallowed) RMSD and RMSF result from MD simulations showed that the uPA-ligand complex was in a stable state and the free energies of isorhamnetin, rhamnetin, and kaempferol were stronger than quercetin. Compounds 1 and 2 were isolated using classical column chromatography and TLC methods. Yellowish white crystals were produced with the yield of Compounds 1 and 2 of 41.37% and 46.05%. The melting points of compounds 1 and 2 are 159-161.10C and 172-174.30C. Identification of Compound 1 by column chromatography, TLC, and two-dimensional TLC. Compound 2 was purified with ethyl acetate and identified by TLC. Identification by FT-IR, Compound 1 showed a peak at 3994 cm-1 presenting of an OH group, but this peak did not appear in Compound 2. The 1H-NMR spectrum of Compound 1 at H-6, 8, 5', 6', 2', CH3, and OH. 13C-NMR spectrum of carbon signal with four carbonyl and methyl signals. 1H-NMR Spectrum of Compound 2 at H-6, 8, 5', 6', 2', and CH3. The 13C-NMR spectrum of carbon signal with five carbonyl and methyl signals. Mass spectroscopy results (ESI-MS) Compound 1 m/z calculated value was C23H18O11 [M+Na] + 493.38 and compound 2 m/z calculated value was C25H20O12 [M+Na] + 535.09. The S score of the two compounds is lower than quercetin due to the reduced hydrogen bonding of the OH group. Prediction of absorption, distribution, and excretion of both compounds was better when compared to quercetin. Compounds 1 and 2 were predicted as inhibitors and non-inhibitors of CYP1A2 and the LD50 toxicity value was < 5000 mg/kg. Compounds 1 and 2 were isolated using the classical column chromatography method. Yellowish white crystals were produced with yields of compounds 1 and 2 of 41.37% and 46.05%. The melting points of compounds 1 and 2 are 159-161.10C and 172- 174.30C. Identification of compound 1 with TLC eluent DCM: acetone (9.0: 1.0 v/v) Rf value = 0.48 and compound 2 with DCM eluent: acetone (9.5: 0.1 v/v) Rf value = 0.43. Identification by FT-IR, compound 1 showed a peak at 3994 cm-1 from the OH group but did not appear in compound 2. 1H-NMR spectrum of compound 1 at H-6, 8, 5', 6', 2', CH3, and OH. 13C-NMR spectrum of carbon signal with four carbonyl and methyl signals. 1H-NMR spectrum of compound 2 at H-6, 8, 5', 6', 2', and CH3. 13C-NMR spectrum of carbon signal with five carbonyl and methyl signals. Mass spectroscopy results (ESI-MS) of compound 1 calculated m/z value C23H18O11 [M+Na]+ 493.38 and compound 2 calculated m/z value C25H20O12 [M+Na]+ 535.09. The anchoring score of the two compounds is lower than that of quercetin due to the reduced hydrogen bonding of the OH group. Prediction of absorption, distribution and excretion of both compounds was better when compared to quercetin. Compounds 1 and 2 were predicted as inhibitors and non-inhibitors of CYP1A2 and the LD50 toxicity value was < 5000 mg/kg. The in vitro test results for uPA inhibitor, quercetin, compounds 1, and 2 showed a slope that met the requirements, which was between 1.30 - 2.31. The response is related to slope and concentration. When the slope increases, the concentration increases. The IC50 values of compounds 1 and 2 were more negative when compared to quercetin as the parent compound and the three other hits compounds. Based on the results of the study, it can be concluded that compound 2 (3,7,3',4',5-pentaacetyl quercetin) is the most powerful inhibitor of the uPA enzyme and can be developed in structural design as a new uPA inhibitor.