Synthesis of a Cinnamic Acid Derivative and Bioactivity as an Anticancer Based on Result Quantitative Structure Activity Relationship (QSAR) Analysis

Synthesis of compound (E)-4-(3-oxo-3-(phenethylamino)prop-1-en-1-il)1,2-phenylene diacetate (3) analogue from (E)-3-(3,4-dihydroxyphenyl)-Nphenethylacrylamide as recommendation of analysis result of QSAR was done. In this synthesis, we used caffeic acid and it was used as starting material through reaction of acetylation, chlorination, and amidation. Acetylation was carried out by using acetic anhydride in pyridine at room temperature for 4 hours. Chlorination by using thionyl chloride reagent in dimethylphormamide in conditions of 80oC for 4 hours, and continued with amidation using phenethylamine in dichloromethane solvent at room temperature for 1 hour. The structure of each reaction product phase was identified by using FT-IR spectrophotometer. The amidation phase also identified by using 1HNMR, 13C-NMR, HSQC and HMBC. Acetylation produces white crystalline solids with melting point 182-184oC, and amidation produces compound 3 such us white crystalline with melting point 170-173oC. The results of bioactivity test on murine leukaemia P-388 cells showed that the bioactivity of compound 3 was clarified the strongest (IC50 = 0,5 μg /mL) so it was very potentially used as anticancer drug.

(E)-3-(3,4-dihydroxyphenyl)acrylate acid as an anticancer potential has the potential to be developed, but on the other hand, the synthesis process does not The design of new compounds is usually done by using the Quantitative Structure-Activity Relationship (QSAR) method. The QSAR methodology is used to systematically analyze the properties that accompany chemical structures of compounds in relation to available biological data (Simon-Hettich et al., 2006;Tropsha and Golbraikh, 2007).
The results of the preliminary analysis using QSAR in this study stated that the current position of the compound (E)-3-(3,4-dihydroxyphenyl)acrylate acid was at position numbers 5, 6, and 10 as shown in Figure 1. Addition of electron withdrawal groups or the electron-pushing group at this position allows the bioactivity of compounds to increase or decrease. Therefore, it is necessary to conduct research by synthesizing several compounds containing groups whose induction and mesomeric properties differ in that position. Based on the results of the QSAR analysis, the research was chosen to synthesize the target compound, namely (E)-4-(3-oxo-3-(phenethylamino)prop-1-en-1-yl)-1,2 phenylene diacetate (3), as shown in Figure 2. Inside compound 3, there is an acetyl group as the withdrawal group at position 6 and 7. Several cytotoxic activities of cinnamic acid derivatives have been tested against murine leukaemia P-388 cells using the MTT method. Therefore, the activity of compound 3 obtained in this study was also determined on the test cells and the method.
The acetylation stage 1.0 gram (6.0 mmol) of caffeic acid was put into a threeneck round bottom flask, then added 3.0 mL of pyridine and 1.8 ml (17.0 mmol) of acetic anhydride. The reaction mixture is stirred using a stirrer for ± 2 hours at room temperature. Next, the reaction mixture was added to 50.0 ml of cold distilled water while stirring. The formed white precipitate is filtered, washed with distilled water and dried in a desiccator to obtain a dry white solid. The solid is recrystallized from a methanol solvent then filtered to obtain a filtrate, and allowed to stand in the refrigerator until a white crystal is formed. Subsequently, the solids were tested for their melting point and purity through TLC analysis with three kinds of eluent systems. The pure crystals obtained were analyzed by FT-IR spectroscopy. IR

Chlorination Stage
The acetylation stage compound of 1.2 grams (5.0 mmol) was dissolved in 15 mL benzene and added with SOCl2 0.5 mL (7.2 mmol). The reaction mixture was refluxed for 4 hours, and nitrogen gas drained for 7 minutes. After reflux, the reaction mixture was cooled until room temperature and evaporated until a yellow solid was obtained.

Amidation Stage
The chlorination stage compound was added with 0.4 mL (4.0 mmol) of phenethylamine, 0.1 ml (0.9 mmol) of pyridine and 0.4 ml (2.9 mmol) of triethylamine. The mixture was then dissolved in 30.0 ml dichloromethane and sterilized for 1 hour, washed with HCl then NH4Cl, dried using anhydrous

Anticancer Bioactivity Test
Bioactivity test of synthesis compound against murine leukaemia P-388 cells was carried out by the MTT method. The target compound in various concentrations and Artonin-E as a comparison was added to murine leukemia P-388 cells which were then incubated for 48 hours. Subsequently, the sample was added to the colouring reagent ([3-(4,5-dimethyltiazo-2-il)-2,5diphenyltetrazoliumbromide) and re-incubated for 4 hours.
After adding stop solution reagents, sample uptake was measured using a microplate reader at λ = 540 nm, then the number of murine leukemia P-388 cells that were inhibited by the sample was calculated to obtain an IC50 value

Result and Discussion
Each product in the synthesis stages was tested using an FT-IR spectrometer, except for chlorination product that was not purified. Specifically for compound 3, the characterization was continued by using 1 H-NMR spectrometers, 13 C-NMR, HSQC, and HMBC.

Synthesis of compound 1
At this stage, a white crystalline product with a melting point of 182-184oC and a yield of 77% was obtained. The comparison of FT-IR spectra of (E)-3-(3,4dihydroxyphenyl) acrylate acid precursor compound ( Figure 1) and acrylic (E)3-(3,4-diethoxyphenyl) acid (compound 1) showed the success of the acetylation reaction.
The FT-IR spectrum of compound 1 shows the presence of a typical carbonyl (C=O) ester group with a sharp absorption band and strong intensity at wave number of 1762.94 cm-1 and supported by the presence of C-O acetyl ester bonds in the region of 1190.08 cm-1 and 1016, 49 cm-1. The absence of OH phenolic absorption band at 3437.15 cm-1 is a sign that the compound has undergone an acetylation reaction, both in the phenolic group and in the carboxylic group. FTIR spectra data of precursor compounds and compound 1 are summarized in Table 1.

Synthesis of compound 2
The product of the synthesis reaction of compound 2 was

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View Article Online used without further purification because it is thought to be very reactive to water, even with water vapour in the air can cause Cl to be substituted with OH again. Therefore, the yellow solid obtained in this reaction stage was continued to the amortization stage in situ.

Synthesis of compounds 3
At this stage, a white crystalline product with a melting point of 170-173oC obtained. In the FT-IR spectrum, there is a sharp absorption band with moderate intensity at 3309.85 cm-1 which is an amide II absorption band (N-H strain) which is supported by absorption band at 1544.  Table 2. The 13 C-NMR spectrum of compound 3 (Figure 4) shows that there are 19 signals representing 21 C atoms because there are two signals representing the positions of carbon atoms that are identical in their chemical environment, namely C-17 and C-21 and C-18 and C-20.
Signals that appear in the highest shift region, namely at d 168.3 ppm, 168.2 ppm, and 165.5 ppm each come from two groups of C=O carbonyl phenolic esters of acetate (C-10 and C-12) and C=O amide group (C-1).
The 1 H-NMR spectrum of compound 3 ( Figure 5) indicates the existence of a spin olefin system with trans conformation at d 7.54 ppm (1H, d, J = 15.6 Hz, H-3) and 6.23 ppm (1H, d, J = 12.6 Hz, H-2). Two types of methyl protons in the phenethyl group each appear as tetraplets at d 3.65 ppm and triplets at d 2.88. Three aromatic protons of the phenolic group and five protons of the phenyl group all give signals that appear in the region of d 7.17 -7.34 ppm, while the methyl protons of the two acetyl groups appear as two singlet signals at d 2,293 and 2.91 ppm with integration values each of 3 (total of 6). The proton group (-CO)N-H (amide) appears at d 5.63 ppm.

Bioactivity Assay Against P-388 Leukemia Cells
Bioactivity study of compound 3 against P-388 leukemia cells resulted IC50 of 0.5 µg/mL. The categories in cytotoxic compounds according to the US National Cancer Institute consist of 4 categories, namely very toxic categories if IC50 values ≤ 20 µg/mL, moderate or sufficient cytotoxics active if the IC50 value is in the range of 21-200 µg/mL, cytotoxic is weak if the IC50 value is in the range 201-500 µg/mL and if the IC50 value ≥ 500 µg/mL, it is included in the non-toxic category (Abdel-Hameed et al., 2012 ). In other words, the smaller the IC50 value produced by a compound, the greater the cytotoxic activity.
Based on the above categories, the results of the bioactivity test of compound 3 are categorized as very toxic to P-388 leukaemia cells, because the IC50 value is ≤ 20 µg/ml.
The intense activity of compound 3 is thought to be related to the low polarity of the compound, which makes it possible to penetrate cell walls easily and provide inhibition of leukaemia cell growth. This is different from caffeic acid, which has a higher polarity, so it is difficult to penetrate the cell wall and cause the smaller concentration of caffeic acid to be adsorbed by cells.
In the conversion of hydroxyl groups, caffeic acid into ester groups and the conversion of carboxylic acid groups to amides resulted in a decrease in polarity of the compound and its stability also increased, thus allowing the bioactivity of compounds to increase. This is reflected by the IC50 value of compound 3 against murine leukaemia P-388 cells of 0.5 µg / mL, while caffeic acid gives an IC50 of 54.31 µg / mL (Son, 2002) Comparing the results of previous studies on the bioactivity of amide analogue compounds against murine leukaemia P-388 cells, namely phenethyl trans-3-(4hydroxy-3-methoxyphenyl)acrylate with IC50 of 10.79 μg/mL and trans-3-(4-hydroxy-3-methoxyphenyl)-Nphenethylacrylamide with IC50 of 29.14 μg/mL (Firdaus et al., 2016), the activity of compound 3 is higher than the two compounds.
This fact shows that in addition to the p-hydroxycinamide group, amide groups also affect the bioactivity of cinnamic acid derivative.