INTRODUCTION:

Curcumin and its analogous compounds have biological activities like antioxidant, anti-inflammatory, hepatotoxic protective, antiviral, and antibacterial activities^1–11. Value-added the use of curcumin and its analogs is their low toxicity¹². However, even though it has effects and safety, curcumin still has several weaknesses among other low solubility in water, low bioavailability, unstable, and intense color of curcumin ^1,13–18. The most likely modification is to substitute the hydrogen (H) on the phenyl group of both flanks. The synthesis of curcumin analogs was carried out by Claisen-Schmidt condensation, namely condensation of aromatic aldehyde compounds with ketones.^5,19–23

The term Green Chemistry brings about changes in conventional chemical synthesis processes. Green Chemistry makes a synthetic method more efficient, shortens synthetic route, uses less hazardous solvents, and minimizes waste^24–27. In conventional reactions, the hot plate method is used as a heating source, while for green chemistry reactions, microwave irradiation is another method used as a heating source in the chemical synthesis process. The basic mechanisms in the synthesis by microwave irradiation are dipolar polarization and conduction. The synthesis of curcumin analog compounds by microwave irradiation has been carried out by obtaining large yields (24,25). Dibenzylidenecyclohexanone (HGV), dibenzylidenecyclopentanone (PGV), and 1,5-diphenyl-1,4-pentadien-3-on (GVT) (Fig. 1) containing mainly hydroxy groups at the para position on the aromatic ring (R2), chlorine (R1 and R3) are known to have anti-inflammatory, antioxidant, and antibacterial activities (28). It takes a long time to obtain synthetic products, namely 1 to 2 weeks with a small yield (60-70%) (21). This study aims to reduce the synthesis time of HGV-6, PGV-6, and GVT-6, as well as to determine the toxic effects.

Figure 1. Chemical structure of HGV-6, PGV-6 and GVT-6 (5)

MATERIALS AND METHODS:

Chemicals:

3,5-dicloro-4-hydroxybenzaldehyde, acetone, cyclohexanone, cyclopentanone, tetrahydrofuran, hydrochloric acid, ethyl acetate, n-Heksane, dichloromethane, chloroform, methanol, ethanol and aquadest acquired from Sigma-Merck and analytical grade.

Instrument:

BUCHI Melting Point B-540 with a temperature gradient at 5°C/min was used for the melting point test. The purity of the synthesized compound was measured using HPLC Elite La-Chrome®. JEOL® spectrophotometer 500 MHz was used to measure the ¹H-NMR Spectrum.

Procedure synthesis of HGV-6, PGV-6, and GVT-6:

In each porcelain crucible added 3,572 mmol of 3,5-dicloro-4-hydroxybenzaldehyde, cyclohexanone (0,185 mL; 1.786mmol), cyclopentanone (0,311mL; 3,009 mmol), acetone (0,183mL; 2,487mmol), and 0.2ml of concentrated hydrochloric acid respectively. Excellent yields of the product were obtained when the reaction mixture was irradiated for 10 minutes with 160 Watt. Recrystallization performed by acetone. TLC and melting point tests were carried out to find out the purity of product qualitatively.

Purity test by HPLC:

Purity test of the product synthesis was using HPLC. Products was analyzed at 100ppm concentration. The analysis was carried out at 350nm UV wavelength and the mobile phase of acetonitrile: water (80:20) with a flow rate of 1ml/min, a pressure of 100 psi, and an injection volume of 20μL on column C18. HPLC analysis results (Table 1) shown that the synthesis results are pure.

Table 1: Results of purity test

Compound	Retention Time (Min)	Area (%)
HGV-6	2.31	99
PGV-6	1.30	100
GVT-6	1.32	100

2,6-bis-(3’,5’-dicloro-4’-hydroxybenzylidene)-cyclohexanone

Orange yellow powder; yield 82.35%; Rf = 0.3, n-Hexane: CHCl3 (2:5); mp. 200.1–201.2^oC; IR γ (cm-1) (KBr): 3518 (-OH bonded); 3155 (=C-H stretching); 2924 (-C-H stretching); 1681 (C=O stretching); 1581 and 1489 (C=C stretching); 1620 (C=C stretching); 933 (C-H bending); 1165 and 1188 (C-O stretching); 1249 (chloro-substituted benzene). MS (EI-MS, m/z)= 444 [M^+·]; 344;308; 281; 252; 231; 217; 199; 178; 164;136;115; 101 (base-peak); 75; 63; 53; 39. ¹H-NMR (500 MHz, ppm, DMSO-d6): δ (ppm): 3,62(s,2H,-CH₂, H₄); 3,28(s,4H,-CH₂, H_3,5): 8,25(s,2H,-CH, H_7’): 8,34(s,4H,-CH, H_2’,6’): 11,44(s,2H,-OH).

2,6-bis-(3’,5’-dicloro-4’-hydroxybenzylidene)-cyclopentanone

Orange yellow powder; yield 77.29%; Rf = 0.32, n-Hexane: CHCl3 (2:5); mp. 262.1–262.9°C; IR γ (cm-1) (KBr): 3518 (-OH bonded); 3155 (=C-H stretching); 2924 (-C-H stretching); 1681 (C=O stretching); 1581 dan 1489 (C=C stretching); 1620 (C=C stretching); 933 (C-H bending); 1165 and 1188 (C-O stretching); 1249 (chloro- substituted benzene). MS (EI-MS, m/z )= 430[M+·] = 395; 330; 294; 260; 231; 200; 179; 165; 144; 115; 101(base-peak); 75; 51; 39. ¹H-NMR (500 MHz, ppm, DMSO-d6): δ (ppm):3,03(s,4H,-CH₂ H₃_,4); 7,31 (s,2H, H₇_’); 7,68 (s,4H,H_2’_,6’); 10,80 (s,2H,-OH, H_4’).

2,6-bis-(3’,5’-dicloro-4’- hidroxyfenyl)-1,4-pentadine-one

Yellow powder; yield 79.61%; Rf = 0.25, n-Hexane: CHCl3 (2:5); mp. 254.0–255.1°C; IR γ (cm-1) (KBr): 3518(-OH bonded); 3224(=C-H stretching); 3093(-C-H stretching); 1689(C=O stretching); 1651(C=C stretching); 1581 dan 1489 (C=C stretching); 979 (C-H bending); 1165(C-O stretching); 1280 (chloro- substituted benzene). MS (EI-MS, m/z )= 404[M+]; 404; 304; 278; 250; 238; 225; 197; 169; 162; 145; 133; 109; 97; 83; 73(base-peak); 49; 47. ¹H-NMR (500 MHz, ppm, DMSO-d6): δ (ppm) : 7,23(d, J=16,CH,H_8,7); 7,63 (d, J=16,CH,H_7,11); 7,821(s,2H,CH (H_6,4); 9,267(s,-OH).

RESULT AND DISCUSSION:

The conventional chemical syntheses method generally requires longer heating times, complicated equipment settings, higher process costs, and excessive use of solvents/reagents during the synthesis process. During this process, many health and safety issues arise for workers. Besides that, it is bad for the environment due to its waste. Application of microwave irradiation in the synthesis we can significantly increase the efficiency, minimize solvents, and cut synthetic stages and minimize waste as much as possible. Microwave-assisted synthesis is considered an important approach towards green chemistry, as this technique is more environmentally friendly. Because of its ability to pair directly with reaction molecules, and by-passing thermal conductivity leading to rapid temperature rises, microwave irradiation has been used to enhance many organic syntheses.

Microwave irradiation can be used as a heat source; it is highly efficient and can be used to significantly reduce the reaction time of a variety of synthetically useful chemical transformations. Thus, microwave-assisted synthesis is better than conventional synthesis: it is more energy-efficient and can increase the yield of synthetic products. The synthesis of curcumin analog mono-carbonyl compounds with microwave-assisted gives optimal results, where the yield of HGV-6, PGV-6, and GVT-6 is higher than conventional synthesis methods. The advantages of the synthesis of curcumin analog mono-carbonyl compounds with microwave-assisted are faster reaction, better yield and higher purity, energy-saving, uniform and selective heating, environmentally friendly, and reproducibility.

Figure 2: The percentage of zebra larvae mortality

Acute toxicity results:

The acute toxicity testing of HGV-6, PGV-6, and GVT-6 compounds was carried out to determine the effect of their toxicity on zebrafish larvae. The toxicity test of a material can be carried out through testing of fish or fish larvae, be it freshwater, estuary, or marine fish (29–33). The effect of a toxic substance is determined by a factor of the length of time of exposure and concentration. The parameter used to evaluate the potential toxicity of HGV-6, PGV-6, and GVT-6 compounds are LC₅₀. The LC₅₀ value of a compound is a manifestation of the toxicity potential of a related compound or drug.

The concentration used in the acute toxicity testing was 2mg/ml; 1mg/ml; 0.5mg/ml; 0.25mg/ml; 0.125mg/ml and 62.5mg/ml. Dimethylsulfoxide (DMSO) is used as a solvent because the sample has low water solubility. Observation of the acute toxicity testing conducted on larvae is the number of larvae deaths during 72 hours. The percentage of larval mortality over 72 hours is shown in the figure 2. Based on this figure, it can be seen that at a concentration of 2mg/ml GVT-6 shows the highest percentage of larvae mortality, which is (95±2.4)%. The percentage of larval mortality due to administration of GVT-6 was much higher than the HGV-6, PGV-6, and Rifampicin. This shows that the change in the form of mono-carbonyl curcumin to acetone in GVT-6 increases the percentage of larval mortality.

The calculation to determine the LC₅₀ value used probit analysis. Probit analysis is a regression analysis to determine the concentration-response relationship (percentage of deaths) in order to obtain a straight line so that the LC₅₀value can be determined more accurately. The LC₅₀value is used to determine the toxicity potential of the HGV-6, PGV-6, GVT-6, and Rifampicin compounds against zebrafish shown in Table 2. The value of toxicity Categories in Larvae can be seen in table 3. Based on the table, it can be concluded that HGV-6, PGV-6, and GVT-6 compounds have low toxicity values so it is safe to be used as a drug candidate.

Table 2. Potential toxicity of HGV-6, PGV-6, GVT-6, and Rifampicin against zebrafish based on probit analysis.

Comp.	LC₅₀ Value (µg / ml) ± SEM	Category
HGV-6	12280,35±46,45	Weak
PGV-6	10591,47±10,75	Weak
GVT-6	7798,22±63,33	Weak
Rifampicin	743653,54±98,22	Weak

Table 3. Toxicity Categories in Larvae (34)

LC₅₀Value - 72 Hours (mg / L)	Category
< 1	Very high
1 – 10	High
10 – 100	Moderate
>100	weak

CONCLUSION:

HGV-6, PGV-6, and GVT-6 compounds can be synthesized by assisted-microwave irradiation at 160Watt for 10 minutes. Synthesis yield of HGV-6, PGV-6, and GVT-6 is 82.35%; 77.3%; and 79.6% respectively. Acute toxicity testing results show that compounds have low toxicity so that it is safe to use as a drug candidate.

ACKNOWLEDGEMENT:

The authors are grateful to Medicinal Chemistry Department, Faculty of Medical, and Universitas Tanjungpura, for facilities this research. Department of Animal health science, Institute biology of Leiden, and Leiden university for acute toxicity testing.

CONFLICT OF INTEREST:

The authors declare no conflict of interest and contribute equally.

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Received on 04.11.2020 Modified on 24.12.2020

Research J. Pharm. and Tech. 2021; 14(9):4591-4594.

DOI: 10.52711/0974-360X.2021.00798