INTRODUCTION:

Coronary Heart Disease (CHD) is the deadliest disease worldwide¹. Globally around 110 million men and 80 million women have coronary heart disease². Coronary heart disease is caused by plaque buildup on the walls of the arteries that supply blood to the heart. Plaque buildup will cause the inside of the artery to narrow and block blood flow³. Several studies show that the use of anti-thrombosis agents such as aspirin and clopidogrel has side effects. The use of aspirin causes a bleeding risk (gastrointestinal disorder) and the pharmacological effect on platelet aggregation is weak⁴. The use of high doses of clopidogrel only achieves modest pharmacodynamic effects⁵. Moreover, clopidgrel profuse bleeding and does not reduce the rate of cardiovascular death, non-fatal myocardial infarction, or stent thrombosis⁶. Therefore the development of new drugs as anti thrombotic are important.

Clopidogrel acts as an antiplatelet agent by exerting its inhibitory effects on the P2Y₁₂ receptor⁷ and a recent study showed a significant increase in the probability of gastrointestinal bleeding while using clopidogrel, as well as a higher risk of bleeding while combining clopidogrel with aspirin⁸. P2Y₁₂ is a receptor that that activates platelets and GIIb/IIIa receptors glycoproteins bind fibrinogen, connect platelets⁹ and reduce platelet aggregation¹⁰. Many compounds have been reported to have anti-thrombotic activity through P2Y₁₂ receptor, including is ferulic acid¹¹. Vanillin has structural similarities with ferulic acid : -OH, - OCH3, aromatic ring, and carbonyl. Previous in silico studies demonstrated that vanillin derivatives possess the ability to inhibit COX-1¹². The vanillin applied has a synthetic. The distinction between synthetic and natural vanillin is determined by the origin of the chemical compound. Synthetic vanilla is usually more affordable to produce compared to original vanilla due to the use of synthetic¹³.

Vanillin was reported to have anti-platelet aggregation activity in vitro and in vivo induced by arachidonic acid, but lower than the control^14,15. Previous research reported that vanillin derivatives showed anticancer properties in the MCF-7 cell line¹⁶, specifically targeting breast cancer. In addition, vanillin derivatives have been found to inhibit zinc corrosion¹⁷. Furthermore, vanillin derivatives showed antibacterial properties against Bacillus subtilis and Micrococcus luteus¹⁸, as well as Staphylococcus aureus and Escherichia coli¹⁹. So, in this study, vanillin was chosen as the lead compound whose structure would be modified to increase the activity. We propose to synthesize three compounds, and followed by identification using FTIR and UV-Vis spectrophotometer, ¹H-NMR and ¹³C-NMR spectrometers, melting point apparatus, and screening their anti trombotic activity by clotting time and bleeding time methods.

MATERIALS AND METHODS:

Molecular Docking Study:

The compounds are evaluated for their enzymatic targeted relevant to specific therapies and are predicted their activity using molecular docking method²⁰. In this study the receptor used was a macromolecule from P2Y₁₂, on https://www.rcsb.org (RCSB) namely PDB ID: 4NTJ²¹ with a native ligand identity (AZJ) in chain B. All test compounds structure as ligand were drawn in two dimension (2D) using the ChemDraw version 20.1.1 program²², then it was changed to a three dimension (3D) structure, and saved in pdb format. The energy minimization of the 3D molecular structures were performed using the Avogadro program²³ with the MMFF94 calculation which aims to obtain the most stable conformation before the simulation of ligand-receptor, then the optimized structures were save the file in pdb format. The validation and docking simulation were set on gridcenter X: 16.599, Y: 99.698, Z: 50.077, 100 independent genetic algorithms (GA Runs), the population size 150, the maximum number of energy evaluations 2.500.000 (medium) and the maximum 27.000 generations, the maximum number of active sites 1, the gene mutation rate 0.02 and the crossover rate 0.8 used in the genetic algorithm method.

Synthesis:

4-formyl-2-methoxyphenyl benzoate (V1):

In a round bottom flask, add vanillin (1.97 x 10^-3mol, 300mg) in 0.5ml THF under cold condition. Benzoyl chloride (2.62mmol, 0,3ml), THF 0.5ml and 0,4 ml TEA were added to the mixture and stir until homogeneous. The mixture was irradiated in a microwave oven at 200W for 7,5 minutes (the mixture was tested by TLC every 30sec). The product was poured into a glass funnel after which it was washed with 5% Na₂CO₃ solution water, then recrystallized with hot ethanol.

4-formyl-2-methoxyphenyl 4-methylbenzoate (V2):

In a round bottom flask, add vanillin (1.97 x 10^-3mol, 300mg) in 0.5ml THF under cold condition. P-methylbenzoyl chloride (2.62mmol, 0,34ml), THF 0.5 ml and 0,4ml TEA were added to the mixture and stir until homogeneous. The mixture was irradiated in a microwave oven at 200 W for 5minutes (the mixture was tested by TLC every 30sec). The product was poured into a glass funnel after which it was washed with 5% Na₂CO₃ solution, then recrystallized with hot ethanol.

4-formyl-2-methoxyphenyl 4-(trifluoromethyl) benzoate (V3):

In a round bottom flask, add vanillin (1.97 x 10^-3mol, 300mg) in 0.5ml THF under cold condition. P-trifluoromethyllbenzoyl chloride (2.62mmol, 0,38ml), THF 0.5 ml and 0,4 ml TEA were added to the mixture and stir until homogeneous. The mixture was irradiated in a microwave oven at 200W for 7,5minutes (the mixture was tested by TLC every 30 sec). The product was poured into a glass funnel after which it was washed with 5% Na₂CO₃ solution, then recrystallized with hot ethanol.

Purification and Structure Identification:

All synthesis compounds were tested for purification using TLC and melting point apparatus. Tested the phenol moiety with FeCl₃ solution. Afterwards, the structure was further identified through the FTIR and UV-Vis spectrophotometer, ¹H-NMR and ¹³C-NMR spectrometer.

Anti Trombotic Activity:

The anti-thrombotic activity test was conducted by the clotting time and bleeding time method using healthy adult male mice aged 8-12 weeks and weighing 25-35g, adapted and fed for one week²⁴. The mice divided randomly into five groups, each group consisting of six mice. All mice were tested for their blood coagulation time on (day 0) and put into the mounting. Blood clotting time was calculated by placing the mice on the observation table. The mice tails which were cleaned with 70% alcohol, then stabbed with a scalpel as far as 2 cm from the edge of the tail with a puncture depth of 2 mm (bleeding test). The dripping blood is then dripped into a glass object and observed every 15 seconds to determine the beginning of fibrin formation (clotting test). Afterwards, the sample solution was orally administered with three doses i.e. 40mg, 80mg, and 160mg, as well as the control positive (aspirin 80mg) and negative control (0.5% CMC Na solution) for a duration of seven consecutive days. These dose selection refers to the dose of aspirin as an antiplatelet agent from 75 to 150 mg²⁵. A dosage of 40mg is employed to determine if the test substance has the ability to exhibit antiplatelet activity at low dosages. The selection of the 80mg dose depended on its efficacy as an antiplatelet agent, whereas the choice of the 160mg dose was intended to evaluate the potency of the test compound²⁶. On the eighth day, the blood clotting time in the test animals was reevaluated using the established technique.

RESULT AND DISCUSSION:

Molecular Docking Study:

Docking validation on P2Y₁₂ was conducted by re-docking the receptor and native ligand (AZJ). Removing water molecules before to the validation process is performed in order to enhance the stability of the interactions²⁷. Furthermore, the ligand performs energy minimization using Avogadro with MMFF94, which attempts to provide the most stable conformation and increased flexibility. The validation results were 0.87 Å with x,y,z dimensions of 40,40,42 and the coordinates of the center grid box (X= 16.599, Y= 99.698, Z= 50.077). Setting the center grid box aims to determine the binding site of the interaction between the ligand and the receptor²⁸.

Molecular docking methodologies have been utilized to discover novel bioactive compounds. These candidates are evaluated an ranked using methods such as a scoring function to identify the optimal binding²⁹. A lower ∆G value indicates a stronger level of stability in the conformation of the ligand and receptor³⁰. The new study used the Topliss method to find the best molecular docking positions for 17 of vanillin derivatives to bind to the P2Y12 receptor. The results can be seen in Table 1. The three best compounds for the P2Y₁₂ receptors are 4-4((4-formyl-2-methoxyphenoxy) carbonyl)benzoic acid (-7.89), 4-formyl-2-methoxyphenyl 4-bromobenzoic (-7.38), and 4-formyl-2-methoxyphenyl 4-methylbenzoate (-6.67). The modified structure of vanillin shows a lower ∆G value than clopidogrel as control.

Table 1. Moleculer docking results of vanillin analogs with P2Y₁₂enzyme.

Compounds	P2Y₁₂
Compounds	∆G (kcal/mol)	Inhibition Constant (uM)
Vanillin	-4.48	523.95
Native Ligand	-11.77	2.37
Clopidogrel	-6.11	33.19
R= alkyl
4-formyl-2-methoxyphenyl 4-acetate	-5.37	114.85
4-formyl-2-methoxyphenyl 4-propionate	-5.58	81.63
4-formyl-2-methoxyphenyl butyrate	-5.32	125.34
4-formyl-2-methoxyphenyl pentanoate	-5.42	107.15
4-formyl-2-methoxyphenyl hexanoate	-5.37	114.93
4-formyl-2-methoxyphenyl heptanoate	-5.77	58.47
4-formyl-2-methoxyphenyl octanoate	-5.65	72.02
4-formyl-2-methoxyphenyl nonanoate	-6.24	26.64
R= substituted aromatic ring with donating substituents
4-formyl-2-methoxyphenyl benzoate	-6.66	13.16
4-formyl-2-methoxyphenyl 4-methoxybenzoate	-6.52	16.63
4-formyl-2-methoxyphenyl 4-methylbenzoate	-6.67	12.86
4-formyl-2-methoxyphenyl 4-hydroxybenzoate	-6.10	33.85
R= substituted aromatic ring with withdrawing substituents
4-formyl-2-methoxyphenyl 4-(trifluoromethyl)benzoate	-6.27	25.54
4-formyl-2-methoxyphenyl 4-fluorobenzoate	-6.47	17.98
4-formyl-2-methoxyphenyl 4-chlorobenzoate	-6.57	15.40
4-4((4-formyl-2-methoxyphenoxy)carbonyl)benzoic acid	-7.89	1.64
4-formyl-2-methoxyphenyl 4-bromobenzoate	-7.38	3.87

In addition, the value of the inhibition constant (Ki) also plays an important role. This value indicates the strength of the inhibition of a compound with the receptor; the smaller the Ki value, the stronger the inhibition that occurs³¹. In the data obtained, all of the vanillin derivatives show a much lower Ki value than vanillin, and all of vanillin derivatives have good inhibition against target receptor, as shown at Figure 1. Figure 2 demostrated the visualization of the amino acids involved in the ligand-receptor. The number of hydrophobic interactions and the presence of hydrogen bonds determine the intensity of binding between the ligand and the receptor.

Figure 1. Amino Acid Interaction of a) Native Ligand, b) Clopidogrel, c) Vanillin

Figure 2. Amino Acid Interaction of Three Best Compounds: a) 4-4((4-formyl-2-methoxyphenoxy)carbonyl)benzoate; b) 4-formyl-2-methoxyphenyl 4-bromobenzoate; c) 4-formyl-2-methoxyphenyl 4-methylbenzoate

Based on our previous research, there are three best compounds that have the potential as anti-thrombotic agent by COX-1 inhibition, namely 4-formyl-2-methoxyphenyl benzoate, 4-formyl-2-methoxyphenyl- 4-fluorobenzoate, and 4-formyl-2-methoxyphenyl 4-(trifluoromethyl)benzoate³². In this results, there are three selected compounds to be synthesized, namely 4-formyl-2-methoxyphenyl benzoate (V1), 4-formyl-2-methoxyphenyl 4-methylbenzoate (V2), and 4-formyl-2-methoxyphenyl 4-(trifluoromethyl)benzoate (V3).

Synthesis:

Microwaves are widely used in the synthesis of organic compounds. The advantage of using microwaves is that the heating rate is faster than conventional heating method, so that it will accelerate the reaction and produce more products³³. A polar group that is given by microwave irradiation will produce a dielectric heat effect³⁴. The targeted derivatives can be synthesized more quickly using microwave conditions compared with conventional reactions³⁵.

Figure 3. Reaction Mechanism of Nucleophilic Acyl Substitution

The average percentage of compound synthesis results are V1 (73%), V2 (67%), and V3 (74%). The difference in the percentage of synthesis results due to several factors such as functional groups attached on benzoyl chloride.

Purification Identification:

A compound is said to be pure if it produces one spot on the TLC plate³⁶. The purity test of the synthesized compound by TLC was carried out using three different types of eluents, namely n-hexane:chloroform: methanol (3:1:1); n-hexane:chloroform:ethanol (2:2.5:0.5); n-hexane:ethyl acetat (4:2). All TLC plates showing one spot with different Rf value among those vanillin derivatives, as can be seen in Figure 4. So that it can be concluded that the compound synthesized are pure vanillin derivatives by TLC method.

a b

Figure 4. TLC test using eluents a) n-hexane:chloroform:methanol (3:1:1), b) n-hexane:chloroform:ethanol (2:2.5:0.5), c) n-hexane:ethyl acetate (4:2).

Furthermore, a qualitative test using FeCl₃ solution was used to identify the presence of phenol groups in a compound. Based on (Figure 5), it reported that vanilin showed blue color when reacting with FeCl₃ solution, it means vanillin contained phenol moiety. Whereas the vanillin derivatives performed no color change, which means the vanillin derivatives were not contained the phenol moiety³⁷.

The melting points of vanillin derivates were determined using an open capillary tube, by observing the temperature at which the substance melts in that capillary tube³⁸. Based on the melting point distance, most of the pure compounds melt in a narrow temperature range of 1-2ºC³⁹. All results in Table 2 indicate that all vanillin derivatives were pure. So that the identification of the structure could be carried out instrumentally.

Figure 5. Qualitative Test using FeCl₃ solution

Structure Identification with UV-Vis Spectrophotometer:

In this study, a UV-VIS spectrophotometer was used to determine the maximum wavelength of a vanillin derivatives at 200-400 nm. The results of the identification of vanillin and its derivatives (V1, V2, V3) can be seen in Table 3.

Table 3. Intrepretation of UV-Vis Wavelength

Compounds	Wavelength (λ max)
Vanillin	308
V1	306
V2	306
V3	306

It shows the vanillin spectrum has a wavelength (λ max) at 308nm, whereas its derivatives (V1,V2, and V3) have a maximum wavelength at 306 nm, a shift towards shorter waves (hypsochromic). That shift indicates a change in structure of vanillin derivatives comparing with vanillin. The chromophore group of a compound plays an important role in the wavelength. The longer the chromophore group, the greater the wavelength⁴⁰. It can be concluded that the addition of the benzene structure, methyl group, and CF₃ group to the initial vanillin structure does not make a significant difference to the shift in wavelength.

Structure Identification with FTIR Spectrophotometer:

The results of the structural identification of vanillin and its derivatives (V1,V2, V3) using FTIR spectrophotometer can be seen in Table 4. Based on the interpretation of the IR spectrum, V1,V2, and V3 has a different spectrum from vanillin.

Table 4 showed that there are spectra differences at wave number 3186.70 cm^-1 in the presence of –OH phenolic moiety of vanillin, while it was not found at its derivatives. In the spectrum of V1-V3 have peaks of the carbonyl ester (C=O) at wave number 1738.04; 1736.04; and 1746.37- cm^-1, and C-O ester bonds at wave number 1025.58; 1020.01; 1115.20 cm^-1, whereas those peaks not found in the spectrum of the vanillin. In addion V3 has peak at wave number 548.61 cm^-1of C-F₃moiety⁴¹. The differences spectra among vanillin and its derivatives (V1; V2; and V3) indicates that the acylation reaction of vanillin was successful. The phenolic moiety of vanillin changed become ester moiety.

Structure Identification by ¹H-NMR Spectrometer:

The results of ¹H-NMR spectra of V1,V2, and V3 and their interpretation can be seen at Table 5. The V1 has 12 protons, namely 5 protons in the ester aromatic ring, 3 protons of the vanillin aromatic ring, 3 methoxy protons bound to the vanillin aromatic ring, and 1 proton of the aldehyde group. So, it can be concluded that the resulting compound of V1 is 4-formyl-2-methoxyphenyl benzoate.

Table 2. Melting Point of Synthesis Compounds

Compounds	Melting point (^OC)			Average (^OC)
Compounds	Replication 1	Replication 2	Replication 3	Average (^OC)
V1	76-78	77-79	77-79	76.6–78.6
V2	106-108	105-107	106-108	105.6–107.6
V3	89-91	90-91	90-91	89.6–91

Table 4. Intrepretation of IR Spectrum

Functional groups	Wave Number (cm^-1)
Functional groups	References⁴¹	Vanillin	V1	V2	V3
C=C aromatic	1450-1600	1465.01	1454.41	1465.2	1465.53
C=O aldehyde	1670-1730	1667.75	1701.48	1701.27	1703.10
C=O esters	1735-1780	-	1738.04	1736.04	1746.37
C-H aromatic	2800-3100	2857.69	2801.43	2810.16	2841.69
C-O eter	1070-1150	1124.42	1075.12	1067.05	1081.30
C-O esters	1000-1280	-	1025.58	1020.01	1115.20
Alkyl C-H	2810-2850	2857.69	2832.80	2810.16	2841.69
O-H phenolic	3200-3550	3186.70	-	-	-
C-F₃	515-850	-	-	-	548.61

Table 5. Interpretation of ¹H-NMR Identification

Compounds	Protons	Chemical Shift (ppm)		Multiplicity	Integration
Compounds	Protons	References⁴²	Vanilin derivatives	Multiplicity	Integration
V1	H aromatic	6.5-8.0	8,21	Doublet	2H
			7.62	Triplet	1H
			7.52	Triplet	4H
			7.35	Doublet	1H
	H aldehyde	9.7-10.0	9.98	Singlet	1H
	H methoxy	3.8-4.5	3.89	Singlet	3H
V2	H aromatic	6.5-8.0	8.09	Doublet	2H
			7.52	Triplet	1H
			7.33	Triplet	4H
	H methyl	2,4-2,7	2.46	Singlet	3H
	H methoxy	3,8-4,5	3.88	Singlet	3H
	H aldehyde	9,7-10.0	8.33	Singlet	1H
V3	H aromatic	6,5-8,0	7.79	Doublet	2H
			7.54	Doublet	2H
			7.37	Doublet	2H
			8.21	Doublet	1H
	H aldehid	9,7-10,0	9.99	Singlet	1H
	H methoxy	3,8-4,5	3.89	Singlet	3H

V2 has a total of 14 protons, i.e 4 protons at the aromatic ester ring, 3 protons at the vanillin aromatic ring, 3 methoxy protons bound to the ester aromatic ring, 1 proton in the aldehyde group, and 3 protons from the methyl group. So, it can be concluded that the resulting compound of V2 is 4-formyl-2-methoxyphenyl 4-methylbenzoate.

V3 has a total of 11 protons, i.e. 4 protons in the ester aromatic ring, 3 protons in the vanillin aromatic ring, 3 methoxy protons attached to the vanillin aromatic ring, and 1 proton in the aldehyde group. So it can be concluded that the resulting compound of V3 is 4-formyl-2-methoxyphenyl 4-(trifluoromethyl)benzoate.

Based on the results of the structural identification test using ¹H-NMR spectrometer, it was concluded that the three vanillin derivatives compounds (V1;V2;V3) were successfully synthesized using the microwave irradiation method.

Structure Identification with ¹³C-NMR Spectrometer

The results of ¹³C-NMR identification of V1,V2, V3 and their interpretation spectra can be seen in Table 6. Based on data of the ¹³C-NMR spectra from V1, it is known that there are 15 carbons, i.e one C aldehydes at 191.2ppm; one C esters at 165.2ppm, 12 C aromatics at 152.2ppm-110.9ppm; one C of methoxy moiety at 56.2ppm. So, it can be concluded that the resulting compound is 4-formyl-2-methoxyphenyl benzoate.

Spectra ¹³C-NMR of V2 showed that there are 16 carbons namely one C aldehyde at chemical shift of 191.2ppm; one C ester at 164.3ppm, 12 C aromatic at position 152.3ppm - 110.8ppm; one C methoxy at 56.2ppm, and one C methyl at 21.9ppm. So it can be concluded that the resulting compound is 4-formyl-2-methoxyphenyl 4-methylbenzoate.

Whereas, the V3 spectra demonstrated 16 carbons namely one C aldehyde with a chemical shift of 191.1ppm; one C ester at 163.0ppm, 12 C aromatic at 152.0ppm-111.0 ppm. Furthermore, one C methoxy at 56.2 ppm, and one C of CF₃ at 122.2ppm. So it can be concluded that the resulting compound is 4-formyl-2-methoxyphenyl 4-(trifluoromethyl)benzoate. Based on the results of the structural identification test using ¹³C-NMR, the three vanillin derivatives were successfully synthesized using the microwave method.

Anti-Trombotic Activity:

Anti thrombotic assay was carried out in vivo at three different doses 40mg, 80mg, and 160mg. Beside that, there were two control groups of positive control using aspirin, and negative control contained 0.5% CMC Na solution. Each group has five test male mice (Mus musculus), to avoid the hormonal effect of system of female mice. The body weight of mice in the range of 25-35 grams.

Table 6. Interpretation of ¹³C-NMR Identification

Compounds	Structure	Absorption Relative Position	Chemical Shift (ppm)		Carbon Position
Compounds	Structure	Absorption Relative Position	References⁴¹	Vanilin Derivatives	Carbon Position
V1		C aromatic	100-170	110.9	7
				123.6	3
				124.9	4
				128.7	12 and 14
				128.8	10
				130.4	11 and 15
				133.9	13
				135.3	2
				145.3	5
				152.2	6
		C esters	160-185	165.2	9
		C aldehyde	182-215	191.2	1
		C methoxy	50-90	56.2	8
V2		C aromatic	100-170	110.8	7
				123.7	3
				124.9	4
				126.1	10
				129.4	12 and14
				130.5	11 and 15
				135.2	2
				144.8	13
				145.4	5
				152.3	6
		C esters	160-185	164.3	9
		C methoxy	50-90	56.2	8
		C aldehyde	182-215	191.2	1
		C methyl	0-40	21.9	16
V3		C aromatic	100-170	130.8	10
				125.8 – 125.7	11 and 15
				124.9	4
				124.8	14
				123.5	3
				111.0	7
		C esters	160-185	163.0	9
				152.0	6
				144.8	5
				135.5	13
				135.4	12
				132.1	2
		C aldehyde	182-215	191.1	1
		C methoxy	50-90	56.2	8
		CF₃	116-156	122.2	16

CONCLUSION:

The vanillin derivatives demonstrated greater in silico activity than the comparators vanillin and clopidogrel, as well as greater interactions in free energy (∆G) values and Ki Inhibition values with the P2Y₁₂receptor. Three selected compounds such as 4-formyl 2-methoxyphenyl benzoate (V1), 4-formyl 2-methoxyphenyl 4-methylbenzoate (V2), and 4-formyl 2-methoxyphenyl 4-(trifluoromethyl)benzoate (V3) has been successfully synthesized using the microwave irradiation method as confirmed by several structural identifications. Additionally, these compounds can contribute as promising anti-thrombotic agents.

CONFLICT OF INTEREST:

The authors declare no potential conflict of interest.

ACKNOWLEDGMENTS:

The authors would like to thank Directorate of Research, Technology and Community Service (DRTPM) Republic Indonesia through PTM 2022 research grant for financial supports No. 893-UN3.15/PT/2022.

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Received on 03.04.2024 Revised on 07.08.2024

Accepted on 10.10.2024 Published on 10.04.2025

Available online from April 12, 2025

Research J. Pharmacy and Technology. 2025;18(4):1625-1633.

DOI: 10.52711/0974-360X.2025.00233

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