Hydroxytyrosol thiodipropionic acid ester with antioxidant activity and a method of preparing the same

Abstract

A compound having the following formula I: ##STR00001##
is disclosed. A method of preparing the compound of formula I is also disclosed.

Claims

1. A compound having the following formula I: ##STR00018##

2. A method of preparing the compound of claim 1, comprising: reacting the compound of formula II with the compound of formula III to obtain the compound of formula I: ##STR00019##

3. The method of claim 2, wherein the reaction of the compound of formula II with the compound of formula III comprises the following steps: dissolving the compound of formula II and the compound of formula III in an organic solvent to form a reaction mixture under nitrogen atmosphere; adding a dehydrating agent to the reaction mixture; heating the reaction mixture at 50-60 C. for 8-10 hours under sonication; removing the organic solvent from the reaction mixture to obtain a crude product; and purifying the crude product on a microporous resin with a mixture of methanol and water as an eluent.

4. The method of claim 3, wherein the organic solvent is acetonitrile, THF, or DMF.

5. The method of claim 4, wherein the organic solvent is DMF.

6. The method of claim 3, wherein the molar ratio of the compound of formula II and the compound of formula III is 2:1 to 2.2:1.

7. The method of claim 6, wherein the molar ratio of the compound of formula II and the compound of formula III is 2.2:1.

8. The method of claim 3, wherein the dehydrating agent is DCC, concentrated sulfuric acid, or EDC.

9. The method of claim 8, wherein the dehydrating agent is DCC.

10. The method of claim 3, wherein the reaction mixture is heated at 60 C.

11. The method of claim 3, wherein the reaction mixture is heated for 8 hours.

12. The method of claim 3, wherein the mixture of methanol and water has a volume ratio of 1:1.

Description

(1) In the drawings:

(2) FIG. 1 is the .sup.1HNMR spectrum of the hydroxytyrosol thiodipropionic acid ester.

(3) FIG. 2 is the .sup.13CNMR spectrum of the hydroxytyrosol thiodipropionic acid ester.

(4) FIG. 3 shows the DPPH radical scavenging activity of the hydroxytyrosol thiodipropionic acid ester and control samples.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

(5) Reference will now be made in detail to embodiments of the present invention, example of which is illustrated in the accompanying drawings. The following examples illustrate the present invention, but the present invention is not limited to the following examples.

(6) In the present invention, hydroxytyrosol is structurally modified by reacting with thiodipropionic acid to obtain a hydroxytyrosol thiodipropionic acid ester. The synthesis method of the hydroxytyrosol thiodipropionic acid ester is described. The antioxidant activity is also measured. The hydroxytyrosol thiodipropionic acid ester can be used as a new type of antioxidant additive for food, medicine and health care products.

Example 1

Preparation of Hydroxytyrosol Thiodipropionic Acid Ester (bis(3,4-dihydroxyphenethyl) 3,3-thiodipropanoate) (Formula I)

(7) ##STR00006##

(8) 95 mg (0.62 mmol) hydroxytyrosol and 50 mg (0.28 mmol) 3,3-thiodipropionic acid were placed in a 100 mL reactor. 50 mL DMF was added to form a reaction mixture under nitrogen atmosphere. 115 mg (0.56 mmol) DCC was then added to the reaction mixture. The reaction mixture was heated at 60 C. under sonication and nitrogen atmosphere for 8 hours. After thin layer chromatography (TLC) indicated that the reaction was complete, the reaction mixture was cooled down to room temperature, concentrated under reduced pressure to obtain a crude product. The crude product was purified by an adsorption purification process using a D-101 microporous resin column and eluting with methanol and water (1:1) mixture. The elution was collected and concentrated to obtain 73.6 mg of purified hydroxytyrosol thiodipropionic acid ester, a yield of 58.4%.

(9) .sup.1H-NMR (400 MHz, DMSO-d.sub.6) (ppm): 9.15 (4H, s), 7.87 (1H, s), 7.52 (1H, d), 7.18 (1H, d), 6.746.52 (2H, m), 5.33 (1H, d), 4.634.62 (4H, m), 2.752.71 (4H, m), 2.15 (4H, m), 1.941.91 (4H, m); .sup.13C-NMR (75 MHz, DMSO-d.sub.6) (ppm): 174.73, 145.37, 143.79, 130.74, 119.90, 116.77, 115.87, 63.07, 34.55, 28.64; MS (ESI) for (M+H).sup.+: 451.1.

Example 2

Preparation of Hydroxytyrosol Thiodipropionic Acid Ester (bis(3,4-dihydroxyphenethyl) 3,3-thiodipropanoate)

(10) ##STR00007##

(11) 95 mg (0.62 mmol) hydroxytyrosol and 50 mg (0.28 mmol) 3,3-thiodipropionic acid were placed in a 100 mL reactor. 50 mL acetonitrile was added to form a reaction mixture under nitrogen atmosphere. 115 mg (0.56 mmol) DCC was then added to the reaction mixture. The reaction mixture was heated at 50 C. under sonication and nitrogen atmosphere for 10 hours. After thin layer chromatography (TLC) indicated that the reaction was complete, the reaction mixture was cooled down to room temperature, concentrated under reduced pressure to obtain a crude product. The crude product was purified by an adsorption purification process using a D-101 microporous resin column and eluting with methanol and water (2:1) mixture. The elution was collected and concentrated to obtain 55.1 mg of purified hydroxytyrosol thiodipropionic acid ester, a yield of 43.7%.

Example 3

Preparation of Hydroxytyrosol Thiodipropionic Acid Ester (bis(3,4-dihydroxyphenethyl) 3,3-thiodipropanoate)

(12) ##STR00008##

(13) 95 mg (0.62 mmol) hydroxytyrosol and 50 mg (0.28 mmol) 3,3-thiodipropionic acid were placed in a 100 mL reactor. 50 mL DMF was added to form a reaction mixture under nitrogen atmosphere. 110 mg (0.6 mmol) EDC was then added to the reaction mixture. The reaction mixture was heated at 60 C. under sonication and nitrogen atmosphere for 8 hours. After thin layer chromatography (TLC) indicated that the reaction was complete, the reaction mixture was cooled down to room temperature, concentrated under reduced pressure to obtain a crude product. The crude product was purified by an adsorption purification process using a D-101 microporous resin column and eluting with methanol and water (1:2) mixture. The elution was collected and concentrated to obtain 49.2 mg of purified hydroxytyrosol thiodipropionic acid ester, a yield of 39.0%.

Example 4

Preparation of Hydroxytyrosol Thiodipropionic Acid Ester (bis(3,4-dihydroxyphenethyl) 3,3-thiodipropanoate)

(14) ##STR00009##

(15) 95 mg (0.62 mmol) hydroxytyrosol and 50 mg (0.28 mmol) 3,3-thiodipropionic acid were placed in a 100 mL reactor. 50 mL THF was added to form a reaction mixture under nitrogen atmosphere. 115 mg (0.56 mmol) DCC was then added to the reaction mixture. The reaction mixture was heated at 60 C. under sonication and nitrogen atmosphere for 8 hours. After thin layer chromatography (TLC) indicated that the reaction was complete, the reaction mixture was cooled down to room temperature, concentrated under reduced pressure to obtain a crude product. The crude product was purified by an adsorption purification process using a D-101 microporous resin column and eluting with methanol and water (2:1) mixture. The elution was collected and concentrated to obtain 58.6 mg of purified hydroxytyrosol thiodipropionic acid ester, a yield of 46.5%.

Example 5

Preparation of Hydroxytyrosol Thiodipropionic Acid Ester (bis(3,4-dihydroxyphenethyl) 3,3-thiodipropanoate)

(16) ##STR00010##

(17) 95 mg (0.62 mmol) hydroxytyrosol and 50 mg (0.28 mmol) 3,3-thiodipropionic acid were placed in a 100 mL reactor. 50 mL acetonitrile was added to form a reaction mixture under nitrogen atmosphere. 5 mL concentrated sulfuric acid was then added to the reaction mixture. The reaction mixture was heated at 60 C. under sonication and nitrogen atmosphere for 8 hours. After thin layer chromatography (TLC) indicated that the reaction was complete, the reaction mixture was cooled down to room temperature, concentrated under reduced pressure to obtain a crude product. The crude product was purified by an adsorption purification process using a D-101 microporous resin column and eluting with methanol and water (1:1) mixture. The elution was collected and concentrated to obtain 40.7 mg of purified hydroxytyrosol thiodipropionic acid ester, a yield of 32.3%.

Example 6

Preparation of Hydroxytyrosol Thiodipropionic Acid Ester (bis(3,4-dihydroxyphenethyl) 3,3-thiodipropanoate)

(18) ##STR00011##

(19) 95 mg (0.62 mmol) hydroxytyrosol and 50 mg (0.28 mmol) 3,3-thiodipropionic acid were placed in a 100 mL reactor. 50 mL DMF was added to form a reaction mixture under nitrogen atmosphere. 115 mg (0.56 mmol) DCC was then added to the reaction mixture. The reaction mixture was heated at 50 C. under sonication and nitrogen atmosphere for 8 hours. After thin layer chromatography (TLC) indicated that the reaction was complete, the reaction mixture was cooled down to room temperature, concentrated under reduced pressure to obtain a crude product. The crude product was purified by an adsorption purification process using an HP-20 microporous resin column and eluting with methanol. The elution was collected and concentrated to obtain 38.6 mg of purified hydroxytyrosol thiodipropionic acid ester, a yield of 30.6%.

Example 7

Preparation of Hydroxytyrosol Thiodipropionic Acid Ester (bis(3,4-dihydroxyphenethyl) 3,3-thiodipropanoate)

(20) ##STR00012##

(21) 95 mg (0.62 mmol) hydroxytyrosol and 50 mg (0.28 mmol) 3,3-thiodipropionic acid were placed in a 100 mL reactor. 50 mL acetonitrile was added to form a reaction mixture under nitrogen atmosphere. 57.8 mg (0.28 mmol) DCC was then added to the reaction mixture. The reaction mixture was heated at 50 C. under sonication and nitrogen atmosphere for 8 hours. After thin layer chromatography (TLC) indicated that the reaction was complete, the reaction mixture was cooled down to room temperature, concentrated under reduced pressure to obtain a crude product. The crude product was purified by an adsorption purification process using an XAD-2 microporous resin column and eluting with methanol and water (4:1) mixture. The elution was collected and concentrated to obtain 52.7 mg of purified hydroxytyrosol thiodipropionic acid ester, a yield of 41.8%.

Example 8

Preparation of Hydroxytyrosol Thiodipropionic Acid Ester (bis(3,4-dihydroxyphenethyl) 3,3-thiodipropanoate)

(22) ##STR00013##

(23) 86 mg (0.56 mmol) hydroxytyrosol and 50 mg (0.28 mmol) 3,3-thiodipropionic acid were placed in a 100 mL reactor. 50 mL acetonitrile was added to form a reaction mixture under nitrogen atmosphere. 115 mg (0.56 mmol) DCC was then added to the reaction mixture. The reaction mixture was heated at 60 C. under sonication and nitrogen atmosphere for 10 hours. After thin layer chromatography (TLC) indicated that the reaction was complete, the reaction mixture was cooled down to room temperature, concentrated under reduced pressure to obtain a crude product. The crude product was purified by an adsorption purification process using a D-101 microporous resin column and eluting with methanol and water (1:1) mixture. The elution was collected and concentrated to obtain 66.9 mg of purified hydroxytyrosol thiodipropionic acid ester, a yield of 53.1%.

Example 9

Preparation of Hydroxytyrosol Thiodipropionic Acid Ester (bis(3,4-dihydroxyphenethyl) 3,3-thiodipropanoate)

(24) ##STR00014##

(25) 95 mg (0.62 mmol) hydroxytyrosol and 50 mg (0.28 mmol) 3,3-thiodipropionic acid were placed in a 100 mL reactor. 50 mL DMF was added to form a reaction mixture under nitrogen atmosphere. 106 mg (0.56 mmol) EDC was then added to the reaction mixture. The reaction mixture was heated at 60 C. under sonication and nitrogen atmosphere for 10 hours. After thin layer chromatography (TLC) indicated that the reaction was complete, the reaction mixture was cooled down to room temperature, concentrated under reduced pressure to obtain a crude product. The crude product was purified by an adsorption purification process using a D-101 microporous resin column and eluting with methanol and water (1:1) mixture. The elution was collected and concentrated to obtain 57.0 mg of purified hydroxytyrosol thiodipropionic acid ester, a yield of 45.2%.

Example 10

Preparation of Hydroxytyrosol Thiodipropionic Acid Ester (bis(3,4-dihydroxyphenethyl) 3,3-thiodipropanoate)

(26) ##STR00015##

(27) 95 mg (0.62 mmol) hydroxytyrosol and 50 mg (0.28 mmol) 3,3-thiodipropionic acid were placed in a 100 mL reactor. 50 mL DMF was added to form a reaction mixture under nitrogen atmosphere. 106 mg (0.56 mmol) EDC was then added to the reaction mixture. The reaction mixture was heated at 60 C. under sonication and nitrogen atmosphere for 8 hours. After thin layer chromatography (TLC) indicated that the reaction was complete, the reaction mixture was cooled down to room temperature, concentrated under reduced pressure to obtain a crude product. The crude product was purified by an adsorption purification process using an HP-20 microporous resin column and eluting with methanol and water (4:1) mixture. The elution was collected and concentrated to obtain 36.2 mg of purified hydroxytyrosol thiodipropionic acid ester, a yield of 28.7%.

Example 11

Preparation of Hydroxytyrosol Thiodipropionic Acid Ester (bis(3,4-dihydroxyphenethyl) 3,3-thiodipropanoate)

(28) ##STR00016##

(29) 95 mg (0.62 mmol) hydroxytyrosol and 50 mg (0.28 mmol) 3,3-thiodipropionic acid were placed in a 100 mL reactor. 50 mL acetonitrile was added to form a reaction mixture under nitrogen atmosphere. 5 mL concentrated sulfuric acid was then added to the reaction mixture. The reaction mixture was heated at 60 C. under sonication and nitrogen atmosphere for 8 hours. After thin layer chromatography (TLC) indicated that the reaction was complete, the reaction mixture was cooled down to room temperature, concentrated under reduced pressure to obtain a crude product. The crude product was purified by an adsorption purification process using an XAD-2 microporous resin column and eluting with methanol and water (1:1) mixture. The elution was collected and concentrated to obtain 45.2 mg of purified hydroxytyrosol thiodipropionic acid ester, a yield of 35.9%.

Example 12

Preparation of Hydroxytyrosol Thiodipropionic Acid Ester (bis(3,4-dihydroxyphenethyl) 3,3-thiodipropanoate)

(30) ##STR00017##

(31) 86 mg (0.56 mmol) hydroxytyrosol and 50 mg (0.28 mmol) 3,3-thiodipropionic acid were placed in a 100 mL reactor. 50 mL DMF was added to form a reaction mixture under nitrogen atmosphere. 110 mg (0.6 mmol) EDC was then added to the reaction mixture. The reaction mixture was heated at 60 C. under sonication and nitrogen atmosphere for 8 hours. After thin layer chromatography (TLC) indicated that the reaction was complete, the reaction mixture was cooled down to room temperature, concentrated under reduced pressure to obtain a crude product. The crude product was purified by an adsorption purification process using a D-101 microporous resin column and eluting with methanol and water (1:1) mixture. The elution was collected and concentrated to obtain 48.4 mg of purified hydroxytyrosol thiodipropionic acid ester, a yield of 38.4%.

Example 13

(32) DPPH Radical Scavenging Activity Assay

(33) Experimental Principle:

(34) 2,2-Diphenyl-1-picryl hydrazyl (DPPH) is an organic compound composed of a stable organic radical. In the DPPH molecule, due to the presence of multiple electron-withdrawing NO.sub.2 and large it bonds of the benzene ring, nitrogen free radical is stabilized. Its methanol solution is purple and has a maximum absorption peak at 517 nm. After the addition of an antioxidant, DPPH captures an electron to be paired with the free electron, and the purple fades and turns into a yellow substance. The absorption at 517 nm disappears, and the degree of fading is quantitatively related to the number of electrons it captures. Based on this principle, a spectrophotometer is used to detect the change of the absorbance of the DPPH radical and the sample solution, and the ability of the sample to provide hydrogen atoms and scavenge free radicals can be measured.

(35) Experimental Method:

(36) (a) Preparation of DPPH solution:measuring exact amount of 2,2-diphenyl-1-picryl hydrazyl (DPPH) and dissolving in methanol to prepare a 0.210.sup.4 mol/L DPPH solution, stored at 0 C. in dark.

(37) (b) Preparation of sample solutions: Vc (vitamin C, positive control), hydroxytyrosol thiodipropionic acid ester, hydroxytyrosol (control), thiodipropionic acid (control), and a mixture of hydroxytyrosol and thiodipropionic acid (control) were dissolved in methanol and diluted in a concentration gradient. The sample solutions are shown in Table 1.

(38) TABLE-US-00001 TABLE 1 Sample Solutions No. Samples Concentrations (g/ml) Vc Vitamin C 1.648, 8.242, 16.484, 37.969, 65.938, 131.875, 263.750, 527.500, 1055.000 A hydroxytyrosol 1.648, 8.242, 16.484, 37.969, 65.938, thiodipropionic 131.875, 263.750, 527.500, 1055.000 acid ester B hydroxytyrosol 1.648, 8.242, 16.484, 37.969, 65.938, 131.875, 263.750, 527.500, 1055.000 C thiodipropionic 1.648, 8.242, 16.484, 37.969, 65.938, acid 131.875, 263.750, 527.500, 1055.000 D hydroxytyrosol 1.648, 8.242, 16.484, 37.969, 65.938, and 131.875, 263.750, 527.500, 1055.000 thiodipropionic acid (2:1) physical mixture

(39) (c) Specific steps:

(40) Measuring the scavenging activity of the sample solutions: 2 mL of the sample solutions (table 1) at each concentration gradient was taken, 2 mL 0.210.sup.4 mol/L DPPH solution was added, the mixture was mixed and reacted at room temperature in dark for 30 minutes, and methanol was then added to adjust final volume. The absorbance A.sub.i was measured at 517 nm. 2 mL control solution and 2 mL methanol were mixed, and the absorbance A.sub.j was measured. 2 mL DPPH solution and 2 mL methanol were mixed, and the absorbance A.sub.0 was measured. The results are shown in Table 2.

(41) TABLE-US-00002 TABLE 1 Absorbance Concentrations/(g/mL) Sample Absorbance 1.648 8.242 16.484 37.969 65.938 131.875 263.750 527.500 1055.000 Vc Ai 0.195 0.190 0.172 0.124 0.066 0.056 0.053 0.064 0.051 Aj 0.065 0.066 0.063 0.060 0.057 0.051 0.051 0.063 0.050 Ao 0.197 A Ai 0.232 0.214 0.178 0.153 0.114 0.085 0.065 0.053 0.050 Aj 0.055 0.063 0.053 0.066 0.060 0.059 0.057 0.047 0.045 Ao 0.262 B Ai 0.257 0.252 0.234 0.197 0.167 0.135 0.111 0.128 0.115 Aj 0.040 0.041 0.051 0.031 0.032 0.047 0.047 0.069 0.058 Ao 0.237 C Ai 0.273 0.266 0.253 0.242 0.238 0.224 0.217 0.223 0.203 Aj 0.034 0.031 0.030 0.035 0.042 0.044 0.048 0.056 0.050 Ao 0.247 D Ai 0.223 0.198 0.182 0.168 0.112 0.102 0.094 0.078 0.071 Aj 0.063 0.059 0.048 0.065 0.062 0.066 0.069 0.062 0.060 Ao 0.229

(42) (d) The scavenging activity of the sample solution and control solutions is calculated according to the following calculation formula and shown in Table 3 and FIG. 3.
Scavenging activity (%)=100[1(A.sub.iA.sub.j)/A.sub.0]

(43) TABLE-US-00003 TABLE 3 DPPH Radical Scavening Activity Concentration/ Scavenging Activity (%) (n = 3) (g/mL) Vc A B C D 1.648 34.01 32.44 8.44 3.24 30.13 8.242 37.06 42.37 10.97 4.86 39.30 16.484 44.67 52.29 22.78 9.72 41.48 37.969 67.51 66.79 29.96 16.19 55.02 65.938 95.43 79.39 43.04 20.65 78.17 131.875 97.46 90.01 62.87 27.13 84.28 263.750 98.98 96.95 73.00 31.58 89.08 527.500 99.49 97.71 75.11 32.39 93.01 1055.000 99.49 98.09 75.95 38.06 95.20

(44) As shown in Table 3 and FIG. 3, hydroxytyrosol thiodipropionic acid ester (A) has obvious DPPH radical scavenging activity in a concentration dependent manner. Specifically, the DPPH radical scavenging activity ranges from 32.44% at 1.648 g/mL to 98.09% at 1055 g/mL. Hydroxytyrosol thiodipropionic acid ester (A) has better DPPH radical scavenging activity than hydroxytyrosol (B), thiodipropionic acid (C), and a 2:1 physical mixture of hydroxytyrosol and thiodipropionic acid (D) at same concentration. Hydroxytyrosol thiodipropionic acid ester (A) has similar DPPH radical scavenging activity to vitamin C. Accordingly, hydroxytyrosol thiodipropionic acid ester can be used a food and cosmetic antioxidant additive, and has a wide application prospect.

(45) It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.