Hydroxytyrosol isophorone diisocyannate derivative with antioxidant activity and a method of preparing the same

Abstract

A compound having the 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): ##STR00006##

2. A method of preparing the compound of claim 1, comprising: reacting a compound of formula (II) with a compound of formula (III) to obtain the compound of formula (I): ##STR00007##

3. The method of claim 2, the reaction of the compound of formula (II) with the compound of formula (III) comprises the following steps: placing the compound of formula (II) and the compound of formula (III), in a molar ratio of 2.4:1 to 2.6:1, in a reactor; adding a solvent and a catalyst under nitrogen atmosphere to obtain a reaction mixture; heating the reaction mixture at 50-75 C. for 1-4 hours under magnetic stirring; concentrating the reaction mixture under reduced pressure to give a crude product; and purifying the crude product by flash chromatograph.

4. The method of claim 3, wherein the solvent is toluene, ethyl acetate or acetonitrile.

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

6. The method of claim 3, wherein the molar ratio of the compound of formula (II) and the compound of formula (III) is 2.6:1.

7. The method of claim 3, wherein the catalyst is triethylamine or 4-dimethylaminopyridine.

8. The method of claim 7, wherein the catalyst is triethylamine.

9. The method of claim 3, wherein the reaction mixture is heated at 75 C.

10. The method of claim 3, wherein the reaction mixture is heated for 4 hours.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention.

(2) In the drawings:

(3) FIG. 1 shows the scavenging activity of the sample and control solutions at different concentrations.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

(4) 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.

Example 1

Preparation of a Hydroxytyrosol Isophorone Diisocyanate Derivative: 3,4-dihydroxyphenethyl ((5-(((3,4-dihydroxyphenethoxy)-carbonyl)amino)-1,3,3-trimethylcyclohexyl)methyl)carbamate

(5) In a 100 mL three-necked flask, 87.9 mg (0.57 mmol) of hydroxytyrosol and 31 L (0.22 mmol) of triethylamine were dissolved in 50 mL of toluene under nitrogen atmosphere. 50 mg (0.22 mmol) of isophorone diisocyanate was slowly added dropwise to the reaction solution. After isophorone diisocyanate was added, the temperature was raised to 75 C., and the reaction was carried out for 4 hours. Thin layer chromatography was used to monitor the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel column chromatography, ethyl acetate:petroleum ether=3:10 as eluent, and the eluent was concentrated under reduced pressure and dried to obtain 79.7 mg of the titled compound, a yield of 68.32%.

(6) 3,4-dihydroxyphenethyl ((5-(((3,4-dihydroxyphenethoxy)-carbonyl)amino)-1,3,3-trimethylcyclohexyl)methyl)carbamate: .sup.1H-NMR (400 MHz, DMSO-d.sub.6) (ppm): 8.46 (2H, b), 6.90 (2H, s), 6.836.71 (4H, m), 5.57 (4H, s), 4.62 (4H, t), 3.75 (H, m), 2.992.81 (6H, m), 1.841.67 (4H, m), 1.631.12 (5H, m), 1.01 (6H, s); .sup.13C-NMR (400 MHz, DMSO-d.sub.6) (ppm): 159.3, 157.2, 148.6, 146.2, 130.5, 125.8, 117.4, 116.9, 64.6, 49.6, 47.5, 43.8, 33.6, 28.1, 24.5, 20.1; MS (ESI) for (M+H).sup.+: 531.3.

Example 2

Preparation of 3,4-dihydroxyphenethyl ((5-(((3,4 dihydroxyphenethoxy)-carbonyl)amino)-1,3,3-trimethylcyclohexyl)methyl)carbamate

(7) In a 100 mL three-necked flask, 87.9 mg (0.57 mmol) of hydroxytyrosol and 31 L (0.22 mmol) of triethylamine were dissolved in 50 mL of ethylacetate under nitrogen atmosphere. 50 mg (0.22 mmol) of isophorone diisocyanate was slowly added dropwise to the reaction solution. After isophorone diisocyanate was added, the temperature was raised to 50 C., and the reaction was carried out for 2 hours. Thin layer chromatography was used to monitor the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel column chromatography, ethyl acetate:petroleum ether=3:10 as eluent, and the eluent was concentrated under reduced pressure and dried to obtain 54.9 mg of the titled compound, a yield of 47.02%.

Example 3

Preparation of 3,4-dihydroxyphenethyl ((5-(((3,4 dihydroxyphenethoxy)-carbonyl)amino)-1,3,3-trimethylcyclohexyl)methyl)carbamate

(8) In a 100 mL three-necked flask, 81.7 mg (0.53 mmol) of hydroxytyrosol and 31 L (0.22 mmol) of triethylamine were dissolved in 50 mL of acetonitrile under nitrogen atmosphere. 50 mg (0.22 mmol) of isophorone diisocyanate was slowly added dropwise to the reaction solution. After isophorone diisocyanate was added, the temperature was raised to 60 C., and the reaction was carried out for 4 hours. Thin layer chromatography was used to monitor the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel column chromatography, ethyl acetate:petroleum ether=3:10 as eluent, and the eluent was concentrated under reduced pressure and dried to obtain 62.8 mg of the titled compound, a yield of 53.78%.

Example 4

Preparation of 3,4-dihydroxyphenethyl ((5-(((3,4 dihydroxyphenethoxy)-carbonyl)amino)-1,3,3-trimethylcyclohexyl)methyl)carbamate

(9) In a 100 mL three-necked flask, 87.9 mg (0.57 mmol) of hydroxytyrosol and 2.5 mg (0.02 mmol) DMAP (4-dimethylaminopyridine) were dissolved in 50 mL of toluene under nitrogen atmosphere. 50 mg (0.22 mmol) of isophorone diisocyanate was slowly added dropwise to the reaction solution. After isophorone diisocyanate was added, the temperature was raised to 60 C., and the reaction was carried out for 1 hour. Thin layer chromatography was used to monitor the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel column chromatography, ethyl acetate:petroleum ether=3:10 as eluent, and the eluent was concentrated under reduced pressure and dried to obtain 30.0 mg of the titled compound, a yield of 25.69%.

Example 5

Preparation of 3,4-dihydroxyphenethyl ((5-(((3,4 dihydroxyphenethoxy)-carbonyl)amino)-1,3,3-trimethylcyclohexyl)methyl)carbamate

(10) In a 100 mL three-necked flask, 87.9 mg (0.57 mmol) of hydroxytyrosol and 2.5 mg (0.02 mmol) of DMAP were dissolved in 50 mL of ethyl acetate under nitrogen atmosphere. 50 mg (0.22 mmol) of isophorone diisocyanate was slowly added dropwise to the reaction solution. After isophorone diisocyanate was added, the temperature was raised to 75 C., and the reaction was carried out for 2 hours. Thin layer chromatography was used to monitor the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel column chromatography, ethyl acetate:petroleum ether=3:10 as eluent, and the eluent was concentrated under reduced pressure and dried to obtain 65.5 mg of the titled compound, a yield of 56.15%.

Example 6

Preparation of 3,4-dihydroxyphenethyl ((5-(((3,4 dihydroxyphenethoxy)-carbonyl)amino)-1,3,3-trimethylcyclohexyl)methyl)carbamate

(11) In a 100 mL three-necked flask, 87.9 mg (0.57 mmol) of hydroxytyrosol and 31 L (0.22 mmol) of triethylamine were dissolved in 50 mL of toluene under nitrogen atmosphere. 50 mg (0.22 mmol) of isophorone diisocyanate was slowly added dropwise to the reaction solution. After isophorone diisocyanate was added, the temperature was raised to 50 C., and the reaction was carried out for 1 hour. Thin layer chromatography was used to monitor the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture was concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel column chromatography, ethyl acetate:petroleum ether=3:10 as eluent, and the eluent was concentrated under reduced pressure and dried to obtain 39.5 mg of the titled compound, a yield of 33.84%.

Example 7

(12) The Antioxidant Activity of the Hydroxytyrosol Isophorone Diisocyanate Derivative Measured by a DPPH Radical Scavenging Activity Assay

(13) 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 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.

(14) Preparation of DPPH solution: measuring exact amount of 2,2-diphenyl-1-picryl hydrazyl (DPPH) and dissolving in toluene to prepare a 0.2 mmoL/L DPPH solution, stored at 0 C. in dark.

(15) Preparation of test solution: Vc (vitamin C, positive control), hydroxytyrosol isophorone diisocyanate derivative (sample) and hydroxytyrosol (control). The sample solution was subjected to gradient dilution with toluene, and two sets of controls were separately dissolved in a test tube with a certain amount of toluene to prepare the same concentration gradient as the sample. The corresponding two groups of control solutions were obtained (gradient settings are shown in Table 1).

(16) TABLE-US-00001 TABLE 1 Dilution gradient of the test solution Number Test solution Concentration gradient/(mg/mL) Vc Vc 0.06, 0.12, 0.48, 1.92, 3.84, 7.68, 15.36, 23.04, 34.56 A Hydroxytyrosol isophorone 0.06, 0.12, 0.48, 1.92, 3.84, diisocyanate derivative 7.68, 15.36, 23.04, 34.56 B Hydroxytyrosol 0.06, 0.12, 0.48, 1.92, 3.84, 7.68, 15.36, 23.04, 34.56

(17) Specific Steps:

(18) Sample liquid absorbance measurement: Take 2 mL of sample solution (Table 1: Vc and B), add 2 mL of DPPH solution with concentration of 2*10.sup.4 mol/L, mix and react in the dark at room temperature for 30 min, adjust to zero with toluene, and measure at 517 nm. The absorbance A.sub.i was simultaneously measured for the absorbance A.sub.j of 2 mL of toluene mixed with 2 mL of the sample solution and the absorbance A.sub.0 of 2 mL of DPPH solution mixed with 2 mL of toluene (The experimental results are shown in Table 2).

(19) TABLE-US-00002 TABLE 2 Absorbance test results of each test solution Concentration/(mg/mL) Sample Absorbance 0.06 0.12 0.48 1.92 3.84 7.68 15.36 23.04 34.56 Vc A.sub.i 0.814 0.549 0.246 0.100 0.060 0.050 0.025 0.014 0.012 A.sub.j 0.005 0.003 0.002 0.002 0.003 0.006 0.005 0.007 0.006 A.sub.0 0.944 A A.sub.i 0.975 0.953 0.843 0.615 0.319 0.155 0.128 0.122 0.110 A.sub.j 0.003 0.006 0.005 0.005 0.008 0.015 0.007 0.009 0.010 A.sub.0 1.062 B A.sub.i 0.990 0.989 0.903 0.868 0.807 0.730 0.492 0.425 0.351 A.sub.j 0.020 0.031 0.015 0.022 0.026 0.037 0.030 0.034 0.025 A.sub.0 1.108
Clearance calculation: clearance rate (%) [1(A.sub.iA.sub.j)/A.sub.0]*100%

(20) TABLE-US-00003 TABLE 3 DPPH clearance rate experiment results Concentration/ Clearance rate/% (n = 3) (mg/mL) Vc A B 0.06 14.35% 8.45% 12.44% 0.12 42.15% 10.80% 13.52% 0.48 74.20% 21.06% 19.87% 1.92 89.63% 42.53% 23.64% 3.84 94.01% 70.69% 29.55% 7.68 95.32% 86.84% 37.41% 15.36 97.88% 88.59% 58.30% 23.04 99.22% 89.36% 64.72% 34.56 99.39% 90.62% 70.59%

(21) The experimental results are shown in FIG. 1 and Tables 1 to 3. The antioxidant activity of the hydroxytyrosol isophorone diisocyanate derivative (A) showed a concentration-dependent relationship. As the concentration increases, the ability of the compound A to scavenge DPPH radicals is enhanced. In the concentration range, the DPPH free radical scavenging rate can reach up to 90.62%, and its antioxidant activity is basically the same as that of the positive control group (34.56 mg/mL) with positive antioxidant activity. Compared with the control group in which hydroxytyrosol (B) was added alone, the hydroxytyrosol isophorone diisocyanate derivative (A) at the same concentration was mostly superior in scavenging DPPH radicals. At a higher concentration, the antioxidant activity of the hydroxytyrosol isophorone diisocyanate derivative (A) was much higher than the equivalent concentration of the control group to which hydroxytyrosol (B) was added.