Hydroxytyrosol 4-methoxycinnamic acid ester with antibacterial 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 method of preparing a compound of formula (I), comprising: reacting a compound of formula (II) with a compound of formula (III) to obtain the compound of formula (I): ##STR00004## wherein the reaction of the compound of formula (II) with the compound of formula (III) comprises the following steps: placing the compound of formula (II), a catalyst, and 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF.sub.4]) in a reactor under nitrogen atmosphere, the catalyst being 12-molybdosilicic acid hydrate (H.sub.6Mo.sub.12O.sub.41Si); adding the compound of formula (III) to the reactor to form a reaction mixture, the compound of formula (II) and the compound (III) having a molar ratio of 1:1.1; heating the reaction mixture at 30 C. for 8 hours; placing the reaction mixture in a separating funnel to separate a crude product; purifying the crude product by recrystallization in methanol to obtain the compound of formula (I); and recycling 1-butyl-3-methylimidazolium tetrafluoroborate.

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 results of in vitro antibacterial activity of hydroxytyrosol 4-methoxycinnamic acid ester against drug-resistant bacteria MARS 18-222.

(4) FIG. 2 shows the results of in vitro antibacterial activity of Cefazolin sodium against drug-resistant bacteria MARS 18-222.

(5) FIG. 3 shows the results of in vitro antibacterial activity of hydroxytyrosol against drug-resistant bacteria MARS 18-222.

(6) FIG. 4 shows the results of in vitro antibacterial activity of 4-methoxycinnamic acid against drug-resistant bacteria MARS 18-222.

(7) FIG. 5 shows the results of in vitro antibacterial activity of hydroxytyrosol 4-methoxycinnamic acid ester against drug-resistant bacteria MARS 18-575.

(8) FIG. 6 shows the results of in vitro antibacterial activity of hydroxytyrosol against drug-resistant bacteria MARS 18-575.

(9) FIG. 7 shows the results of in vitro antibacterial activity of 4-methoxycinnamic acid against drug-resistant bacteria MARS 18-575.

(10) FIG. 8 shows the results of in vitro antibacterial activity of hydroxytyrosol 4-methoxycinnamic acid ester against drug-resistant bacteria MDR-PA 18-174.

(11) FIG. 9 shows the results of in vitro antibacterial activity of hydroxytyrosol against drug-resistant bacteria MDR-PA 18-174.

(12) FIG. 10 shows the results of in vitro antibacterial activity of 4-methoxycinnamic acid against drug-resistant bacteria MDR-PA 18-174.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

(13) 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 Compound (E)-3,4-dihydroxyphenethyl 3-(4-methoxyphenyl)acrylate (Compound of Formula I)

(14) In a 200 mL three-necked flask, 100.1 mg (0.65 mmol) of hydroxytyrosol and 124.0 mg (0.65 mmol) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) were dissolved in 50 mL of acetonitrile under nitrogen atmosphere. 126.7 mg (0.71 mmol) of 4-methoxycinnamic acid was dissolved in 20 mL of acetonitrile, and slowly added dropwise to the reaction liquid by a separatory funnel. After the completion of the dropwise addition, the temperature was raised to 80 C., and the reaction was carried out for 10 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The concentrated solution was washed in water, extracted with chloroform, dried and concentrated, and a crude product was obtained. The crude product was further purified by silica gel column chromatography, petroleum ether:ethyl acetate=1:3 as eluent, and the eluent was concentrated under reduced pressure and dried to obtain the title compound, 145.8 mg, a yield of 75.36%.

(15) .sup.1H-NMR (400 MHz, DMSO-d.sub.6) (ppm): 7.75 (2H, d), 7.42 (1H, s), 6.94 (2H, d), 6.76 (1H, s), 6.72 (1H, d), 6.64 (1H, d), 6.32 (1H, s), 5.32 (2H, s), 4.48 (2H, t), 3.75 (3H, s), 2.63 (2H, t); .sup.13C-NMR (400 MHz, DMSO-d.sub.6) ( ppm): 167.8, 155.6, 140.5, 127.5, 126.2, 123.5, 118.8, 112.2, 111.9, 110.2, 61.3, 50.8, 30.9.

Example 2: Preparation of Compound (E)-3,4-dihydroxyphenethyl 3-(4-methoxyphenyl)acrylate

(16) In a 200 mL three-necked flask, 100.1 mg (0.65 mmol) of hydroxytyrosol and 124.0 mg (0.65 mmol) EDC were dissolved in 50 mL of toluene under nitrogen atmosphere. 126.5 mg (0.71 mmol) of 4-methoxycinnamic acid was dissolved in 20 mL of toluene, and slowly added dropwise to the reaction liquid by a separatory funnel. After the completion of the dropwise addition, the temperature was raised to 60 C., and the reaction was carried out for 12 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The concentrated solution was washed in water, extracted with chloroform, dried and concentrated, and a crude product was obtained. The crude product was further purified by silica gel column chromatography, petroleum ether:ethyl acetate=1:3 as eluent, and the eluent was concentrated under reduced pressure and dried to obtain the title compound, 131.7 mg, a yield of 64.08%.

Example 3: Preparation of Compound (E)-3,4-dihydroxyphenethyl 3-(4-methoxyphenyl)acrylate

(17) In a 200 mL three-necked flask, 100.1 mg (0.65 mmol) of hydroxytyrosol and 124.0 mg (0.65 mmol) EDC were dissolved in 50 mL of ethyl acetate under nitrogen atmosphere. 126.5 mg (0.71 mmol) of 4-methoxycinnamic acid was dissolved in 20 mL of ethyl acetate, and slowly added dropwise to the reaction liquid by a separatory funnel. After the completion of the dropwise addition, the temperature was raised to 60 C., and the reaction was carried out for 12 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The concentrated solution was washed in water, extracted with chloroform, dried and concentrated, and a crude product was obtained. The crude product was further purified by silica gel column chromatography, petroleum ether:ethyl acetate=1:3 as eluent, and the eluent was concentrated under reduced pressure and dried to obtain the title compound, 125.1 mg, a yield of 61.23%.

Example 4: Preparation of Compound (E)-3,4-dihydroxyphenethyl 3-(4-methoxyphenyl)acrylate

(18) In a 200 mL three-necked flask, 100.1 mg (0.65 mmol) of hydroxytyrosol and 124.0 mg (0.65 mmol) EDC were dissolved in 50 mL of acetonitrile under nitrogen atmosphere. 139.0 mg (0.78 mmol) of 4-methoxycinnamic acid was dissolved in 20 mL of acetonitrile, and slowly added dropwise to the reaction liquid by a separatory funnel. After the completion of the dropwise addition, the temperature was raised to 70 C., and the reaction was carried out for 12 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The concentrated solution was washed in water, extracted with chloroform, dried and concentrated, and a crude product was obtained. The crude product was further purified by silica gel column chromatography, petroleum ether:ethyl acetate=1:3 as eluent, and the eluent was concentrated under reduced pressure and dried to obtain the title compound, 139.5 mg, a yield of 68.28%.

Example 5: Preparation of Compound (E)-3,4-dihydroxyphenethyl 3-(4-methoxyphenyl)acrylate

(19) In a 200 mL three-necked flask, 100.1 mg (0.65 mmol) of hydroxytyrosol and 124.0 mg (0.65 mmol) EDC were dissolved in 50 mL of toluene under nitrogen atmosphere. 151.6 mg (0.85 mmol) of 4-methoxycinnamic acid was dissolved in 20 mL of toluene, and slowly added dropwise to the reaction liquid by a separatory funnel. After the completion of the dropwise addition, the temperature was raised to 80 C., and the reaction was carried out for 8 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The concentrated solution was washed in water, extracted with chloroform, dried and concentrated, and a crude product was obtained. The crude product was further purified by silica gel column chromatography, petroleum ether:ethyl acetate=1:3 as eluent, and the eluent was concentrated under reduced pressure and dried to obtain the title compound, 128.1 mg, a yield of 62.70%.

Example 6: Preparation of Compound (E)-3,4-dihydroxyphenethyl 3-(4-methoxyphenyl)acrylate

(20) In a 200 mL three-necked flask, 100.1 mg (0.65 mmol) of hydroxytyrosol and 124.0 mg (0.65 mmol) EDC were dissolved in 50 mL of ethyl acetate under nitrogen atmosphere. 139.1 mg (0.78 mmol) of 4-methoxycinnamic acid was dissolved in 20 mL of ethyl acetate, and slowly added dropwise to the reaction liquid by a separatory funnel. After the completion of the dropwise addition, the temperature was raised to 70 C., and the reaction was carried out for 10 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The concentrated solution was washed in water, extracted with chloroform, dried and concentrated, and a crude product was obtained. The crude product was further purified by silica gel column chromatography, petroleum ether:ethyl acetate=1:3 as eluent, and the eluent was concentrated under reduced pressure and dried to obtain the title compound, 118.7 mg, a yield of 58.10%.

Example 7: Preparation of Compound (E)-3,4-dihydroxyphenethyl 3-(4-methoxyphenyl)acrylate

(21) In a 100 mL three-necked flask, 100.1 mg (0.65 mmol) of hydroxytyrosol, 126.5 mg (0.71 mmol) of 4-methoxycinnamic acid and 12.0 mg (0.007 mmol) silicomolybdic acid were dissolved in 50 mL of 1-butyl-3-methylimidazolium tetrafluoroborate under nitrogen atmosphere. After full dissolution, the temperature was raised to 30 C. and the reaction was carried out for 8 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture system was allowed to separate into layers to give a crude product. The crude product was recrystallized with 50 mL methanol and dried to obtain the title compound, 174.6 mg, a yield of 85.51%.

Example 8: Preparation of Compound (E)-3,4-dihydroxyphenethyl 3-(4-methoxyphenyl)acrylate

(22) In a 100 mL three-necked flask, 100.1 mg (0.65 mmol) of hydroxytyrosol, 126.5 mg (0.71 mmol) of 4-methoxycinnamic acid and 12.0 mg (0.007 mmol) silicomolybdic acid were dissolved in 50 mL of 1-butyl-3-methylimidazolium tetrafluoroborate under nitrogen atmosphere. After full dissolution, the temperature was raised to 50 C. and the reaction was carried out for 10 hours. Thin layer chromatography was used to track the reaction to completion, heating was stopped, and the protective device was removed. The reaction mixture system was allowed to separate into layers to give a crude product. The crude product was recrystallized with 50 mL methanol and dried to obtain the title compound, 163.0 mg, a yield of 79.83%.

Example 9: Antibacterial Activity Test of the Compound of Formula I

(23) The minimal inhibitory concentration (MIC) of the compound as determined by a microbroth dilution method with gentamicin, cefazolin sodium and ceftriaxone sodium as positive control.

(24) The experimental strains included methicillin-resistant Gram-positive bacteria: methicillin-resistant Staphylococcus aureus MRSA 18-222, 18-575; multiple drug-resistant Gram-negative bacteria: vancomycin-resistant enterococci VRE 18-80,18-94, multidrug-resistant Pseudomonas aeruginosa MDR-PA 18-1774,18-202, carbapenem-resistant Acinetobacter baumannii CR-AB 18-183,18-560. All the experimental strains were donated by Huashan Hospital affiliated to Fudan University (Institute of antibiotics, Fudan University) and used after routine identification.

(25) Preparation of Test Strains:

(26) Preparation of MHB medium: 20.0 g MHB medium was added to 1 L distilled water, boiled until completely dissolved, packed in conical bottles and sterilized at 121 C. for 15 min.

(27) The experimental strain was cultured to the logarithmic growth phase: under aseptic condition, the experimental strain was inoculated into 100 mL MHB medium and incubated in a constant temperature and humidity incubator at 37 C. for 20-22 hours.

(28) Preparation of storage solution: weigh the sample to be tested, dissolve it with 1% DMSO solution, prepare a storage solution with a concentration of 2560 g/mL, weigh a positive reference substance, dissolve it with aseptic distilled water, and configure a storage solution with a concentration of 2560 g/mL.

(29) Preparation of Bacterial Suspension:

(30) Under aseptic condition, the experimental strains cultured to logarithmic growth phase were adjusted to 0.5 MCF turbidity standard with MHB medium and diluted according to 1:10, and the bacterial suspension with concentration of 10.sup.6 CFU/mL was prepared for standby.

(31) Dilution of storage solution and inoculation of experimental strain: under aseptic condition, the storage solution was diluted to 256 g/mL solution. Take a sterile 96-well plate, add 200 L MHB medium to the 12th well, and add 100 L MHB medium to each well. Add 100 L of positive control solution to the first well, mix well, and suck 100 L from it and discard. Add 100 L of the compound sample solution to the second well, mix well, and then pipette 100 L to the third well. After mixing, pipette 100 L to the fourth well, and dilute to the 11th well in this way. Finally, 100 L was pipetted from the 11th well and discarded. The 12th hole was the growth control without drugs. So far, the concentration of the positive reference substance is 128 g/mL, the concentrations of the sample solution are 128, 64, 16, 8, 4, 2, 1, 0.5, 0.25 g/mL respectively. Then, 10 L of the prepared bacterial suspension is added to each well, so that the final concentration of the bacterial liquid in each well is 510.sup.5 CFU/mL.

(32) Incubation: Cover the 96-well plate inoculated with the experimental strains, and incubate in a constant temperature and humidity box at 37 C. for 20-22 hours.

(33) Interpretation of the MIC endpoint: The concentration that can completely inhibit the growth of bacteria in a 96-well plate under a black background is the lowest inhibitory concentration of the sample against the bacteria.

(34) In FIGS. 1-10, the twelve wells represent twelve groups, from left to right, positive, 128 g/mL, 64 g/mL, 32 g/mL, 16 g/mL, 8 g/mL, 4 g/mL, 2 g/mL, 1 g/mL, 0.25 g/mL, 0.0625 g/mL, Negative. FIG. 1 shows the in vitro antibacterial activity of hydroxytyrosol 4-methoxycinnamic acid ester against drug-resistant bacteria MARS 18-222. FIG. 2 shows the in vitro antibacterial activity of Cefazolin sodium against drug-resistant bacteria MARS 18-222. FIG. 3 shows the in vitro antibacterial activity of hydroxytyrosol against drug-resistant bacteria MARS 18-222. FIG. 4 shows the in vitro antibacterial activity of 4-methoxycinnamic acid against drug-resistant bacteria MARS 18-222. FIG. 5 shows the in vitro antibacterial activity of hydroxytyrosol 4-methoxycinnamic acid ester against drug-resistant bacteria MARS 18-575. FIG. 6 shows the in vitro antibacterial activity of hydroxytyrosol against drug-resistant bacteria MARS 18-575. FIG. 7 shows the in vitro antibacterial activity of 4-methoxycinnamic acid against drug-resistant bacteria MARS 18-575. FIG. 8 shows the in vitro antibacterial activity of hydroxytyrosol 4-methoxycinnamic acid ester against drug-resistant bacteria MDR-PA 18-174. FIG. 9 shows the in vitro antibacterial activity of hydroxytyrosol against drug-resistant bacteria MDR-PA 18-174. FIG. 10 shows the in vitro antibacterial activity of 4-methoxycinnamic acid against drug-resistant bacteria MDR-PA 18-174. The results are summarized in Table 1.

(35) TABLE-US-00001 TABLE 1 Minimum Bacteriostatic Concentration of Test Compound and Positive Controls (g .Math. mL.sup.1) Strain MRSA VRE MDR-PA CR-AB Sample 18-222 18-575 18-80 18-94 18-174 18-202 18-183 18-560 Hydroxytyrosol 4- 4 32 >128 >128 8 >128 >128 >128 methoxycinnamic acid ester Gentamicin 128 2 0.0625 >128 0.0625 0.0625 >128 >128 Cefazolin sodium >128 >128 32 >128 8 128 >128 >128 Ceftriaxone sodium >128 >128 8 >128 128 16 >128 >128 Hydroxytyrosol >128 >128 >128 >128 >128 >128 >128 >128 4-methoxycinnamic >128 >128 >128 >128 >128 >128 >128 >128 acid

(36) According to the experimental results of FIG. 1-10 and Table 1, hydroxytyrosol and 4-methoxycinnamic acid have no inhibitory effect on drug-resistant bacteria, while hydroxytyrosol 4-methoxycinnamic acid ester derivative has strong inhibitory effect on drug-resistant Gram-positive bacteria MRSA (MIC=4 g/mL) and MDR-PA (MIC=8 g/mL), even stronger than that of positive control drugs. In summary, the hydroxytyrosol 4-methoxycinnamic acid ester of the invention can be used as an antibacterial candidate for Gram-positive bacteria methicillin-resistant Staphylococcus aureus and multi-drug-resistant Pseudomonas aeruginosa, and further preclinical research.