JPH01311048A - Phenylpropenoic acid derivative - Google Patents

Abstract

NEW MATERIAL:1-Carboxy-2-(3,4-dihydroxyphenyl)ethyl (E)-3-[3- hydroxy-4-[[2-(3,4-dihydroxyphenyl)-1-methoxycarbonyl]vinyloxy]phenylp henyl]- propenoate of formula. USE:Useful as an anti-inflammatory agent having improved activity. PREPARATION:KEIGAI (leaf and stalk of Schizonepeta tenuifolia) is extracted by infusing with a cold mixture of acetone and water. The mixing ratio of acetone to water is usually 7:3 (V/V), 3-4 liter of the mixture is used in one extraction step of 1kg of KEIGAI and the cold extraction is repeated 3-4 times to obtain an extracted liquid. The acetone in the extracted liquid is distilled off under reduced pressure to obtain an aqueous solution, which is washed 4-6 times with chloroform. The obtained aqueous layer is extracted with ethyl acetate, the extracted liquid is concentrated under reduced pressure and the extract is separated and purified by column chromatography to obtain the objective compound of formula.

Description

DETAILED DESCRIPTION OF THE INVENTION "Field of Industrial Application" The present invention relates to a novel phenylpropenoic acid derivative isolated from Heisheng. More specifically, the present invention relates to a novel phenylpropenoic acid derivative represented by the following formula (1). The compound of formula (1) is expected to be used as an anti-inflammatory agent.

"Conventional technology" Schizonepeta tenuifolf
a BRIQ, ) is a plant belonging to the Labiatae family, and is one of the herbal medicines that are included in Chinese herbal medicines such as Jumi-Ketoku-Dou.

It is already known that the essential oil obtained by steam distilling the ethyl acetate extract of Hi-Shou has anti-inflammatory effects, and 1-prebon has been reported as its active ingredient (Joji Yamahara et al., Pharmaceutical Journal, 100). (see Vol. 713, 1980)
. It has also been reported that Hisheng contains monoterpene glycosides and flavonoid glycosides (M,
Kubo et al., Chea+icalPharmaceut
ical Bulletin, Volume 34, Page 3097, 1
986).

"Problems to be Solved by the Invention" The present inventors conducted various studies to isolate a novel compound having anti-inflammatory effects from Hi-Sho. An object of the present invention is to isolate and provide a novel compound having anti-inflammatory effects from Hi-Shang.

"Means for Solving the Problems" As a result of various studies on the single m purification method of the flying component, the chemical structure of the isolated component, and the anti-inflammatory effect, the present inventors have determined that the trimer structure of caffeic acid has the following formula: (I) The present invention was completed by successfully isolating a novel phenylpropenoic acid derivative represented by (I) and confirming the strong anti-inflammatory effect of this compound.

Hereinafter, a single N purification method for the compound of the present invention will be explained.

First, Hi-Sho is extracted by cold immersion in a mixed solvent of acetone and water. The acetone-water mixing ratio is usually 7:3 (v/v), and an amount of 3 to 41 per kg of flying water is used for one extraction.
The cold immersion extraction is repeated 3 to 4 times to obtain an extract.

Next, acetone in the extract is distilled off under reduced pressure to obtain an aqueous solution, which is washed 4 to 6 times with chloroform.

Next, the aqueous layer obtained above is extracted 5 to 6 times with ethyl acetate to obtain an ethyl acetate extract.

Next, the extract is concentrated under reduced pressure to obtain an extract, which is separated and purified by column chromatography several times as described below.

First, a porous polymer (e.g. Diaion) IP-2
0 [manufactured by Mitsubishi Kasei Corporation] is subjected to column chromatography to obtain both fractions containing the compound of the present invention. A mixed solvent of water and methanol is used as the elution solvent, and elution is carried out while increasing the methanol concentration sequentially. The compound of the present invention is mainly eluted with a water-methanol mixed solvent of about 3 Nia (v/v).

Next, the above fractions containing the compound of the present invention are collected, and the compound of the present invention is separated by column chromatography packed with Sephadex. Sephadex Lll-20 (manufactured by Pharmacia) is usually used for cefazo-nx, and the elution solvent is water-methanol in a ratio of 4:6 to 3 (v/
v) Use a mixed solvent.

Furthermore, porous polymers such as Toyopearl 1 (W-4
Column chromatography packed with 0 (manufactured by Toso) [elution solvent, methanol-water 1:1 (v/v)
It is purified using a mixed solvent, and finally purified by high performance liquid chromatography and/or recrystallization.

For example, in high-performance liquid chromatography columns, Y
MC-Pack 5343 [2cm x 25
cm, manufactured by Yamamura Chemical Laboratory Co., Ltd.] using ? :8 It is preferable to use a mixed solvent of 1% formic acid aqueous solution and acetonitrile in a ratio of 3:2 (v/v) for the eluate.

In addition, in the above-mentioned Diaion) IP-20 column chromatography, caffeic acid, rosmarinic acid, hesveridin, luteolin, and benzofuranylpropenoic acid conductors are obtained from both fractions before the compound of the present invention is eluted. .

``Effect of the invention'' The strength of the anti-inflammatory effect is due to the oxidoreductase Ig! is 3
α-Hydroxysteroid dehydrogenase Efl:
1.1.1.50 (hereinafter abbreviated as 3α-)1sD) [T, M, P
enning et al., Proceedings of the
National Academy of Sciences
ce of the United 5tat
Es of Hachimerica, Volume 80, 4
504, 1983, and Ohe et al., Japanese Society of Pharmacological Pharmaceutical Sciences, 34th Annual Pension (Osaka) Lecture Abstracts, p. 16, 1986], the above-mentioned enzyme inhibitory activity of the compounds of the present invention was measured according to these methods. , showed extremely strong activity. That is, the compound of the present invention has a trimeric structure of caffeic acid, but rosmarinic acid [L, Grac
za et al., Archive der Pharmazie (
Weinheim, Germany), 318 volumes,
p. 1090, 1985],
It also showed much stronger activity than aspirin, a typical anti-inflammatory analgesic. Therefore, the compounds of the present invention can be expected to act as anti-inflammatory agents with stronger activity.

The activity of caffeic acid was also tested, but it was weaker than that of the compound of the present invention.

The anti-inflammatory effects of the compounds of the present invention will be explained below by giving test examples.

Test Examples The following tests were carried out in accordance with the method described in the 34th annual meeting of the Japanese Society of Pharmacological Sciences (Osaka), collection of lecture abstracts, p. 16, 1988.

1. Sample・Compound of the present invention (I) ・Rosmarinic acid (control compound) ・Caffeic acid (control compound) ・Aspirin (control compound) 2. Test method Preparation of enzyme (3α-H3D) solution SD male and female rat liver was extracted and mixed with a homogenate solution (100mM potassium phosphate buffer, 250mM potassium phosphate buffer, 3 times the weight).
(consisting of mM sucrose, 1mM dithiothreitol, and 1mM sodium ethylenediaminetetraacetate) was added and homogenized, and then incubated at 10,000 G and 4°C for 30 minutes.
Centrifugation was performed for a minute to obtain a supernatant. This supernatant was further added to 1000
Centrifuge for 60 minutes at 00G and 4°C, remove the supernatant (sidesill fraction), and add the enzyme solution (3α-H5D solution).
I got it. This was diluted 2.5 times with 100 mM potassium phosphate buffer (hereinafter referred to as PBS) having a pH of 6.0 to obtain an enzyme solution for assay.

2) Measurement of enzyme (3α-)150) inhibitory activity First, the following operations 1 to 2 were performed to determine the decrease (S) in the absorbance at 340 tv caused by the enzyme reaction in the presence of the sample.

■Sample, PBS 2. On+I1. Dissolve the sample at 37°C to prepare a sample solution.

■ Add 0.2 mJ2 of the above assay enzyme solution to the sample solution, incubate for 10 minutes, and add nicotinamide adenine dinucleotide phosphate (NADPH) in PBS.
Add 0.2 mm of M solution (NADPH?!A 9mM) and incubate for an additional 10 minutes.

(2) Add 0.1 mm of an acetonitrile solution of 4-nitroacetophenone (concentration 25 mM), measure the absorbance at 340r+a+ after 30 seconds and 10 minutes, and determine the decrease in absorbance (Sl).

■ Repeat steps ■ to ■ in exactly the same way except adding 0.1 mj2 of acetonitrile instead of the acetonitrile solution of 4-nitroacetophenone in ■ above, and calculate the decrease in absorbance when the substrate (4-nitroacetophenone) is not present (S
, ).

■ Take the difference between S1 and 52 to find the decrease in absorbance (S) that occurs due to the enzyme reaction in the presence of the sample (i.e., S =
S+-32).

On the other hand, the decrease in absorbance (C) that occurs due to the enzyme reaction when no sample is added is determined. That is, PBS was used instead of the sample solution in 2 above. Except for using Omu, perform steps 1 to 2 in the same manner as above to determine the value CI corresponding to S and the value C2 corresponding to S2, and determine the absorbance decrease value (C) when no sample is added. .

Next, the enzyme inhibition rate by the sample was calculated using the following formula.

Then, the enzyme inhibition rate at various concentrations of the specimen was determined, and the 50% enzyme inhibition concentration (hereinafter referred to as
IC5°) was calculated.

3. Test results The results are shown in Table 1.

Table 1 "Example" Hereinafter, the present invention will be explained in more detail with reference to Examples.

The compound of the present invention was detected and collected by high performance liquid chromatography under the following conditions.

Column: YMC-Pack A312 [ODS,
6mm x 15cm.

Yamamura Chemical Laboratory Co., Ltd.] Eluent: 1% formic acid-acetonitrile 5:2 (v/
v) Mixed solvent flow rate: 1rnll/min Column temperature: 40°C Detection wavelength: UV 280nm Under these conditions, the lithium condensation time of the compound of the present invention is approximately 2
It is 0.4 minutes.

Example 1 Compound (1) of the present invention...1-carboxy-2-(3
,4-dihydroxyphenyl)ethyl(E)-3-[3
-Hydroxy-4-[[2-(3,4-dihydroxyphenyl)-1-methoxycarbonyl]vinyloxy]phenyl]propenoate: 10 kg of dried flying flower spikes from Korea were mixed with acetone and water in a 7:3 ratio.
(v/v) mixed solvent for 6 hours at 40 liters, 6 hours at 30 Il, and 6 hours at 31 liters in total, and these extracts were combined and concentrated under reduced pressure to obtain a concentrated aqueous solution of 1OJ2. Ta. After washing this 5 times with 101 chloroform each time,
Each tOIL was extracted five times with ethyl acetate to obtain an ethyl acetate extract. Ethyl acetate was distilled off under reduced pressure to obtain 80 g of extracted extract. 80 g of this extracted extract was subjected to column chromatography [filling material: Diaion HP-20 manufactured by Mitsubishi Kasei Corporation, 1.311], eluted with solvents with increasing concentrations of methanol as shown below, and the eluate was fractionated. 35 fractions were obtained.

l water 2002
Water-methanol (5:1)
6003-9 Water-methanol (3:2)
5001O~23 each Water-methanol (1:l) 50024~32 each
Water-methanol (3nia) 50033 each
~35 Methanol
Each of the 500 fractions with fraction numbers 24 to 29 was collected and the solvent was distilled off under reduced pressure to obtain 8.82 g of a residue containing the compound of the present invention. This was subjected to column chromatography packed with Sephadex LH-20 (manufactured by Pharmacia) 160 mJ2, and after flowing a mixed solvent of water-methanol 1:1 (v/v) at 1.2°C, water-methanol 3 : 7 (
v/v) mixed solvent at 700 mA. 400 mJ:l of the eluate initially eluted at the beginning of elution with water-methanol (v/v) was collected, and the solvent was distilled off under reduced pressure. Next, 1.14 g of the obtained residue was added to Sephadex LH.
It was applied to a column packed with -2061 mJ2 and eluted with a mixed solvent of water-methanol 4:6 (v/v).
00＋＋＋Jl! A fraction of 600 mJ2 was collected after effluent. The solvent was distilled off under reduced pressure, and the resulting residue (450 mg) was further purified by column chromatography packed with a porous polymer, Toyovar IW-40 (manufactured by Toso) 531λ. Elution solvent: water-methanol 1:1
(v/v) mixed solvent was used. Elution solvent 420aJ2
A fraction of 210 mu was collected after effluent, and the solvent was distilled off under reduced pressure to obtain 200 mg of the crude compound of the present invention. Finally, this is subjected to preparative high performance liquid chromatography [column: Y
MC-Pack 5343, 20 mm x 25 cm, manufactured by Yamamura Chemical Laboratory Co., Ltd., eluent: 1% formic acid-acetonitrile 3:2 (v/v) mixed solvent,
Flow rate: 13.5 rnfl1 min, detection: U V 2
Purification was carried out using 80 nml to obtain 80 mg of the compound of the present invention. When this is recrystallized from water, brown powdery crystals are obtained. The physical property analysis values are shown below.

Melting point = 120-122°C 11V(MeO)l)no+(10g6): 202(
4,72), 214sh (4,47).

230sh(4,34), 290(4,40), 3
32(4,47).

IR(niBr)cm-': 3413,1701,1
633.1609゜' H-NMR (DMSO-d6)
δppm: 2.911H, dd, J-8, 5 and 14
．． 2Hz), 3.01 (1)1. dd, J-4,
0 and 14.21(z).

3.69 (3H, s), 5.04 (1) 1. dd, J
-4,0 and 8.5H2).

6.36(1)1. d, J-18,0Hz), 6.
53 (11 (, d, J-8, 2Hz), 6.65 (IH
, d, J-8, 2H2), 6.68(1)1. d, J-
8.2Hz), 6.69 (ill, brs)
, 8.74 (1)1. d, J-8, 2) IZ)
.

7.03-7.09 (2H, m), 7.20-7.24
(2H, I), 7.28 (IH, s), 7.50 (1)
1. d, J-16,0H2).

"C-NMR (DMSO-d,) δppm: 3B,
3,52.2,73.3°114.2. 115.5.
115.6. 115.7. 115.9°115.
8. 117.5. 120.1. 120.6. 1
23.4°123.5. 127.6. 127.8.
128.8. 136.5°144.0. 145.
0. 145.3. N6.7. 147.0°14
B, 0. 163.4. 165.8. 171.0°MS m/z (k): 575 (M+Na)” (6),
553 (Mho) Responsibility 7).

355 (ioo).

Claims (1)

[Claims] (1) Below formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) A phenylpropenoic acid derivative represented by