JPS6157594A - Galactosphingolipid and its production - Google Patents

Abstract

NEW MATERIAL:A compound of formula I (n is 10, 11; R<1> is H, methyl, acetyl; R<2> is H, methyl). USE:Medicine for gastric ulcer, hypertension, allergic diseases, stenocardia and arrhythmia. It has hypotensive, coronary vasodilative and decarboxylase-inhibitory actions. PREPARATION:Sponge (Chondropsis sp.) is frozen with liquid nitrogen and crushed at a temperature lower than -150 deg.C. Then, the product is freeze-dried into a fine powder and the powder is extracted with an organic solvent such as alcohol or acetone, preferably at room or lower temperature. Then, the extract is concentrated to give the compound of formula II where R<1>, R<2> in formula I are H.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a compound of the general formula (I) (where n represents 10 or 11, R1 represents a hydrogen atom, a methyl group or an acetyl group, and R2 represents a hydrogen atom) or a methyl group) and a method for producing the same.

The above compounds and sponges containing them as main components
dropsis sp.) has inhibitory effects on histidine decarboxylase (HDC), lowers blood pressure, and dilates coronary vessels, so it is expected to be used as a medicine for gastric ulcers, hypertension/allergies, angina pectoris, and arrhythmia. It is a substance that is

[Conventional technology]

Since the living environment of marine organisms is significantly different from that of terrestrial organisms, it is expected that novel compounds with new physiological activities that cannot be obtained from terrestrial organisms can be obtained from marine organisms. In fact, many compounds with novel skeletons have been discovered from marine organisms. For example, the present inventors have obtained a substance having a vasodilatory effect from a sponge (Xestospongia exigua) (Chuyo et al., 26th Natural Organic Compound Symposium, Abstracts, p. 110, 1983). In addition, Nakamura et al.
-Obtaining receptor blocking active substances [Eiji Nakamura et al., Tet
rahedron Letters+23+, 555
5+ (I982)).

For glycolipids, purple sea urchin (Anthocidaris)
crass-isptna) to Kitayo et al. (Chem,
Pharm, Bull, (Tokyo) 27, 1
934 (I979)), and the green alga Anaosa (
Ulva pertusa) to Fushiya et al.
Biol, Chem.

39, 2021 (I975)) have discovered sulfonoglycolipids.

[Problems to be solved by the present invention]

The present inventors planned the development of a medicinal substance with a new skeleton and conducted research on marine animals.In particular, among marine animals, they conducted extensive research on lower marine organisms belonging to the phylum Porifera. Chond sponges collected around Boat Phillip in Victoria.
ropsis sp.) has a histidine decarboxylase (HDC) inhibitory effect, a coronary vasodilator effect, and an antihypertensive effect.

Since this substance has the above-mentioned pharmacological effects, it is considered to be an effective medicinal substance for allergies, gastric ulcers, cerebral circulation disorders, hypertension, angina pectoris, arrhythmia, etc., and research was conducted to find the substance that exhibits its activity.

[Means for solving problems]

The galactosphingolipid according to the present invention is represented by the general formula (I) (wherein n represents 10 or 11, R1 represents a hydrogen atom, a methyl group, or an acetyl group, and R2 represents a hydrogen atom or a methyl group). This substance has antihypertensive, coronary vasodilatory, and histidine decarboxylase inhibitory effects.

The galactosphingolipid of general formula (I) according to the present invention can be produced as follows.

That is, the compound represented by the general formula (Ia) (in which n represents 10 or 11)
sp, ) is frozen using liquid nitrogen or liquid air, for example, and then crushed using a crusher or homogenizer at -150°C or lower, and then freeze-dried to obtain a finely divided product. This finely divided product is extracted with an organic solvent according to a conventional method. As the extraction solvent, for example, alcohol, acetate, acetone, etc. are preferable. The extraction operation can be carried out according to conventional methods, but is preferably carried out at room temperature or below.

By extracting and then concentrating in this way, α-
It is possible to obtain an extract that has a histidine decarboxylase inhibitory activity of about 172 of fluoromethylhistidine, and also has coronary vasodilatory and antihypertensive effects. The main component of this extracted extract is a substance represented by the above-mentioned structural formula (Ia).

This extracted extract can be further refined using the method described below.

The extract obtained as described above can be purified by, for example, chromatography using one or a combination of adsorption chromatography, partition chromatography, ion exchange chromatography, and gel filtration chromatography. , or by combining these chromatography with solvent separation, recrystallization, etc.
It can also be manufactured. If necessary, further purification can be carried out using high performance liquid chromatography.

The compound of general formula (Ia) thus obtained is a new substance.

Next, by methylating this galactosphingolipid (Ia), R' = CO in the general formula (I)
Galactosphingolipid (Ib) of CH3 can be produced. This methylation was performed using the Hakomori method (S,
1. Hakomori, J., Biochem,
Preference is given to using the anion of dimethyl sulfoxide and the methyl halide according to J.D. + 55173-, p. 205, 1964).

It goes without saying that if ethyl halide is used in place of methyl halide, a galactosphingolipid of general formula (I) in which groups R1 and R2 are ethyl groups can be obtained.

Furthermore, by acetylating the galactosphingolipid of the general formula (Ia), R1=C
OCH3, galactosphingolipid (Ic) with R2=H
can be obtained. This acetylation can be carried out by a conventional method, but it is preferable to use acetic anhydride or acetyl chloride as the acetylating agent. Similar to this acetylation, other acylated galactosphingolipids can be obtained by using acylating agents such as propionic anhydride, pentanoyl chloride, trifluoroacetic anhydride or benzoyl chloride.

The compounds (Ib) and (Ic) thus obtained
) is also a new substance.

〔Example〕

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but it goes without saying that the scope of the present invention is not limited to these Examples.

Production Example 1 Production of Compound (Ia) A sponge (Chondropsis sp.) was frozen in liquid nitrogen and pulverized using a pulverizer in liquid nitrogen.

This was freeze-dried into a fine powder.

100 g of fine powder was extracted with 1.5N ethanol at room temperature, and the solvent was removed under reduced pressure to obtain 4.1 g of crude extract.

2.37 g of the crude extract thus prepared was dissolved in a mixed solvent of chloroform:methanol:water=5:6:4, and 1.60 g of the active fraction was obtained from the lower layer. This product was subjected to silica gel column chromatography using an elution solvent of chloroform:methanol = 20:1-3:
1) Chloroform:methanol = 3 as active fraction
:291 mg of both doses of 1 were obtained. In order to further purify the active fraction obtained here, silica gel column chromatography (elution solvent: chloroform: methanol-5:
1), then Toyo Pearl HW-40 (Toyo Soda Kogyo ■
, course) column chromatography (sol + im solvent:
The substance of the present invention was purified using chloroform:medanol=1:1) as a white powder (187 mg (yield: 0.32)).
%) was obtained.

The resulting compound (in formula Ia n = 10 and n =
The physical properties of 11) were as follows.

(a) Properties: White powder (b) Mass spectrum (secondary ionization method, m/z): 8
94 (M(n-+o)+Na), 90B (
M(n-+i)+Na)” (c) Infrared spectrum (K
Br, cm-1): 3304.2927°284
9, 1656. 1544. 1466, 1076
, 1047(d)H-NMR spectrum (heavy pyridine, δ2.270 MHz); 0.85 (
d, J=7.3 [1z, 6H.

t, 311), 1.25 (m), 1.46 (m
, 111) , 2.17 (m, 211) , 2.67
(ddd, J-14,3,8,1,7,211z.

18) , 3.01 (ddd, J= 14.3,7
．． 8,2.011z,1)I),3.99(t,
J=5.4,1)1) ,4.10 (dd, J=8.
1゜3.0. LH), 4.2-4.6 (m),
-4,75(dd, J= 10.8.6.OH2,LH
), 4.87 (d, J=6.Ofiz), 5.2
6 (m, IH), 5.71 (dt, J=15
．． 4,8.1) 1z, IH) 5.94 (dL, J
= 15.4.8.1Hz, LH), 8.54(d
, J = 9.5 Hz, 11() (e) 13C-NMR spectrum (heavy pyridine, δ
-167,5 units +12): 11.98.14.6
8.19.77.23.19.25.92.27.64
．． 27.93.2B, 46.29.86.30.10
, 30.25.30.47.30.56.32.40
．． 33.31.34.87.35.26.36.8B
, 37.12.39.52.51.39.62.51.
69.97.71.83.72.21.72.32.7
4.26.74.49,? 5.82.104.86.
126.60.133.67.175.61 (c) Thin layer chromatography (Merck & Co., Kieselgel
60P 254) Rf value (migration rate) = 0.3 (methanol; methylene chloride - 15:85) -0,25 (chloroform; methanol: H20 = 7:3:1) (g) High performance liquid chromatography retention time = 9 minutes (Column: 5hodex KF-8
01° Solvent: Tetrahydrofuran, flow rate 1 m1/min) 1146 g of fine powder prepared in the same manner as in Production Example 1 was extracted using methylene chloride 41 to remove impurities, and then extracted using ethanol 42.

The ethanol extract was concentrated to obtain 24.6 g of crude extract.

13.42 g of this crude extract was subjected to silica gel column chromatography to obtain 2.30 g of crude powder (solvent: chloroform:methanol = 10:L to 7:1).

This coarse powder was recrystallized using methanol/methylene chloride to obtain the target compound Ia as a white powder with a concentration of 1.365
g (yield 0.22%) was obtained.

The physical properties of the obtained compound were similar to those of Production Example 1.

Production Example 3 Production of Ethanol Extract of Sponge (Chondropsis sp.) Sponge (Chondropsis sp.) was frozen in liquid nitrogen, pulverized using a grinder in liquid nitrogen, and then freeze-dried to form a fine powder.

After 41 g of methylene chloride was added to 1146 g of this fine powder and impurities were first removed, 41 g of ethanol was added to the residue and extracted at room temperature. By removing ethanol under reduced pressure, 24.6 g of the desired ethanol extract was obtained.

This extract contained the compounds of formula (Ia) with n = 10 and 11 as main components.

Production Example 4 Production of Octamethylgalactosphingolipid (Compound Ib) Anhydrous ether or anhydrous hexane was added to 40 mg of sodium hydride (50% oil suspension) to remove the oil, and the mixture was dried under reduced pressure. Dimethyl sulfoxide (DMS◯) was added to this sodium hydride powder at 0.5° C. and the mixture was heated and stirred at 60 to 70° C. for 2 hours under an argon atmosphere.

This solution was cooled to room temperature, and the galactosphingolipid 10 represented by the general formula (Ia) produced in Production Example 1 was
+ng of DMSO solution 1- was added and stirred for 1.5 hours.

Next, methyl bromide 1- was added to the reaction solution, and the mixture was stirred at 0° C. for 2 hours while shielded from light.

Water was added to the reaction solution, extracted with methylene chloride, and the extract was dried and concentrated under reduced pressure.

The obtained oil was subjected to 20 g silica gel column chromatography and eluted with methanol:methylene chloride (2:98) to obtain the title compound 10-.

The physical properties of this product were as follows.

IR spectrum (film, cm-'): 29
26.2855.1q49.1466.11l108N
Spectrum (CDC13, δ-1270MHz):
0.85 (d, J= 7.6 Hz, 6H)
, 0.88 (L, J-7,5Hz, 31
1) , 1.97 (+, 3H) , 2.28 (
+w, 2H), 2.97 (s, 3H), 3.
30 (s, 311), 3.35 (s, 311)
, 3.38 (s, 311) , 3.42 (s
, 4H) , 3.49 (s, 6H) , 3.59
(s, 3H), 3.80 (dd, J= 11.
3.0.8flz), 3.97 (brt, J
=3.3 Hz, III), 4.20 (m,
2B), about 5.45 (+a, 4H) Production Example 5 Galactosphingolipid (Ia
) 3hg was dissolved in three volumes of anhydrous pyridine, 3-acetic anhydride was added thereto, and the mixture was allowed to stand at room temperature for 14 hours.The reaction mixture was then poured into water and extracted with methylene chloride.

After drying the methylene chloride layer over magnesium sulfate, the solvent was distilled off under reduced pressure to obtain 31 mg of the title compound.

The physical properties of the obtained compound were as follows.

NMR spectrum (CDCl3, δP, 360M1
lZ): 0.85 (d, J=6.311z,
6H), 0.88 (t, J-6,0Hz, 3H
), approximately 1.24 (CII2 x n), 1.51
(m, IH) , 1.85 (m, IH) , 1.97
(s, 311), 2.03 (s, 3H), 2.0
5 (s, 3H), 2.06 (s, 31(), 2.
09 (s, 3H) , 2.15 (s, 3H) , 2
．． 22 (s, 311), 2.27 (t, J8.4
Hz, 1■), '2.38 (brd, LH),
3.67 (dd, J= 10.8.3.5112.l
11), 3.86 (dd.

J = 10.8, 3.0Hz, LH), 3.
90 (td, J-6, 5°0.9 Hz), 4.1
3 (d, J-6, 5H2, 21), 4.33 (
dddd, 110.4.44 (d, J=7.5Hz
, IH), 4.92 (dt, J-9,3+3.5Hz
+LH), 5.00 (dd.

J = 10.7.3.5112. LH), 5
．． 10 (dd, J-10, 7°7.5Hz, IH)
, 5.13 (4d, J”9.3.8.2Hz, 1ll
), 5.15 (t, J = 6.0Hz, LH), 5
．． 26 (dt, J -15,3,7,0Hz, 1
11), 5.36 (dd, J = 3.5.1
．． 0Hz, IH), 5.48 (dL, J =
ts, 3. e, euZ, to ), 6.82 (d,
J=9.0 Hz, Nu) 13cm NMR spectrum (CDCI, , δ2.67.8 MHz)
:11.62 (q), 14.31 (q),
19.42 (q), 20.82.22.84 (
q) , 25.07 (t) , 27.28 (
t) , 27.39 (t) , 27.59 (t
), 28.15 (d), 29.8B, 31.
97 (t), 32.09 (t), 32
．． 31 (t), 32.77 (t), 3
4.57 (d), 36.80 (t),
39.20 (t), 48.24 (d)
, 61.13 (t) , 66.27 (t)
, 67.03 (d) , 68.68 (d)
, 70.86 (d) , 72.07 (d
), 72.63 (d), 73.90 (
d), 100.59 (d), 123.88 (
d), 134.34 (d), 169.0
5 (s), 169.47 (s), 169.
60 (s), 169.63 (s), 169
．． 69 (s), 169.76 (s), 16
9.94 (s), 170.31 (s)0,
2 X 10-6 moles of dithiothreitol (DTT)
, 0.1 X 10-” moles of pyridoxal-5′
- Partially purified HDC preparation from rat stomach (T, WaLanabe et al., A
advance in the bioscience,
33.93-106 (I982)) 0.02 mg and 0.1 mff of the test solution prepared by dissolving the test compound in a 0.1% ethanol aqueous solution were added, preincubated at 37°C for 1 hour, and then mixed with 2.5 10 −3 mol of histidine O, lyae was added and further incubated at 37° C. for 2 hours. 60% perchloric acid aqueous solution 0.05-
The reaction was stopped by adding 20% of the total amount of histamine produced, and the amount of histamine produced was measured.

Histamine was quantified using the 5hare method (J, Phar
+++.

exp, Ther, 127.182 (I959
)).

The results obtained are shown in Table 1.

Table 1 Test sample name I C5g (+mg/J
) Ethanol extract 2.8X10 (Production example 3
) α-Fluoromethyl 1.0X10 histidine 2) Ethanol extraction method for the sponge CChondropsis sp.
y) The hearts of guinea pigs were removed immediately after being bludgeoned to death using Langendorff's method (J).
, PharmacolMethods 2; 14
3 (I979)), 95%02+5%C
02 mixed gas is vented and kept at 37℃ using Taleb-Hensele3t liquid at a constant flow rate (6J/win)? I played vL. Perfusion pressure was measured with a pressure transducer. All test compounds were dissolved in 0.9% physiological saline and administered directly into the coronary artery through a rubber tube connected to the aortic cannula.
t), the data shown in Table 2 were obtained.

(Left below) Table 2 Test compound Dose (P9/heart ff1) Response rate (
%) Ethanol extract 10 40
(Production Example 3) 〃25 65 #100 100 Papaverine
33 100 Experimental Method Male Wistar runts weighing 230-330 g were anesthetized with a subcutaneous injection of urethane (I, 25 g/kg) and fixed in a dorsal position. An incision was made in the neck, a tube was inserted into the common carotid artery, and blood pressure was measured from this tube via a pressure transducer. The pulse wave also drives a heart rate monitor to measure heart rate.
Continuous recording was made on a polygraph at the same time as blood pressure.

The test substance was dissolved in dimethyl sulfoxide at a dose of 0.1@g/100 g body weight and administered into the femoral vein.

■ As shown in Table 3, the compound produced in Production Example 2 (
When Ia) was administered at 1,0,5,0 and 10 mg/kg, blood pressure decreased in a dose-dependent manner, but heart rate hardly changed.

This effect was seen 2 minutes after administration and lasted for more than 5 minutes.

(Margin below)

Claims (1)

[Claims] 1. General formula (I) ▲ Numerical formulas, chemical formulas, tables, etc. ▼ (I) (In the formula, n represents 10 or 11, and R^1 is a hydrogen atom, a methyl group, or an acetyl group. and R^2 represents a hydrogen atom or a methyl group). 2. General formula (I a) characterized by extracting a sponge (Chondropsis sp.) or its finely divided material with an organic solvent and then separating it to collect the necessary fraction (I a) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (I a) A method for producing a galactosphingolipid having the formula (n represents 10 or 11). 3. The production method according to claim 2, wherein the extraction solvent is acetone, methyl acetate, ethyl acetate, methanol, ethanol, propanol or isopropanol. 4. The production method according to claim 2, wherein the separation operation is chromatography.