JPS58106000A - New triterpene - Google Patents

Abstract

NEW MATERIAL:A compound of the formula (R1 is H, acyl; R2 is lower alkyl) or its lactone. USE:Since it has hypolipemic acivity, it is used as a medicine. PREPARATION:A plant, Antidesma pentarum, its whole parts, or its leaves, brances, barks and/or roots, are extracted with alcohol and a mixture thereof with water and the extract is subjected to additional solvent extraction and column chromatographic treatment to collect the triterpene of the formula. The purification by means of chromatography with Cephadex LH-20 is particularly preferred.

Description

[Detailed description of the invention] The present invention relates to a novel triterpene, more specifically.

Plants, Shimaya-q Hiha (Ant idesmap)
The present invention relates to a novel triterpene belonging to the veol family extracted and isolated from the genus Entandrum and its reader. These triterpenes have blood lipid-lowering effects,
Useful as medicines, the invention therefore also relates to pharmaceutical compositions containing said triterpenes.

The triterpene of the present invention is a compound represented by the formula: Of 2 R200CCIIB [wherein R1 means hydrogen or acyl, R2 means hydrogen or lower alkyl] or an acid compound of the formula [■] (R1 and It is a lactone in which both R2 are hydrogen).

In formula [■], the acyl group represented by Rt is acetyl, propionyl, butyryl, isobutyryl, valeryl,
Examples include alkanoyl h having 2 to 5 carbon atoms such as isovaleryl.

In addition, two lower argyl groups represented by R2 include carbon atoms of 1 to 6 such as methyl, ethyl, propyl, butyl, etc.
Examples include alkyl groups.

Representative examples of the triterpenes of the present invention include the following.

Compound-1: Lactone represented by formula [11].

Compound-2 A compound of the formula [■] in which R1 and R2 are both hydrogen.

Compound-3=A compound of formula [I] in which R1 is hydrogen and R2 is methyl.

Compound-42 Formula [I] where R1 is acetyl and R2 is methyl
compound.

Of these, Compound-1 and Compound-2 are triterpenes extracted and isolated from Shiyamahiha;
3 is a methylated derivative of compound-2, and compound-4 is an acetylated derivative of compound-3.

Thus, the triterpenes of the present invention include Shimayamahi...
It includes those extracted and isolated from these and their alkylated and/or acylated derivatives.

The triterpene of the present invention can be extracted from a Japanese cypress tree by solvent extraction of the entire plant, leaves, branches, bark and/or roots.

Extraction solvents include alcohols such as methanol, ethanol, 1J1-propanol, isopropanol, and n-butanol, and mixtures of water and alcohol.

The target triterpene can be isolated and purified by further treating the extract obtained with (H) by conventional methods such as solvent extraction and column chromatography. In particular, purification is preferably carried out by column chromatography using Sephadex I, I (-20).

The alkylation and/or acylation derivative of the obtained triterpene can be carried out according to a conventional method using a suitable alkylating agent or acylating agent under conditions that do not affect the skeletal structure of the triterpene.

The triterpene of the present invention has a blood lipid lowering effect.

The hypolipidemic effect of the compound can be demonstrated in the following manner.

(1) Blood is collected from the tail of a rat that has been given cholesterol feed to induce hypercholesterolemia, and the amount of serum cholesterol is measured. Then, the test drug is orally administered twice. Blood was collected again 5 hours after the second administration.
The amount of serum cholesterol is measured, and the difference (A) from the value before administration of the test drug is calculated. Pour distilled water instead of the test drug4
The serum cholesterol level of Group C (control group) was measured in the same manner, and the difference (B) before and after administration was calculated. Compare the difference between A and B (A-B) [7. Calculate the serum cholesterol inhibition rate c% according to the following formula.

-B Inhibition rate c%) - x 100 According to this method, the lipid-lowering effect of Compound-1 and Compound-2 was tested and the results are shown in Table 1. For comparison, Table 1 shows clofibrate, which is known as a drug for treating hypercholesterolemia.
The results of a similar test using

Table 1 (n=5) (2) 4-week-old Wistar (CWister) rats (
Male) contains 2% cholesterol, 1% bile acid, and 12% hydrogenated oil.
feed for 3 days. 1 day during this period
An aqueous suspension of the test drug is orally administered once. Then -
The animals were fasted overnight, and the next morning they were sacrificed, blood was drawn, and the liver was removed. Total cholesterol (TC) of the obtained serum samples, IIDL-
Cholesterol (HD■-.C) and triglyceride (TG) are measured with an autoanalyzer. In addition, liver was lyophilized and extracted with impropanonil, and total cholesterol (TC) and doliglyceride (TG'r
Measure.

Table 2 shows the results of testing Compound-1 according to this method. For comparison, Table 2 also shows the results of a similar test for drug-administered groups (control group, clofibrate-administered group, and normal rats without C-cholesterol loading).

As shown in Table 2, administration of Compound-1 at doses as low as 10/kq per day resulted in a decrease in serum total cholesterol and an increase in IIDI,-cholesterol.
c Index, (TC-11DL-C)/II
A significant improvement in DL・C] was observed. In addition, significant increases in cholesterol and glycerides in the liver were also significantly suppressed. Note that liver hypertrophy due to administration of Compound-1 was not observed.

(3) 5-week old Spragu
An aqueous suspension of the test drug was orally given once a day to male e-Dawley rats, and immediately after administration on the third day.
C.Fast. After 5 hours of stratification, serum total cholesterol (TC) was determined as in the previous Ht (2) test.
, tlDL, -near 1/sf tff full (Ilr)L
-C) Hepatic total cholesterol (TC) and triglycerides (TG') and liver total cholesterol (TC) and triglycerides (TG')
) to measure.

Table 3 shows the results of testing Compound-1 according to this method. For comparison, Table 3 also shows the results of a similar test for a control group not treated with drugs and a group treated with clofibrate Li.

As shown in Table 3, administration of Compound-1 at a daily dose of 80M97kq reduced serum total cholesterol and I
There was a trend towards an increase in IDL-cholesterol and therefore a clear improvement in the atherogenic index. In addition, for administration of compound-1! No liver Ω hypertrophy was observed.

The present invention also includes a lipid-lowering composition containing the triterpene as an active ingredient. Such pharmaceutical compositions include, for example:
It can be in the form of conventional pharmaceutical preparations for oral or parenteral administration, such as tablets, capsules, granules, fine granules, powders, syrups, herbal medicines, injections, etc., and its formulation involves the usual methods. Pharmaceutical excipients, diluents, etc. are used. Furthermore, the injection preparation may appropriately contain a usual pH adjusting agent, isotonizing agent, and the like.

The dose of the triterpene varies depending on the type of compound used, the age of the patient, the severity of the disease, etc., but usually
It is about 0 to 1000I1111/kq.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 10 kti of branches and leaves of Shiyamahiha are cut into strips and extracted twice with methanol 401. The extracts are combined and concentrated under reduced pressure to remove methanol.

Add 21 parts of water to the residue, and add the same amount of ethyl acetate. The mixture was thoroughly stirred, and after being allowed to cool overnight, the ethyl acetate layer was separated. This ethyl acetate extraction procedure is repeated. The separated ethyl acetate Jtu were combined and concentrated under reduced pressure to remove ethyl acetate. 110 g of oily residue are obtained.

Add this oil to 3 times the amount of silica gel (Kiese 1 manufactured by Merck & Co.)
ge1 6Q, 70-23o mesh, 330g). Add this to silica gel (Kieselgel)
6Q, 70-230 mesh) was loaded onto a 4kq column and subjected to column chromatography, and eluted with n-hexane-ethyl acetate C85:15) mixture 8j. The eluate is concentrated under reduced pressure to obtain 22.6 g of a fraction containing the Compound-1, which is a lactone represented by formula [■].

These two portions are mixed again with 67.8 g of 3 times the strength of silica gel. This was placed on a silica gel 900f column and subjected to column chromatography.
Elution is carried out with 2.211 l of a mixture of ethyl acetate (10:1) and then with 1.5 l of a mixture of n-hexane-ethyl acetate (5:1). The eluate from a mixture of n-hexane and ethyl acetate (5:1) was concentrated under reduced pressure to obtain 5.3 g of a fraction containing Compound-1. Both parts were dissolved in a small amount of methanol-chloroform (1:1) mixture, and Sephadex Ll-1-2
Chromatography was performed on a column of Qr (trade name: Pharmacia AI 3.5 ooy) and eluted with methanol.

The eluates from the 21st and 41st eluates to the 31st eluate were collected and concentrated under reduced pressure to obtain 256 ml of crude crystals of Compound-1. The crude crystals were recrystallized from acetone to form compound-187.
get.

Melting point = 186-191℃ Elemental analysis value: Calculated value as C80I-14602
(Wholesale: C,82,1'8 i H,10,
57 experimental value c%): C,'81.58;I(,10,
84 mass spectrum: I (R-MS calculated value = 438°3498 experimental value: 4
B8.8501 FD-MS (MI438 CM-002) 394 IRc■]cl! 8 Niji max=18IO11635,
1380,880crn-1 NMR (CDC18): δ = 0.78 f8
H.

Pm 5), 0.92 (311,s), Q, 97
(311, s).

1.0.8 (811,s), 1.48 (8
11,s), 1.68 (8H,s"+, 4
．． 88 (IH, dd, J = 7Hz.

6Hz), 4.58 (I II, dd,
J=2I(Z, lHz,)4.70(IH,d,
J=211z'+color reaction When heated after spraying 10'@L acid on a silica gel thin layer chromatography plate, a red color appears. Liberman-Purhiart (Li
(Ebermann-Burchard) Positive reaction Example 2 15 kq of branches and leaves of Shiyama Hiha were cut into thin pieces,
Extract twice with methanol 601. The extracts are combined and concentrated under reduced pressure to remove methanol.

Add 31 parts of water to the residue, and to this add the same amount of ethyl acetate. The mixture was thoroughly stirred and, after being allowed to stand overnight, the ethyl acetate layer was separated. This ethyl acetate extraction operation is repeated. The separated ethyl acetate/L layers are combined and concentrated under reduced pressure to remove ethyl acetate, yielding 140 g of an oily residue.

Add this oil to silica gel (Kiesel 1 Ge 16 manufactured by Merck & Co.)
0.70-230 mesh) is miscible with 4209. Cholera Silica Kel (KieSeIge160.70-230
The mixture was subjected to chromatography on a 6 kq column (mesh) and eluted with 12 parts of a mixture of n-hexane and ethyl acetate (85:15) and then with 10 parts of ethyl acetate. The ethyl acetate eluate was concentrated under reduced pressure to obtain 2.98f of the above compound-2, which is represented by the formula [E] where kl and 2 are both hydrogen.

This fraction was added to silica gel (Kieselgel16) again.
(0.230-400 mesh) mix with about 9 g. Add this to silica gel (Kieselgel 6Q, 230~
400 mesh) was placed on a 200 g column and subjected to chromatography, and n-hexane-7ton (10:
1) Mixed liquid 2.81. Then, n-hexane-acetone (
7:1) Elute with a mixture of 0.81. The eluate with n-hexane-acetone (7:1”l mixture) was concentrated under reduced pressure,
Fraction 710 "i" containing compound-2 is obtained.

Both obtained fractions were dissolved in methanol-chloroform (3:1) mixture 4-, and Hitachi Gel (Nippon Seimitsu 3019) 150
chromatography on a column of g and eluting with methanol. The eluate from the elution start point 1.21 to point 1.81 was collected and concentrated under reduced pressure to obtain a fraction containing compound-2, 380
get ~. This was dissolved in methanol 2#I/Sephadex L II-20 (trade name Pharmacia AB).
Chromatography on a 200 g column and elution with methanol. Elution start 640m/90m from eye
The eluate at 0m/eye was collected and concentrated under reduced pressure to obtain compound-2.
Obtain fraction 170.
Platten RP-18), methanol-
When developed with acetonitrile (5:1) mixture, R)0.
A spot of compound-2 is observed at the f:f position of 3].

B1) Both methanol-acetonitrile C5:
1) Dissolve the mixed solution in 1 kg and add C + 8-lb Sori Gel (Whatman C1lROC11RO, 1
3 to 24 μm), developed and eluted with a methanol-acetonitrile C5:1) mixture, collected the eluate from 25 (1+f! to 280-), and concentrated under reduced pressure. Crude crystal 1 of compound-2
14''+9 is obtained. The crude crystals are recrystallized from methanol to obtain white plate-like compound -273.

Melting, 1: 298-300°C (decomposition) Elemental analysis value: Cs 0) 14 a Calculated value as O8 c%): C, 78,94i [■ + 10.53 experimental value c%): C, 76, 27iH , 11,02 mass spectrum: El-M5 456 IRccl! 4 Niji max=8630.1680.16
85.880m-1 CDC18+DMSO, l to NMRC: ]'I
: δ = 0.72 (811,s), 0.76
(311,sPm 'r, 0.92 (311,S), 1.00
(311, s).

1.14 ('8L1..s), 1.64 (
811,s), 3.32 (IH,m), 4.5
2 (llL dd, J =211z.

11(z'r, 4.65 (III, d,
J = l fiz '+ Color reaction: BCG positive Example 3 Compound-250■ obtained in Example 2 was dissolved in methanol, an ether solution of diazomethane was added in excess,
Leave at room temperature for 1 hour. After the reaction is complete, excess diazomethane is treated with acetic acid and evaporated under reduced pressure. The obtained product was recrystallized from methanol to obtain a compound of the formula r'■, where hydrogen is hydrogen and R2 is methyl (above compound-3) 45. Get IIK/. Yield 87.4%.

Melting point: 199-202°C Elemental analysis value: Calculated value (%) as C81H4805:
679.14; I-1,10,64 experimental value (%):
C,79,71il(,11,24 Mass spectrum: +11-M5(M)470 IR: ν =8625.1718.16C
C14max 35+880c7n' NMR (CDC1s): δ = 0.65
(8H.

Pm 5), 0.78 (all, s), 0.
94 r3I(,s).

1.02 (311,s), 1.24 (81
1,s), 1.66 (311,s)+3.
64 (3)1. s), 4.06 (l II
, m, W = 8Hz), 4.56
(IH, dd.

1/2 J Engineering 2IIZ, 1111), 4.68 1
111+d+J"211z) Example 4 +J+il 3211g of the compound obtained in Example 3 was dissolved in 5ml of pyridine, 5ml of acetic anhydride was added and left at room temperature for 16 hours. The product was reconstituted from methanol. Crystallize to obtain a compound of the formula [■] where R1 is acetyl and R2 is methyl (
The above compound-4) 17■ is obtained.

Yield 800%.

Melting, 6: l7q~】78℃ Elemental analysis value: Calculated value as C88H5006):C
,79,14; IL 10.64 experimental value excellent): C,
79,71, II, 11°24 mass spectrum: El-M≦CM”+512 IRcc14 Niji max=1735,1720,171
0.1635,1240.880m-1HMR(cDc
/a): δ = 0.66 (311°Pm 5), 0°78 (811,s), 0.98
(3H, s).

1.04 (all, s), 1.14 (3)
1. s),, 1.68 r3H,s), 2.
08 (8I(,s), 8.65 (3H,s),
4.56 (III, dd, J = 21
1z, IIIz), 4.69 (il
l, d, J=211z'+, 5.3
Qrl) (, m, W = 311z) 1/2 Patent applicant Fujisawa Pharmaceutical Co., Ltd. Agent Masukenshi Aoyama 葆? 712 people

Claims (1)

[Scope of Claims] [In the formula, R1 means hydrogen or acyl, and R2 means hydrogen or lower alkyl] or a lactone thereof. (2) The compound of item (1) above which is a lactone. (3) The compound of the above item (1), which is represented by the formula (I) in which Rs and 2 are both hydrogen. (4) The compound of the above item (1), which is represented by the formula [I] in which R1 is hydrogen and R2 is methyl. ). (5) A compound of the above (1), which is represented by the formula r11 in which R1 is acetyl and R2 is methyl. (6) Formula: C112 , [wherein R1 is hydrogen or acyl and R2 is hydrogen] or lower alkyl] or a lactone thereof as an active ingredient.