JPH04288095A - Complemental activity-inhibiting agent - Google Patents

Abstract

PURPOSE:To provide a complemental activity-inhibiting agent containing a specific beta-boswellic acid derivative as an active ingredient, having a good complemental activity-inhibiting activity and effective for treating autoimmune diseases such as systemic erythematoides and articular rheumatism. CONSTITUTION:The petroleum ether extract of a mastic is applied to a silica gel column gel chromatography and subsequently eluted with a hexane/acetone solvent. The fifth fraction is further eluted and purified with a negative phase gel using a methanol/water solvent to produce an approximately 1:1 mixture of alpha- and beta-boswellic acid acetates. The mixture is recrystallized from a hexane/ acetone mixture solvent and further subjected to a high performance liquid chromatography to separate the alpha-boswellic acid acetate from the beta-boswellic acid acetate. The beta-boswellic acid acetate is hydrolyzed to provide the objective compound (salt) of the formula (R1, R2 are H, alpha-hydroxyl group, or R1 and R2 are O together; R3 is H, acetyl).

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention provides a complement activity inhibitor containing a beta-boserinic acid derivative such as beta-boserinic acid, 11-keto-beta-boserinic acid, or 11-alpha-hydroxy-beta-boserinic acid as an active ingredient. It is related to.

0002

[Prior Art] Complement is a group of about 20 proteins that have important functions in the immune system of the body in the removal of exogenous foreign substances, and they play their roles in a delicate balance. When activated, these components become chemical mediators, which are said to cause various physiological effects. It is known that if this balance is disturbed, various abnormal pathological conditions, such as autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, occur.

[0003] In addition to systemic disorders caused by foreign blood transfusions and inflammation caused by Streptococcus pneumoniae, there is a recent theory that psoriasis is also associated with complement abnormalities.

[0004]Currently, there are no drugs known that are sufficiently effective against the above-mentioned diseases, and there are no drugs available for the treatment of diseases caused by excessive activation of these complements. Anti-complement activity inhibitors may be useful.

[0005] In recent years, such activity has been found in some polysaccharides that are components of herbal medicines, but polysaccharides are generally mixtures, making it difficult to identify and develop them as pharmaceuticals.

[0006]

[Problem to be solved by the invention] Wagner of West Germany discovered strong anti-complementary activity in a mixture of alpha and beta bosueric acid, which is a component of frankincense, a herbal medicine that has been used as a fragrance and anti-inflammatory analgesic since ancient times [H ．． Wagner, P.
lanta Medica, 55, 235 (1989
)], but it was extremely difficult to separate this mixture, and it was unclear which substance had the true activity. Therefore, it was thought that by solving this problem, it would be possible to develop anti-inflammatory agents for new autoimmune diseases and the like using a mechanism that suppresses abnormal complement activation.

[0007] As a result of extensive research to solve the above problems, the present inventors succeeded in separating alpha and beta bosueric acids, and found that only beta bosueric acid has activity, and that sodium salts etc. It has been found that the same activity is also observed in salts of and boserinic acid derivatives such as 11-keto-beta-boserinic acid and 11-alpha-hydroxy-beta-boserinic acid, and the present invention has been completed.

That is, the present invention provides the following formula I (wherein,
R1 and R2 are hydrogen atoms, α-hydroxyl groups, or R
1 and R2 together represent an oxygen atom, and R3 represents a hydrogen atom or an acetyl group. ) or a pharmacologically acceptable salt thereof as an active ingredient.

Hereinafter, the compound represented by formula I will be referred to as a compound of formula.

Among the compounds of the formula, the compound in which R1 and R2 are hydrogen atoms and R3 is an acetyl group is beta-boserinic acid, and the present inventors separated it from alpha-boserinic acid and discovered its anti-boserinic acid for the first time. Complement activity has been confirmed, and compounds with other formulas are known substances described in the literature, but anti-complement activity has not been clarified, and this is the first clarification by the present inventors. be.

A compound of the formula can be obtained, for example, as follows.

[0012] That is, frankincense [Boswellia]
Carteri, B. bhawdajiana,B
．． resin exuded from the trunk of B. neglecta] or Indian frankincense [B. serrata resin] selected from petroleum ether, hydrocarbons such as pentane, hexane, and cyclohexane, lower alcohols such as methanol, ethanol, and propanol, and organic solvents such as acetone, methyl ethyl ketone, methylene chloride, chloroform, and ethyl acetate. Extract by heating to a temperature below the boiling point of the solvent used, or by using a mixed solvent with a higher temperature than 0°C.
The extract is obtained by ultrasonic extraction at room temperature.

[0013] The solvent is distilled off from this extract, and the extract is subjected to column chromatography or high performance liquid chromatography once or several times, and the liquid is separated to obtain fractions. At this time, hydrocarbons such as petroleum ether, pentane, hexane, cyclohexane, etc. may be used alone as an elution solvent, or acetone,
A mixture of solvents such as ethyl acetate, chloroform, methylene chloride, tetrahydrofuran, and alcohols can be used.

The fraction thus obtained is further subjected to recrystallization or high performance liquid chromatography using a reverse phase gel such as ODS-silica gel or porous polymer gel as an adsorbent, and is then subjected to methanol, tetrahydrofuran, acetonitrile, The compound of the formula is obtained by fractionating the eluted fraction with an aqueous solvent and recrystallizing it.

[0015] If the fraction obtained as described above contains a fraction having an ester bond such as acetic acid, the compound of the formula can also be obtained by carrying out commonly used alkaline hydrolysis. .

As the solvent for recrystallization, water, lower alcohols, acetone, ethyl acetate, tetrahydrofuran, petroleum ether, pentane, hexane, cyclohexane, methylene chloride, chloroform, etc. alone or a mixed solvent of more than one may be used.

Next, a specific example of the production of the compound of the formula will be shown.

Specific Example 1 Frankincense Petroleum Ether Extract 117
g was subjected to 1Kg silica gel column chromatography, eluted with a hexane/acetone solvent, and the fifth fraction was further chromatographed on an ODS-based reverse phase gel (Fuji-G
el Q-3) with a methanol/water solvent, an approximately 1:1 mixture of alpha and beta bosueric acid acetates was obtained. This was crystallized from a hexane/acetone mixed solvent to obtain 873 mg of a colorless crystalline substance. This mixture was applied to a preparative HPLC column [D-ODS-10 (YMC, solvent system; THF:C
H3CN:H2O=3:12:1, flow rate: 3ml/mi
n)]. By distilling off the solvent of the fraction eluting at Rt of about 115 minutes and the fraction eluting at around 125 minutes, 220 mg of alphaboserynic acid acetate having the following physicochemical properties and formula I in which R1 and R2 are hydrogen were obtained. 280 mg of beta-boroseric acid acetate in which the atom R3 is an acetyl group was obtained.

Alphaboserynic acid acetate (recrystallized from methanol, colorless needle crystals) Melting point: 218-219°C [α]D +66.1° (c=0.3, CHCl3)
Mass spectrum EI-MS m/z: 498 [M]+, 218, 20
3 Infrared absorption spectrum IRmax cm-1:
2972, 2944, 2860, 1744, 1730,
1705,1242 Proton nuclear magnetic resonance spectrum (δ ppm in
CDCl3): 0.84, 0.87, 0.87, 0
．． 90, 1.01, 1.19, 1.12 (each
3H, s), 2.09 (3H, s), 5.20 (1H,
t, J=3.4Hz), 5.30(1H, t, J=2.
5)

Beta-boseric acid acetate (recrystallized from methanol, colorless needle crystals) Melting point: 227-228°C [α]D=+62.3° (c=0.3, CHCl3) Mass spectrum EI-MS m/ z:498[M]+,218,20
3 Infrared absorption spectrum: IRmax cm-1
:2976, 2920, 2860, 1746, 1730
, 1702, 1240 Proton nuclear magnetic resonance absorption spectrum (δ ppm
in CDCl3): 0.80 (3H, d, J = 6.3Hz), 0.81 (3
H, s), 0.91 (3H, s), 0.93 (3H, d
, J=6.1Hz), 1.05, 1.12, 1.25(
each 3H, s), 2.10 (3H, s), 5.
15 (1H, t, J = 3.5Hz), 5.31 (1H,
t, J=2.5Hz)

Specific Example 2 Beta-boseric acid acetate 400 obtained in Specific Example 1
mg was dissolved in 25 ml of 5% potassium hydroxide/methanol and refluxed on a water bath for 8 hours, then 25 ml of 2N hydrochloric acid was added to the reaction solution, and the mixture was extracted three times with ether. The ether layers were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off. The residue was subjected to preparative HPLC (D-ODS-10
column, THF-CH3CN-H2O=3:12:1)
By recrystallizing from hexane-acetone, 288 mg of beta-boserinic acid having the following physical and chemical properties and in which R1, R2 and R3 in formula I are hydrogen atoms was obtained as a white crystalline substance. .

Melting point: 236-238°C [α]D=+100.7° (c=0.3, CHCl3)
Mass spectrum EI-MS m/z: 456 [M+], 441, 23
8,218,203 Infrared absorption spectrum: IRmax cm-1:
3440,2944,1696 Proton nuclear magnetic resonance spectrum (δ ppm in
CDCl3): 0.79 (3H, d, J = 5.9H
z), 0.81, 0.90, (each 3H, s)
,0.92(3H,d,J=6.1Hz),1.04,
1.09, 1.35 (each 3H, s), 4.0
8 (1H, t, J = 2.7Hz), 5.14 (1H, t
, J=3.6Hz)

Specific Example 3 15 mg of beta-boserinic acid obtained in Specific Example 2 was taken and 0
．． The mixture was dissolved in 3 ml of 05M sodium hydroxide/methanol by shaking well, concentrated under reduced pressure to the extent that it did not dry up, and then ether was added to obtain sodium beta-boroserinate in the form of a white powder having the following physical and chemical properties.

Melting point: >290°C [α]D=+51.4°
(c=0.2, MeOH) Infrared absorption spectrum:
IRmax cm-1: 3432, 1634, 155
2,1348,880

Specific Example 4 100 g of Indian frankincense was ground, extracted twice with 2 liters of petroleum ether at room temperature, and further extracted twice with 2 liters of chloroform, each of which was concentrated under reduced pressure to remove the solvent, and 24.0 g of each was extracted.
and 27.5 g of extract were obtained. Chloroform extract 1
0 g was subjected to 300 g silica gel column chromatography, sequentially mixed with hexane and 2 to 30% acetone, and finally eluted with acetone alone and fractionated into 13 fractions. Of these, 1.89 g of the 6th and 7th fractions combined was subjected to column chromatography on 57 g of silica gel and eluted with hexane/ethyl acetate = 4:1. This fifth fraction was further divided into hexane/acetone 5:
Purification was repeated by silica gel column chromatography using a mixed solvent of 1 to obtain a substance that absorbs ultraviolet light, which was recrystallized from hexane/acetone and has the physical and chemical properties shown below, and R1 and R2 in formula I are the same. Thus, 23.1 mg of colorless prismatic crystals of 11-keto-betabobosueric acid-3-O-acetate in which oxygen atom and R3 are acetyl groups were obtained.

Melting point: 270-276°C [α]D=+77.5° (c=0.44, CHCl3)
Mass spectrum EI-MS m/z: 512 (M+), 273 (base peak), 2
32 High resolution EI-MS m/z: Calculated value 512.3501 C32H48O5 actual value 512.3506 Ultraviolet absorption spectrum UVmax nm (ε):
252 (11200) infrared absorption spectrum: IR
max cm-1: 3300-2500, 1730,
1695,1650,1615,1250 Proton nuclear magnetic resonance spectrum (δ ppm in
CDCl3): 0.80 (3H, d, J=6.2H
z), 0.83 (3H, s), 0.95 (3H, br.
s), 1.15, 1.20, 1.24, 1.35 (each
3H, s), 2.10 (3H, s), 2.41 (1H
, s), 5.31 (1H, br.s), 5.56 (1H
,s)

Specific example 5 11-keto-beta bosueric acid-3 obtained in specific example 4
500 mg of -O-acetate was dissolved in 25 ml of 5% potassium hydroxide/methanol solution, heated under reflux on a water bath for 5.5 hours, allowed to cool, then the reaction solution was diluted with 100 ml of water and acidified with hydrochloric acid to remove the precipitated white precipitate. Extraction was performed three times with 100 ml portions of ether. The extracts were combined, washed twice with saturated saline, dried over anhydrous magnesium sulfate, and the solvent was distilled off. 511 mg of colorless acicular crystals of 11-keto-betaboboserinic acid in which R3 is a hydrogen atom were obtained.

Melting point: 192-193°C [α]D= +131° (c=1.0, CHCl3)
Mass spectrum EI-MS m/z: 470 (M+), 455, 27
3,232 High resolution EI-MS m/z: Calculated value 470.3391 C30H46O4 actual value 470.3400 Ultraviolet absorption spectrum UVmax nm (ε):
250 (11400) infrared absorption spectrum: IR
max cm-1: 3500-2400, 1695,
1650,1615 proton nuclear magnetic resonance spectrum (δ
ppm in CDCl3): 0.79 (3H
, d, J=6.2Hz), 0.82(3H, d, J=5
．． 6), 0.95, 1.13, 1.19, 1.31, 1.35 (each 3H, s), 2.4
3 (1H, s), 4.08 (1H, br.s,), 5.
55 (1H, s)

Specific Example 6 The eighth fraction following the fraction from which the compound obtained in Specific Example 4 was obtained contained almost a single compound and was recrystallized from hexane/acetone to obtain 176.4 mg of colorless needle crystals. In addition, 349 mg of the mother liquor was subjected to re-column chromatography using 10.5 g of silica gel, eluted with hexane/ethyl acetate 4:1 to 2:1, and fractionated into 6 fractions.
The second and third fractions were recrystallized from hexane/acetone to obtain 69 mg of the same substance as colorless needles. These have the physical and chemical properties shown below, and in formula I, R1 is a hydrogen atom, R2 is an α-hydroxyl group, and R3 is an acetyl group. Ta.

Melting point: 160-166°C [α]D=+11.3° (c=0.21, CHCl3)
Mass spectrum EI-MS m/z: 514 (M+), 496, 23
4 (base peak) high resolution EI-MS m
/z: Calculated value 514.3658 C32H50O5 actual value 5
14.3680 Infrared absorption spectrum: IRmax cm-1:
3640, 3300-2500, 1750, 1730,
1280 proton nuclear magnetic resonance spectrum (δ ppm
in CDCl3): 0.86 (3H, d, J=
6Hz), 0.88, 0.93, 1.07, 1.11, 1.22,
1.25 (each 3H, s), 1.63 (1H,
d, J = 8.8Hz), 2.10 (3H, s), 4.27 (1H, dd, J = 9.2, 3.3Hz), 5
．． 18 (1H, d, J = 2.9Hz), 5.30 (1H
,br. s)

Specific Example 7 Take 50.3 mg of 11-alphahydroxy-betabobosueric acid-3-O-acetate obtained in Specific Example 6, dissolve it in 6 ml of 5% potassium hydroxide/methanol, and heat under reflux on a water bath for 6 hours. After cooling, methanol was distilled off under reduced pressure. The residue was dissolved in 20 ml of ether, washed with 15 ml each of 2N hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine, and then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the product was recrystallized from hexane/ethanol to obtain a 11-alphahydroxy-beta bolus having the physicochemical properties shown below, in which R1 and R3 in formula I are hydrogen atoms and R2 is an α-hydroxyl group. 26.3 mg of colorless crystals of eric acid were obtained.

Melting point: 171-178°C [α]D=+63.9° (c=0.33, CHCl3)
Mass spectrum EI-MS m/z: 472 (M+), 454 (M+
-H2O), 234 (base peak) High resolution EI-MSm/z: Calculated value 472.3552 C30H48O4 Actual value 472.3556 Infrared absorption spectrum: IRmax cm-1:
3690,3000-2500,1720 proton nuclear magnetic resonance spectrum (δ ppm in CDCl
3): 0.81 (3H, s), 0.85 (3H, d, J
=5.5Hz), 0.93 (3H, br.s), 1.07, 1.10, 1.18, 1.37 (each
3H, s), 1.61 (1H, d, J=8.8Hz)
, 4.08 (1H, br.s), 4.26 (1H, dd, J=9.2, 3.3Hz), 5
．． 18 (1H, d, J=2.9Hz)

Reference Example Alphaboserynic acid acetate 42 obtained in Specific Example 1
25 ml of 5% potassium hydroxide/methanol was added to 0 mg, and after refluxing on a water bath for 8 hours, 25 ml of 2N hydrochloric acid was added to the reaction solution, and the precipitated white precipitate was extracted with ether. The ether layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain a white powder. By recrystallizing this from methanol, 312 mg of alphaboserinic acid having the following physicochemical properties was obtained as colorless scaly crystals.

Melting point: 140/190-230°C (double melting point) [α]D=+108.7° (c=0.3, CHCl
3) EI-MS m/z: 456 (M+), 441, 259, 238, 218, 2
03 Infrared absorption spectrum: IRmax cm-
1:3400,2944,2856,1720 proton nuclear magnetic resonance spectrum (δ ppm in CD
Cl3): 0.84, 0.87, 0.87, 0.89,
1.00, 1.15, 1.35, (each 3H,
s), 4.08 (1H, t, J=2.6Hz), 5.2
0 (1H, t, J=3.4Hz)

Next, the fact that the compound of the formula has excellent anti-complement activity and is useful as a complement activity inhibitor will be explained with reference to experimental examples.

Experimental Example 1 Anti-complement activity was measured using Mayer's method on a scale of 1/5 (Mayer, MM Experiment
ntal Immunochemistry, 2nd
．． P. 133). That is, rabbit anti-sheep red blood cells were used as the antibody, sheep red blood cells were used as the antigen, and human CPD-preserved plasma was used as the complement source. Since the compound of the formula is poorly soluble in water, it is dimethyl sulfoxide (DMSO).
It was dissolved to a concentration of 0 mM and added to gelatin-Bernard buffer to a final concentration of 50 μM. Diluted plasma at 0.25, 0.3, 0.35, 0.4, 0.5, 0
．． Change the volume to 7 ml, mix with sensitized sheep red blood cells and distilled water, incubate at 37°C for 2 hours, cool on ice to stop the reaction, 2.
After centrifugation at 500 rpm for 5 minutes, the absorbance of the supernatant at 414 nm was measured at each diluted plasma concentration. Also, D
1 μl of MSO was used as a control. The results are shown below. In the table, CB indicates mechanical hemolysis.

Table 1 (Anti-complement activity of compounds obtained in specific examples)

Table 2 (Concentration dependence of the compound obtained in Example 2)

Table 3 (Concentration dependence of the compound obtained in Example 3)

Table 4 (Concentration dependence of the compound obtained in Example 5)

As is clear from these results, it was demonstrated that the compound of the formula has anti-complement activity.

Next, the dosage and formulation of the compound of the formula will be explained.

The compounds of the formula can be administered to animals and humans either neat or with conventional pharmaceutical carriers. The dosage form is not particularly limited and may be selected and used as necessary, and may include oral dosage forms such as tablets, capsules, granules, fine granules, and powders, and parenteral dosage forms such as injections and suppositories. It will be done.

[0044] In order to exert the desired effect as an oral agent, although it varies depending on the age, weight, and severity of the disease of the patient, it is usual for adults to take 10 to 3 g of the compound of the formula in divided doses several times a day. It seems appropriate to take .

[0045] Oral preparations include, for example, starch, lactose, sucrose,
It is manufactured using conventional methods using mannitol, carboxymethyl cellulose, cornstarch, inorganic salts, etc.

[0046] In addition to the excipients mentioned above, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, fragrances, etc. may be used in this type of preparation as appropriate. Can be done. Specific examples of each are shown below.

[Binder] Starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch,
Methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, macrogol.

[Disintegrant] Starch, hydroxypropyl starch, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, carboxymethyl cellulose, low substituted hydroxypropyl cellulose.

[Surfactant] Sodium lauryl sulfate,
Soy lecithin, sucrose fatty acid ester, polysorbate 80.

[Lubricant] Talc, waxes, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol.

[Fluidity promoter] Light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate.

The compounds of the formula can also be administered as suspensions, emulsions, syrups, and elixirs, and these various dosage forms may contain flavorings and coloring agents. .

[0053] In order to exert the desired effect as a parenteral agent, it depends on the age, weight, and severity of the disease of the patient;
Normally for an adult, 5 to 500 mg per day by weight of the compound of formula
Intravenous injection, intravenous drip, subcutaneous injection, and intramuscular injection are considered appropriate.

This parenteral preparation is manufactured according to a conventional method, and diluents generally include distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, and polyethylene glycol. etc. can be used. Furthermore, a bactericide, a preservative, and a stabilizer may be added as necessary. In addition, from the viewpoint of stability, this parenteral preparation may be filled into a vial or the like, then frozen, water removed by ordinary freeze-drying techniques, and a liquid preparation prepared from the freeze-dried product immediately before use. Furthermore, isotonizing agents, stabilizers, preservatives, soothing agents, etc. may be added as appropriate.

Other parenteral preparations include liquid preparations for external use, liniments such as ointments, suppositories for intrarectal administration, etc.
Manufactured according to conventional methods.

Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto in any way.

Example 1 According to the above recipe, ① to ② were uniformly mixed and compressed using a tablet machine to obtain tablets each weighing 200 mg. One tablet contains 20 mg of the compound obtained in Example 2, and adults should take 5 to 15 tablets a day in several doses.

Example 2 According to the above recipe, ■, ■, and part of ■ were uniformly mixed, compressed and molded, and then pulverized, the remaining amounts of ■ and ■ were added, mixed, and compressed using a tablet machine. Molded tablet 200mg
tablets were obtained. One tablet contains 20 mg of the compound obtained in Example 3, and adults should take 5 to 15 tablets a day in several doses.

Example 3 ■, ■, and ■ were uniformly mixed according to the above recipe, and the mixture was homogenized by a conventional method, granulated using an extrusion granulator, dried and crushed, and then ■ and ■ were mixed. Then, compression molding was performed using a tablet machine to obtain tablets each weighing 200 mg. One tablet contains 20 mg of the compound obtained in Example 5, and adults should take 5 to 15 tablets a day in several doses.

Example 4 According to the above recipe, ① to ② were uniformly mixed, compression molded using a compression molding machine, crushed using a crusher, and sieved to obtain granules. 1 g of this granule contains 100 mg of the compound obtained in Example 7, and adults should take 1 to 3 g in several doses per day.

Example 5 According to the above recipe, ① to ② were uniformly mixed and made into a paste. After granulation using an extrusion granulator, the mixture was dried and sieved to obtain granules. 1 g of this granule contains 100 mg of the compound obtained in Example 2, and adults should take 1 to 3 g in several doses per day.

Example 6 ■~■ were mixed uniformly according to the above recipe, and 200
mg was filled into No. 2 capsules. One capsule of this preparation contains 20 mg of the compound obtained in Example 3, and adults should take 5 to 15 capsules a day in several doses.

Example 7 According to the above recipe, ■ was dissolved in ■ and ■, and the solutions of ■ and ■ were added to emulsify to obtain an injection.

Claims (1)

[Claims] Claim 1: The following formula I (wherein R1 and R2 are a hydrogen atom or an α-hydroxyl group, or R1 and R2 together represent an oxygen atom, and R3 represents a hydrogen atom or an acetyl group). A complement activity inhibitor containing the represented compound or a pharmacologically acceptable salt thereof as an active ingredient.