JPH03184993A - Bile acid derivative, production thereof and diagnosticum for enterobacteria - Google Patents

Abstract

NEW MATERIAL:A compound shown by formula I (R<1> and R<2> are H, OH or sulfonyloxy). EXAMPLE:Ursodeoxycholyl-p-aminobenzoic acid-3alpha-monosulfate. USE:A diagnosticum for enterobacteria. PREPARATION:A compound shown by formula II (R<1>' and R<2>' are H or OH) is reacted with an acylating agent to give a compound shown by formula III (R<1a> and R<2a> are H or acyloxy; A is acyl), which is hydrolyzed under a weakly alkaline condition to give a compound shown by formula IV. This compound is reacted with a sulfonating agent to give a compound shown by formula V, which is finally hydrolyzed with an alkali to give a compound shown by formula I wherein group R<1> is R<1>' and group R<2> is R<2>'.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] This invention relates to a novel bile acid derivative, a method for producing the same, and a diagnostic agent for intestinal bacteria containing the bile acid derivative as an active ingredient.

[Prior art and problems to be solved by the invention] Conventionally, methods for measuring the activity of intestinal bacteria include administration of 1C-labeled glycocholic acid, or 14C-labeled glycocholic acid.
Breath test by administration of Kinroll labeled with (“CO
t breath test) was conducted. However, this method uses the radioactive isotope 14c as a tracer and requires special and expensive equipment for the measurement.
2. Requires special equipment to adsorb t and a scintillation counter to count I4C.
It has serious drawbacks in this respect. For this reason, this breath test is generally difficult to accept as a routine clinical test. On the other hand, Wolgemuth
) et al. have developed a conjugate (PABA-C) of cholic acid (hereinafter abbreviated as CA) and p-aminobenzoic acid (hereinafter abbreviated as PABA).
A) and discloses that the PABA-CA conjugate is an effective substance for measuring the activity of intestinal bacteria (US Pat. No. 4,171,352). The structural formula of the PABACA conjugate is:

It is expressed as The principle of Woorghemus et al.'s method is as follows. That is, orally administered PABA-CA
In the intestinal tract, cholic acid (CA) is produced by the action of intestinal bacteria.
and p-aminobenzoic acid (PABA), and all released PABA is absorbed from the intestinal tract and excreted in the urine. Therefore, by measuring the amount of P and 'lBA excreted in urine within a certain period of time after administering PABA-CA, it is possible to know the activity level of bacteria in the field, and this substance uses radioactive isotopes. It is considered to be an excellent diagnostic agent that uses PABA as a tracer.

The present inventors also discovered that urtdesoxycholic acid (UD
We synthesized a conjugate of CA) and PABA and reported that the conjugate was effective for measuring the activity of bacteria in the field (
History of Medicine, Vol. 13g (3), p. 211, 1986). However, the above PABA-CA conjugate and PA
BA-UDCA conjugates have the property that a considerable amount of them are actively absorbed from the intestinal tract before being hydrolyzed by local bacteria, and therefore the amount of urinary PABA excreted from these conjugates is entirely absorbed from the intestine. This is not due to the action of local bacteria, and does not completely indicate the activity level of local bacteria.

[Means for Solving the Problems] Therefore, as a result of intensive research into a type of diagnostic agent that passes through the intestinal tract without being actively absorbed from the intestinal tract, - General formula (I): (wherein, Rl, R2 are hydrogen atoms, hydroxyl groups, or sulfonyloquino groups) The new compound has the property that only PABA released through intestinal bacteria is absorbed from the intestinal tract without being actively absorbed from the intestinal tract. However, it was found to be significantly superior in diagnosing intestinal bacterial activity.

That is, according to the present invention, a novel bile acid derivative (I
) and its salts are provided.

Bile acid molecules forming the core of compound (I) of this invention include ursodeoxycholic acid (3α, 7β-dihydroxycholanic acid) and cholic acid (3α, 7α).

12α-trihydroxy-cholanic acid), chenodeoxycholic acid (3α, 7α-dihydroxy-cholanic acid), deoxycholic acid (3α, 12α-dihydroxycholanic acid), lithocholic acid (3α-hydroxycholanic acid), etc. Among these, ursodeoxycholic acid, which is frequently used as a gallstone dissolving agent in daily clinical practice, is particularly preferred.

Formula (I) does not show a steric structure, but the formula (I) of this invention
The compounds include stereoisomeric compounds based on the bile acids mentioned above. For example, the three-dimensional structure of ursodeoxycholic acid is expressed as follows.

Further, according to the present invention, a novel diagnostic agent for intestinal bacteria is provided.

The compound of this invention can be produced by various methods, but typical orientations are as follows.

l) Reacting an acylating agent with a compound represented by the general formula (If) (wherein R1' and R!' are hydrogen atoms or hydroxyl groups), which is a condensation product of bile acid and p-aminobenzoic acid. , the general formula (I) (wherein R", R" are hydrogen atoms or acyloxy groups,
A is a compound represented by an acyl group), and this is hydrolyzed under weak alkaline conditions to produce a compound represented by the general formula (IV) -to= (wherein R''' and R2a have the same meanings as above) was obtained, and then reacted with a sulfonating agent to obtain the general formula (
■): (wherein R'' and R'' have the same meanings as above), and finally subjected to alkaline hydrolysis to give the general formula (Ia): (wherein R1' and R9' have the same meanings as above) (same meaning) is produced.

Further, a compound represented by the formula (Ib): (wherein RIb, R'' is a hydrogen atom or a sulfonyloxy group) is produced by reacting the above compound (n) with a sulfonating agent.

The acylating agent is preferably one that protects the hydroxyl group during sulfonation and is easily eliminated by alkaline hydrolysis. Examples include acetyl chloride, benzoyl chloride, acetic anhydride, and the like.

Examples of the sulfonating agent include chlorosulfonic acid, sulfuric acid, sulfuryl chloride, and the like, with chlorosulfonic acid being preferred. The reaction is carried out in pyridine solvent at room temperature.

Hydrolysis of compound (I[I) is carried out under weak alkaline conditions, such as an alcoholic solution of alkali carbonate (eg, sodium carbonate, potassium carbonate).

Next, the hydrolysis of compound (V) is carried out under stronger alkalinity, using, for example, a highly concentrated aqueous solution of alkali hydroxide (sodium hydroxide, potassium hydroxide).

The hydrolysis reaction is usually carried out at room temperature, and the reaction time is from several hours to several tens of hours.

The carboxyl group of compound (+) may form a salt, and examples thereof include pharmaceutically acceptable salts, such as alkali metal (sodium, potassium) salts, alkaline earth metal (calcium) salts, and ammonium salts.

3- According to one aspect of the present invention, there is provided an enterobacteriaceal diagnostic agent comprising a bile acid derivative of general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

When the "diagnostic agent for intestinal bacteria" of this invention is orally administered to mammals including humans, the bile acid derivative (I) is converted into bile acid sulfate and p-aminobenzoic acid by intestinal bacteria in the intestine. (PABA), and free PABA is substantially absorbed from the intestinal tract and excreted in the urine. By quantifying the amount of PABA in urine, the activity of intestinal bacteria can be evaluated. PABA in urine can be easily quantified by conventional methods.

Moreover, the compound (I) of this invention is a compound with extremely low toxicity, and is extremely safe even when administered to humans several times.

The dosage of the compound (I) of this invention varies depending on body weight and symptoms, but is usually about 100 to 500 m
g, preferably about 200 to 300 a+g.

The diagnostic agent of this invention is usually formulated and used.+4- It will be done.

To formulate the compound of the second invention, it is prepared into a dosage form such as a tablet, granule, powder, capsule, or liquid by a conventional method in the technical field of pharmaceutical preparation. That is, as a method for preparing a solid preparation for oral use, after adding excipients of herbal medicine and, if necessary, a binder, a disintegrant, a lubricant, a coloring agent, a flavoring agent, etc., tablets are prepared by a conventional method. , sugar-coated tablets, granules, powders, capsules, etc.

Examples of excipients include lactose, cornstarch, white sugar,
Examples of binders include glucose, sorbitol, crystalline cellulose, etc., and disintegrants include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylcellulose, hydroquinpropyl starch, polyvinylpyrrolidone, etc. Examples of lubricants include starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin, etc. Examples of lubricants include magnesium stearate, talc, polyethylene glycol, glue, and hydrogenated vegetable oil. Coloring agents that are permitted to be added to medicines include cocoa powder, peppermint, aromatic acid, peppermint oil, and flavoring agents.
Examples include cybernetic powder, cinnamon powder, etc. These tablets and granules may be coated with sugar coating, gelatin coating, or other coatings as appropriate.

Liquid formulations may also be in solution, syrup, topical form, or provided as a dry product that can be reconstituted with either water or a suitable vehicle before use. You can.

Such liquid preparations are prepared by adding pharmaceutically acceptable additives such as buffers, suspending agents, emulsifiers, stabilizers, preservatives, etc. in a conventional manner. Buffers include sodium citrate, sodium acetate, sodium phosphate, etc. Suspending agents include sorbitol syrup, methyl cellulose, etc. Emulsifiers include lecithin, gum arabic, etc. Stabilizers include sodium sulfite, sodium bisulfite,
6. Preservatives such as sodium thiosulfate include benzoic acid, sorbic acid, and sodium dehydroacetate.

Next, the pharmacological effects of compound (I) of this invention will be shown.

(Leaving space below) 7 Experimental Example 1 Enzyme ice melting using chorylgrin hydrolase (A) Experimental method A bile acid derivative was dissolved in a 125 mmol acetate buffer containing 10 mmol of ethylenediaminetetraacetic acid disodium salt and 20 mmol of 2-mercaptoethanol. (pi(
5, 6) to a concentration of 2.0.1, 0.0.5.0.2.0.1 mmol. Clostridium verfringens (Clostridium)
Cholylglycine hydrolase (manufactured by Sigma) derived from P. erfringens was added to a final concentration of 2 mg/ml, and cultured at 37°C.
30, 45. PAB released by enzymatic ice thawing after 60 minutes
The amount of A was quantified using a urinary PABA measurement reagent kit for PFD (manufactured by Eisai Co., Ltd.). For Compound I, the amount of cholic acid liberated was measured.

The following bile acid derivatives were used as substrates.

Compound I: Glycocholic acid 8 Compound 2: Ursodeoxycoryruparaamine amene, fragrant compound 3, Urtudeoxycoryruvala aminobenzoic acid-3α-monosulfate Compound 4: Urtudeoxycoliruvala aminobenzoic acid-3α , 7β-disulfate (3 and 4 of the above compounds are the compounds of this invention.) (B) Experimental results From the enzymatic ice-thawing experiment using cholylglycine hydrolase for compounds 1 to 4, Lineweber-Park (Lineweber-Paak)
The M/% Ellis constants determined by the (Eaver-Burk) formula are 1.3.60 mmol for compound and 3.60 mmol for compound 2, respectively.
, 3.71 mmol, compound 3.0.31 mmol, compound 4, o, 23 mmol, and the compounds of this invention, 3, 4. was found to have 10 times higher substrate affinity than compound 1.2.

Experimental Example 2 Ice-melting experiment of bile acid derivatives using various intestinal bacteria (A) Experimental method Experimentally, the four types of bile acid derivatives shown in (I) were each
It was dissolved in AM broth (manufactured by Hinaga Pharmaceutical Co., Ltd.) at a concentration of 0.3 mmol/village, sterilized, and then inoculated with various intestinal bacteria. Anaerobic bacteria were cultured anaerobically in an anaerobic jar for 7 days, and aerobic bacteria were cultured under aerobic conditions for 3 days, and then the progress of the ice-melting reaction was determined using thin-layer chromatography. Ta. That is, 10 μg of the cultured medium was spotted on a Kieselgel 60F□4 (Merck > TI, C plate) and benzene-dioxane-acetic acid (+5:
5:2), the bile acid derivatives were developed by spraying with a 5% phosphomolybdic acid-ethanol solution, and PABA was developed by
Its presence or absence was confirmed using 4 nm ultraviolet light.

Judgment was made based on the disappearance of raw bile acid derivatives and the appearance of free bile acids and free PABA.

(B) Experimental results The results are shown in Table 2.

Experimental Example 3 Absorption experiment using an inverted intestinal tube (A) Experimental method In order to clarify the absorption dynamics of various bile acid derivatives in the intestinal tract, the following absorption experiment was conducted using an inverted intestinal tube.

Spragu tree weighing 200g
eDawley) After fasting for 12 to 24 hours from a male rat, the small intestine was removed under ether anesthesia, and the region from the origin of the jejunum to the terminal ileum was divided into four equal parts.
．． It was set at 3.4. An inverted intestinal tract was created by inverting the mucosal side and the lamina of each segment obtained. Next, a 0,1-concentration bile acid derivative solution heated to 37°C was poured into the cavity on the capsule side, and this was immersed in a bile acid derivative solution at the same temperature and concentration. After incubation for 60 minutes while passing gas, the concentration of bile acid derivatives in the solution inside and outside the intestinal tract was measured.

Note that Compounds 1 to 4 described in Experimental Example 1 were used as bile acid derivatives.

Compound 1 was measured using a bile acid measurement reagent (Kyokuto Pharmaceutical Co., Ltd.), and compounds 2 to 4 were measured using concentrated hydrochloric acid 0 and ly to each solution Ly and Q.
P was generated by adding Q and hydrolyzing it at 100°C for 1 hour.
Obtained from A B A I. Quantification of PABA was performed by BratLon-Marchall.
The measurement was carried out based on the absorbance value at 550 nm after the coupling reaction.

Note that Krebs-Ringer bicarbonate buffer containing 0.3% glucose was used as the bile acid derivative solution.

(B) Experimental results The results are shown in Figure 1 (each experiment was performed five times).

Experimental Example 4 Animal administration experiment (A) Experimental method
eDawley) Compounds 2 and 3.4 were orally administered at 0.019 mmol each to male rats, and the amount of urinary PABA excreted within 6 hours immediately thereafter was measured using a urinary PABA measurement reagent kit for PFD. The rats were divided into the following three groups based on the pretreatment method. (10 animals in each group)) Control group: Untreated group 11) Antibiotic treatment group, a group in which various antibacterial agents were orally administered twice a day for 3 days in advance for the purpose of antiseptic intestinal tract. The single dose consists of 2011 g of tinidazole (TDZ), 30,000 units of polymyxin B (PL), and kanamycin (K
M) 50 years old, clindamycin (CLDM) Ion.

111) Bacterial overgrowth group: Stagnan is surgically placed in the small intestine for the purpose of overgrowth of intestinal bacteria.
t 1ntestinal 1oop) was prepared (
B) Experimental results The results are shown in Figure 2.

Discussion The results of Experimental Example (I) show that Compound 3 and Compound 4, which are compounds of the present invention, are better as substrates for cholylglycine hydrolase than the comparative Compounds I and 2. Furthermore, in hydrolysis by intestinal bacteria, both of compounds 3 and 4, which are compounds of this invention, are compound! , showed the same tendency as compound 2.

5- On the other hand, in intestinal absorption experiments of various bile acid derivatives using an inverted intestinal tube, Compound 2 was efficiently absorbed into the intestinal tract by active transport from the distal ileum (segment 4), similar to taurocholic acid, which is one of the human bile acids. In contrast, it has been revealed that the compound of the present invention does not undergo active absorption from the small intestine.

In the final oral administration experiment to rats, the amount of urinary PABA excreted was significantly lower in the antibiotic-treated group that underwent intestinal embalming for all compounds, indicating that the bacterial overgrowth group had an abnormal proliferation of intestinal bacteria. showed a significantly high value. Furthermore, in the untreated group, Compound 3 had slightly better sensitivity than Compound 4.

From the above experimental results, it was found that by using the compound of this invention, unlike conventional breath tests, commercially available PFD By measuring the amount of urinary PABA excreted using a urinary PABA measurement reagent kit, the state of intestinal bacteria can be easily evaluated. Furthermore, Compound 2 (urtudeoxycollilupara-aminobenzoic acid) is considered to be an ideal testing method because it takes into consideration the shortcoming of active absorption from the small intestine. This is what we provide.

Next, examples of this invention will be shown.

[Example] Reference Example 1 Ursodeoquincholic acid p-aminobenzoic acid 5.0 g of ursodeoquincholic acid was dissolved in 25 μm of dioxane, and 3.6× of tri-n-butylamine was added. While stirring at 10°C. , 1.5 μg of ethyl chloroformate was added and reacted for 15 minutes. To this was added a solution of 2.0 g of p-aminobenzoic acid dissolved in 15×Q of IN sodium hydroxide solution, and the mixture was incubated for 30 min at room temperature.
Stir for a minute. That water test is 30zf! The mixture was made acidic by adding IN and IN hydrochloric acid, and extracted with ethyl acetate. The ethyl acetate solution was washed with water, dried over anhydrous sodium sulfate, the solvent was distilled off, the resulting residue was separated and purified by silica gel column chromatography, and dried to give wurtudeoxycollirubaraminobene, folic acid. 3.29 (yield 49%) was obtained.

Example I Urtudeoxycorylu-p-aminobenzoic acid (Method 1) Ursodeoxycorylu-p-aminobenzoic acid obtained in Reference Example 1 above 3. Og was dissolved in 6 mQ of anhydrous pyridine, 6 mQ of acetic anhydride was added, and the mixture was stirred at room temperature for 24 hours. After adding water, the mixture was extracted with ethyl acetate. The ethyl acetate solution was washed with water, dried over anhydrous sodium sulfate, and then evaporated to dryness to obtain ursodeoquine colyl-p-aminobenzoic acid-3α,7β-diacetyl compound 2.89 (yield: 80%).

Next, the above diacetyl derivative 1.09 was dissolved in 1.32% potassium carbonate-methanol solution 22.5x (!), stirred at room temperature for 8 hours, acidified by adding water and IN hydrochloric acid, and extracted with ethyl acetate. The ethyl solution was washed with water, dried over anhydrous sodium sulfate, and then dried to obtain 0.729 (yield: 77%) of ursodeoquine colyl-p-aminobenzoic acid-7β-acetyl compound.

A solution of l011 (l) of anhydrous pyridine containing 2.0 g of the above compound was added at 0°C to l0 containing 11iI2 of chlorosulfonic acid.
A solution of zQ in pyridine was added, and the mixture was stirred at room temperature for 2 hours. Thereafter, water and formic acid were added to make the mixture acidic, and the mixture was extracted with n-butanol. The n-butanol solution was made weakly alkaline by adding aqueous ammonia, and the solvent was distilled off. The resulting residue was separated and purified by silica gel column chromatography, and dried to give wurtudeoxycorylu p-aminobenzoic acid-3.
64 g (yield 68%) of ammonium salt t of α-sulfate-7β-acetyl compound was obtained.

Next, the above compound 1.09 was dissolved in methanol 10xQ, and a 25% aqueous sodium hydroxide solution IO! After stirring at room temperature for 18 hours, water and IN hydrochloric acid were added to make the solution acidic, and the mixture was extracted with n-butanol. Aqueous ammonia was added to the n-butanol solution to make it slightly alkaline, and the solvent was distilled off. The resulting residue was separated and purified by silica gel column chromatography and dried to obtain 0.51 g (yield 51%) of the ammonium salt of the title compound in powder form.

Melting point: 186-190°C 9-IR (KBr) cm, 2920.2860 1665 1610°525 NMR (d6 DMSO, δ); 0.61113H,s) 0.88(3H,s) 0.93(31, d, J・6) 3.9-4.1 (2H, m) 7.7o (zu, d, J=9) 7.87 (2H, d, J=9) 10.19 (II(, s ) (Method 2) An anhydrous pyridine solution of 1011Q containing compound 2.09 obtained in Reference Example 1 above was poured into a pyridine solution containing 0.5xQ of chlorosulfonic acid.After 2 minutes, water was added to stop the reaction. , acidified with IN hydrochloric acid, and extracted with n-butanol. Aqueous ammonia was added to the n-butanol solution to make it slightly alkaline, and the solvent was distilled off. The resulting residue was separated and purified by silica gel column chromatography, and dried to dryness. to obtain powdery ammonium salt o of the title compound, 9 gg (yield: 4
0%) was obtained.

X Yakkoi i 30 Ursodeokinoculyllupara-aminobenzoic acid Compound (II[) obtained in Reference Example 1 above 2.0! ? 20x(l
Anhydrous pyridine solution was added dropwise to a birinone solution containing 2 g of chlorosulfonic acid, and the mixture was stirred at room temperature for 24 hours.
- Aqueous ammonia was added to the butanol solution to make it weakly alkaline, and the solvent was distilled off. The resulting residue was separated and purified by silica gel column chromatography and dried to give a powdery ammonium salt of the title compound 139 (yield: 46 %) was obtained.

Melting point: 177-183°C IR(KBr) cm-': 2920.28
60,1660,1605°525 N M R (d s DMSO, δ); 0.6
1 (3H, s) 0.89 (3H, S) 0.94 (3H, d, J・6) 3.9 to 4.2 (2H, m) 7.70 (2H, d, J=9) 7.87 (2H, d, J・9) to, 18 (lH, s) 1 not

[Brief explanation of drawings]

Figure 1 shows the absorption behavior of various bile acid derivatives in the inverted intestinal tract (rat). Figure 2 shows the amount of PABA excreted in rat urine by various bile acid derivatives. 32 Figure 2

Claims (1)

[Claims] 1. General formula (I): ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (In the formula, R^1 and R^2 are hydrogen atoms, hydroxyl groups, or sulfonyloxy groups) The indicated bile acid derivatives and their pharmaceutically acceptable salts. 2. A bile acid derivative and a pharmaceutically acceptable salt thereof according to claim 1, wherein in the general formula (I), R^1 is a hydroxyl group or a sulfonyloxy group, and R^2 is a hydrogen atom. 3. General formula (II): ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) (In the formula, R^1' and R^2' are hydrogen atoms or hydroxyl groups) When an acylating agent is added to the compound represented by By reacting, the general formula (III): ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (III) (In the formula, R^1^a and R^2^a are hydrogen atoms or acyloxy groups, and A is an acyl group) A compound represented by is hydrolyzed under weakly alkaline conditions to form the general formula (IV): ▲There are mathematical formulas, chemical formulas, tables, etc.▼(IV) (In the formula, R^1^a, R^2^ a has the same meaning as above), and then reacted with a sulfonating agent to form the general formula (V): ▲There are numerical formulas, chemical formulas, tables, etc.▼(V) (where R^1^a , R^2^a has the same meaning as above), and finally it is subjected to alkaline hydrolysis to form the general formula (I a): ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (I a) (Formula A method for producing a bile acid derivative or a salt thereof, which comprises obtaining a compound represented by (in which R^1' and R^2' have the same meanings as above) and converting it into a corresponding salt if necessary. 4. The compound of general formula (II) according to claim 3 is reacted with a sulfonating agent to form formula (I b): ▲There are numerical formulas, chemical formulas, tables, etc.▼( I b) (In the formula, R^ 1^b, R^2^b is a hydrogen atom or a sulfonyloxy group) A method for producing a bile acid derivative or a salt thereof, which comprises obtaining a compound represented by: 5. A diagnostic agent for intestinal bacteria, comprising the bile acid derivative of general formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.