JPH07304675A - Amyloid beta-protein aggregation/deposition inhibitor - Google Patents

Abstract

PURPOSE:To obtain an inhibitor excellent in amyloid beta-protein aggregation/ deposition inhibitory activity, thus useful as a therapeutic/preventive agent for Alzheimer's disease, containing, as active ingredient, a specific rifamycin derivative. CONSTITUTION:This inhibitor contains, as active ingredient, a compound of formula I (R<1> and R<2> each is H or a 2-7C lower acyl; R<3> to R<7> each is H or a 1-6C lower alkyl; (n) is 1 or 2), a compound of formula II, or a salt thereof [e.g. 3-(4-benzylhomopiperazin-1-yl)rifamycin SV of formula III]. The dose of this inhibitor is, in terms of the active ingredient, pref. 10-1000 (esp. 100-600)mg/ adult a day.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an inhibitor of amyloid β protein aggregation and / or deposition. For more details,
The present invention relates to an amyloid β protein aggregation and / or deposition inhibitor containing rifamycins as an active ingredient.

[0002]

2. Description of the Related Art Amyloid β protein is a major component forming the amyloid core of senile plaque, which is one of the characteristic pathological changes observed in the brain of Alzheimer's disease patients.
A peptide consisting of 3 amino acids, which is produced by enzymatic degradation of transmembrane amyloid precursor protein APP (abbreviation of Amyloid Protein Precursor) (eg, MORI, H et al. (1992) THE JOURNAL OF BIOLOGICAL CHEMISTRY).
Vol. 267, pp. 17082-17086, LANSBURY, PT
Jr. (1992) BIOCHEMISTRY Volume 31 6865-6
870, SISODIA, SS et al. (1990) SCIENCE 248, 492-495, MULLAN, M. et al. (1993)
Year) TRENDS IN NEUROSCIENCE, Vol. 16, pp. 398-403 etc.). From experiments using chemically synthesized amyloid β protein and the like, such amyloid β protein has a strong self-aggregating property (eg, JARRETT, JT et al. (1993) CELL No. 73).
Volume 1055-1058, BURDICK, D. et al. (1992)
Year) THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 267, pages 546-554, FRASER, PE et al. (1992) BIOC
HEMISTRY, Vol. 31, p. 10716-10723, etc.), and that aggregated amyloid β protein may directly exhibit cytotoxicity to nerve cells, and enhances susceptibility to cell damage caused by excitatory amino acids. (For example, PIKE, CJ et al. (1993) THE JOUR
NAL OF NEUROSCIENCE Vol. 13, pp. 1676-1687,
NATTSON, MP et al. (1993) TRENDS IN NEUROSCIEN
CE 16: 409-414 etc.). In particular,
The partial peptide β25-35 of the amyloid β protein (amino acid sequence GSNKGAIIGLM) exhibits neurotoxicity, and one of the action points of β25-35 is the mitochondrial electron transfer system of neurons, and the MTT (3-
(4,5-dimethylthiazol-2-yl) -2,5-diph
phenyltetrazolium bromide) The ability to know the intensity of cytotoxicity by measuring its reducing activity.
Have already been reported (Yankner et al., Science, 250,
279-282, 1990; Kaneko et al., Neurochemistry, 32,
148-149, 1993). Furthermore, amyloid β
It is known that senile plaque formation due to protein deposition in the brain is a pathological change that appears earlier than neurofibrillary tangle, which is another characteristic pathological change in the brain of Alzheimer's disease patients ( For example, SEJKOE, DJ (1991)
NEURON Vol. 6, pp. 487-498, RUMBLE, B. et al. (198
9th) THE NEW ENGLAND JOURNAL OF MEDICENE No. 320
Pp. 1446-1452). That is, in Alzheimer's disease, the mechanism of onset that amyloid β protein is aggregated / deposited in brain tissues to form senile plaques, resulting in neuronal cell death and dementia is predominant.

Therefore, a drug which inhibits aggregation and / or deposition of amyloid β protein is expected to be useful as a therapeutic drug and / or a preventive drug for Alzheimer-type dementia. However, there are no reports of low molecular weight compounds having such inhibitory activity.

Further, rifamycin S, which is related to the present invention,
It is also known that a derivative in which the 3-position of rifamycin SV is substituted with a 4-substituted homopiperazin-1-yl group inhibits amyloid β protein aggregation or deposition and is effective in treating or preventing Alzheimer's dementia. Not not.

[0005]

DISCLOSURE OF THE INVENTION The present invention relates to amyloid β
It is intended to provide a drug that inhibits protein aggregation and / or deposition. Further, the present invention relates to amyloid β
It is an object of the present invention to provide a therapeutic agent or preventive agent for anti-Alzheimer's disease, which is based on the action of inhibiting protein aggregation and / or deposition.

[0006]

Means for Solving the Problems As a result of diligent studies of the possibility of a drug that inhibits agglutination and / or deposition of amyloid β protein, the present inventors have found that rifamycins according to the present invention The present invention has been completed, and it was found that it has an activity of inhibiting deposition and / or has an action of suppressing neuronal toxicity caused by amyloid β protein.

[0007]

That is, the present invention has the following formula [I]:

[0008]

[Chemical 3]

Alternatively, the following formula [II]

[0010]

[Chemical 4]

[Wherein R₁And R₂Are the same or different
And hydrogen atom or C₂~ C₇Represents a lower acyl group,
R₃~ R₇Are the same or different and are a hydrogen atom or C
₁~ C ₆Represents a lower alkyl group, n represents an integer of 1 to 2
You ] Rifamycin derivative represented by
Containing a pharmaceutically acceptable salt as an active ingredient
It is an inhibitor of aggregation and / or deposition of β-protein.

The ring system numbering described herein is used essentially correspondingly, eg, based on that described in the specification of JP 60-12358 A.

As used herein, the term "lower" refers to a group to which it is attached, for example, an alkyl group having a chain (straight or branched) chain having 1 to 6 carbon atoms and a cyclic moiety unless otherwise specified. It is used in the sense that it can have any shape. Therefore, specific examples of the C _{1 to} C ₆ lower alkyl group in the formulas [I] and [II] include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t
ert-butyl, pentyl, neopentyl, hexyl,
Cyclopropyl, cyclohexyl, cyclopropylmethyl and the like can be mentioned.

In the formula [I] or the formula [II], R ₁ and R ₂ are the same or different and each represents a hydrogen atom or C ₂ -C.
₇ represents a lower acyl group. The C ₂ -C ₇ lower acyl group means a group composed of the above lower alkyl group and a carbonyl group, and specific examples thereof include C ₂ -C ₅ lower acyl groups such as acetyl and pivaloyl. Among these, the case where R ₁ and R ₂ are hydrogen atoms can be mentioned.

In the formula [I] or the formula [II], R ₃ to
R ₇ is the same or different and is a hydrogen atom or C ₁ -C
₆ represents a lower alkyl group. Specific examples of C ₁ -C ₆ lower alkyl group, the C ₁ -C ₆ lower alkyl group like can be mentioned and, among them methyl, ethyl, C ₁ -C ₃ lower alkyl groups propyl preferred It is mentioned as a thing. Among these, R _{3 to} R ₇ are preferably hydrogen atoms, C _{1 to} C ₃ lower alkyl groups substituted at the 2, 4, and 6 positions, and particularly hydrogen atoms. Although n represents an integer of 1 to 2, it is more preferable that n = 1.

R _{1 to} R ₇ , n of formula [I] or formula [II]
As a preferred combination of R ₁ and R ₂ in the formula [I] and the formula [II], each of R ₃ and R ₃
R ₇ is a hydrogen atom and n = 1.

Since the compounds of formula [I] or formula [II] have several asymmetric centers, isomers such as optical isomers and diastereoisomers exist. The present invention includes the corresponding pure optical isomers, pure diastereomers, racemates,
All diastereomeric or ceramitic mixtures are included.

The rifamycins of the present invention, due to the acidic or basic groups present in the molecule,
Also included are pharmaceutically acceptable salts which may be formed with known bases (eg sodium, potassium) or acids (eg inorganic or organic acids), if present.
Specific examples thereof include salts prepared using hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, oxalic acid, malonic acid, fumaric acid, ascorbic acid, lactic acid, tartaric acid, glutamic acid, methanesulfonic acid and the like.

Suitable examples of the compound include those having the following structures.

[0020]

[Chemical 5]

[0021]

[Chemical 6]

The rifamycin derivative according to the present invention is
It can be easily synthesized by a known synthesis method. (References: JP-A-60-12358 and JP-A-2-3)
6597, JP-A-64-66188, JP-A-1-149790, JP-A-2-304090, JP-A-2-56487, and JP-A-3-169.
No. 884, No. 4-159283, No. 4-230688, and No. 4-247088.
Issue).

The amyloid β protein aggregation and / or deposition inhibitor of the present invention is preferably used as a amyloid β protein aggregation and / or deposition inhibitor by incorporating a pharmaceutically acceptable carrier. Can be used. Examples of the pharmaceutically acceptable carrier in this case include the same as the below-mentioned excipients. The compounding amount of the compound of the present invention and the carrier in this case depends on the dose of the active ingredient as described below, but is not particularly limited and can be selected within a wide range. Usually, the rifamycins of the present invention are ˜70 wt%, preferably 5 to 50 wt%. The obtained composition is further prepared by a known method using suitable excipients and the like, oral capsules such as soft capsules, hard capsules, tablets, granules, powders, suspensions, solutions, syrups, and injections. It is provided as an agent, a suppository or an external preparation.

Examples of such excipients include vegetable oils (for example, corn oil, cottonseed oil, coconut oil, almond oil, peanut oil, olive oil, etc.), oily esters of medium-chain fatty acid glyceride oil, mineral oil, glycerin such as tricaprylin, triacetin, etc. Esters, alcohols such as ethanol, physiological saline, propylene glycol, polyethylene glycol, petrolatum, animal fats and oils, cellulose derivatives (crystalline cellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), polyvinylpyrrolidone, dextrin, lactose, mannitol, Examples include sorbitol and starch.

The rifamycins or pharmaceutically acceptable salts thereof of the present invention are useful as amyloid β protein aggregates and / or prophylactic agents thereof.

The dose of the active ingredient depends on the degree of disease, age of the patient, etc., but is usually about 10 to 1000 mg / day / person, preferably 100 to 600 mg / day / person.
It is preferable to formulate so as to satisfy such conditions.

The test method used in the present invention is as follows.

Test Method for Amyloid β Protein Aggregation Inhibitory Activity A dye called thioflavin T (ThT) is amyloid β.
It has been reported that it binds to the β-sheet structure of aggregated proteins such as proteins and emits new fluorescence (482 nm) that was not shown in the free state (Harry LeVine III, 19
93, Protein Science 2, 404-410). The intensity of fluorescence is proportional to the degree of aggregation of bound proteins. Therefore,
The amyloid β protein aggregation inhibitory activity of the drug can be examined by measuring the degree of aggregation of the amyloid β protein in the presence of the drug by measuring the fluorescence intensity of ThT binding thereto. Specifically, 10 μl is taken from a sample solution of amyloid β protein containing a certain drug, and this is added to 1 ml of a ThT solution dissolved in 50 mM sodium phosphate buffer (pH 6.0) at a concentration of 3 μM and stirred. . After stirring, the spectrofluorometer (JASC
(Made by O), excitation wavelength 450 nm, fluorescence wavelength 482
The fluorescence of the solution is measured in nm. Measurements over time,
If the increase in fluorescence is suppressed for a certain period of time in the absence of a drug, that is, as compared with a solution containing only amyloid β protein, the drug is determined to have amyloid β protein aggregation inhibitory activity.

[0029]

EXAMPLES The present invention will be described in detail below with reference to examples and examples. The rifamycin derivative used in the examples is
JP-A-2-36597, JP-A-1-149790
Japanese Patent Laid-Open No. 2-56487 and Japanese Patent Laid-Open No. 2-56487.
According to the method described in the specification such as 304090 publication, rifamycin S derived from commercially available rifamycin SV by a known method was synthesized as a starting material.

Reference Example 1 3- (4-benzylhomopiperazin-1-yl) rifa
Synthesis of mycin SV (Compound 1) After dissolving 300 mg of rifamycin S and 756 mg of 4-benzyl homopiperazine in 1 mL of dioxane,
The solvent was distilled off, and the solution was left as it was at 40 ° C. for 15 hours. The reaction solution was diluted with 100 mL of ethyl acetate and washed twice with 50 mL of 10% aqueous citric acid solution, and then the solvent was evaporated. Dissolve the obtained dark red crystals in 30 mL of ethanol,
% Aqueous ascorbic acid solution (30 mL) at room temperature
Stir for minutes. 150 mL of ethyl acetate was added to the reaction solution, the organic layer was separated, the organic layer was washed with saturated brine and dehydrated with anhydrous magnesium sulfate, and then the solvent was distilled off. The obtained red crystals were purified by silica gel column chromatography (solvent / ethyl acetate: methanol = 9: 1) to give 3-
248 mg of (4-benzylhomopiperazin-1-yl) rifamycin SV (Compound 1) was obtained as red crystals. mp. 178-179 ° C MS (SIMS) [M + H] ⁺ 886 (C ₄₉ H ₆₃ N ₃ O
₁₂ )

Reference Example 2 2- (4-benzylhomopiperazin-1-yl) rifa
Synthesis of mycin S (compound 2) 3- (4-benzylhomopiperazin-1-yl) rifamycin SV 94 mg was dissolved in methylene chloride 5 mL,
100 mg of fine powder of manganese dioxide was added and vigorously stirred at room temperature for 30 minutes. Solid components were removed by filtration through Celite and the filtrate was concentrated to obtain 71 mg of 3- (4-benzylhomopiperazin-1-yl) rifamycin S (Compound 2) as black crystals. mp. 143-145 ° C MS (SIMS) [M + H] ⁺ 884 (C ₄₉ H ₆₁ N ₃ O
₁₂ )

[Reference Example 3] 3- [4- (2-phenethyl) homopiperazine-1-i
Synthesis of rifamycin SV (compound 3) Rifamycin S384 mg and 4- (2-phenethyl) homopiperazine 879 mg were dissolved in dioxane 3 mL, the solvent was distilled off, and the mixture was left standing at room temperature for 3 days. The reaction solution was diluted with 200 mL of ethyl acetate and washed twice with 30 mL of 10% aqueous citric acid solution, and then the solvent was evaporated. The obtained dark red crystals were dissolved in 30 mL of ethanol, 30 mL of a 20% aqueous ascorbic acid solution was added, and the mixture was stirred at room temperature for 10 minutes. 200 mL of ethyl acetate in the reaction solution
Was added to separate the organic layer, and the organic layer was washed with saturated brine and dehydrated with anhydrous magnesium sulfate, and then the solvent was distilled off. The obtained orange crystals were purified by silica gel column chromatography (solvent / ethyl acetate: methanol = 9: 1) to give 3- [4- (2-phenethyl) homopiperazin-1-yl] rifamycin SV ( 340 mg of compound 3) was obtained as orange crystals. mp. Partially melted at 170-180 ℃, blackened at 185 ℃

[Reference Example 4] 3- [4- (2-phenethyl) homopiperazine-1-i
Synthesis of 3- [4- (2-phenethyl) homopiperazin-1-yl] rifamycin SV (137 mg) with methylene chloride (5 m)
It was dissolved in L, 100 mg of manganese dioxide fine powder was added, and the mixture was vigorously stirred at room temperature for 30 minutes. Solid components were removed by Celite filtration, and the filtrate was concentrated to 3- [4- (2-
100 mg of phenethyl) homopiperazin-1-yl] rifamycin S (compound 4) was obtained as black crystals. mp. 150-152 ° C

Reference Example 5 Synthesis of Amyloid β Protein (Peptide β1-35) In the following examples, the amyloid β protein used to examine the inhibitory activity against amyloid β protein aggregation was:
Of the amyloid β protein sequence consisting of about 40 amino acids, peptide β1-35 consisting of the amino terminal 35 residues was synthesized.

The peptide was synthesized by using the peptide synthesizer (manufactured by Applied Biosystems) by the solid phase method using Fmoc-amino acid as a raw material. After completion of the synthesis, the peptide was cleaved from the resin and C ₁₈
This was purified by reverse phase high performance liquid chromatography (manufactured by Waters) using a column. The obtained β1-
It was confirmed by a peptide sequencer (manufactured by Applied Biosystems) that 35 peptides had the target amino acid sequence. Β1-obtained in this way
The 35 peptides were lyophilized and used in the examples below.

[Examples 1 to 4] (1) Rifamycin derivatives obtained in Reference Examples 1 to 4 were each added to dimethyl sulfoxide (DMSO) at 10 mg / mg.
The solution was dissolved in the solution so that the volume of the solution became 1 ml to obtain a rifamycin derivative solution (Examples 1 to 4; corresponding to compound numbers of Reference Examples 1 to 4, respectively).

On the other hand, the β1-35 peptide obtained in Reference Example 5 was twice dissolved in deionized water to a concentration of 2 mg / ml, passed through a 0.22 μm filter, and then 0.2 M sodium phosphate buffer solution was added. (PH 7.4) was mixed at a ratio of 1: 1 to obtain a β1-35 peptide solution having a final concentration of 1 mg / ml. 20 solutions of this solution in 15 Eppendorf tubes
Aliquots of 0 μl were dispensed.

Next, 10 mg / ml of each rifamycin derivative solution was dispensed with the β1-35 peptide solution 3
2 μl each was added to each tube, and each β-35 peptide solution containing 3 rifamycin derivatives (Examples 1 to 4)
Was obtained (final concentration of rifamycin derivative 100 μg / m
l).

DMSO was added to the remaining 3 tubes out of the 15 tubes into which the β1-35 peptide solution was dispensed.
Was added in 2 μl increments, and this DMSO-containing β1-35 peptide solution was used as a control (Control Example 1).

Β1-3 containing these rifamycin derivatives
5 peptide solutions (Examples 1 to 4) and DMSO-containing β1
-35 peptide solution (Control Example 1) was allowed to stand at room temperature, and according to the above-mentioned test method, a part of each solution was taken over time (every week) to determine the degree of aggregation of β1-35 peptide by fluorescence intensity. Was measured as an index. The results are shown in FIG. 1 and FIG.
Shown in.

(2) In the DMSO-containing β1-35 peptide solution (control example 1), as shown in FIG. 1, fluorescence was increased 14 days after the peptide was dissolved, that is, peptide aggregation occurred. Fluorescence increased further on day 2
It can be seen that almost the equilibrium is reached on the 8th day.

(3) A β1-35 peptide solution containing a rifamycin derivative when the fluorescence intensities of the DMSO-containing β1-35 peptide solution (Control Example 1) on the 21st and 28th days were set to 100 (implementation) The relative intensities of fluorescence in Examples 1 to 4) are shown in FIG. In the figure, the vertical axis represents the relative intensity (%) with respect to Control Example 1, and the horizontal axis represents the compound numbers contained in Examples 1 to 4. Also, the darkly dotted bar graph shows the 21st day, and the lightly dotted bar graph shows the 28th day.

As is apparent from FIG. 2, all four rifamycin derivatives suppressed the aggregation of β1-35 peptide. The above results indicate that the rifamycin derivative of the present invention is effective in suppressing aggregation of amyloid β protein. Further, from this, it is expected that the rifamycin derivative of the present invention suppresses deposition of amyloid β protein due to aggregation of amyloid β protein.

[Brief description of drawings]

FIG. 1 shows the aggregate formation of amyloid β protein over time for Control Example 1. In the figure, the vertical axis represents the fluorescence intensity (4
50 nm / 482 nm), and the horizontal axis shows the incubation time (days).

FIG. 2 shows the amyloid β protein aggregation inhibitory activity on day 21 (dark dot bar graph) and day 28 (light dot bar graph) for Examples 1 to 4 as fluorescence of the corresponding control example 1. The result measured as relative intensity with respect to intensity is shown. In the figure, the vertical axis represents relative intensity (%) with respect to Control Example 1, and the horizontal axis represents the rifamycin derivative (compounds 1 to 4) contained in the rifamycin derivative-containing β1-35 peptide solution of Examples 1 to 4. The compound number of is shown.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication location C07D 307: 92) (72) Inventor Noriaki Endo 4-3-2 Asahigaoka, Hino-shi, Tokyo Teijin shares Company Tokyo Research Center

Claims (4)

[Claims] 1. The following formula [I]:Alternatively, the following formula [II][In the formula, R₁And R₂Are the same or different and are hydrogen atoms
Or C₂~ C₇Represents a lower acyl group, R₃~ R₇Is
The same or different, hydrogen atom, or C₁~ C ₆Low grade
It represents an alkyl group, and n represents an integer of 1 to 2. ]]
Rifamycin derivative or its pharmaceutically acceptable
Of amyloid β protein containing a salt
Collection and / or deposition inhibitors. 2. In the formula [I] or the formula [II], R ₁
And R ₂ is a hydrogen atom, an amyloid β protein aggregation and / or deposition inhibitor containing the rifamycin derivative or the pharmaceutically acceptable salt thereof according to claim 1 as an active ingredient. 3. In the formula [I] or the formula [II], R ₃
To R ₇ each represents a hydrogen atom, the amyloid β protein aggregate and / or the rifamycin derivative according to claim 1 or 2 containing a pharmaceutically acceptable salt thereof as an active ingredient.
Or a deposition inhibitor. 4. A rifamycin derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein n is 1 in the formula [I] or the formula [II], as an active ingredient. An amyloid β protein aggregation and / or deposition inhibitor contained.