Angiotensin-converting enzyme inhibitor, pharmaceutical composition, and food

Abstract

To provide a novel angiotensin-converting enzyme (ACE) inhibitor, and a pharmaceutical composition and food containing the ACE inhibitor.SOLUTION: An angiotensin-converting enzyme inhibitor contains a compound represented by general formula (1), a pharmaceutically acceptable salt thereof or a solvate thereof as an active ingredient, where R1 is H, a C1-5 alkyl group, or a C1-5 haloalkyl group; R2 is H, a C1-5 alkyl group, a C1-5 haloalkyl group, or a C1-5 alkoxy group; X is a single bond or a divalent linking group; and n is an integer of 1-4.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an angiotensin-converting enzyme inhibitor, a pharmaceutical composition, and a food.

Background Art

[0002] Angiotensin-converting enzyme (hereinafter also referred to as ACE) is a factor that raises blood pressure. By inhibiting the action of ACE, a blood pressure-inhibiting effect is expected.

[0003] As ACE inhibitors, peptides and terpenoids have been proposed (see Patent Documents 1 to 2). In addition, the present inventors have found that a fermented product of abalone liver has ACE inhibitory activity (see Patent Document 3).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0005] From the viewpoint of excellent medicinal effects and the like, an ACE inhibitor based on a new mechanism of action is required. Therefore, an object of the present invention is to provide a novel ACE inhibitor, a pharmaceutical composition, and a food containing the novel ACE inhibitor.

Means for Solving the Problems

[0006] The inventors have found homarine as an ACE inhibitory active ingredient contained in an aqueous extract from a fermented product of abalone visceral powder, and completed the present invention. That is, the present invention is as follows. [1] An angiotensin-converting enzyme inhibitor containing, as an active ingredient, a compound represented by the following general formula (1), a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0007] [Chemical formula]

[0008] [In general formula (1), R 1 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a haloalkyl group having 1 to 5 carbon atoms. R 2 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, a haloalkyl group having 1 to 5 carbon atoms, or an alkoxy group having 1 to 5 carbon atoms. X represents a single bond or a divalent linking group. n represents an integer of 1 to 4.] [2] The angiotensin-converting enzyme inhibitor according to [1], containing, as an active ingredient, a compound represented by the following general formula (1-1-1) or the following general formula (1-2-1), a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0009] [Chemical formula]

[0010] [In general formula (1-1-1) and general formula (1-2-1), R 1 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a haloalkyl group having 1 to 5 carbon atoms.] [3] A pharmaceutical composition containing the angiotensin-converting enzyme inhibitor according to [1] or [2]. [4] A food containing the angiotensin-converting enzyme inhibitor according to [1] or [2]. [Effects of the Invention]

[0011] According to the present invention, a novel ACE inhibitor, as well as a pharmaceutical composition and a food containing the novel ACE inhibitor, can be provided. [Brief Description of the Drawings]

[0012]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0013] [Angiotensin-Converting Enzyme Inhibitor] In one embodiment, the present invention provides an angiotensin-converting enzyme inhibitor containing, as an active ingredient, a compound represented by the following general formula (1), a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0014]

Chemical formula

[0015] [In general formula (1), R 1 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a haloalkyl group having 1 to 5 carbon atoms. R 2 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, a haloalkyl group having 1 to 5 carbon atoms, or an alkoxy group having 1 to 5 carbon atoms. X represents a single bond or a divalent linking group. n represents an integer of 1 to 4.]

[0016] R 1 and R 2 Specific examples of the alkyl group having 1 to 5 carbon atoms for R

[0017] R 1 and R 2Examples of the haloalkyl group having 1 to 5 carbon atoms include an alkyl fluoride group, an alkyl chloride group, and an alkyl bromide group. R 1 and R 2 include those in which at least one hydrogen atom in the above-described alkyl group is replaced by a halogen atom.

[0018] Examples of the alkoxy group having 1 to 5 carbon atoms in R 2 include those in which the -OR part of R is the same as the alkyl group having 1 to 5 carbon atoms described above in R 2 .

[0019] Examples of the divalent linking group in X include an alkylene group, -O-, -C(=O)-, -NH-, -S-, -S(=O)2-, or a combination thereof, and an alkylene group is preferred. As the alkylene group, a group obtained by removing one hydrogen atom from the alkyl group having 1 to 5 carbon atoms in the above R 1 is preferred. Examples of the alkyl group having 1 to 5 carbon atoms include those the same as those described above in R 1 and R 2 .

[0020] The angiotensin-converting enzyme inhibitor of this embodiment preferably contains, as an active ingredient, a compound represented by the following general formula (1-1) or general formula (1-2), a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0021] [Chemical formula]

[0022] [In general formula (1-1) or general formula (1-2), R 1 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a haloalkyl group having 1 to 5 carbon atoms. R 2 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, a haloalkyl group having 1 to 5 carbon atoms, or an alkoxy group having 1 to 5 carbon atoms. X represents a single bond or a divalent linking group. n represents an integer of 1 to 4.]

[0023] Among those described above, as X, a single bond is preferred, n is preferably 1, and R 2 is preferably a hydrogen atom. That is, as the angiotensin-converting enzyme inhibitor of the present embodiment, it is particularly preferable to contain a compound represented by the following general formula (1-1-1) or general formula (1-2-1), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.

[0024]

Chemical formula

[0025] [In general formula (1-1-1) or general formula (1-2-1), R 1 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a haloalkyl group having 1 to 5 carbon atoms.]

[0026] Among those described above, R 1 is preferably an alkyl group having 1 to 5 carbon atoms, more preferably a methyl group. That is, as the angiotensin-converting enzyme inhibitor of the present embodiment, it is particularly preferable to contain a compound represented by the following formula (1-1-1-1) or formula (1-2-1-1), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.

[0027]

Chemical formula

[0028] Further, in one embodiment, the present invention provides an angiotensin-converting enzyme inhibitor containing a compound represented by the following general formula (2), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.

[0029]

Chemical formula

[0030] [In general formula (2), R 1represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a haloalkyl group having 1 to 5 carbon atoms. R 2 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, a haloalkyl group having 1 to 5 carbon atoms, or an alkoxy group having 1 to 5 carbon atoms. X represents a single bond or a divalent linking group. n represents an integer of 1 to 4.]

[0031] R 1 and R 2 As the alkyl group having 1 to 5 carbon atoms in R and R, specifically, a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, etc. can be mentioned.

[0032] R 1 and R 2 As the haloalkyl group having 1 to 5 carbon atoms in R and R, a fluoroalkyl group, a chloroalkyl group, and a bromoalkyl group can be mentioned, and those in which at least one hydrogen atom in the above-mentioned alkyl group is replaced by a halogen atom can be mentioned. 1 and R 2 As the alkoxy group having 1 to 5 carbon atoms in R, those in which the R part of -OR is the same as the above-mentioned alkyl group having 1 to 5 carbon atoms can be mentioned.

[0033] R 2 As the alkoxy group having 1 to 5 carbon atoms in R, those in which the R part of -OR is the same as the above-mentioned alkyl group having 1 to 5 carbon atoms can be mentioned. 1 As the divalent linking group in X, an alkylene group, -O-, -C(=O)-, -NH-, -S-, -S(=O)2-, or a combination thereof can be mentioned, and an alkylene group is preferable. As the alkylene group, a group obtained by removing one hydrogen atom from the above-mentioned alkyl group having 1 to 5 carbon atoms in R is preferable. As the alkyl group having 1 to 5 carbon atoms, those the same as those mentioned above in R and R can be mentioned.

[0034] Among the above, as X, a single bond is preferable, as n, 1 is preferable, and R 1 1 2 and R 1 Among the above, as X, a single bond is preferable, as n, 1 is preferable, and R

[0035] Among those described above, as X, a single bond is preferred, n is preferably 1, and R 1is preferably an alkyl group having 1 to 5 carbon atoms, and R 2 is preferably a hydrogen atom.

[0036] As the angiotensin-converting enzyme inhibitor of the present embodiment, it is preferable to contain a compound represented by the following general formula (2-1) or general formula (2-2), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.

[0037] [Chemical formula]

[0038] [In general formula (2-1) or general formula (2-2), R 1 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a haloalkyl group having 1 to 5 carbon atoms. R 2 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, a haloalkyl group having 1 to 5 carbon atoms, or an alkoxy group having 1 to 5 carbon atoms. X represents a single bond or a divalent linking group. n represents an integer of 1 to 4.]

[0039] Among the above, as X, a single bond is preferable, n is preferably 1, and R 2 is preferably a hydrogen atom. That is, as the angiotensin-converting enzyme inhibitor of the present embodiment, it is particularly preferable to contain a compound represented by the following general formula (2-1-1) or general formula (2-2-1), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.

[0040] [Chemical formula]

[0041] [In general formula (2-1-1) or general formula (2-2-1), R 1 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a haloalkyl group having 1 to 5 carbon atoms.]

[0042] Among the above, R 1An alkyl group having 1 to 5 carbon atoms is preferred, and a methyl group is more preferred. That is, as the angiotensin-converting enzyme inhibitor of the present embodiment, it is particularly preferable to contain a compound represented by the following formula (2-1-1-1) or formula (2-2-1-1), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.

[0043] [Chemical formula]

[0044] As the angiotensin-converting enzyme inhibitor of the present invention, the above compound may be used in the form of a free body, or may be used in the form of a pharmaceutically acceptable salt. Further, it may be used in the form of a solvate of the free body, or may be used in the form of a solvate of the salt.

[0045] The salt is not particularly limited as long as it is a pharmaceutically acceptable salt. For example, hydrochloride, sulfate, hydrobromide, hydroiodide, phosphate, nitrate, benzoate, methanesulfonate, 2-hydroxyethanesulfonate, p-toluenesulfonate, acetate, propionate, oxalate, malonate, succinate, glutarate, adipate, tartrate, maleate, fumarate, malate, mandelate, etc. may be mentioned. The solvate is not particularly limited as long as it is a pharmaceutically acceptable solvate. For example, hydrate, organic solvent solvate, etc. may be mentioned.

[0046] The angiotensin-converting enzyme inhibitor of the present invention can be used as a blood pressure increasing inhibitor.

[0047] [Pharmaceutical composition] In one embodiment, the present invention provides a pharmaceutical composition containing the above angiotensin-converting enzyme inhibitor and a pharmaceutically acceptable carrier.

[0048] The pharmaceutical composition of this embodiment can be administered orally in the form of, for example, tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions, etc., or parenterally in the form of injections, suppositories, external skin preparations, etc.

[0049] As the pharmaceutically acceptable carrier, those usually used in the formulation of pharmaceutical compositions can be used without particular limitation. More specifically, for example, binders such as gelatin, corn starch, tragacanth gum, gum arabic; excipients such as starch, crystalline cellulose; swelling agents such as alginic acid; solvents for injections such as water, ethanol, glycerin; adhesives such as rubber-based adhesives, silicone-based adhesives, etc. can be mentioned. The pharmaceutically acceptable carrier can be used alone or in combination of two or more.

[0050] The pharmaceutical composition of this embodiment may further contain additives. Examples of additives include lubricants such as calcium stearate, magnesium stearate; sweeteners such as sucrose, lactose, saccharin, maltitol; flavoring agents such as peppermint, perilla oil; stabilizers such as benzyl alcohol, phenol; buffers such as phosphates, sodium acetate; solubilizing agents such as benzyl benzoate, benzyl alcohol; antioxidants; preservatives, etc. The additives can be used alone or in combination of two or more.

[0051] Since the pharmaceutical composition of this embodiment has an effect of suppressing blood pressure elevation, it is suitably used for the treatment or prevention of diseases related to hypertension, such as arteriosclerosis; cerebral infarction such as lacunar infarction, atherosclerotic thrombotic infarction, cerebral embolism; cerebral hemorrhage; subarachnoid hemorrhage; cardiac hypertrophy; angina pectoris; myocardial infarction; kidney disorders, etc.

[0052] (Administration method) The method of administering the angiotensin-converting enzyme inhibitor or the pharmaceutical composition is not particularly limited and may be appropriately determined according to the patient's symptoms, body weight, age, gender, etc. For example, tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc. are administered orally. In addition, injections are administered intravenously alone or in combination with ordinary replenishing solutions such as glucose and amino acids, and if necessary, are also administered intraarterially, intramuscularly, intradermally, subcutaneously or intraperitoneally. Suppositories are administered rectally. Topical skin preparations are applied, affixed or sprayed onto the affected area.

[0053] (Dosage) The dosage of the angiotensin-converting enzyme inhibitor or the pharmaceutical composition varies depending on the patient's symptoms, body weight, age, gender, etc. and cannot be determined unconditionally. However, in the case of oral administration, for example, an active ingredient of 1 μg to 10 g per day, for example, 0.01 to 2000 mg per day may be administered. In the case of injections, for example, an active ingredient of 0.1 μg to 1 g per day, for example, 0.001 to 200 mg per day may be administered. In the case of suppositories, for example, an active ingredient of 1 μg to 10 g per day, for example, 0.01 to 2000 mg per day may be administered.

[0054] [Other Embodiments] In one embodiment, the present invention provides a compound represented by the general formula (1) or (2), a pharmaceutically acceptable salt thereof, or a solvate thereof for suppressing blood pressure elevation.

[0055] In one embodiment, the present invention provides a method for suppressing blood pressure elevation, which includes administering an effective amount of a compound represented by the general formula (1) or (2), a pharmaceutically acceptable salt thereof, or a solvate thereof to a patient in need of prevention or treatment.

[0056] In one embodiment, the present invention provides the use of a compound represented by the general formula (1) or (2), a pharmaceutically acceptable salt thereof, or a solvate thereof for producing a blood pressure elevation inhibitor or a composition for suppressing blood pressure elevation.

[0057] [Food] The food of the present invention contains the angiotensin-converting enzyme inhibitor of the present invention. Examples of the food include confectionery such as candies and jellies; processed products such as kamaboko and sausages; dairy products such as cheese and butter; and beverage foods such as fruit juice drinks such as orange and grape, and yogurt drinks. These foods can be functional foods, foods for specified health use, beauty and health foods, dietary supplements, and the like.

Examples

[0058] Hereinafter, the present invention will be described in more detail with reference to experimental examples, but the present invention is not limited by these examples.

[0059] [Preparation of Lactobacillus casei-fermented abalone viscera sample] L. casei 001 was statically cultured at 37 °C for 1 day using ILS medium. After freeze-drying the abalone viscera, it was pulverized with a household mixer, and the freeze-dried product in powder form was used as abalone viscera powder. A 500 ml Erlenmeyer flask was used for the culture. 2 g of abalone viscera powder and 2 g of glucose were added to 100 ml of 33 mM phosphate buffer (pH 5.0), adjusted to pH 6.8 with 1 M NaOH, and then autoclaved (121 °C, 15 minutes). Thereafter, the preculture solution of L. casei 001 was inoculated at 1% into the main culture medium and statically cultured at 37 °C for 1 day. This was used as the fermented product, and the abalone viscera medium without inoculation of bacteria was used as the unfermented product.

[0060] [Identification of ACE inhibitory substances in L. casei 001-fermented abalone viscera] After adding distilled water to the fermented product and the unfermented product, extraction was performed by shaking at 50 °C for 1 hour at 125 times / min using a water bath. The cultured solution subjected to extraction was centrifuged (12000 rpm, 10 minutes), and the supernatant was collected. This operation was performed until the color of the supernatant became transparent, and the obtained supernatant was dried under reduced pressure to obtain a sample.

[0061] Mass spectrometry was performed using a substance purified from this sample by ultrafiltration or liquid chromatography (see Figure 1). It was revealed that the molecular formula of such a substance is C7H7NO2.

[0062] Furthermore, nuclear magnetic resonance (NMR) analysis was used to identify the structure of this substance. 1 In the spectrum of 1D H-NMR analysis, the protons of the purified substance appeared as a singlet at δ 4.31, and two doublets and two triplets each appeared at 7.89 - 8.65 ppm (see Figure 2). These are characteristic peaks of a 2-substituted aromatic ring substance with the substituent position at o (ortho), and also indicate the presence of N-CH3. Based on these and each data, it was determined that the purified substance is homarine.

[0063] [Measurement of ACE inhibitory activity of homarine] The ACE inhibitory activity of homarine was measured. The ACE inhibitory activity was measured using ACE Kit-WST (Dojindo Laboratories). 10 μL each of the sample, Enzyme working solution, and Substrate buffer were placed in a 96-well plate and mixed, and then pre-incubated at 37°C for 1 hour. Next, 100 μL each of the Indicator working solution was added and stirred with a plate mixer. After incubating at room temperature for 10 minutes, the absorbance at 454 nm was measured. Distilled water was used as the control instead of the sample. Also, distilled water was used as the blank instead of the Enzyme working solution. The inhibition rate was calculated by the following formula.

[0064] ACE inhibition rate (%) = [(control - control blank) - (sample - sample blank)] × 100 / (control - control blank)

[0065] The IC 50 values of homarine and known ACE inhibitors are shown in Table 1. Homalin IC 50 The value was 532 μL / mL, showing strong activity compared to tripeptides and D-mannitol derived from shiitake mushrooms.

[0066]

Table 1

[0067] [Determination of the inhibition mode of homalin] The inhibition mode was determined by measuring the ACE inhibitory activity and estimating the amount of the product His-Leu. Distilled water and homalin were prepared at 0.776 mM and 2.17 mM, and 50 μL of each was dispensed into wells. Subsequently, 100 μL of a 10 mU / mL ACE solution was added to each well, mixed with a plate mixer, and pre-incubated at 37 °C for 10 minutes. Then, 25 μL of 2.5, 5, 12.5, and 25 mM Hip-His-Leu was added to each well and incubated at 37 °C for 40 minutes. After incubation, 50 μL of 1 N NaOH was dispensed into each well to stop the reaction, 10 μL of a 0.2% orthophthalaldehyde solution was added, and the reaction was carried out at room temperature for 15 minutes under light-shielded conditions. Then, 15 μL of a 3.6 M phosphoric acid aqueous solution was added to each well, and the fluorescence intensity was measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. A calibration curve was prepared using serially diluted His-Leu as a standard to calculate the slope and intercept, and the amount of His-Leu generated from the fluorescence intensity obtained by measurement was estimated using these values. Subsequently, the kinetics of ACE activity according to the concentration of homalin was determined by a Lineweaver–Burk plot. The kinetic parameters, the maximum initial velocity Vmax, the Michaelis–Menten constant Km, were calculated, and the inhibition constant Ki was determined from the slope. Ki is an index of the affinity between the enzyme and the inhibitor, and a lower value indicates a stronger affinity of the inhibitor for the enzyme.

[0068] Lineweaver–Burk plots of ACE in distilled water and homarine at 0.776 mM and 2.17 mM are shown in Figure 3. The values of Vmax in distilled water and homarine at concentrations of 0.776 mM and 2.17 mM were 73.9 μM / min, 60.3 μM / min, and 41.2 μM / min, respectively, and were concentration-dependent. Also, the Km values were 12.9 mM, 11.5 mM, and 10.2 mM, respectively, and although they were concentration-dependent, they were close values. The three straight lines in Figure 3 intersect in the third quadrant, and since the intersection point is very close to the x-axis, it was suggested that the inhibition mode of homarine is close to non-competitive inhibition. The slopes of the graphs of distilled water and homarine at concentrations of 0.776 mM and 2.17 mM were plotted, and as a result of calculating Ki from the intersection point with the x-axis, it was 5.00 mM (Figure 4). This indicates that homarine binds to some allosteric site of ACE, reducing the affinity of angiotensin I for the active site, and inhibits the production of angiotensin II by binding to the ACE–angiotensin I complex to form an ACE–angiotensin I–homarine complex. From the above, it was shown that homarine can be a novel ACE inhibitor.

Claims

1. An angiotensin-converting enzyme inhibitor containing, as an active ingredient, a compound represented by the following general formula (1-1-1-1), a pharmaceutically acceptable salt thereof, or a solvate thereof. 【Chemical 1】

2. The angiotensin-converting enzyme inhibitor according to Claim 1, which is a pharmaceutical composition.

3. The angiotensin-converting enzyme inhibitor according to Claim 1, which is a food.

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