JPS6325579B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides a novel substance with the following general formula (): (In the formula, R ₁ represents a hydrogen atom or an acetyl group; R ₂ is a group -CH ₂ OH;

[expression] or ; * indicates that the configuration can be R or S configuration) and a method for producing the same. Previously, the present inventors focused on the inhibitory effect on aminopeptidase and the anticancer enhancing effect, produced a new dipeptide compound with a structure similar to bestatin, and confirmed the physiological activity of these compounds (e.g. 136118). As a result of further expanding this research, the present inventors found that
The present invention was completed by synthesizing bestatin-related substances represented by the above general formula and examining their physiological activities, and confirming that these compounds exhibit aminopeptidase inhibitory and immune-enhancing effects. The target compound () according to the present invention can be obtained as follows. That is, the following general formula (): (In the formula, R ₁ has the above-mentioned meaning; R ₃ is a group -OR ₄ ;

[expression] or R ₄ represents a lower alkyl group or a benzyl group; * indicates that the steric configuration can be R or S configuration) or a protected derivative of a functional group not involved in the reaction is prepared by a conventional method. The protective group may be removed by reduction, if necessary. Compounds () are usually amino acids, dipeptides,
It is the C-terminal ester form of the tripeptide. If this compound () is an ester of an amino acid, it may be, for example, 3R-N-tert-butoxycarbonylamino-2S-hydroxy-4-phenylbutyric acid methyl ester, which is 3R-amino-2S-hydroxy- 4-phenylbutyric acid (J.
(See Antibiotics Vol. 29, No. 5, 600-601 (1976))
It is prepared by converting the amino group of into an N-tert-butoxycarbonylamino group by a conventional method, and then esterifying it with a diazomethane ether solution. For example, when the compound () is a dipeptide ester, the amino group of bestatin (see JP-A-51-1787) is converted into an N-tert-butoxycarbonylamino group by a conventional method, and then esterified in the same manner as above. The resulting (2S,3R)-3-N-tert-butoxycarbonylamino-4-phenylbutanoyl-(S)-leucine methyl ester is mentioned. An example of a tripeptide ester in which the compound () is a tripeptide ester is a compound obtained by condensing N-benzyloxycarbonylbestatin synthesized by a conventional method with an ester of an amino acid using a conventional method in peptide chemistry. The thus obtained compound () can be exchanged into the compound () using a conventional reducing agent which reduces the acid ester to the corresponding alcohol, for example, by reducing the compound () in a hydrous or anhydrous alcohol solution of sodium borohydride at room temperature. After the reduction, the protective group is removed if necessary, and this deprotection can be carried out by known methods such as catalytic reduction using a palladium catalyst, hydrobromic acid in acetic acid, trifluoroacetic acid, hydrochloric acid in an organic solvent, etc. Can be removed by protection methods. A compound in which R ₁ represents an acetyl group in the general formula () can also be produced by acetylating a corresponding compound in which R ₁ is hydrogen by a conventional method. Examples of compounds () of the invention include: (2S,3R)-3-amino-2-hydroxy-
4-phenyl-1-butanol, (2S)-2-N
−[(2′S,3′R)-3′-amino-2′-hydroxy-
4'-phenylbutanoyl]amino-4-methyl-
Pentanol, (2S)-2-N-[(2'S,3'R)-3'-N-acetylamino-2'-hydroxy-4'-phenylbutanoyl]amino-4-methyl-pen Tanol,
(2S)-2-N-[(2′S,3′R)-3′-amino-2′
−
Hydroxy-4'-phenylbutanoyl-S-leucyl]amino-4-methyl-pentanol. Next, the physiological activity of the compound of the present invention will be described. Inhibitory activity test method for aminopeptidase B: Aminopeptidase B inhibitory activity was measured by VKHOPSU et-al, Archives of
Biochemistry and Biophysics 114 , 557 (1966)]
The method was improved. That is, a mixed solution (PH7.0) of 0.8 ml of 0.1 mM arginine-β-naphthylamide, 1.0 ml of 0.1 M Tris-HCl buffer, and 0.7 ml of a solution containing the specimen was heated at 37°C for 3 minutes.
Add 0.2 ml of aminopeptidase B solution purified using Cephadex® G-100 using the oxygen purification method of Hopps et al., react at 37°C for 30 minutes, and add garnet GBC (O
- aminoazotoluene diazonium salt),
1.0M containing Twin® 20 at a concentration of 10%
After adding 0.6 ml of acetate buffer (PH4.2) and leaving it at room temperature for 15 minutes, absorbance a at 525 nm was measured. At the same time, blind absorbance b was measured using only a buffer solution containing no analyte, and the aminopeptidase B inhibition rate was calculated as (ba)/b×100. Result: The inhibition rate was determined using the above method, and it was determined to be 50%.
Inhibition (IC50) was ascribed. The results are shown in Table 1.

[Table] Effect on cell-mediated immunity The effect on cell-mediated immunity was investigated using sheep red blood cells as an antigen and the delayed-type hypersensitivity (DTH) obtained by inoculating them into mouse footpads as an indicator. Test method: ¹⁰⁸ sheep red blood cells were suspended in 0.05 ml of physiological saline and inoculated subcutaneously into the footpad of CDF1 female 8-week-old mice (5 mice per group). At this time, the sample was dissolved in physiological saline, After passing the sample through a Millipore filter, it was administered orally at 0.5 mg/Kg. Four days later, ¹⁰⁸ sheep red blood cells were subcutaneously injected into the other footpad of the mouse.
After 24 hours, the thickness of the footpad of the mouse was measured with a caliper and determined. Results: Shown in Table 2.

[Table] The toxicity of this substance was evaluated for the compounds of Example 2(b) and Example 5(b). That is, when 100 mg/Kg of the test compound was intraperitoneally administered to ICR mice (male, 5 weeks old, body weight 20 g, 6 mice per group), all mice survived in both cases. From the above results, it is clear that the bestatin-related substances of the present invention enhance cell-mediated immunity at low doses. This strong immunostimulatory effect indicates that the substance of the present invention can be widely used as a cancer immunotherapy or an infection preventive agent. When the bestatin-related substance of the present invention is used as an immunostimulant, it may be administered orally, by injection, or as a rectal suppository, and various PH regulators, stabilizers, excipients, etc. may be added to prepare each form of preparation. can do. The dosage varies depending on the symptoms, but for adults, it is recommended to orally administer 50 to 200 mg once per day. Examples of the present invention will be shown below, but the present invention is not limited thereto. Example 1 (a) (2S,3R)-3-N-t-butoxycarbonylamino-2-hydroxy-4-phenyl-1
-Synthesis of butanol N-t-butoxycarbonyl-(2S,3R)-3
- Dissolve 92.8 mg of amino-2-hydroxy-4-phenyl-butyric acid methyl ester in 0.44 ml of ethanol, dropwise add 2.3 ml of ethyl alcohol containing 46.6 mg of sodium borohydride at room temperature, and stir at room temperature for 2 hours after the dropwise addition. do. After the reaction was completed, it was concentrated to dryness under reduced pressure, and 3 ml of ethyl acetate was added to dissolve it to give a 1%
Wash with 2 ml of citric acid aqueous solution, then 2 ml of saturated saline solution.
After washing three times, the solvent layer was dehydrated with anhydrous magnesium sulfate, concentrated to dryness, and 81 mg of crude material was obtained. Add this to 0.5ml of ethyl ether-n-hexane
Recrystallization from 0.5 ml of the mixed solution gave 68 mg of colorless needle crystals. Melting point 113-115℃, m/e282 by mass spectrometry
(M+1) was given. Elemental analysis value ( _C15H23N1O4 , molecular weight _281.39 ) Actual value C: _63.69 H: _8.31 N: 4.70% Calculated value C: 64.00 H: 8.26 N: 4.98% (b) (2S, 3R) - 3-amino-2-hydroxy-
Synthesis of 4-phenyl-1-butanol hydrochloride 68 mg of the compound obtained in step (a) was dissolved in 0.4 ml of anhydrous hydrogen chloride methanol solution under ice cooling, left for 30 minutes, concentrated to dryness, and diluted with ethanol-ether. Recrystallization yielded 36 mg of colorless plate-like crystals. Melting point 123-125
C, [α] ²⁵ _D +28.4 (c=1.0, 1N-HCl) mass spectrometry gave m/e182. Elemental analysis value (C ₁₀ H ₁₆ NO ₂ Cl, molecular weight 217.72) Actual value C: 54.93 H: 7.46 N: 6.40 Cl: 16.0% Calculated value C: 55.30 H: 7.42 N: 6.44 Cl: 16.3% Example 2 ( a) (2S)-2-N- [(2'S, 3'R)-3'-N--
t
Synthesis of -butoxycarbonylamino-2'-hydroxy-4'-phenylbutanoyl]amino-4-methyl-pentanol (2S,3R)-3-Nt-butoxycarbonylamino-2-hydroxy-4- Example 1 440 mg of phenylbutanoyl-S-leucine methyl ester was dissolved in a mixture of 8 ml of ethanol and 8 ml of distilled water.
Treated with sodium borohydride in the same manner as in (a) to obtain 347 mg of crude material. This was recrystallized from ethyl acetate-ether to obtain 230 mg of colorless needle crystals.
Melting point 159.5-160℃, m/e394 by mass spectrometry
(M ⁺ ) was given. Elemental analysis value ( _C21H34N2O5 , molecular weight _394.57 ) Actual value C: _63.92 H: _8.76 N: 7.14% Calculated value C: 63.92 H: 8.70 N: 7.10% (b) (2S)-2- N-[(2′S,3′R)-3′-amino-
Synthesis of 2'-hydroxy-4'-phenylbutanoyl]amino-4-methyl-pentanol 300 mg of the compound of step (a) was dissolved in 3 ml of anhydrous hydrogen chloride methanol solution containing anisole under ice cooling, and the mixture was dissolved for 30 minutes. After standing, it was concentrated to dryness to obtain the title compound hydrochloride.
Obtained 210 mg. The material is hygroscopic and gave an m/e of 295 by mass spectrometry. Furthermore, dissolve this substance in water,
After neutralization with sodium hydrogen carbonate, the mixture was extracted with butanol, washed with water, and concentrated to dryness to obtain the title free base as an oil. The mass spectrometry value of this substance is m/e295 (M+1)
The structure was confirmed by IR and NMR. [α] ²⁵ _D -2.5° (c=1.0 1N-HCl) Example 3 (2S)-2-N-[(2'S, 3'R)-3'-N-acetylamino-2'-hydroxy Synthesis of -4'-phenylbutanoyl]amino-4-methyl-pentanol 42 mg of the free base of the compound obtained in Example 2(b) was dissolved in 0.4 ml of methanol, 0.16 ml of acetic anhydride was added, and the mixture was heated at room temperature. After standing overnight, concentrate and 2 ml of n-butanol.
dissolved in Wash the butanol solution with water (2 ml),
The mixture was washed with 2 ml of a saturated aqueous sodium bicarbonate solution, and then concentrated to dryness to obtain 32 mg of a crude substance. This was recrystallized from ethanol-ether to form colorless needle-shaped crystals.
Got 20mg. Melting point: 188-189℃, mass spectrometry value: m/
Gave e336 (M ⁺ ). Elemental analysis value ( _C18H28N2O4 , molecular weight _336.48 ) Actual value C: _64.14 H: _8.35 N: 8.30% Calculated value C: 64.25 H: 8.40 N: 8.33% Example 4 (2S)-2- Synthesis of N-[(2′S,3′R)-3′-N-acetylamino-2′-hydroxy-4′-phenylbutanoyl]amino-4-methyl-pentanol (2S,3R)- 3-N-acetylamino-2-hydroxy-4-phenylbutanoyl-S-leucine methyl ester was obtained by reducing it with sodium borohydride in the same manner as in Example 2(a). Example 5 (a) (2S)-2-N-[(2'S, 3'R)-3'-N-benzyloxycarbonylamino-2'-hydroxy-4'-phenylbutanoyl-S- Synthesis of [leucyl]amino-4-methyl-pentanol (2'S, 3'R)-3'-N-benzyloxycarbonylamino-2'-hydroxy-4'-phenylbutanoyl-S-leucyl-S -120 mg of leucine benzyl ester was treated with sodium borohydride in the same manner as in Example 1(a) to obtain 100 mg of colorless powder.
Mass spectrometry gave m/e528 (M+1). Elemental analysis value ( _C30H43N2O6 , molecular weight _527.75 ) Actual value C: _67.98 H: 8.03 N: 5.09% Calculated value C: 68.27 H: 8.23 N: 5.31% ₍ b) (2S)-2- N-[(2′S,3′R)-3′-amino-
2'-Hydroxy-4'-phenylbutanoyl-S
Synthesis of -leucyl]amino-4-methyl-pentanol 100 mg of the compound obtained in step (a) was mixed with methanol 1
Dissolve palladium black in a mixture of 0.3 ml and distilled water.
Add 20 mg of the mixture and perform hydrogenolysis overnight at room temperature with hydrogen at 3 atm. After removing the catalyst, concentrate to form a syrup. 2 ml of ethyl acetate and 3 ml of 1N hydrochloric acid were added to this, and the mixture was transferred to the aqueous layer for extraction. The aqueous layer was concentrated to dryness to obtain 26 mg of colorless powder. The melting point was 78-80°C, and the mass spectrometry value gave m/e 408.

Claims (1)

[Claims] 1. General formula: (In the formula, R ₁ represents a hydrogen atom or an acetyl group; R ₂ represents a group -CH ₂ OH; [Formula] or ; * indicates that the steric configuration can be R or S configuration). 2 General formula: (In the formula, R ₁ has the meaning below; R ₃ is a group -OR ₄ ; [Formula] or R ₄ represents a lower alkyl group or a benzyl group; * indicates that the steric configuration can be R or S configuration) or a protected derivative of a functional group not involved in the reaction is prepared by a conventional method. A general formula characterized by reducing; optionally removing its protecting group: (In the formula, R ₁ represents a hydrogen atom or an acetyl group; R ₂ represents a group -CH ₂ OH; [Formula] or ; * indicates that the configuration can be R or S configuration) A method for producing a bestatin-related substance.