JP2804817B2 - Method for producing 3-amino-2-oxo fatty acid derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION INDUSTRIAL APPLICATION The present invention relates to endopeptidase (particularly serine enzyme or
The present invention relates to a method for producing a novel synthetic peptide having an inhibitory activity on enzymes classified as SH enzymes.

[Prior Art] As a low-molecular inhibitory peptide that inhibits endopeptidase, peptide aldehydes such as leupeptin and elastatinal are conventionally known as natural products. It has been synthesized as. These peptide aldehydes can exhibit inhibitory activity by binding to the enzyme and the aldehyde occupying a reactive site with the peptide carbonyl group on the enzyme.
Thompson, RC (Biochemistry, 12
Volume, pp. 47-51 (1973)).

[Problems to be Solved by the Invention] The peptide aldehyde group has an active center carbonyl group at the C-terminus of the peptide chain. Therefore, the molecular structure can be changed to selectively bind to an enzyme. Only on the N-terminal side of the carbonyl group of the active center. The poststatin (compound No. 1 to be described later) that the present inventors previously invented has the following partial structural formula in a peptide chain. And has a novel structure having amino acid residues on both sides of the 3-amino-2-oxocarbonyl group. If the amino acid residues on both sides can be appropriately changed and appropriately combined with the structural part containing this carbonyl group and synthesized,
It is thought that it is possible to produce substances that exhibit selective inhibitory activity against various peptidases.

An object of the present invention is to provide a novel method for producing a physiologically active substance containing the above partial structure. 3-amino-2-
One peptide containing a keto acid has been reported in the past. Hori et al. Reacts an acyl amino acid with an alkyl or benzyl oxalyl chloride to form a 3-amino-
It has been reported that a peptide having ethyl 2-oxo-pentanoate or benzyl ester is synthesized to inhibit elastase (Peptides, Structure and Function.
Proceedings of the Ninth American peptide Symposiu
m, pp 819-822). However, they use the Dakin-West reaction, so that the base of the 3-amino group is racemic, and this method has a problem that only the C-terminal ester can be synthesized.

[Means for Solving the Problems] Accordingly, the present inventors have conducted intensive studies to overcome the above problems, and as a result, as a precursor of a physiologically active substance having the above partial structure, the following partial structural formula (Wherein R ₁ represents a saturated or unsaturated hydrocarbon group) having a 3-amino-2-hydroxy fatty acid derivative (functional groups other than the 2-position hydroxy group are optionally protected). Synthesis, and then oxidizing the 3-amino-2-hydroxy fatty acid derivative to obtain the following partial structural formula (Wherein R ₁ is the same as described above), and the above-mentioned 3-amino-2-oxocarbonyl group is obtained by removing a protecting group of a functional group as necessary. The present inventors have found that a physiologically active substance having the formula (or an intermediate thereof) can be obtained and completed the present invention.

The oxidation reaction of the present invention is preferably performed under mild conditions so as not to adversely affect other than oxidation at the 2-position of the above partial structural formula. For example, DMSO (dimethyl sulfoxide) as an oxidizing agent
Oxidation using is preferred. When DMSO is used, a combination catalyst of a weak base (for example, pyridines with a PKa of about 7.5 or less) and an acid (for example, trifluoroacetic acid or phosphoric acid with a PKa of about 3 or less) as a catalyst, preferably trifluoroacetic acid It is preferable to use a salt of pyridine with a carbodiimide (for example, dicyclohexylcarbodiimide, diisopropylcarbodiimide, ethyldimethylaminopropylcarbodiimide, etc.), acetic anhydride or oxalyl chloride.

The reaction may be carried out using DMSO as a solvent, or in another inert solvent as long as the reaction is not adversely affected. The reaction temperature is preferably in the range of about 0 ° C to 40 ° C. The reaction time varies depending on the conditions, but usually about 3 to 50 hours is sufficient.

As the physiologically active substance (or an intermediate thereof) obtained by the method of the present invention, a partial structural formula wherein an amino acid or a peptide is bonded to the carbonyl group of the partial structural formula [II] (Wherein R ₁ is the same as above, and Y represents a residue obtained by removing hydrogen from the amino group of an amino acid or a peptide or an ester thereof. When Y has a functional group, it may be protected with a protecting group. ), More preferably a peptide of the general formula (Wherein R ₁ and Y are the same as described above, X represents a residue obtained by removing OH from the carboxyl group of an amino acid or a peptide, and may be protected when X has a functional group.) Statins and their analogs.

These poststatins and their analogous compounds have the general formula (Wherein X ′ and Y ′ represent X and Y each having a protected functional group, where X and Y are the same as above, and R ₁ is the same as above.) Can be obtained by oxidizing

Substances represented by the general formula [V] has a substituent corresponding to R ₁ as an amino acid 3-protected - amino-2-hydroxy - be prepared by conventional peptide synthesis method using propionic acid.

If a general liquid phase method is used, for example when catalytic hydrogenolysis is used for the final deprotection, the amino acid benzyl ester or the benzyl ester of the peptide may be combined with e.g. p-methoxybenzyloxycarbonyl-3-amino-2-hydroxy-fatty acid or Condensation agents such as dicyclohexylcarbodiimide, diisopropylcarbodiimide, and N-butoxycarbonyl-3-amino-2-hydroxy-fatty acid are usually dissolved in a solvent usually used for peptide synthesis, such as dimethylformamide, for example, dicyclohexylcarbodiimide, N
Carbodiimides such as-(3-dimethylaminopropyl) -N'-ethylcarbodiimide or a salt thereof, or a condensing auxiliary thereof, for example, 1-hydroxybenzotriazole (HoBt), N-hydroxysuccinimide, etc. Condensation is carried out using a combination, and then a p-methoxybenzyloxycarbonyl group or a t-butoxycarbonyl (Boc) group is removed with a selective deprotecting agent such as trifluoroacetic acid. X '
Can be synthesized by condensing with a usual method.

If trifluoroacetic acid is used for the final deprotection, amino acid t-butyl ester and p-methoxybenzyloxycarbonyl-3-amino-2-hydroxy-fatty acid or benzyloxycarbonyl-3-amino-2
-Hydroxy-fatty acids are condensed in the same manner, and the p-methoxybenzyloxycarbonyl group or the benzyloxycarbonyl group is removed by catalytic hydrogenolysis, and then Y, X or a suitable derivative thereof in the general formula [I] is condensed sequentially. Is also good. If the solid phase method is used, 9-fluorenylmethyloxycarbonyl-aminoacyl-p-
Using an alkoxybenzyl alcohol resin as a starting material, removal of the 9-fluorenylmethyloxycarbonyl (Fmoc) group by base treatment and condensation of the Fmoc-amino acid are repeated sequentially, and finally, a benzyloxycarbonyl-amino acid or a Boc-amino acid, Alternatively, it can be synthesized by condensing a carboxylic acid.

In the general formula [V], when X 'and Y' include amino acids containing a hydroxyl group such as serine or threonine, and when trifluoroacetic acid is used for the final deprotection in the solid phase method and the liquid phase method, each amino acid is used. May be protected as t-butyl ether. When catalytic hydrogenolysis is used for final deprotection in the liquid phase method, each amino acid may be protected as benzyl ether.

Amino acids constituting "amino acids" or "peptides" appearing in the definitions of X and Y generally include α-amino acids, but are not necessarily α-amino acids.

The following formula is used as α-amino acids [Wherein R ₂ represents (1) hydrogen, (2) unsubstituted lower alkyl or (3) substituted lower alkyl (substituents are amino, hydroxy, mercapto, lower alkylthio, carboxylphenyl, hydroxyphenyl, imidazole, indolyl Group). ) The amino acid or formula represented by (Wherein R ₃ represents hydrogen or a hydroxy group).

Specific examples of these amino acids include glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, aspartic acid,
Examples include glutamic acid, lysine, arginine, phenylalanine, tyrosine, histidine, tryptophan, proline, oxyproline and the like.

As the peptide, generally, the above amino acids are 2-3.
A low-polymerized peptide in which about a few are bonded is exemplified.

Further, as the protecting group for the amino group in X or the like,
(1) An acyl group including a phthalyl group and a sulfonyl group; and (2) an oxycarbonyl group protected in a urethane form. For example, the acyl group of (1) includes unsubstituted lower alkylcarbonyl, substituted lower alkylcarbonyl (substituents are halogen atom, nitro, lower alkoxy, phenyl, etc.), benzoyl, phthalylarylsulfonyl, and the like. Those acyl groups having a phenyl group may have a substituent such as lower alkyl, halogen atom, lower alkoxy, nitro, etc. on the phenyl group. Specific examples of these are acetyl, trifluoroacetyl,
Phenylacetyl, phenylbutanoyl, phenylpropionyl, benzoyl and the like can be mentioned.

Examples of the oxycarboxy group protected by urethanization of (2) include unsubstituted lower alkoxycarbonyl and substituted lower alkyloxycarbonyl (substituents as defined in the above (1)
And the acyl groups described above. ). Specific examples include isopropyloxycarbonyl, t-butoxycarbonyl, isopentyloxycarbonyl, benzyloxycarbonyl, and substituted benzyloxycarbonyl (substituents are the same as those described above for the acyl group). .

In addition to the protecting groups (1) and (2), benzyl, substituted benzyl (substituents may be the same as those described above), o-nitrophenylthio, triphenylmethylthio, triphenylmethyl and the like may be present in some cases. Can be used.

Examples of the saturated or unsaturated hydrocarbon group of (2) for R ₁ include a saturated or unsaturated lower aliphatic hydrocarbon group and an aromatic hydrocarbon group. These may have a substituent. Specific examples of these include a lower alkyl group, a benzyl group and a phenyl group.

Examples of the ester for Y include esters with lower alcohol, benzyl alcohol and the like.

In the present invention, examples of the lower alkyl include C ₁ -C ₅ alkyl such as methyl, ethyl, propyl, butyl and pentyl.

Examples of the lower alcohols methanol, ethanol, propanol, alcohols C ₁ -C _5, such as butanol and pentyl alcohol and the like.

In the general formula [V], R is preferably methyl, ethyl, propyl, benzyl, X is L-valine, benzyloxycarbonyl-valine, benzoyl-valine, phenylacetyl-valine, phenylpropionyl-valine,
Examples include a peptide in which phenylbutanoyl-valine, benzyloxycarbonyl-aspartic acid, benzyloxycarbonyl-lysine, or any of these amino acids is condensed with an amino acid such as valine, phenylalanine, proline, or threonine.

Y is D-leucyl-L-valine, glucyl-valine,
L-leucyl-L-valine and esters thereof, for example, lower alkyl esters such as methyl or t-butyl esters or benzyl esters and the like. Table 1 shows the structure, physicochemical properties and endopeptidase inhibitory activity of the endopeptidase inhibitory peptide produced in the present invention.
Are shown together.

The antiprolyl endopeptidase activity was determined according to the method of Yoshimoto et al. [(Biochim. Piophys. Acta) vol. 569, pp. 184-192 (1
979)]. That is, 0.5 mM N-benzyloxycarbonyl-containing 40% dioxane was placed in a test tube.
L-glycyl-L-prolyl-β-naphthylamide 0.2 m
l, a mixed solution obtained by adding 0.5 ml of 0.05 M Tris-hydrochloric acid buffer (pH 7.8) and 0.25 ml of an aqueous solution containing a specimen was heated at 37 ° C. for 3 minutes, followed by Koida, Walter et al. .] Volume 251, 7593
-775 (1976), a method for purifying an enzyme by the method of fractionating ammonium sulfate from pig kidney,
0.05 ml of a prolyl endopeptidase solution purified by AE-Sephadex A-50 column chromatography was added and reacted at 37 ° C. for 30 minutes. After the reaction, Tween-20 was added to 10%, and Fast Garnet GBC salt was added to 0.1%.
Add 1 ml of 1% sodium acetate buffer (pH 4.2) containing 1%
After standing at room temperature for 15 minutes, the absorbance (a) at 525 nm was measured. At the same time, the absorbance (b) of the blind reaction solution using only the buffer solution containing no sample was measured, and the prolyl endopeptidase inhibition rate was calculated from the formula [(ba) / b] × 100.

The anti-elastase inhibitory activity was determined by the method of Mumford, RA et al. [J.
Biol. Chem. 225, 2227 (1980)], anti-cathepsin B
The inhibitory activity was determined by the method of Knight, CG [Biochem. J. 189, 447
-453 page (1980)].

Rf1, Rf2 = TLC, reverse Phase (RP-18), KOAc 5% -citric acid 1%: MeCN = 65: 35 Rf3 = TLC, silica gel F, n-BuOH: AcOH: H20 = 4: 1: 1 Rf4 = TLC, silica gel F, CHCl3: MeOH = AcOH = 90: 10: 5 PPCE = prolyl endopeptidase inhibitory activity, IC50
(Ug / ml) Elast = elastase inhibitory activity, IC50 (ug / ml) Cat-B = cathepsin B inhibitory activity IC50 (ug / ml) Z = benzyloxycarbonyl Boc = t-butoxycarbonyl PB = phenylbutanoyl PP = phenyl Propionyl PA = phenylacetyl Bz = benzoyl-OBzl = benzyl ester-OBut = t-butyl ester-OMe = methyl ester [Examples] Hereinafter, the present invention will be described specifically by way of examples, but the present invention is not limited thereto. Not something.

Example 1 Synthesis of Boc-D-leucyl-L-valine benzyl ester Boc-D-leucine monohydrate 0.749 g (3.00 mmol),
1.252 g (3.30 mmol) of L-valine benzyl ester / p-toluenesulfonate and 0.811 g (6.00 mmol) of N-hydroxylbenzotriazole are dissolved in 15 ml of dichloromethane, and 0.504 ml (3.60 mmol) of triethylamine is cooled on ice.
And 1.108 g (4.20 mmol) of 97% 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide perchlorate were added, and the mixture was stirred for 2 hours and further stirred at room temperature for 4 hours. After completion of the reaction, 30 ml of water and 5 ml of a 1% aqueous citric acid solution were added, and the precipitated crystals were separated by filtration and separated. The organic layer was washed with 20 ml of water, 20 ml of a saturated aqueous solution of sodium hydrogen carbonate and 10 ml of water, dried over anhydrous sodium sulfate, and evaporated to remove crude Boc.
1.390 g of white crystals of -D-leucyl-L-valine benzyl ester were obtained. Then, the crude crystals were purified by column chromatography (silica gel, ethyl acetate: hexane (5: 2)) to obtain 1.233 g of Boc-D-leucyl-L-valine benzyl ester as white crystals. Yield 9
7.8%. mp 82.0-83.0 ° C. ▲ [α] ²⁹ _D ▼ ＋ 35.1 ゜ 、 ▲
[Α] ²⁹ ₄₃₆ ▼ +76.6 ゜ (c1.02, chloroform).

1H-NMR (CDC13) δ 0.85,0.92 (d, d, 3H, 3H, J = 7Hz, CH 3) 0.90-0.98 (d, d, 3H, 3H, J = 2.7Hz, CH 3) 1.36-1.54 ( m, 10H, (CH ₃ ) ₃ COCO, (CH ₃ ) ₂ CH-CHaHb) 1.62-1.76 (m, 2H, (CH ₃ ) ₂ CH-CHaHb) 2.14-2.27 (m, 1H, (CH ₃ ) ₂ CHCH) 4.14 (br, 1H, NHCHCONH) 4.57 (dd, 1H, J = 9.2, 4.2Hz, CONHCHCOO) 4.79 (br, 1H, NH) 5.13, 5.19 (d, d, 1H, 1H, J = 12.0Hz, _{CH 2 Ph) 6.68 (br,} 1H, NH) 7.29-7.40 (m, 5H, Ph) example 2 instead of in example 1 N- hydroxybenzotriazole N- hydroxysuccinimide 0.693 g (6.02 mmol)
In the same manner as above, 0.710 g of Boc-D-leucyl-L-valine benzyl ester was obtained.

Example 3 Synthesis of Threo (2R, 3S), (2S, 3R) -3- (p-methoxybenzyloxycarbonyl) amino-2-hydroxypentanoic acid Threo (2R, 3S), (2S, 3R) -3- To 5.99 g (45.0 mmol) of amino-2-hydroxypentanoic acid, 15.07 g (49.5 mmol) of p-methoxybenzyl S-4,6-dimethylpyrimidin-2-ylthiolcarboxylate, 25 ml of water, 25 dioxane
ml and triethylamine (9.45 ml, 67.5 mmol) were added, and the mixture was stirred at room temperature for 24 hours. After completion of the reaction, 65 ml of water was added, and the mixture was washed twice with 100 ml of ethyl acetate, and the aqueous layer was cooled with an ice bath. The aqueous layer was adjusted to pH 2 by adding 22 ml of 5N hydrochloric acid, and extracted once with 65 ml of ethyl acetate and twice with 30 ml of ethyl acetate. Wash the oil layer with 50 ml of 5% hydrochloric acid 3
And twice with 50 ml of saturated saline, dried over anhydrous sodium sulfate, and evaporated to remove threo (2R, 3S).
9.65 g of (2S, 3R) -3- (p-methoxybenzyloxycarbonyl) amino-2-hydroxypentanoic acid was obtained. Yield 72.1%.

Example 4 Synthesis of erythro (2R, 3S), (2S, 3R) -3- (p-methoxybenzyloxycarbonyl) amino-2-hydroxypentanoic acid Threo (2R, 3S), (2S, 3R) of Example 3 ) -3-Amino-2-hydroxypentanoic acid instead of erythro (2R, 3
S), (2S, 3R) -3-amino-2-hydroxypentanoic acid (5.99 g, 45.0 mmol) was used, and erythro (2R,
3S), (2S, 3R) -3- (p-metroxybenzyloxycarbonyl) amino 2-hydroxypentanoic acid 10.76 g
I got

Example 5 Synthesis of Z (OMe)-(2R, 3S) -3-amino-2-hydroxypentanoyl-D-leucyl-L-valine benzyl ester Boc-D-leucyl-L-valine benzyl ester 18
2 ml of trifluoroacetic acid was added to 9.2 mg (0.450 mmol), the mixture was stirred at room temperature for 40 minutes, the solvent was distilled off, and 2 ml of toluene was further added and the solvent was distilled off twice to obtain D-leucyl as white crystals. -L-valine benzyl ester trifluoroacetate was obtained. Here, (2R, 3S) -3- (p
-Methoxybenzyloxycarbonyl) amino-2-hydroxypentanoic acid 133.8 mg (0.450 mmol), N-hydroxybenzotriazole 91.2 mg (0.675 mmol) and DMF
Add 2 ml to dissolve, and add N-methylmorpholine 50.
4 μ (0.450 mmol) and 97% 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide perchlorate 14
8.3 mg (0.563 mmol) was added, and the mixture was stirred for 2 hours, and further stirred at room temperature overnight. After completion of the reaction, the reaction mixture was diluted with 30 ml of dichloromethane, and 10 ml of a 1% aqueous citric acid solution, 10 ml of water,
The extract was washed successively with 10 ml of a saturated aqueous solution of sodium hydrogencarbonate and 10 ml of water, dried over anhydrous sodium sulfate, and evaporated to give an oily crude Z (OMe)-(2R, 3S) -3-amino-2-.
316.5 mg of hydroxypentanoyl-D-leucyl-L-valine benzyl ester were obtained. Then, the crude product was purified by column chromatography (silica gel, ethyl acetate: hexane (5: 1)) to give Z as white crystals.
(OMe)-(2R, 3S) -3-amino-2-hydroxypentanoyl-D-leucyl-L-parin benzyl ester
197.6 mg were obtained. Yield 73.2%. mp 115.0-115.5 ° C.

▲ [α] ²⁵ _D ▼ + 45.5 ゜, ▲ [α] ²⁵ ₄₃₆ ▼ + 102.9 ゜ (c
1.04, chloroform). 1H-NMR (CDC13) δ 0.80-1.04 (m, 15H, CH ₃ × 5) 1.51-1.80 (m, 5H, CH ₂ × 2, CH (Leu)) 2.19 (m.1H, CH (Val)) 3.70 −3.85 (m, 4H, CH ₃ O, CH (EtAhp)) 4.12 (dd, 1H, J = 6.4, 3.0 Hz, CHOH) 4.52 (dd, 1H, J = 8.4, 4.8 Hz, CH (Val)) 4.49 (m, 1H, CH (Leu )) 4.61 (br d, 1H, J = 6.4Hz, OH) 4.98 (s, 2H, CH 2 -C 6 H 4) 5.08,5.18 (d, d, 2H, J = 12.4Hz, CH ₂ Ph) 5.31 (br d, 1H, J = 8.4Hz, NH (EtAhp)) 6.79 (br d, 1H, J = 8.4Hz, NH (Val)) 6.84-6.92 (m, 2H, C _{6 H 4) 7.07 (br d} , 1H, J = 8.4Hz, NH (Leu)) 7.22-7.40 (m, 7H, C 6 H 4, Ph) except EtAhp implementation of 3-amino-2-hydroxy pentanoic acid example 6 Synthesis of Boc-L-valyl- (2R, 3S) -3-amino-2-hydroxypentanoyl-D-leucyl-L-valine benzyl ester (OMe)-(2R, 3S) -3-amino-2- Hydroxypentanoyl-D-leucyl-L-valine benzyl ester (190.2 mg, 0.317 mmol) Added 0.1ml of trifluoroacetic acid 2 ml, after stirring for 40 minutes at room temperature, the operation of the solvent was distilled off, further evaporated with toluene 2 ml 2
Times to form a white amorphous (2R, 3S) -D
-Leucyl-L-valine benzyl ester / trifluoroacetate (243.6 mg) was obtained. To 219.2 mg (corresponding to 0.258 mmol) of this salt, 65.4 mg (0.301 mmol) of Boc-L-valine, 58.1 mg (0.430 mmol) of N-hydroxylbenzotriazole and D
MF (2 ml) was added to dissolve the mixture, and N-methylmorpholine (32 µ (0.286 mmol) and 97% 1-ethyl-3- (3
-Dimethylaminopropyl) carbodiimide / perchlorate (94.0 mg, 0.357 mmol) was added, and the mixture was stirred for 2 hours and further stirred at room temperature overnight. After completion of the reaction, the reaction mixture was diluted with 30 ml of dichloromethane, and 10 ml of 1% aqueous citric acid solution and 10 ml of water were added.
The mixture was washed successively with 10 ml of a saturated aqueous solution of sodium hydrogencarbonate and 10 ml of water, dried over anhydrous sodium sulfate, and evaporated to give an oily crude Boc-L-valyl- (2R, 3S) -3-amino-2-amine. Hydroxypentanoyl-D-leucyl-L-
520.8 mg of valine benzyl ester were obtained.

Then, the crude product was purified by column chromatography (silica gel, dichloromethane: methanol (24: 1)).
By doing so, Boc-L-valyl- (2R.3
S) -3-Amino-2-hydroxypentanoyl-D-
149.7 mg of leucyl-L-valine benzyl ester were obtained. 82.8% yield. mp 167.0-168.0 ° C.

▲ [α] ²⁶ _D ▼ + 35.7 ゜, ▲ [α] ²⁶ ₄₃₆ ▼ + 81.0 ゜ (c
1.00, chloroform). 1H-NMR (CDC13) δ 0.80-1.03 (m, 21H, CH ₃ × 7) 1.44 (S, 9H, (CH ₃ ) ₃ COCO) 1.52-1.90 (m, 5H, CH ₂ × 2, CH (Leu) 2.11 (m, 1H, CH (Val)) 2.19 (m, 1H, CH (Val)) 3.87 (m, 1H, CHNH (EtAhp)) 3.93 (dd, 1H, J = 5.8, 8.5 Hz, CH (Val) )) 4.10 (dd, 1H, J = 3.4, 7.0Hz, CHOH) 4.36 (m, 1H, CH (Leu)) 4.58 (dd, 1H, J = 5.0, 8.6Hz, CH (Val)) 5.09 (br, 1H, NH (Val)) 5.10,5.20 (d, d, 2H, J = 12.2Hz, CH ₂ Ph) 5.36 (br, 1H, OH) 6.98 (br d, 1H, J = 8.6Hz, NH (Val) ) 7.04 (br d, 1H, J = 8.4 Hz, NH (EtAhp)) 7.20 (br d, 1H, J = 8.4 Hz, NH (Leu)) 7.30-7.40 (m, 5H, Ph) However, EtAhp is 3- Amino-2-hydroxypentanoic acid Example 7 Synthesis of ZL-valyl-L-valyl- (2R, 3S) -3-amino-2-hydroxypentanoyl-D-leucyl-L-valine benzyl ester Boc-L -Valyl- (2R, 3S) -3-amino-2-hydroxypentanoyl-D-leucyl-L-vari Trifluoroacetic acid 2m benzyl ester 145.0 mg (0.228 mmol)
After stirring at room temperature for 40 minutes, the solvent is distilled off, and the operation of adding 2 ml of toluene and distilling off the solvent is performed twice to obtain L-valyl- (2R, 3S) -3-amino acid as white crystals. -2-Hydroxypentanoyl-D-leucyl-L-valine benzyl ester trifluoroacetate was obtained. The salt was made up to 10 ml with ethyl acetate, and the residue after sampling 1 ml was evaporated (corresponding to 0.206 mmol). Here, 54.8 mg (0.218 mmol) of ZL-valine, 42.4 mg (0.314 mmol) of N-hydroxylbenzotriazole and 1.
5 ml was added and dissolved, and N-methylmorpholine 23 was added under ice cooling.
μ (0.206 mmol) and 97% 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide perchlorate 6
After adding 8.0 mg (0.258 mmol) and stirring for 2 hours, the mixture was further stirred at room temperature overnight. After completion of the reaction, the reaction mixture was diluted with 15 ml of dichloromethane, washed successively with 7 ml of 1% aqueous citric acid solution, 7 ml of water, 7 ml of saturated aqueous sodium hydrogen carbonate solution and 7 ml of saturated saline, dried over anhydrous sodium sulfate, and evaporated. Thus, 277.9 mg of crude ZL-valyl-L-valyl- (2R, 3S) -3-amino-2-hydroxypentanoyl-D-leucyl-L-valine benzyl ester was obtained as a partially crystalline amorphous material. Then, the crude product was purified by column chromatography (silica gel, dichloromethane: methanol (40: 1)) to give ZL as white crystals.
-Valyl-L-valyl- (2R.3S) -3-amino-2-
125.9 mg of hydroxypentanoyl-D-leucyl-L-valine benzyl ester were obtained. Yield 79.7%.

mp 222.0-223.0 ° C. ▲ [α] ²⁷ _D ▼ + 9.2 ゜, ▲ [α] ²⁷
₄₃₆ ▼ + 24.4 ゜ (c1.01, chloroform). SI-MS m / z
768 (M + 1 +), 790 (M + Na +). 1H-NMR (CDC1
3) δ 0.76-1.00 (m, 27H, CH ₃ × 9) 1.37 (m, 1H, (CH ₃ —CHaHb) 1.53-1.85 (m, 4H, (CH ₃ ) ₃ CHCH ₂ , CH ₃ HaHb) 1.93− 2.10 (m, 2H, CH × 2 (Val)) 2.17 (m, 1H, CH (Val)) 4.13-4.27 (m, 2H, HNCHCH (OH) CO) 4.41-4.64 (m, 4H, CH × 3 ( Val), CH (Leu)) 5.06,5.15 (d, d, 2H, J = 12.0Hz, COOCH ₂ Ph) 5.06,5.09 (d, d, 2H, J = 12.0Hz, PhCH ₂ OCO) 5.49 (br s , 1H, OH) 5.86 (br, 1H, NH (Val)) 7.09 (br, 1H, NH (Val)) 7.22 (br, 1H, NH (EtAhp)) 7.22-7.39 (m, 10H, Ph × 2) 7.45 (br, d, 1H, J = 7.8 Hz, NH (Leu)) 7.94-8.23 (br, 1H, NH (Val)) where EtAhp is 3-amino-3-hydroxypentanoic acid Example 8 ZL- Valyl-L-valyl- (3S) -3-amino-2
Synthesis of -oxopentanoyl-D-leucyl-L-valine benzyl ester ZL-valine-L-valyl- (2R, 3S) -3-amino-2-hydroxypentanoyl-D-leucyl-L-
19.9 mg (0.103 mmol) of pyridine trifluoroacetate was added to 108.1 mg (0.141 mmol) of valine benzyl ester, 97% 1
-Ethyl-3- (3-dimethylaminopropyl) carbodiimide perchlorate 116.2 mg (0.441 mmol), DMSO 0.
5 ml and 2 ml of benzene were added, and the mixture was stirred at room temperature for 24 hours. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (15 ml) and water (5 m).
and dried over anhydrous sodium sulfate, and then the solvent was distilled off to obtain a partially gelled crude ZL-valyl-L-valyl- (3S) -3-amino-2-oxopentanoyl-D −
Leucyl-L-valine benzyl ester was obtained. Then, the crude product was subjected to thin layer chromatography (silica gel,
Purification with dichloromethane: methanol (15: 1) yielded ZL-valyl-L-valyl- (3
63.2 mg of S) -3-amino-2-oxopentanoyl-D-leucyl-L-valine benzyl ester were obtained. yield
58.6%.

mp 201.0-202.0 ° C. ▲ [α] ²⁶ _D ▼ + 8.4 ゜, ▲ [α] ²⁶
₄₃₆ ▼ +50.3 ゜ (c0.90, chloroform).

1H-NMR (CDC13) δ 0.77-1.04 (m, 27H, CH ₃ × 9) 1.50-1.85 (m, 4H, CH ₂ CH, CH ₃ —CHaHb) 2.00 (m, 1H, CH ₃ —CHaHb) 2.05- 2.25 (m, 3H, CH × 3 (Val)) 4.06 (m, 1H, CH (Val)) 4.32 (dd, 1H, J = 7.4 Hz, 8.6 Hz, CH (Val)) 4.47−4.63 (m, 2H) , CH (Val), CH ( Leu)) 5.08,5.16 (d, d, 2H, J = 12.0Hz, CH 2 -Ph) 5.10,5.11 (d, d, 2H, CH 2 -Ph) 5.34 (ddd, 1H, J = 4.8,8.2,8.2Hz, CH (EtAop)) 5.57 (br, d, 1H, J = 8.6Hz, NH (Val)) 6.69 (br, d, 1H, J = 8.6Hz, NH (Val) )) 6.74 (br, d, 1H, J = 9.1 Hz, NH (Val)) 6.86 (br, d, 1H, J = 8.2 Hz, NH (EtAhp)) 7.25-7.40 (m, 10H, Ph x 2) 7.48 (brd, 1H, J = 8.8 Hz, NH (Leu)) where EtAhp is 3-amino-2-hydroxypentanoic acid Example 9 Poststatin (L-valyl-L-valyl- (3S) -3
-Amino-2-oxopentanoyl-D-leucyl-L
-Valin) ZL-valyl-L-valyl- (3S) -3-amino-
4-6.9 mg (0.061 mmol) of 2-oxopentanoyl-D-leucyl-L-valine benzyl ester was added to palladium black 6.6.
mg and 12 ml of methanol were added, and the inside of the system was replaced with hydrogen, followed by catalytic reduction at room temperature for 20.5 hours. After the catalyst was filtered off, the filtrate was evaporated to give 40.0 mg of white crystals. 39.7mg of this
HPLC (column: CAPCELL PAK C18, SHISEIDO, 20 ×
250 mm, after elution: 1% citric acid, 0.65% potassium acetate aqueous solution: acetonitrile = 8: 2, flow rate: 8 ml / min, detection wavelength: 25
4 nm), L-valyl-L-valyl- produced by unreacted starting material and partial epimerization
(3R) -3-Amino-2-hydroxypentanoyl-D
-Leucyl-L-valine was separated, and a fraction containing L-valyl-L-valyl- (3S) -3-amino-2-hydroxypentanoyl-D-leucyl-L-valine was collected. After concentrating this fraction at 25 ° C,
The resin was washed with water and eluted with 80% methanol. The solvent was distilled off from the eluate and dried overnight with a vacuum pump to obtain 15.2 mg of poststatin as a white powder. In this poststatin, impurities derived from the CHP20 resin were observed by NMR.
The mixture was eluted with vol% methanol, the eluate was evaporated, and the resulting solid was washed with ether by decantation.
By drying with a vacuum pump, 2.4 mg of white powdered poststatin was obtained. Yield 21.7%. SI-MS m / z 542 (M + 1
+).

1H-NMR (DMSO-d6) δ 0.70-0.95 (m, 27H, CH ₃ × 9) 1.40-1.67 (m, 3H, CHaHbCH (Leu), CH ₃ -CHaHb) 1.61 (m, 1H, CHaHbCH (Leu) ) 1.76 (m, 1H, CH 3 -CHaHb) 1.86-2.10 (m, 3H, CH (Val)) 3.20 (m, 1H, CH (Val)) 4.06 (m, 1H, CH (Val)) 4.27 (m , 1H, CH (Val)) 4.43 (m, 1H, CH (Leu)) 4.95 (m, 1H, CHCOCO) 7.98 (br d, 1H, J = 8.4Hz, NH (Val)) 8.10 (br d, 1H , J = 8.9Hz, NH (Val)) 8.36 (d, 1H, J = 6.6Hz, NH (EtAhp)) 8.56 (d, 1H, J = 8.7Hz, NH (Leu)) where EtAhp is 3-amino- 2-hydroxypentanoic acid However, there is a broad peak at δ 2.80-3.90 derived from water (including amino group and carboxyl group).

Example 10 Synthesis of ZD-leucyl-L-valine t-butyl ester 1.000 g (3.77 mmol) of ZD-leucine, L-valine t
-Butyl ester hydrochloride 0.719 (3.43 mmol) and N-
0.927 g (6.00 mmol) of hydroxylbenzotriazole was dissolved in 17 ml of dichloromethane, and 0.530 ml (3.79 mmol) of triethylamine and 97% 1-ethyl-3- (3
After adding 1.265 g (4.80 mmol) of -dimethylaminopropyl) carbodiimide perchlorate, the mixture was stirred for 2 hours, and further stirred at room temperature for 14 hours. After completion of the reaction, a saturated aqueous solution of sodium hydrogen carbonate 7 ml, water 7 ml, a 1% aqueous citric acid solution 7 ml and water 7 ml
And dried over anhydrous sodium sulfate, and then the solvent is distilled off to obtain an oily crude ZD-leucyl-L-valine t.
1.932 g of butyl ester were obtained. Then, the crude crystals were purified by column chromatography (silica gel, dichloromethane: methanol (400: 3)) to obtain 1.267 g of ZD-leucyl-L-valine t-butyl ester as white crystals.

87.9% yield.

▲ [α] ²⁸ ₄₃₆ ▼ + 66.3 ゜, ▲ [α] ²⁸ _D ▼ + 31.1 ゜ (c
1.06, chloroform).

Example 11 Erythro (2R, 3R), (2S, 3S) -Z (OMe) -3-amino-2-hydroxypentanoyl-D-leucyl-L-
Synthesis of valine t-butyl ester ZD-leucyl-L-valine t-butyl ester 50
21.2 mg of palladium black and 5 ml of methanol were added to 3.9 mg (1.198 mmol), and the inside of the system was replaced with hydrogen and then catalytically reduced at room temperature for 3 hours. After removing the catalyst by filtration, the filtrate was evaporated to obtain oily D-leucyl-L-valine t-butyl ester. Here, erythro (2R, 3R), (2S, 3S) -3- (p
-Methoxybenzyloxycarbonyl) amino-2-hydroxypentanoic acid (391.8 mg, 1.318 mmol), N-hydroxylbenzotriazole (324.1 mg, 2.399 mmol) and dichloromethane (8 ml) were added and dissolved. 3- (3-Dimethylaminopropyl) carbodiimide perchlorate (442.1 mg, 1.677 mmol) was added, and the mixture was stirred for 2 hours and further stirred at room temperature overnight. After completion of the reaction, the reaction mixture was diluted with a saturated aqueous solution of sodium hydrogen carbonate (8 ml), water (8 ml), 1%
The extract was washed successively with 8 ml of citric acid aqueous solution and 8 ml of water, dried over anhydrous sodium sulfate, and evaporated to remove crude erythro (2R, 3R), (2S, 3S) -Z (OMe) -3-. 728.1 mg of amino-2-hydroxypentanoyl-D-leucyl-L-valine t-butyl ester were obtained. Then, the crude product was purified by column chromatography (silica gel, dichloromethane: methanol (100: 1)) to obtain an erythro (2R, 3R), (2S, 3S)-as an amorphous solid.
Z (OMe) -3-amino-2-hydroxypentanoyl-D-leucyl-L-valine t-butyl ester 666.9m
g was obtained. 98.4% yield. SI-MS m / z 566 (M + 1
+).

Example 12 ZL-phenylalanyl (2RS, 3RS) -3-amino-
Synthesis of 2-hydroxypentanoyl-D-leucyl-L-valine t-butyl ester Erythro (2R, 3R), (2S, 3S) -Z (OMe) -3-amino-2-hydroxypentanoyl-D-leucyl -L
-Valine t-butyl ester (340.9 mg, 0.603 mmol) was dissolved in methanol (4 ml), palladium black (50.7 mg) was added in three portions every few hours, and the mixture was subjected to catalytic reduction in a hydrogen atmosphere at room temperature for 32 hours. After removing the catalyst by filtration, the filtrate was evaporated to remove erythro (2R, 3R), (2S, 3S) -3-amino-2.
-Hydroxypentanoyl-D-leucyl-L-valine t-butyl ester was obtained. Half of this amount is ZL
-100.8 m (0.337 Lmmo) of phenylalanine, 81.9 mg (0.606 mmol) of N-hydroxylbenzotriazole and 10 ml of dichloromethane were added, and 97% 1-ethyl- was added under ice cooling.
3- (3-dimethylaminopropyl) carbodiimide.
After adding 111.3 mg (0.422 mmol) of perchlorate and stirring for 2 hours,
Stirred at room temperature for another 18 hours. After addition of 2.0 ml of DMF and further stirring for 22.5 hours, the reaction mixture was diluted with 9 ml of a saturated aqueous solution of sodium hydrogen carbonate, 9 ml of water, 9 ml of a 1% aqueous solution of citric acid and 9 ml of water.
And dried over anhydrous sodium sulfate, and the solvent was distilled off to obtain an oily crude ZL-phenylalanyl- (2R
(S, 3RS) -3-Amino-2-hydroxypentanoyl-
D-leucyl-L-valine t-butyl ester 255.6 mg
I got This crude product was used for the next reaction without purification.

Example 13 Synthesis of ZL-phenylalanyl- (RS) -3-amino-2-oxopentanoyl-D-leucyl-L-valine t-butyl ester Crude ZL-phenyl obtained in Example 12 Alanyl-
(2RS, 3RS) -3-Amino-2-hydroxypentanoyl-D-leucyl-L-valine benzyl ester 255.6m
g to pyridine / trifluoroacetate 25.8mg (0.134mmo
l), 97% 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide perchlorate 211.6 mg (0.803 mmo
l), 0.65 ml of DMSO and 2 ml of benzene were added, and the mixture was stirred at room temperature for 20 hours. After the completion of the reaction, the yellow reaction mixture was diluted with 6.5 ml of ethyl acetate, washed with 7 ml of water, dried over anhydrous sodium sulfate, and the solvent was distilled off.
244.4 mg of -L-phenylalanyl- (RS) -3-amino-2-oxopentanoyl-D-leucyl-L-valine t-butyl ester were obtained. Then, the crude product was purified by column chromatography (silica gel, dichloromethane: methanol (100: 1)) to obtain an amorphous solid of ZL-phenylalanyl- (RS) -3-amino-2-oxopentane. 113.7 mg of Noyl-D-leucyl-L-valine t-butyl ester were obtained. Yield 63.5%. SI-M
S m / z 681 (M + 1 +).

Example 14 ZL-phenylalanyl- (RS) -3-amino-2-
Synthesis of oxopentanoyl-D-leucyl-L-valine ZL-phenylalanyl- (RS) -3-amino-2
-Oxopentanoyl-D-leucyl-L-valine t-
To 105.9 mg (0.156 mmol) of butyl ester, 2 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 90 minutes. The solvent was distilled off, and 2 ml of toluene was further added to distill off the solvent. -L-phenylalanyl- (RS) -3-amino-2-oxopentanoyl-D
-Leucyl-L-valine (106.5 mg) was obtained. Then, the crude product was purified by column chromatography (LH-20, methanol) to obtain Zr as an amorphous solid white crystal.
70.5 mg of -L-phenylalanyl- (RS) -3-amino-2-oxopentanoyl-D-leucyl-L-valine were obtained. Yield 72.6%. SI-MS m / z 625 (M + 1 +), 64
7 (M + Na +).

Example 15 Synthesis of Z (OMe)-(2RS, 3RS) -3-amino-2-hydroxypentanoyl-D-leucine t-butyl ester 0.896 g of D-leucine t-butyl ester hydrochloride (4.
(00 mmol) to (2RS, 3RS) -3- (p-methoxybenzyloxycarbonyl) amino-2-hydroxypentanoic acid
1.309 g (4.40 mmol), 1.081 g (8.00 mmol) of N-hydroxylbenzotriazole and 20 ml of dichloromethane were added, and 0.62 ml (4.43 mmol) of triethylamine and ice-cooled were added.
97% 1-ethyl-3- (3-dimethylaminopropyl)
1.476 g (5.60 mmol) of carbodiimide / perchlorate was added, and the mixture was stirred for 2 hours and further stirred at room temperature for 15 hours. After completion of the reaction, the reaction mixture was washed with a saturated aqueous solution of sodium hydrogencarbonate 8 ml,
After washing sequentially with 8 ml of water, 8 ml of 1% citric acid aqueous solution and 8 ml of water, drying over anhydrous sodium sulfate and distilling off the solvent, crude Z (OMe)-(2RS, 3RS) -3-amino-
2.122 g of 2-hydroxypentanoyl-D-leucine t-butyl ester were obtained. Then, the crude product was purified by column chromatography (silica gel, dichloromethane: methanol (100: 1)) to give Z (O
1.858 g of Me)-(2RS, 3RS) -3-amino-2-hydroxypentanoyl-D-leucine t-butyl ester were obtained. 99.6% yield. SI-MS m / z 467 (M + l).

Example 16 Synthesis of Boc-L-valyl- (2RS, 3RS) -3-amino-2-hydroxypentanoyl-D-leucine t-butyl ester Erythro (2R, 3R), (2S, 3S) -Z (OMe ) -3-Amino-2-hydroxypentanoyl-D-leucine t-
303.9 mg (0.651 mmol) of butyl ester in 4 ml of methanol
And 43.4 mg of palladium black was added in three portions every few hours, followed by catalytic reduction in a hydrogen atmosphere at room temperature for 15 hours. After the catalyst was filtered off, the filtrate was evaporated to give erythro (2R, 3R), (2S, 3S) -3-amino-2-hydroxypentanoyl-D-leucine t-butyl ester. Here, Boc-L-valine 155.9m (0.718mmol), N-
176.1 mg (1.303 mmol) of hydroxylbenzotriazole
And 3.5 ml of dichloromethane, 499.4 mg (2.605 mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added under ice cooling, and the mixture was stirred for 2 hours.
Stirred at room temperature for an additional 16.5 hours. After diluting the reaction mixture with 7 ml of dichloromethane, the mixture was diluted with 6 ml of 1% aqueous citric acid solution and water.
6 ml, washed successively with 6 ml of a saturated aqueous solution of sodium hydrogencarbonate and 6 ml of water, dried over anhydrous sodium sulfate and evaporated to remove the crude crude Boc-L-valyl- (2RS, 3
RS) -3-Amino-2-hydroxypentanoyl-D-
Leucine t-butyl ester was obtained. This crude product was used for the next reaction without purification.

Example 17 Synthesis of Boc-L-valyl- (RS) -3-amino-2-oxopentanoyl-D-leucyl t-butyl ester Boc-L-valyl- (2RS, 3RS) -3-amino-2- Hydroxypentanoyl-D-leucyl t-butyl ester (0.651 mmol) and pyridine / trifluoroacetate (69.5 mg)
(0.360 mmol), dicyclohexylcarbodiimide 408.8
mg (1.982 mmol), 1.3 ml of DMSO and 2.5 ml of benzene were added, and the mixture was stirred at room temperature for 18 hours. After completion of the reaction, the yellow reaction mixture was diluted with 10 ml of ethyl acetate, and the generated DCC urea was separated by filtration. The organic layer was washed with 10 ml of water, dried over anhydrous sodium sulfate, and evaporated. Ethyl acetate (5 ml) was again added to the obtained oil, and the precipitated DCC urea was separated by filtration. The filtrate was evaporated to give crude Boc-L-valyl- (RS) -3-.
Amino-2-oxopentanoyl-D-leucyl t-butyl ester-L-valine benzyl ester was obtained. Then, the crude product was purified by column chromatography (silica gel, dichloromethane: methanol (100: 1), dichloromethane: ethyl acetate (12: 1 to 10: 1) to give Boc-L-valyl- (white crystals) as white crystals. 178.9 mg of RS) -3-amino-2-oxopentanoyl-D-leucyl t-butyl ester were obtained, SI-MS m / z 534 (M + 1).
+).

Example 18 Synthesis of (2R, 3S) -3- (9-fluorenylmethyloxycarbonyl) amino-2-hydroxypentanoic acid (2R, 3S) -3-Amino-2-hydroxypentanoic acid
To 0.759 g (5.70 mmol), 1.199 g (1
Aqueous solution of 4.28 mmol) in 20 ml of water and dioxane 20
and add 9-fluorenylmethyl chloroformate under ice-cooling.
A solution prepared by dissolving 1.623 g (6.27 mmol) in 10 ml of dioxane was added to 30
Dropped in minutes. After completion of the dropwise addition, the mixture was stirred at room temperature for 2 hours, and diluted with 100 ml of water.
Washed once with 75 ml each. Add 2.8 ml of 6N hydrochloric acid to the aqueous layer and adjust the pH.
After adjusting to 1.5, the mixture was extracted three times with 50 ml of ethyl acetate. The oil layer was washed with 50 ml of saturated saline, dried over anhydrous sodium sulfate, and the solvent was distilled off to obtain crude (2R, 3S) -3- (9-fluorenylmethyloxycarbonyl) amino-2-hydroxypentanoic acid. 1.622g was obtained. Ethyl acetate
After crystallizing by adding 4 ml and n-hexane 4 ml, the crystals are collected by filtration, washed with ethyl acetate-n-hexane (1: 1) solution 2 ml, and dried to give a white solid (2R, 3S)-. 3- (9
-Fluorenylmethyloxycarbonyl) amino-2-
0.999 g of hydroxypentanoic acid was obtained. mp 160-161.5 ° C.
Then, 0.999 g of the N-protected amino acid was added to 4 ml of ethyl acetate.
And stirred for 40 minutes while suspending at a bath temperature of 50 ° C.
-Hexane (4 ml) was added, and the crystals were collected by filtration. The crystals were washed with 2 ml of an ethyl acetate-n-hexane (1: 1) solution, and dried to give (2R, 3S) -3- (9-fluorenylmethyloxycarbonyl) amino-2-hydroxypentane as white crystals. 0.866 g of acid was obtained. Recrystallization yield 86.7%.

▲ [α] ²⁶ _D ▼ -30.2 ゜, ▲ [α] ²⁶ ₄₃₆ ▼ -63.0 ゜ (c
1.01, methanol). mp 162-1650C. FAB-MS m / z
356 (M + 1 +).

1H-NMR (CDC13) δ 0.96 (dd, 3H, J = 7.4,7.4Hz, CH 3) 1.69 (m, 2H, CH 2) 3.25 (br, COOH, OH) 4.01 (m, 1H, CHNH) 4.18 ( dd, 1H, J = 6.6,6.6Hz, ArCHAr) 4.24 (br s, 1H, CHOH) 4.40,4.41 (d, d, 1H, 1H, J = 6.6,6.6Hz, ArCHCHaHb) 5.13 (d, 1H, J = 9.6 Hz, NH) 7.20-7.80 (m, 8H, Ar) On the other hand, the amorphous 0.548 obtained by distilling off the solvent from the crystallization filtrate and the washing solution was purified by column chromatography (silica gel, chloroform: methanol: Acetic acid (95: 5: 1)) to give (2R, 3S)-
0.307 g of 3- (9-fluorenylmethyloxycarbonyl) amino-2-hydroxypentanoic acid was obtained.

Example 19 Synthesis of Boc-L-Val-L-Val-L-3-amino-2-hydroxypentanoyl-D-Leu-L-Val resin 0.5 g of Fmoc-L-valyl-p-alkoxybenzyl alcohol resin (Containing 0.345 mmol valine) (purchased from Watanabe Chemical) with dimethylformamide (3 x 6 ml x 1 min)
Swelled in.

Next, 6 ml of 20% piperidine / DMF was added and the mixture was stirred for 3 minutes. After the waste liquid, the piperidine treatment was repeated twice (the second time was stirred for 3 minutes, and the third time was stirred for 20 minutes). After waste liquid, DMF (3 × 6ml × 1
Minutes). Then N-methylpyrrolidone (3 × 6 ml
X 1 minute). Next, Fmoc-D-Leu366mg (3 equivalents), HoBt 140mg (3 equivalents), and diisopropylcarbodiimide 0.265ml (3 equivalents) were dissolved in 6ml of N-methylpyrrolidone and stirred for 3 hours. After the waste liquid, wash with N-methylpyrrolidone (3 × 6 ml × 1 minute), and after the waste liquid, DMF (3 ×
(6 ml x 1 minute).

Next, piperidine deprotection was performed as described above, and Fmoc
368 mg of -3-amino-2-hydroxy-pentanoic acid, HOB
140 mg of t and 0.265 ml (3 equivalents) of diisopropylcarbodiimide were dissolved in 6 ml of N-methylpyrrolidone, and the mixture was stirred for 3 hours. After washing and piperidine deprotection and washing in the same manner, Fmoc-L-Val (351 mg) and Boc-L-Val (22
5 mg) were condensed.

Example 20 Oxidation of Boc-L-Val-L-Val-L-3-amino-2-hydroxypentanoyl-D-Leu-L-Val resin After washing the above resin with DMF (3 × 6 ml × 1 minute) ) After washing with DMSO (3 x 6 ml x 1), 125 mg of pyridine-trifluoroacetate and 0.45 ml of acetic anhydride dissolved in 6 ml of DMSO were added, and the mixture was stirred for 18 hours. DMSO after waste liquid (3 x 6 ml x 1 min), D
After washing sequentially with MF (3 × 6 ml × 1 minute) and methanol (3 × 6 ml × 1 minute), it was dried under vacuum for 2 hours.

Example 21 Synthesis of L-Val-L-Val-L-3-amino-2-oxopentanoyl-D-Leu-L-Val The dry resin of Example 20 was purified using TFA: phenol 95: 5 (3 ×
(6 ml × 60 minutes). The solution resulting from this treatment was evaporated in vacuo and the residue was dissolved in 3 ml of methanol and applied to a column of Sephadex LH-20 (300 ml) swollen with methanol. Developed with methanol, the first eluting fractions exhibiting UV absorption were collected and evaporated in vacuo. The obtained residue (139 mg) was dissolved in an upper layer (1 ml) and a lower layer (1 ml) of a mixture of n-butanol, acetic acid and water 100: 1: 100, and subjected to centrifugal countercurrent chromatography manufactured by Miki Engineering Co., Ltd. to perform chromatography using the lower layer as a mobile phase. The fractions exhibiting UV absorption were collected and evaporated in vacuo to give the title compound as a colorless powder, 82.9.
mg was obtained.

Example 22 In the same manner as in Examples 19, 20, and 21, using 0.5 g of Fmoc-L-valyl resin as a starting material, 3-fold equivalents of Fmoc-amino acid were sequentially condensed, and then oxidized and deprotected to obtain L-valyl. -L-
Prolyl-3-amino-2-oxopentanoyl-D-
124.6 mg of leucyl-L-valine were obtained. Physical properties are shown in Table 1.

Example 23 23.6 mg of L-valyl-L-prolyl-3-amino-2-oxopentanoyl-D-leucyl-L-valine obtained in Example 22 was dissolved in 2 ml of methanol, and 30 mg of 4-phenylbutanoic anhydride was obtained. Of methylene chloride and 0.050 ml of triethylamine were added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was acidified with acetic acid, and Sephadex LH20 filled with methanol.
And purified using 20.9 mg of phenylbutanoyl-L-valyl-L-prolyl-3-amino-
2-oxopentanoyl-D-leucyl-L-valine was obtained.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // A61K 38/55 A61K 37/64 (72) Inventor Machiko Nagai 1-7-10 Chuocho, Meguro-ku, Tokyo Daikawabe 2-207 (58) Fields investigated (Int. Cl. ⁶ , DB name) C07B 41/06 C07C 227/00-233/00

Claims (1)

(57) [Claims] 1. A compound of the general formula [IV] (Wherein, R ₁ represents a saturated or unsaturated hydrocarbon group;
Represents a residue obtained by removing OH from the carboxyl group of an amino acid or a peptide; when X has a functional group, it may be protected; and Y represents a residue obtained by removing hydrogen from the amino group of an amino acid or a peptide or an ester thereof. Y represents a group
When has a functional group, it may be protected by a protecting group. A method for producing a 3-amino-2-oxo fatty acid derivative represented by the following formula: (Wherein X ′ and Y ′ represent X and Y each having a protected functional group, where X and Y are the same as above, and R ₁ is the same as above.) Wherein the protective group of the functional group is removed, if necessary, to produce a 3-amino-2-oxo fatty acid derivative.