JPS6034938A - Alpha-aminofluoroketone - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates generally to fluoroketones, and more particularly to fluoroketones.
-Relating to aminofluoroketones.

Proteases are an important class of enzymes that are involved in many diseases,
For example, but not exclusively, it is associated with pulmonary emphysema: Mitrnen, C-ed. (1972), Pulmonary Emphysema an.
d Proteolysis +Academic P
ress, New Y'ork;'I'uri
no, G-M-.

Rodriguez, J. R., + Greenb.
aum, LoM-, and Mandl, I, (1
974), Am, J. Med, 57,493: and Hance, A. J., & Crysta.
l, )(, Cr, (1975).

Am, R, Re5p, Disease 112, 65
See 7. Leukocyte elastase is believed to be responsible for much of the tissue destruction in emphysema. Different proteases are involved in the same or similar pathologies. Another protease, cathepsin G, is present in the lungs and has the ability to digest elastin fibers.

Protease inhibitors (including naturally occurring and synthetic) act on the reactive sites of proteases to inhibit their enzymatic activity.

In recent years, researchers have reported the synthesis of many synthetic inhibitors. Peptide chloromethyl ketones (α-amino-chloromethyl-ketones) have been synthesized and some of these compounds have been found to inhibit porcine pancreatic elastase, cathepsin G and human leukocyte elastase.

Thompson, RlC-* and Bloout,
E-R, (1973).

), Biochemistry 12.47;
Powers, J+C+1Gupton, B
-F,, Harley, A, D,, NishN1
5hi, N- and Witley, R-J-(1
977) T Biocbem-Biophys, A
cta-525, 1,56; Lively, M.O.
, + and Powers, J.C. (1978) +
BloChem, Bj, ophys-Acta+5
25, 171; Yoshimura, T-,
Barber, L, N-.

and Powers, J.C. (1982), J.
, Biol, Chem.

257, 5077; and Teshima, T-
, Cr1ffin, J.C;.

and Powers, J.C. (1982) t
J. Biol-Chem.

257, see 5085.

α-Amino chloromethyl ketones have also been synthesized and used to study the physiological and pathological roles of elastase. Janoff, A.

Blondin+J++ 5andhaus+R-A,
, Mo5ser, A., and Malemud, C+
(1975), Proteases and B.
j, oro-gical Control (I(ei
ch, ”'-+, Rifkin, D-B, + and Shaw, E, co-eds.), pp. 603-620, Co1
d Spring Harbor Laborato
ry, New York.

α-amino chloromethyl ketone has shown no promise as an inhibitor in treating disease.

These are strongly a-electronic and non-selectively alkylate non-target molecules present under in vivo conditions.There is a strong desire to develop inhibitors that do not cause such non-selective alkylation. ing. It was expected that fluoroketones would have the desired weak alkylating effect. A discussion of F/(J ratios in various conversion reactions is provided by Hudlicky IM.

Plemun Press, New York.

Attempts to synthesize α-aminofluoroketones have so far been unsuccessful: Powers, =jC0(19
77 edition), Vol, 4, 65, 178 pages, Mar
cel Dekker.

See New York.

Such synthetic methods have generally been attempted by conventional substitution reactions in which CI of α-aminochloromethylketone is replaced with FVC. As a result, the 4th part of the amine was destroyed and it was a failure. In some synthetic examples, KF
and 18-crown-6-ether (Aldrich
Chemica I Company, Mj 1w
aukee.

Wisconsin) used a small excess of K to replace Gl on the peptide chloromethyl ketone with F.
A method of using Ff: and refluxing the solution for 1 G to 12 hours was attempted, but no displacement occurred.

In the synthesis of α-aminochloromethylketone, aminodiazomethylketone is treated with HCl to synthesize the desired final product. Attempts to synthesize amine fluoromethyl ketones by this method (attempts using HF instead of HCl) proved unsuccessful. Therefore, the existence of the peptide 9α-aminofluoroketone was previously unknown. Providing such types of compounds would be conducive to technological progress.

The object of the present invention is to provide a new class of compounds, α-aminofluoroketones.

Another object of the invention is to provide α-amine fluoromethyl ketones.

Yet another object is to provide α-aminofluoroketones having a single amino acid residue or residues of from 5 to 6 amino acids.

Yet another object is to provide a method for the synthesis of α-aminofluoroketones.

Yet another object is to provide a method of irreversibly inhibiting serine or cysteine proteases.

Yet another object is to provide a method of irreversibly inhibiting serine proteases.

To achieve the above and other objects, in the process of the present invention, which is described herein as an embodiment and more broadly, the a-aminofluoroketone of the present invention is prepared by formula 1 (a), I (b), I (c) Reed or I
(d): (wherein R1 and R2 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, a substituted alkyl group having 1 to 6 carbon atoms, an aryl group, or the number of carbon atoms in the alkyl moiety) is selected from the group consisting of 1 to 4 alkylaryl groups, n represents an integer from 1 to 4, X represents a blocking group at the terminal of the peptide, and Y represents an amino acid residue or a group consisting of 1 to 6 amino acids. (representing a peptide chain residue).

According to another aspect of the invention, formula 1 (a) −1(d)
The α-aminofluoroketone represented by is synthesized by suspending an N-acylamino acid or its peptide derivative in an inert solvent with about 2 equivalents of fluoroacetic anhydride. This solvent is added in an equal amount to the approximate weight of the N-acylamino acid or peptide derivative thereof. A tertiary amine is then added in an amount equivalent to about 2 equivalents of the N-acylamino acid or Niptito9 derivative and cooled to a temperature of about 0°C. Thereafter, a catalytic amount of a substituted 4-dialkylaminopyridine catalyst is added to synthesize the desired ketone.

According to another aspect of the invention, a sample containing a protease is prepared under protease inhibiting conditions by formula 1(a), r
A method of inhibiting a protease is provided, which comprises contacting the compound represented by (bl, I (c) i or 1(d)) in an amount sufficient to inhibit the protease.

The α-aminofluoroketones of the present invention irreversibly inhibit cysteine proteases and serine proteases. The ketone of the present invention does not have a strong electron affinity,
Therefore, non-target molecules are not indiscriminately alkylated under various conditions in vivo or in vitro. The monoaminofluoroketones of the present invention are applicable to the production of therapeutically effective protease inhibitors. More specifically, the α-aminofluoroketone of the present invention is cathepsin B.

H, C, G, R; elastase; trypsin; plasma kallikrein; glandular kallikrein; plasmin; plasminogen activator; useful for inhibiting other serine and cysteine proteases, including but not limited to do not have.

The present invention provides novel α-aminofluoroketones, methods for their synthesis, and methods for irreversibly inhibiting proteases. Inhibition is defined herein as the initial binding of an inhibitor (α-aminofluoroketone) to the recognition site of a protease, followed by the irreversible covalent binding of α-fluorometelcutone to the active site of the enzyme. be done.

Table 1 below shows the meanings of the abbreviations used in this specification.

Table 1 AFC-Aminofluorocoumarin AAa-Alanine Arg-Arginine Am1no ACid CH2F-α-Amino acid fluoroketone Asp-Aspartate Bz-Benzoyl 13oc-t-Nodoxycarbonyl BzA-Benzylruvirolidine CHN-Diazomethylketone Cys- Cystine GO2-t-butyl-j-butoxycarbonyl C0
2-Et-EthoxycarbonylGzy-Gly7ineL(11-LeucineLys-Lysine1eO8uc-MethogiasuccinylPro-ProlinePir)-PyRcholic acidPhe-PhenylalaninePh-Phenyl5er-SerineTos-)Tulu2-Carbo Benzoxy θ′-aminofluoroketone of the present invention (d, formula 1%
(: (In the formula, R1 and R2 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, a substituted alkyl group having 1 to 6 carbon atoms, an aryl group, and an alkyl moiety having 1 carbon atom. n represents an integer from 4 to 4, X represents a blocking group at the terminal of the peptide 9, and Y represents an amino acid residue or a peptide chain of 6 to 6 amino acids. (representing a residue).

In this specification, a substituted alkyl group refers to a hydroxyl group, an amino group,
It is a group in which a guanidino group, a carboxyl group, or a mercapto group is bonded to an alkyl group having 1 to 6 carbon atoms.

Suitable R peptide end blocking groups (X), i.e. deep peptide protecting groups, are known in the art. In this specification, the 9-terminal Norotsuki/g group may be bonded to an amino acid or may be bonded to a peptide 8 chain. Appropriate peptide 9-terminal ProBiology (Gr
ass, E+ and Me: Lenh', fe'r
-J-+ co-ed.), Vol. 3 (1981)
, Academic Press.

Listed in New York.

Or preferably, an acetyl group, a benzoyl group, a carbobenzoxy group, a glutaryl group, a t-butoxycarbonyl group, a succinyl group, a methoxysuf/nyl group, a D-Pro
, D-ValSD-Aha, and D-Phe-C.

More preferably, X is selected from benzoyl, t-butoxycarbonyl, methoxysuccinyl or carbobenzoxy.

R2 is preferably a hydrogen atom.

Y is preferably an amino acid or the residue of a peptide chain of 1 to 4 amino acids.

Compounds represented by formulas 1(c) and 1(d) above are preferred.

The α-aminofluoroketone t of the present invention is synthesized by forming a C--C bond, but not by forming a C--F bond.

In one embodiment, α- of formulas 1 (a) to J (d)
Aminofluoroketones are prepared by suspending an N-acylamino acid or its takuzotide derivative together with about 2 molar equivalents of fluoroacetic anhydride in an amount of an inert solvent approximately equal to the weight of said N-acylamino acid or its peptide derivative. It is synthesized by making it cloudy. Non-limiting examples of suitable solvents include benzene, toluene, tetrahydrofuran, dioxane, chloroform, dichloromethane, ethyl acetate, and the like. A preferred solvent is benzene. Two equivalents of the tertiary amine are then added to the solution while cooling to about 0° C. to control the rate of oxacylone formation. Non-limiting examples of tertiary amines include triethylamine, N-methylmorpholine, triethylenediamine, and the like. Preferred is triethylamine. 4-' of catalyst amount)
Add alkylaminopyridine catalyst and finish cooling. Non-limiting examples of 4-dialkylaminopyridines include 4-dimethylaminopyridine and 4-pyrrole/pyridine. 4-') Methylaminopyridine is preferred. Severe carbon dioxide gas generation occurs. The resulting solution was stirred at room temperature for about 2 hours, then a water-immiscible solvent was added for about 1 hour.
Dilute to 0 medium. Non-limiting examples of solvents include benzene, toluene, chloroform, dichloromethane, ethyl acetate, and the like. A preferred solvent is benzene. The resulting solution is washed with dilute acid, followed by dilute base, and then saturated NaCl, followed by e.g.
The α-aminofluoroketone is separated from impurities by drying with S04. The solvent is distilled off to purify the α-aminofluoroketone.

The Oo-aminofluoroketones of formula 1(a) and formula I(b) can be further combined with tert-butylfluoroacetate or
l&te)'e, an activated N-urethane protected amino acid;
It can be synthesized by reacting in a non-nucleophilic solvent at about -10 to OoC for about 1 hour. Activated amino acids are defined as acylating agents. The reaction mixture is cleaved by treating with Oo and then extracted with a water-immiscible solvent and washed with dilute base and then water. The resulting organic solution is dried, the solvent is distilled off, and the α-aminofluoroketone is removed. The ketones thus produced can also be used as precursors for α-aminofluoroketones of the formulas ](c) and 1(d).

Non-limiting examples of preferred erulates include erulate derivatives of tertiary arylethyl fluoroacetate and of pencil fluoroacetate. Elerate can be prepared in a conventional manner by treatment with potassium tertiary oxide and a strong base such as sodium hydride in an inert solvent. A preferred method of activating amino acids is
Examples include, but are not limited to, a mixed carboxylic anhydride method, a symmetrical anhydride method, a carbodiamide method, and a carbonyldiimidazole method.

The present invention is useful for the irreversible inhibition of monoaminofluoroketones a:, l:, cysteine and serine proteases. A non-limiting example of a protease is cathep7B
-H, L, G, R; elastase, trino7, plasma kallikrein, glandular kallikrein, plasmin, plasminogen activator and others.

When the α-aminofluoroketone of the present invention is brought into contact with protease inhibiting conditions in vitro, it forms a covalent bond with protease as an inhibitor due to the nucleophilic substitution of the fluorine atom. This covalent bond is formed in the active portion of the protease. A reversible enzyme-inhibitor complex is formed at the enzyme active site prior to covalent inactivation of the protease. In each case the active site of the protease is alkylated.

Although each cysteine protease and Seri/protease have different geometries, the reactive sites of the proteases (the sites that are alkylated by the inhibitor) are essentially the same. 2 at this protease-reactive site, α
- substitution of F in the aminofluoroketone occurs.

Even if the ketone of the present invention is of one amino acid, k
It may be of petite 9 chains. Therefore, the geometry of the ketone can be altered so that it can match that of the protease.

However, in each case the ketone and protease reactive sites are the same.

The method of inhibiting cysteine protease or serine protease of the present invention comprises α of the above formulas 1(a)-1(d).
- contacting an aminofluoroketone with a protease under protease inhibiting conditions in an amount sufficient to inhibit the reactive sites of the protease. Protease inhibition conditions refer to a pH of about 4-10, preferably about 6.
-9, most preferably at a pH of about 6-8.
The relative concentrations of α-aminofluoroketone and protease are adjusted to about 1:1 to about 60=1 in a ratio of about 20-
It refers to incubation at a temperature of 37°C, preferably about 25°C. The relative ratio of ketone inhibitor to protease will vary depending on the time over which inhibition occurs as well as the particular protease/ketone combination. The protease may be in a purified state, present in a sample for analysis or in a sample of homogenized tissue.

Various aspects of the present invention will be illustrated by the following Examples, but the scope of the present invention is determined by the claims and is not limited to these Examples.

Table 2 shows the formula 1 (al-I(
The substituents of the α-aminofluoroketone in d) are shown below.

Compound -X- 1, 8z-AIaCH2F B7゜2, Z-Phe A1aCH2F Z3, Boa-
ProCHFC:02Et Boa4, MeO3uc
-Phe-Ala-Ala-Phe-Phe-Val-
LeuCH2F MeO3ue5, HCL, ProC
H2F H6, Boc-A1aCHFCO2-t-butyl B
oa7, TFA L A1aC82F H8, MeO
8uc-Ala-Ala-Pro-A1aCH2F M
eO3uc9, Z-Ala-PheCH2F ZQ,
Z-Phe-LeuCH2F Zl, Boc-Gl
y-AspCH2F' Z2, Boc-D-Va 1
-Leu-ArgCH2F B (1゜3, Boc-Pr
o-NHCH(C6H,,)COCH2F BOC4,
Mc-1O8uc-C1y-NHCH(C6H5)CO
CH2F MeO8uc5, Z-Ala-NHGI (
(a(.CH2a(2CH2C6H5)Ca)[3H2
F Z6, Z-Gly-PipCHFCO2Et Z
7, Boc-Gly PheOf(FCH2GH2C
H2CH(CH3) 2 B OCs, Z Phe
A1aGHFG6Hs ZQ, Z-Gly-Let+
CHFGH2CiH2CH20H2C6H5Zo,Z
A 1 a-GlyCHFGHz OH (GH3)
2 Zl, Boc-ProCHFCO2-t-but
yl Boa methyl H Phe methyl H co2Et h h CO2-t-butylmethyl H Ala-Ala-Pro methyl H Ala pencil H Phe isobutyl H Gly carboxymethyl H ILVa 1-Leu 3-guanidinopropyl Pro
Cyclohexyl H Glv Phenyl H Ala 4-phenylbutyl H G1y4CO2Et Gly Benzyl 4-Methyl is butyl Phe Methyl Phenyl G1v Inbutyl 4-phenylbutyl Ala H Isobutyl 3 002-1-Dutyl Example IBz-Synthesis of AlaCH2F [3-(N- benzoylamino)-1-fluoro-2-
Phtano/] N-benzoylalanine CB, 25g, 42.8myn
ol) and full 7-mouth acetic anhydride (11,8,9,85,
6 mmol) were mixed and treated with 10 types of benzene. Triethylamine (11,c++, 85.61=1.m0
1) was added to obtain a bath liquid, which was cooled in a water bath. 4-dimethylaminopyridine (0.26g, 2.15 mr7LO
1) was added and the water bath was removed. Immediate intense CO2
0 occurrences occurred. The solution was stirred at room temperature for 2 hours. Benzene (100ml) k;'-fc. organic RWf
I N HCd (2 x 50 ml) was washed with saturated NaHOO (2 x 50 ml), anhydrous MgS
Dry with O4. The solvent was distilled off, and the resulting oil was passed through a silica gel (60 meso/unit) column (2.5X 80crr).
t). δ7. -AAaCH2F''f chloroform. The chloroform was distilled off and the residue was triturated with petroleum ether to give a solid melting at 67-69°C.

According to 'HNIviR (CDC71!3), Ttvs
The peaks shifting from: δ1.5
1(3) L a.

-CH3)L 5-07 (2H,6,JHF-47-
4Hz, -CH2F).

5.09 (IH, m, -GH-), 6.88 (
l) (, float S.

NH'), 7.52 (3H, m, aromatic), 7.74
(2H, m, aromatic).

According to 13ONMR (CDC13), the peaks emerging from TMS were as follows: δ16.9(-CH
3,51,7,-0H-), 83.7(-CH2F
Jo,,, m18.4.3Hz), 127.2 (aromatic), 128.6 (aromatic) 9130.2 (aromatic), 1
32.1 (Aromatic).

F NMR (CD(J3)) performed without control showed trigilet; 1JHF='17
', 4) 1z.

IR (according to KBrBr fret J751(':In(
The absorption peaks of 1632cIrL (ketone carbonyl) and 1632cIrL (ζdocarbonyl) were shown.

Analysis value Total Ml direct C for C11H12FNO□
63,15, H5,78, N6.69. F9.08 measurement value G62.42. H5, 80, N6.52. F'
9.37 Example 2 Z-Ph e-A13a GH2F
Synthesis of 'C3-(N-pencyloxycarponylphenylalanylamito')-1-fluoro-2-zutanone]
N--? ndyloxycarbonylphenylalanyl-alanine (3,0!1, 8.11 ynmolJ)
and fluoroacetic anhydride (2.24 g, 16.22
mmol) and treated with benzene (30 mJ). Triethylamine (1,64d, 16.22
yn7nQ7) was added to obtain a solution. 4-') Methylaminopyridine (50η, 0.41 mmol) was added. The solution was stirred at room temperature for 2 hours.

Benzene (100mlV) was added. Organic solution IN
HCl (2x50m), saturated NaHCO3 (2x5
0d).

Wash with saturated NaCl (2X!5 omJ), m water and dry with MgSO4. The solvent was evaporated and the residue was passed through a 2.5×60 crrL column of silica gel (60 mesh).

Z Ph e I'-13a GH2F was eluted with 2% methanol in chloroform. The solvent was evaporated and the residue was crystallized from ether to give 0.24 g of product, melting point 129-131°C.

Shift peak from TMS of 1HNMFt (CDC73): δ1.24 (3H, d of d, -〇H
-qa) -3,06(2H,a.

Ph 'H2c), 4.51 (2H,m, -CH
+ -GH-), 4.91 (2H, dofd, JH
F=47.4Hz, -0H2F), 5.09 (2
H.

s, Ph-%-〇), 7.23 (5H,s, C6
dan, -CH2-C).

7.32 (5H, s, G6H5-OH2-0).

13CNMR (CDC13) TMS shift peak: δ16.6 (CH3), 38.7 (Ph-C
H2-C), 51.1 (N-CH-CH3),
56.4 (rtr-cH-cH2-ph), 67,
2 (Ph-CH20).

83,6 (d, J, m185.6 Hz, 0H2F
), 126.9 (d, aromatic), 128.1
(d, aromatic), 128.6 (d', aromatic, 1
29.4 (aromatic), 136.4 (aromatic).

Example 3 Synthesis of BOCP ro(1'HFG02 k t [2-(2'-ethoxycarbonyl-2'-fluoroacetyl)-Nt-butoxycarbonylpyrrolidine]ethyl fluoroacetate (2°09, 18.9 mmo
A solution of x-f l) in ether (20d) (D NaH (50% dispersion) (o, 91.!9°18,9 mm011>
was added to the suspension. The reaction mixture was stirred at room temperature for 3 hours, then cooled to -15°.

Mixed anhydride of Boc-Pro-OH [N-methylmorpholif (2,1d, 18.9 mm011) f: Boc-Pro-OH (
4.1g, 18.9 mmall) to -15°
isobutyl chloroformate (2,45rd, 1
8.9 mrnolJ) and stirring for 5 minutes] was filtered into the cooled Ehrlate suspension and stirred at -15° for 10 minutes.

The reaction was then warmed to room temperature for 1 hour. 2 to 5 times the solution
NH2So4f was poured into the containing water. The organic solvent was distilled off, and the aqueous layer was extracted with ethyl acetate. The ethyl acetate solution was diluted with saturated NaHCo3. Wash with saturated Nac7 and anhydrous M
Dry with gSO4. Ethyl acetate was distilled off and the residue was passed through a silica gel column. The product was eluted with chloroform. The solvent was distilled off, and 1.08 g of oil was obtained.
7 Tori: (3H, M, - egg, CH3 + ladle) +
5.44 (IH, d of d.

-CHF-+JHF=46.9 Hz).

13CNMI ((CDC13) shift peak from TMS: a 14.1 (OH2C5H3), 23.6
(N6.), 28.3 (-C(CH3)3)62
．． 5 (CH,0H3), 77.1 (a, , Jo
F=168.5Hz.

-CHF-).

Example 4 MeO8uc-Phe-Ala-Ala, -
Phe-Phe-Val"e uGH2F ():)
Synthesis [3-(N-me)succinylphenylalanylalanylalanylphenylalanylphenylalanylvalylamide)-1-fluoro-5-methyl-2-hexano/
]N-methylmorpholif (0,i3'+ 1.14y
, zmo7) in tetrahydrofuran (10 ml) containing MeO8uc-Pbe-AA'a-
AI! a-Phe-Phe-OH (0,82 &, 1.1
Inbutyl chloroformate (0,15
d, 1.14 mmall >f: Added. The mixture was stirred at -10' for 2 min and then dissolved in pre-cooled HO2-VaA LeuCH2F in dimethylformamide (Group 5).
(o, 339, 1.14 mm07) was added, followed by N-methylmorpholine (0, 1, 3 ml, 1.
14 mmol) f-added. The mixture was stirred at -10° for 1 hour and then stirred and stirred at room temperature overnight. The mixture was filtered and the solvent was distilled off. The residue was passed through a column of 2.5×45 cIrL silica gel (60 mesh) and the product was eluted with 2% methanol in chloroform. The solvent was evaporated and the residue was triturated with ether to give a solid material in 24% yield.

Example 5 Synthesis of HCl-ProOH2F [2-fluoroacetylpyrrolidine hydrochloride] 2- (2'-t-
Butoxycarbonyl-2'-fluoroacetyl)-N-
t;-Butoxycarbonylpyrrolidine (1.4g, 4
．． 2 mmall) in dichloromethane (genus 20) and 5.5 N in dioxane (20a) for 1 h at room temperature.
Treated with HO2. Pour the solution into ether (200ml)
Added to. The solid content was collected by filtration, washed with ether, and dried under vacuum. HNfB is characterized by the absence of t-butyl group and −
CH2F showed retention of Dazure Soto.

Example 15 Synthesis of Boc = AAaCHFCO2-t-Sotyl [:3-(N-butoxycarbonyl)-1
-Fluoro-1-t,-butoxycarbonyl-2-butanone] in potassium tertiary sotoxide (1,19 g, 10.6 mm011) in THF (25 ml) at 0°;
Tertiary butyl fluoroacetate (1,4
The water bath was removed and the yellow solution was stirred at room temperature for 20 minutes. Then add the solution to 1
Cool to 0°, during which time add Boc-Ala OH (2g
, 10.6 mmolJ) of mixed anhydride was prepared.

The mixed anhydride reaction mixture was filtered into the cooled Ellate solution.

The resulting solution was stirred at room temperature for 1 hour, then 2.5N H
2SO4f: Poured into the containing crushed ice. THF=i was evaporated and the aqueous residue was extracted with ethyl acetate (2×5M). The organic layer was washed with saturated NaHCO3 and brine, then dried over MgSO4. The solvent was evaporated and the residue was chromatographed on silica gel.

The product was eluted with 2% ethyl acetate in chloroform.
2.4 g (78%) of oil was obtained.

1H NMR (CDC13) showed d, d at δ5.39 (CHI', JJ=46.9 Hz).

The 13c N dish (CDCA3) showed δ77, d in l (-C:HF-, JoF=172.1 Hz).

Example 7 Synthesis of TFA L Alac'H2F [L-
1-Fluoro-3-amino-2-butanone trifluoroacetate salt] Boc AIJ a GHFCO2-t-Sochi# (
1g, 2.46mm011') was treated with trifluoroacetic acid for 1-2 hours. The solvent was distilled off and the residue was dissolved in ether.
Solid material (0.35L 6
5%).

1HNMR (D20) showed d at δ5.48 (-C
H2F.

JHF = 46.4 H7).

Example 8 MeO8uc-Ala-AAa-Pro-A
, gaCH2F 1-(N-methoxysuccinylalanylalanylzolorylamide)-1-fluoro-2-butanone] Following the method of Example 1, MeO was used instead of N-benzoylalanine.
8uc-Aha, -Ala-Pro-Ala-OH'x
using MeO8uc-AAa-Aha-Pro-Al
aCH,, F fc was synthesized.

Example 9 Synthesis of Z-Ala PheGH2F (3-(
N-pencyloxy7carbonylalanylamito'')-1
-Fluoro-3-phenyl-2-phtatsuno] Following the method of Example 1, ZA was substituted for N-benzoylalanine.
Using Aa-Phe-OHi, Z-Ala-ph e
CH2F was synthesized.

Example 1 Synthesis of oz-Phe-Le11CH2 [3-(
N-benzyloxycarbonylphenylalanyl amyl
')-1-fluoro-5-methyl-2-hexa7/] Following the method of Example 1 and using Z-Phe-Leu-OH in place of N-benzoylalanine, 2-Phe
-LeuCH2F f was synthesized.

Example 11 Synthesis of Boc-G/y-AspCH2F [
:3-(N-1-butoxycarbonylglycylamide)''
)-1-Fluoro-4-carboxy-2-butanone] Following the method of Example 1, using Boc-Gly-0-BzAAsp-OH in place of N-benzoylalanine,
The obtained Boc-GAy-0-BZIAspCH2F was catalytically hydrogenated to synthesize Boc-GAy-AspGH2F.

Example 12 Bac-D-Va,f? -Leu-Arg
Synthesis of CH2F [3-(Nt-zthoxycarbonyl-〇-valyl-L-loidylamyl')-1-fluoro-
6-guanidino-2-hexanone] Following the method of Example 1, replacing N-benzoylalanine with BOC-D-Va6-Leu-No2Arg-OH
Boc-D-Val-Leu-(N
o2) Catalytic reduction of Ar gGH2F to Boc-D-V
al-Leu-ArgG) 12F f was synthesized.

Example 13 Boc-Pro-NHCH (C6H1,)
Synthesis of COCH2F (3-(N-t;-buttoky7carponylzolorylamito'')-1-fluoroco(-cyclohexyl-2-propanone) Following the method of Example 1, ) Boc-Pro-NHGH (G6
H1□) COCH2F ftr: Synthesized.

Example 14 MeO8uc-GAy-NHOH (C6H
5) Synthesis of GOGH2F' (3-(N-methoxysuccinylglinolamide)-1
-Full 71-1:l-3-7Eur-2-Propanone] Following the method of Example 1, using MeO8uc-CAy-noenylglycine in place of N-pensoylalanine, MeO8uc-Ay-NHCH (C, H5) COC
H2F was synthesized.

Example 15z-Ala-NHCH(CH2CH2CH2
Synthesis of CH2C6H5)-COCH2F (3-(N-benzyloxycarbonylalanylamide)-1-fluoro-7-phenyl-2-heptanone) Following the method of Example 1, replacing N-benzoylalanine with Z -AA!a-6-phenylnorleucine,
Z-A7a-NHCH(CH2CH2CH2CH2C6
H5)-COCH2F was synthesized.

Example 16 Z-GA! y P I T) OHF'00
Synthesis of 2 k t C2-C2'-ethoxycarbonyl-
2'-Fluoroacetyl)-N-(N/-benzyloxycarbonylglycyl biveridine] Following the method of Example 3, replacing Boc-Pro-OH with K Z-CAy -Hininia!J 7 e e was used to synthesize z-e6y-PipCHFGO□Et.

Example 17 Boc-GAy-PheC; HFGH20
Synthesis of H2CH20H(C)13)2 [2-(Nt-ptoxylponylglycylamito-4-fluoro-8-methyl-1-phenyl-3-nonanone] Following the method of Example 1 , using Boc-(Jy Phe-OH in place of N-benzoylalanine and 2-fluoro-6-methylhebutanoic anhydride in place of fluoroacetic anhydride, Boc-Gly-PheCHFCH2
CH2CH2CH(CH3)2 was synthesized.

Example 18 Z-Phe-A7aCHFC6H5 (7)
Synthesis [3-(N-penzyloxylponylphenylalanylamito)-1-fluoro-1-phenyl-2-
Butanone] Following the method of Example 2, instead of fluoroacetic anhydride, 2
- Using fluoro-2-phenylacetic anhydride, Z-
Phe-AlaCHFC6H5f was synthesized.

Example 19z-Gly-LeuCHF'CH2CH2C
Synthesis of H2CH2C6H5 C7-CN-benzyloxycarbonylglycylamide)-5-fluoro-9-methyl-1-phenyl-6-decanone] Following the method of Example 1, instead of fluoroacetic anhydride, 2- using fluoro-6-phenylhexanoic acid,
Z-Gly-Leu- instead of N-benzoylalanine
Z-Gay-LeuCHF'CH2C using OH
H2CH2CH2C6H5 was synthesized.

Example 20 Z-Aha-GlyCHFCH2CH(C
H3) Synthesis of 2 [1-(N-benzylox7carponylalanylamito5)-3-fluoro-5-methyl-2-
Hexanone] Following the method of Example 1, using Z-Ala-G4 y-OH instead of N-chondzoylalanine and 2-fluoro-4-methylpentanoic acid instead of fluoroacetic anhydride. Using anhydride, Z-Ala-Gly-
CHF'CH2CH(CH3)2 was synthesized.

Example 21 Boc-ProCHFCO2-t-phthyl tD synthesis (2-(2'-t-butoxycarbonyl-2'
-Fluoroacetyl)-N-t-7')oxycarbonylpyrrolidine] Synthesis of Boc-ProCHFCO2-t-butyl following the method of Example 3 and using t-butylfluoroacetate in place of ethyl fluoroacetate. did.

Example 22 Z-Phe-AlaC, an α-aminofluoromethyl ketone for the cysteine protease cathepsin B
The irreversible inhibitory effect of H2F was inhibited by Z-P, a known inhibitor.
h e-A13 a OHN 2 and Z-Ph e
Compared with Ala OH2Glj. Cathezosin B used for comparison was isolated and purified from human and rat livers.

For measurement of enzyme activity, synthetic oligo R peptide substrate Bz-V
an-Lys-Lys-Arg-AF (3 in 10mM
Fluorescence of released AFC was detected at pH 6.5 using 1 mM TT and activator salt EDTA.

The unit of enzyme activity is 50% glycerol phosphate buffer 1
Standardized for 00fflJ. standard concentration of enzyme (i
onM) with each inhibitor (50 nM) at 25° 3.5.1
The residual activity units of the purified enzyme were determined by prewarming for 0.15, 20 and 30 minutes, and then activity measurements using the same synthetic substrate and conditions for each test Zampuno+, Jc. The amount of enzyme activity inhibited by each inhibitor was determined by subtracting the residual activity value from the control activity value for each inhibitor.

The resulting take was 0.1 μM Z-Phe AlaC
We have shown that H2F is an effective inhibitor of purified cathepsin B. Z Ph e A13a GH2F I, (
The inactivation rate constant) was 9.2 ± 1.3 × 10 s, and the K□ (inhibitor dissociation constant) was 0.57 ± 0.09 μM
and K3/Ni1 (inhibitor specific constant) is 16,200
M S ; I = 0.40 (0.10 μM
), in Z-Phe-AlaGHN2 = 4
．． 1±1.7X10S, -7゜4±3,0μM
, -1-1° and 3/ni = 546M S , I-2,5-0,
It was 50 μM. The inhibitor specificity constant (K3/Nitech) of Z-Phe Ala GH2F is Z-Phe AlaC
It was 30 times larger than HINz.

Example 23 α-aminofluoromethylketone Z-Phe-Aj? a
The ability of OH2F to irreversibly inhibit serine protease, elastase from human leukocytes and porcine pancreas, was demonstrated by the known inhibitor Z-Ph e-Ala C'H2.
CaC12 (10mM)'& Q, compared to F.
elastase (0) in l M'I'ES pH 8,2
, 3 μM) was added to the synthetic substrate MeO8uc-AAa.
-Ala-Pro-Va e-MNA. The liberated MNA groups were quantified by fluorometry. A standard unit concentration of the enzyme with each concentration of the respective inhibitor;
℃ for the specified time, i.e. 5°15: (0 and 60 min prewarming, then for all test samples respectively.
Activity was measured using the same synthetic substrate as in the control without inhibitor. The amount of enzyme activity inhibited by each inhibitor was determined by subtracting the residual activity from the control activity value for each inhibitor. The data obtained showed that Z-Phe-A7aCH2F inhibited elastase under alkaline conditions.

Example 24 According to the method of Example 22, the cysteine protease cathepsi 7L is converted to Z-Ph e Pb e GH2F
was found to be inhibited under the same conditions and at the same concentration ratio of protease to ketone inhibitor.

Example 25 Following the method of Example 22, the cysteine protease cathepsyL was found to be more inhibited by Z PheA7aCHzFVC under the same conditions and at the same protease to ketone inhibitor concentration ratio.

Example 26 Following the method of Example 23, a plasminogen activator of serine protease was prepared.

It was found to be inhibited by MeO3uc-(Jy-G/y-ArgCH2F under the same conditions and at a protease concentration of 50 μM and a ketone inhibitor concentration of 0.1 mM.

Example 27 According to the method of Example 23, the serine protease plasma kallikrein is isolated from the ketone inhibitor D-PrO-Phe-
It was found to be inhibited by ArgCH2F under the same conditions and at a protease concentration of 10 μM and a ketone inhibitor concentration of 0.1 mM.

Example 28 According to the method of Example 23, the serine protease glandular kallikrein is synthesized with the ketone inhibitor D-VaA-Leu-A.
It was found that r gcH2F was inhibited at a protease concentration of 10 μM and a ketone inhibitor concentration of 0.1 mM.

Example 29 According to the method of Example 23, the serine protease trypsin was found to be inhibited by the ketone inhibitor Z-LysCH2F IC at a protease concentration of 10 μM and a ketone inhibitor concentration of 0.1 mM. It was done.

Example 30 Each of the following inhibitors: Z-Phe-AlaGHN2; Z-
Phe-AlaGHC1; and Z Phe klaG
IM of rat pancreas, pancreas, liver and kidney tissue homogenization against cathezozone B using H2F f
Measure the damage rate.

The tissue was homogenized and heated at 13.00 rpm for 25 min.
Centrifuge for a minute and separate the supernatant. A portion of the supernatant was diluted with water to 0.01 μM at 6°C. The method of Example 22 was repeated. As shown in Table 3, the inhibition of cathepsin B by the three inhibitors was compared for each tissue sample.

Results are expressed as τ (half-time of inactivation or inhibition). The smaller the half time, the greater the inhibitory ability of the inhibitor. The concentration of the inhibitor was 0.10 μM, and the reaction temperature was 25° C.

Table 3 Inhibitor τ (Notes Mame) Pancreas - CHN226.4 minutes -CH2CA! 74.2 -0H2F 1.4.2 Pancreas-〇HN215.8 -CH2G/21.4 Liver-〇HN222.4 -0H2G1 66.5 -0H2F 8.5 Kidney-CHN220.1 -aH2c176.6 10H2F 7.9 The above description of preferred embodiments is merely illustrative.

It will therefore be understood that these descriptions are not intended to limit the invention to the scope thereof, and that modifications and variations may be effected in light of this disclosure. The embodiments described above are intended to most clearly explain the principles and practical applications of the invention and to enable those skilled in the art to modify the invention and use it in various embodiments to suit the particular application desired. selected and described herein. Therefore, the scope of the invention is limited only by the scope of the claims.

Patent Applicant Enzyme Systems Products, Inc. First Page Continuing Procedures Amendment Book Patent Office Official Shiga Character Number 1 Case Display Showa? 2nd year patent application No./A/G? No. 2 Name of invention Guamino? rv"J Jibtonro, Relationship with the amended person's case Patent applicant's address: 1- Preliminary/Recommendation/Site: Glazing/7
°Rosocks I, Kobo V On the other hand, 7th 4 Attorney's Procedures Amendment Written August 1982/Date 2 Name of the invention α-amine fluoroketone 6 Relationship to the case of the person making the amendment Address of the patent applicant 4. Contents of Amendment 6 by Agent (1) Change “arylethyl” in line 6, page 25 of the specification to “
butyl”.

that's all

Claims (1)

[Claims] (1) The following formula 1 (a), l (b), I (c) or I (d): (wherein R1 and R2 are each independently a hydrogen atom, the number of carbon atoms selected from the group consisting of an alkyl group having 1 to 6 carbon atoms, a substituted alkyl group having 1 to 6 carbon atoms, an aryl group, and an alkylaryl group in which the alkyl moiety has 5 to 4 carbon atoms, and n is an integer of 5 to 4; Representation,
X represents a blocking group at the end of the peptide, and Y
is an amino acid residue or 1 to 6 amino acids is a proline residue) α-aminofluoroketone. (2) The compound according to claim 1, wherein the substituted alkyl group is an alkyl group having 1 to 6 carbon atoms substituted with a hydroxyl group, an amine group, a guanidino group, a carboxyl group, or a mercapto group. (3) X is an acetyl group, a benzoyl group, a carbobenzogysi group, a glutaryl group, a t-butoxycarbonyl group, a succinyl group, a methoxysuccizonyl group, a D-proline residue,
The compound according to claim 1, which is a D-valine residue, a D-loinine residue, or a D-phenylalanine residue. (4) The compound according to claim 1, wherein X is a benzoyl group, a t-butoxycarbonyl group, a methoxysuccinyl group, or a carbobenzoxy group. (5) The compound according to claim 1, wherein R2 is a hydrogen atom. (6) The compound according to claim 1, wherein Y is an amino acid residue or a peptide chain residue of 1 to 4 amino acids. (7) The compound according to claim 1, which is Bz-AlaCH2F. (8) The compound according to claim 1, which is Z-Phe-AlaCH2F'. (9) The compound according to claim 1, which is Boc-ProCHFCO2h:t. (10) Boc-AAa (3HFlj○2-1.-phthylteal specific FF compound according to claim 1. Ql) MeO3uc-AAa-Aha-ProAla
The compound according to claim 1, which is CH2F'. Q2) MeO8uc-Phe-Ajl! a-PheP
The compound according to claim 1, which is he-Val-L-, qCH2F. (13) The following formula! (aj, I(b), I(c)t or I(c+): (wherein, R1 and R2 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a C1 to C6 alkyl group, selected from the group consisting of 6 to 6 substituted alkyl groups, aryl groups, and alkylaryl groups in which the alkyl moiety has 1 to 4 carbon atoms, n represents an integer of 1 to 4, and X represents a blocking group at the terminal of Takuputide. and Y represents an amino acid residue or a peptide chain residue of 1 to 6 amino acids. A method of irreversibly inhibiting a protease, the method comprising contacting a protease with a substance sufficient to inhibit the protease. ) The method according to claim 13, wherein the protease is a serine protease. (I6) The protease inhibition condition is such that the protease
14. The method of claim 13, comprising contacting the aminofluoroketone at a pH of about 4-10. (17) Protease inhibition conditions inhibit protease from α-
14. The method of claim 13, comprising contacting with an aminofluoroketone at a pH of about 6-9. 14. The method of claim 13, wherein the protease inhibiting conditions comprise contacting the protease with the alpha-aminofluoroketone at a temperature of about 20-37[deg.]. 14. The method of claim 13, wherein the α-protease inhibition conditions comprise contacting the protease with the α-aminofluoroketone at a temperature of about 25°. (20) α-aminofluoroketone is Z-Phe-A
, 6aCH2F. 21. The method according to claim 20, wherein the protease is catezosin B. (2) The method according to claim 20, wherein the protease is elastase. (23) The α-aminofluoroketone is Z-Phe-P
h eGH2F, and the protease is cathepsin L.
14. The method according to claim 13. (24) α-aminofluoroketone is M60Suc-
14. The method of claim 13, wherein CTly-CTly-ArgCH2F is a protease 75 cup plasminogen activator. (c) α-Aminofluoroketone is D-Pro-P
14. The method according to claim 13, wherein h e-A r gGH2F and the protease is plasma kallikrein. (26) α-Aminofluoroketone is D-Val-L
14. The method of claim 13, wherein e u Ar gCH2F and the protease is glandular kallikrein. (27) α-aminofluoroketone is Z Ly s O
14. The method according to claim 13, wherein the protease is H2F and the protease is tridicin. (c) (a) An N-acylamino acid or its R peptide derivative is suspended in an inert solvent with about 2 equivalents of fluoroacetic anhydride, wherein the solvent (b) adding to the product from step (a) about 2 equivalents of the N-acylamino acid or its Rzotide derivative, while cooling the temperature to about 0°; adding a triamine and (c) to the product from step (b) a catalytic amount of substitution 4-
Formula I (a), I (bl, I (cj or step (d)) by adding a dialkylaminopyridine catalyst to form an organic solution containing the desired ketone and impurities
:(In the formula, R1 and R2 each independently represent a hydrogen atom,
selected from the group consisting of an alkyl group having 1 to 6 carbon atoms, a substituted alkyl group having 1 to 6 carbon atoms, an aryl group, and an alkylaryl group having 1 to 4 carbon atoms in the alkyl moiety, and n is 1 to 4. , X represents a blocking group at the end of the peptide, and Y represents an amino acid residue or a peptide chain residue of 1 to 6 amino acids) 29. The method of claim 28, further comprising the step of total purification of the organic solution from step (c) to isolate the alpha-aminofluoroketone from impurities. 00) The method according to claim 28, wherein the inert solvent in step (a) is benzene. 8. The method according to claim 5, wherein the tertiary amine in step (b) of OI) is triethylamine. (3-substituted 4-:) alkylaminopyridine is 4-
Claim 28 which is dimethyl-aminopyridine
The method described in section. 03) α-Aminofluoroketone is L z-AIJ
29. The method according to claim 28, wherein a is CH2F and the N-acylamino acid is N-benzoylalanine. (34) α-aminofluoroketone is Z-Phe-A
29. The method according to claim 28, wherein 7aCH2F and the R-petito9 derivative is N-benzyloxycarbonylphenylalanyl-alanine. C39α-aminofluoroketone is MeO8uc-A
Aa-Ala-Pro-AA! acH2F, and the peptide derivative is MeO8uc-Aha-Ala-Pr.
29. The method of claim 28, wherein o-Ala-OH.