JPS63145299A - Tetrapeptide methyl ketone inhibitor of virus protease - 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 certain peptide methyl ketones useful as inhibitors of viral proteases, and specifically as specific inhibitors of protein processing by picornavirus proteases.

Proteases are enzymes that cleave proteins at specific peptide bonds. In living systems, highly specific proteases and complementary protease inhibitors mediate or control a wide range of biological functions. For example, proteases cleave precursors in post-translational processing of polypeptides to form active proteins, leading to thymodene activation cascade reactions, such as blood coagulation.
It provides the mechanism for fibrinolysis and the immune system's complement response, and mediates the transport of selected proteins across biological membranes. Therefore, proteases act as specific inhibitors of protease activity! It corresponds to the question of Hiroji of Ichikan nosaka of the scaled Jikiyoshi.

Coded by Filth Denom (cncode
cl) Proteases play an important role in viral reproduction. Viral proteases cleave large precursor polypeptides produced by infected cells into smaller protein components or subunits, which are then assembled to form functional viral structures. Lonotsky
(Loz i tsk i i) et al. ('7 Subekhi
Sobremenoi bio mouth! e-C former Sovrem, Bi
ol, ), 93: 352-362 (1982)) examined the role of protein hydrolysis in the reproduction of χ6 and ff+i mammalian viruses and reviewed the literature on virus protease inhibitors. .

Picornaviruses are common in humans and other ll+ff 2L!
i! It is an important type of virus pattern origin in JJ products. This species includes poliovirus, rhinovirus, hepatitis A (1+epatitiq^), and hoof'-and-moutb disease.
se) etiological agents (eLiologicaFle
nts) is a virus. During the replication of picornaviruses, the viral mRNA is transported through successive passages of liposomes (
virus m by continuous passage)
It is translated along the RNA molecule, producing a linear protein product that is digested by virus-specific proteases before the protein/liposome complex is separated.
It is cleaved at a site selected by specific proteases.

Many researchers have sought 9-specific inhibitors of picornavirus protease activity. Kofunt (KoranL)
(7'r-f Le of Pyralasis (Jeng V, l Eng 11) L Chang = 751-759 (1972)) is a study of protein processing of poliovirus and echovirus-12 using simple amino acid chloromethyl ketone derivatives. Discloses inhibition.

In particular, Furand has shown [IN] inhibition by tolylsulfonylphenylalanylchloromethylketone (TPCK) and tolylsulfonyllysylchloromethylketone (TLCK). Summers
rsj et al. (Journal of Pyralology 10 = 8
80-884 (1972)) are also TPCK, TLC
The inhibition of protease cleavage of large poliovirus-specific polypeptides by K and D- and L-isomers of carbobenzyloxyphenylalanylchloromethylketone (Z PCK ) is disclosed. In a follow-up report, Colant et al. (Proc, Nati, Δ
cad, sci, USA)'/6: 2 9
9'2-2995 (1979)) is carbopencyl oxyleucyl chloromethyl ketone (Z L
discloses the inhibition of poliovirus protein processing by CK).

Various peptide derivatives are known that have the ability to inhibit protease activity. “Proteolytic enzyme haloketone inhibitor” “l1aloketone Inhibi
tors of Promeolytic Enzym
es'' (Weinstein!)
74 Emo, Peptide and Tarto-1977, page 65
~178, Marcel Dek
Bowers (Ker), New York)
rs) reviewed the literature on inhibition of protease activity by haloketone derivatives of amino acids and peptides. Powers and the others Biokimi Dakimakura, Biofuino Chikara, Acta ([
liochim, Biohs, ^eta) 480:
246-261 (1977) demonstrate the inhibition of the bacterial protease subtilisin BPN' by a series of peptide chloromethyl ketones. Among the compounds tested, acetyl-1-phenylalanyl-L-glycyl-7-ranyl-mono-leucylchloromethylketone (AcPh-Gly-A
l a -Lcu CH2Cl ) was the most potent inhibitor. Related compounds, methoxysuccinyl-L-7x, 7ranyl, L-glynes, monoalanyl, monoleucylchloromethylketone (MeO8u
c -Ph e -Gly -Ala Le u
CH2Cl) was released in November 1981 as a product bulletin (1:1zym6 Sys).
temsProducts).

Ito et al., Biochemical and Biophysical Research Co., Ltd. (Biocl+em.

Biophys, Res, Co1Ila+un, )49
: 343-349 (1972) describe experiments involving the inhibition of the digestive protease, chymotrypsin, by certain peptide aldehydes.

Itov et al. also reported that tripebutynol methyl ketone, Ac
Inhibition of chymotrypsin by Leu -Leu -Ph e CH3 was tested. However, 600q
No inhibition was observed at inhibitor concentrations of /mβ.

Finally, Fittkau et al.
llaba) Rope Edition, Peptide 1982 (cleGruytcr, New York, 1983
), “Synthesis and Properties of Peptide Ketones” (5yntl+esis and Prop
erties of PeptideKetones”
) Thermoact/Mr. vulgaris (T) + ermoact by certain peptide methyl ketones with odor
cermitase (ther+++1t), a thermostable serine protease of
discloses the inhibition of Ase.

It was found to be an inhibitor of picornavirus protease activity. These compounds are suitable therapeutic agents for treating viral infections in mammals.

The present invention is based on the following formula %formula%() During the ceremony, A1 is Ala, Val, Leu, Hele.

Phe, Tyr, Gly, Pro, Ser and 'l''
A2 is an amino acid residue selected from the group consisting of hr, A2 is an amino acid residue selected from the group consisting of Ala, Val, Leu, He and Gl, A3 is At aS Va l , Lcu, If e .

An amino acid residue selected from the group consisting of Phe, Tyr, and Gly, R'hawk is the N-terminal protecting group, and R2 is methyl, isopropyl, isobutyl, 4-hydroxybenzyl, or -CH2CII□CRco1! , where R' is amino, methoxy, ethoxy,
benzyloxy or alkyl having 1 to 6 carbon atoms, provided that when A1 is Ala, A2 is Gly, A' is Phe and R2 is isobutyl, -2 is R'i, tB
or physiologically acceptable salts thereof.

The present invention also provides antiviral compositions comprising one or more of the compounds described above, and methods of using the compositions in the treatment of picornavirus infections in mammals.

The compounds of the present invention are selected tetrapeptide methyl ketones that are capable of inhibiting virus-specific protease activity.

Amino acid residue or ami/! The following abbreviations of $1 apply throughout this specification: Ala ``L-Alanine Gly'' Glycine Gln '' L-Glutamine Glu ' L-Glutamate Leu
"L-leucine lie' L-inleucine Lys"
L-lysine Pbc' L 7z = Lualanine Pro
'L-ProlineSer' L
-Serine Thr'L-Threonine"l'yr' L-Tyrosine Vat °
L-valine Throughout this specification, 'N*N-terminal protecting group arylcarbonyl, alkylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, aralpoxycarbonyl, arylsulfonyl or alkylsulfonyl. Please use other known materials. Gross and Meienhofer, The Peptides, Volume 3 (Academic Press, New York, 1981), Pages 3-81 contain a number of suitable amine protecting groups. Are listed. In this specification,
"Alkyl" individually or as part of a larger group means a linear, cyclic or fractional aliphatic moiety having from 1 to 10 carbon atoms; "aryl" means from 6 to 18 carbon atoms;
aralkoxy is an aliphatic substituent and optionally other iI\ substituent, for example 1% of aryl of 7 to 19 carbons with one or more alkyl, alkoxy, nitro or halo crystals.

As used herein, "halo 6" means C1 or Br.

Examples of suitable N-terminal protecting groups R1 include polmyl, acetyl, trifluoroacetyl, pennoloxycarbonyl (carbobenzoxy), substituted benzyloxycarbonyl, tert-butyloxycarbonyl, isopropyloxycarbonyl, allyloxycarbonyl, 7 Taloyl, benzoyl, acetoacetyl, chloroacetyl, phenoxycarbonyl, methoxysuccinyl, succinyl, 2t4-nonitoO-/x-nyl, dansyl
syl), p-methoxybenzenesulfonyl, p-)luenesulfonyl, methanesulfonyl and phenylthio.

Certain N-terminal protecting groups R1 are abbreviated throughout this specification as follows: χ: carbobenzoxy f3oc: t-butyloxycarbonyl AC:
Acetyl EL: Ethyl Suc: Succinyl MeO8uc: Methoxysuccinyl DNS
: Dansyl DNP:2,4-
Nonitrophenyl When naming the compounds of the present invention, the C-terminal amino acid moiety -NIICHC- (2) was used as the name of the corresponding amino acid. Therefore, the above formula (1
), R1 is Z, AI is Leu, and A2
The compound in which is a+y, A3 is Phe, and R2 is isobutyl is conventionally named N-carbobenzoxy-L-phenylalanyl-di-glycyl-di-leucyl-di-leucyl methyl ketone. This compound is herein referred to as Z Ph e Gly L
It is abbreviated as eu-LeuCI-1z.

The scope of the invention has been defined by reference to closely related compounds that have been empirically determined to be inhibitors of picornavirus protease activity. These compounds are halomethylketone derivatives of selected tri- and tetrapeptides and are designated by the same nomenclature as above. The following formula (TI) R'-[A', A2ΔI ]-N) IC1ICCII2
In X([)H, AI%A2, A3 and R1 are defined as described above for equation (1). In formula (1■),
X is CI or 13r; ), and n is 0 or 1.

Therefore, in the above formula (L)1, R1 is Z and A1
is Leu, A2 is Gly, A is Phe, l is 1, and ■(4 is isobutyl and X is CI). The compound is conventionally named Z-Glycyl-S-Leucyl-S-Leucyl-Rolomethyl Ketone.
It is abbreviated as Le u -LeuCH2CI.

Preferred peptide methyl ketones of the present invention have formula (1) in which R2 is isobutyl, R' is Z, and Me OS
A compound that is u c , Su c , DNS or DNP. Particularly preferred compounds of the present invention are compounds of formula (1) in which 2 is isobutyl and R1 is Z or MeO8u
c, and AI, , A2 and A are Phe, Gly,
The compound is selected from the group consisting of At a and Leu.

Based on its proven activity in inhibiting the growth of picornaviruses, in formula (1) 1(2 is isobutyl, r(1 is Z or MeO8t+c, A'75'
AI [L or Lcu, A) is Phc or Gly
The compounds in which A3 is Phe or Leu are the most preferred compounds within the scope of the present invention.

Particular examples of compounds within the scope of this invention include:

Z-Phe-Gly-A1 a -LeuCtl,
:MeOSuc-Phe-Gly-Leu
-LeuCll, ;Z -Phe -Gly
-Leu -LcuCII 3 ;^c-Phe-Gl
y-^1a-LeuCII3;5uc-Phe-G
ly-^la-LeuCIl,;MeOSuc
-Phe-Gly-8Ia-LeuCII:
DNS -Phe -GI y-81a-LeuCH
t;DNP-Phe-Gly-^1a-LeuC113
;Z-Leu-Gly-81a-LcuC1lz;Z-
Phe-Gly-Gly-LeuCII*:Z-Phe
-Leu-8l a -LeuCII 3:Z-Phe
-Gly-Phe-LeuCII*;Z-Phe-Gl
y-Ser-LeuCL;Z-1'l+e-Gly-P
ro-LeuCH3:MeOSuc-^1a-11e-
Phe-LeuCII*;Z-Phe-Gly-^1a
-ValCL:Z-Phe-Gly-81a-TyrC
I13; MeOSuc-PI+e-Gly-Leu-G
lu(OMe)CII3MeOSuc-Δla-11e
-Phe-Glu(OMe)Cll,.

Physiologically acceptable salts of compounds of formula , maleic acid, acetic acid, citric acid, succinic acid, etc. Alternatively, salts of free peptidic acids are included within the scope of this invention, including sodium, kaliwam and ammonium salts.

In carrying out the antiviral method of the present invention, the compounds may be used alone, in combination with each other, in combination with other therapeutic agents, or in combination with various inert physiologically acceptable carriers in various dosage forms. It can be used orally or parenterally. The required dosage will vary depending on the compound, the dosage form used and the animal being treated. Typically, therapy is started at a lower dose and the dose is increased until the desired inhibitory effect is achieved.

Compounds of the invention are described by Kettner et al. 62: 56 (1974) and 7f-
can be manufactured by techniques generally similar to those disclosed in .

First, N-protected peptides or amino acids are reacted with about 1 equivalent of N-methylmorpholine and 1 equivalent of isobutyl chloroformate at about -20°C to form a mixed peptide-isobutyric anhydride (+n1xe
d peptide-isobutyric ac
id anhydride). This standard method was developed by Anderson et al., L.
American Chemica/lz-'/Saiaty (J, 8mer, Chem, Soc, ) 89
: 5012 (1967). Secondly,
The resulting mixed anhydride is treated with about 1 equivalent of diazomethane in ketone hydrofuran or other suitable inert aprotic solvent at 0°C to produce the N-protected peptide or amino acid diazomethyl ketone. Third, this compound was purified with HC in absolute ethanol or ether at 0°C.
1 or HBr to generate the N-protected halomethyl ketone. Fourth, convert the methyl ketones to the corresponding N
1 on charcoal as a catalyst from halomethylketones
It can be produced by hydrogenolysis in anhydrous methanol using 0% palladium.

Coupling the deproLected halomethyl ketone with other N-protected peptides or mixed anhydrides of amino acids produced according to the method described above.
Larger peptide halomethyl ketones can be assembled by Deprotection of the N-terminal amine group can be accomplished by treatment with trifluoroacetic acid, anhydrous HF, aqueous HCl"CC treatment, or other methods known to those skilled in the art, using a deprotected methyl ketone rather than a The corresponding peptide methyl ketones can be prepared by the following coupling methods. Specific examples of these methods are disclosed in Examples 1 and 2 below.

General methods of application suitable for producing compounds within the scope of this invention are described in the preceding paragraph of the Examples below. In Synthesis Methods and Example 1, all parts and percentages are by weight and all degrees are in degrees Celsius unless otherwise specified.

General Synthetic Method The N-protected amino acid or peptide about 1-2i9 is dissolved in 20 mβ of tetrahydrofane (THF) and the resulting solution is cooled to -20°C. N-Ethylmorpholine (1 eq.) and isobutyl chloroformate (1 eq.) were added, and after 5 minutes, additional 10- and triethylamine were added to cold THF.
Add equivalent amount. The resulting mixture was diluted with trimethylformamide (
Add immediately to an equivalent amount of amine hydrochloride or trifluoroacetate dissolved in DMF) 5-. The reaction system was then heated for 1 hour.
The resulting mixture is filtered, stirred at -20° C. and then at about 23° C. for 2 hours, and then concentrated to about 5 mβ by evaporating the liquid thereby obtained. The obtained concentrate or residue was dissolved in ethyl acetate,
It is then washed successively with 0.2N hydrochloric acid, 5% sodium bicarbonate solution and saturated aqueous sodium chloride solution. The resulting organic solution is then briefly dried over sodium sulfate, filtered and finally evaporated to yield the crude peptide product.

N-protected ami/PI! and N-hydroxysuccinimide esters of Pepti 1T, Anderson et al., Journal of the American Chemical Society 8.
6: 1839 (1964). The O3u ester was dissolved in a minimum volume of dioxane and the resulting fB solution was dissolved in equal volumes of 1.5 equivalents of triethylamine and the amino acid (1,5
(eq.) or the peptide (1,1 eq.) to form a reaction mixture. After about 5 minutes, if a complete solution is not obtained, a small sample of the reaction mixture can be diluted with water and another sample can be diluted with dioxane. Based on the results obtained, the reaction mixture is then diluted with a suitable solvent (water or dioxane) until complete dissolution. After the reaction has finished, the reaction mixture is acidified with hydrochloric acid and the resulting product is The resulting extract was extracted into ethyl acetate and then diluted with 0.2N hydrochloric acid and then 0.2N in saturated sodium chloride.
Wash with hydrochloric acid. The washed extract is then dried over sodium sulfate, filtered and finally evaporated to dryness to obtain the crude peptide.

3. Others! 'Hitotomo Store - Peptide guncyl, 2,4-dinitrophenyl and methoxysuccinyl transconductors are prepared by reacting selected chloride, fluoride or N-hydroxysuccinimide esters with appropriate peptides. Ru. Acetyl and succinyl derivatives can be prepared from the corresponding anhydrides. The peptide hydrochloride or trifluoroacetate is dissolved in 50% aqueous dioxane at a level of 0.25 mmol/1 nji and the resulting solution is cooled to O'C. The selected coupling reagent (1.0-1.2 equivalents) is dissolved in dioxane and added together with 2 equivalents of sodium bicarbonate. The resulting reaction system is tested according to the disappearance of ninhydrin-positive substances.

4, - Methyl Esute n Sol, Bunyu and Ahi.

N-protected methyl ester in dioxane (1 mmol)
1 ml) and an equal volume of 1. Add OON sodium hydroxide over 30 minutes. The disappearance of starting material is checked by thin layer chromatography. After this reaction has finished, an equivalent of 1. Add OON hydrochloric acid and dilute the solution to 100+aA with water. The product is then extracted into ethyl acetate and the extract obtained is washed with 0.2N hydrochloric acid and then with saturated hydrochloric acid) l) with 0.2N hydrochloric acid in rum. The solvent is then removed by evaporation to obtain the crude carboxylic acid.

5. Convert selected peptides with Boa group to trifluoroacetic acid? 8 Remove the Boc:JL group from the peptides by stirring the resulting solution for 5 minutes at 23° C. 6 Cold ether is then added. If ether is added and a precipitate is obtained, triturate it with ether and lit! do. If no precipitate is obtained, evaporate the ether, add toluene and co-evaporate the remaining trifluoroacetic acid to obtain the deprotected Vepti Y as the trifluoroacetate salt.

Alternatively, the Boc-protected peptide can be dissolved in ethanolic hydrochloric acid (2.0-3.5N) and the resulting solution
C. for about 30 minutes and then the solvent may be evaporated. In all cases, the peptide hydrochloride or
The fluoroacetate is dried under reduced pressure in the presence of solid potassium hydroxide and phosphorus pentoxide overnight.

6.1 Hydrogenation of Nibutyl Esters (Bu) - The butyl peptide esters are dissolved in fluoroacetic acid and the resulting solution is stirred for 1 hour at about 23°C. The solvent is then evaporated and the resulting residue is dissolved in toluene]I)
Dissolve H. After the second toluene evaporation step, the residue is dried under vacuum over solid potassium hydroxide. The crude product is crystallized from a suitable solvent such as toluene or ethyl acetate.

7. Thin layer chromatography (TLC) method 5X10C
Perform TLC treatment using a fluorescent reagent on a J silica gel plate. The spots are visualized by conventional methods using UV light or an iodine bottle.

A zero order solvent system is useful for chromatography in which peptides with free amino groups protected by r3oc groups are exposed to HCI vapor and then stained with ninhydrin.

Methanol: chloroform (1:9) butanol: acetic acid: water (4: 1: 1> ethyl acetate: hexane (8:2) (sigma-resistant) 165: 739 (1
HLe u CHz .HBr was produced by a method substantially similar to that described by JIH (974).

ZPhe-GlyAta-OH was prepared according to the following method. Dissolving the N-hydroxysuccinimide ester of Z-Pbe-OH in nooxane,
About 1 equivalent of H-Gly-Ala-QH1 was added to the resulting solution.
Filter into a solution containing approximately 1.5 grams of triethylamine and water. The resulting reaction mixture is stirred at about 23° C. overnight and then concentrated to about 60% of its original volume by evaporation.

This concentrated solution was then heated 1. until acidic. Diluted with ON hydrochloric acid. The obtained product was extracted into ethyl acetate, and the obtained extract was washed with 0.2N hydrochloric acid and then with a saturated aqueous thorium chloride solution adjusted to 0°ω IN hydrochloric acid. The washed extracts were then dried over anhydrous sodium sulfate and filtered. The solvent is then evaporated from the extract,
The product Z-PI+ e -Gly -AlaOtf was obtained as a foam. The crude product was recrystallized from ethyl acetate.

Z Pl+e Gly Ala-0) (H-L
It was combined with eucl(, ・I-I B r as follows. First, Z-Pl+ e -Gly Al
The mixed anhydride of a -0I-1 is Z-Ph e -Gl
y-Aln-0! ((1,227,2,85 mmol) and N-methylmorpholine (0,32 mB,
2.85 mmol) was dissolved in 35+n of tetrahydrofuran and packaged individually.

The resulting solution was cooled to about -20°C and enriched with 0.251 IIλ (1.90 mmol) of inbutyl chloroformate.

After 10 minutes, the resulting solution was diluted with triethylamine.
(1,90 mmol) of cold trimethylformamide 5I111i! H-LcuCH dissolved in.

・Add to HBr (0.40 g, 1.90 mmol).

The resulting mixture was stirred for 1 hour at -20 °C,
Warm to about 23°C and then stir at about 23°C overnight.

The resulting solution was filtered, then 100 parts of ethyl hexaacetate, evaporated to a volume of approximately 5%, was added to reduce the resulting solution to 5%
sodium bicarbonate, 0.2N hydrochloric acid and saturated chloride)
Continued washing with +7um aqueous solution. After drying over sodium sulfate and filtration to remove undissolved material, the solvent was removed by evaporation to yield a white solid,
This was recrystallized from hot ethyl acetate to give the first product (0
, 2°7g, melting point 133-134°C) and a second product (0.25g, melting point 133.5-134°C).

Thin layer ov thornfish (Me OH: C1-lCl
) (1: 9, V: V)) with 1 soil single spot (
RrO,48), and the spectrum was consistent with the expected structure.

C25l(:+aN+Og: total 1;)-I1m: ca 4.65; l217,12; N10. 40 implementation value:
C64,51; H6,91; NIo, 56 Z-Ph e -Gly -Ala=-Le of Example 1 above
Z-Phe Gly-Leu by a method substantially similar to that described for the preparation of u C1-13
0H (2,232,4,furimimole) to 1(-L
Z-Ph e -Gly -Leu -Leu by binding to cuCH7.I-IBr
CHs were produced. When the product was recrystallized from ethyl acetate, 2.34 g of Z-Ph e -Gly -Leu
-Le uCD3(?yi, 1.', (165,5-
166,5°c) is generated.

Cz2H4<N 406: Calculated value: C66,26; H7,66; H9,66
Experimental values: C66,48; H7,75; H9,66
Z-Ph e -Gly -Leu -Leu
CHa(1.

09.1.72 mmol) in absolute ethanol 100! t
0.5 fI of 10% perlanum on carbon dissolved in lk
and 0.5-4N ethanolic HCI were added.

The resulting mixture was heated to an initial pressure of 40 ps.
i) Hydrogenation was carried out at (276 KPa). The catalyst was removed by ip filtration and the solvent was then evaporated leaving a foam of 0.8
s9 was produced and solidified after addition of ether. The product was washed thoroughly with ether and dried under reduced pressure over solid potassium hydroxide and phosphorus pentoxide.

H-Ph e -Gly -Leu -Leu
CH3.HCI (0.62 g, 1.29 mmol) and N-hydroxysuccinimide of methylsuccinate (0.30 g, 1.29 mmol) were dissolved in 5 mβ of N,N-dimethylformamide, then triethylamine (0, 18+oβ, 1.29 mmol) was added. - After stirring overnight, 5% sodium carbonate aqueous solution 5+oj2 was added and the resulting solution was stirred for 15 minutes. The resulting product was extracted into ethyl acetate. extract with 5% hydrogen carbonate)
+7 Tsum aqueous solution, 0.2N hydrochloric acid and saturated chloride) l
) followed by washing with rum aqueous solution. The washed extract was dried over 17 μm of anhydrous sulfuric acid and filtered. The solvent was then evaporated to give an oily product. When the product is crystallized from ethyl acetate:hexane, Me of 0.68&
O8u c -Ph e-Gly -Leu -L
eu CH3 (melting point 113.5-114°C) was obtained.

c2. H,,N,0,: Calculated value: C62,45; H7,42; N10.0
5 Experimental values: C62,09; H7,82; NIo,
10 Biology of Tetrapeptide Methyl Ketones Selected compounds of the present invention inhibited viral protease activity in two assays. The first assay, referred to as the virus cleavage assay, compares protein trade synthesis patterns (visualized by incorporation of labeled amino acids) in virus-infected He LLL cells grown in the presence and absence of selected test compounds. It is. The second assay, known as the plaque inhibition assay, uses agar-supplemented cell culture medium (
ager-overlaid cell culture
eq) to evaluate the effect of the test compound on viral infectivity. Of course, the toxic effect of the test compound, if any, is important during the incubation period of the plaque inhibition assay. i,
It will be worshiped. Both tests are described in more detail below.

Virus excision 11 In this assay, a sample of growing He1a-0 cells is inoculated with human voriomyrtis virus type 2 (h
uman poliotoyeliLiSvirus
type 2) or human rhinovirus type 1A (1
+umarlrhinovirus type 1Δ)
exposed to After several hours, host cell metabolism was severely inhibited and the added radioactive amino acids were incorporated only into viral proteins. After adding various concentrations of the test compound to the cell samples, fils proteins were incubated at the appropriate mid-cycle of infection (mi
d-cycle) (3-5 hours after initial exposure of cells to virus) for 60 minutes. The cell sample was then treated with 1% (w/v) sodium dodecyl sulfate and 1%
(V/V) containing 2-mercaptoethanol 0,0
IW was dissolved in methane buffer (pH 6,8). Previously, Colant et al., Prosefsinges op National Academy.
Of science of the knee, varnish, knee (P
rOc, Nntl, ^cad, sci, USA) 76:
2992 (1979).
ed) Viral proteins were separated by polyacrylamide del electrophoresis and then detected by autoradiography. A compound was recorded as active at a selected concentration of a test compound if it disrupted the normal mode of processing of viral proteins.

The presence of a test compound exhibiting viral protease inhibitory activity was generally clearly distinguishable by the presence of commercial molecular weight protein species that were not evident in medial cells, corresponding to labeled cell samples.

In this assay, cultured HeLa cells were grown at 60 mm.
confluence in a plastic bird dish (can f I u
ency). Each culture was then grown to approximately 300 plaque-forming units.
The cells were infected with the virus. Poliovirus type 2 and rhinovirus type IA were used in the studies reported below. The viruses used in a given experiment were allowed to absorb into cells for 30 minutes at 34.5°C.

Each test compound was dissolved in ethanol or trimethyl sulfoxide to a concentration 1.00 times the highest concentration tested. The resulting solution was then heated to inactivate fetal bovine serum (regal calfserum).
Matsukoi medium (McC) containing % and agar 0.38%
oy'S medium) and diluted 1:100. Then, a 2-fold dilution was made by placing it in an agar medium.

After adsorption of the virus to the cells, excess virus was washed away and each culture dish was overlayed with 5 ml of agar medium containing a preselected diluent of the compound to be tested. Only agar medium was added to the control medium. Each medium was then incubated at 34.5°C for 2 to 511 hours depending on the virus used, and plaque development was determined as nT capacity.

A plaque is a roughly circular area of dead cells in culture that marks the area where a single plaque-forming unit of the virus first infected a cell. The addition of agar limits the mobility of the virus, so that virus infection is only transmitted between consecutive cells.

All cultures were stained with 1% crystal violet when the plaques in the control cultures were large enough to be easily observed and still relatively separate. Plaques appeared as clear areas in a deep purple field of uninfected cells. Addition of a toxic dose of test compound results in visible cell detachment in culture blood.

In the virus cleavage assay, the following compounds were used at test concentrations of 100% against poliovirus type 2 and leprosy virus type IA.
The activity was shown at μg/mβ. In addition, the compound Z-Pl+
c -Gly -Ala -Leu CH3 and Me O3u c -Ph e -Gly -Le
u −, LeuCH = 50 against poliovirus type 2
It was active in corridor/m1. In this assay, the compound Boc
-Ph e Gly =Ala -LeuC
H3 showed no activity.

X1 eye student 1 3 Z-Phe-Gly-^1a-LeuCl
134 MeO5uc-Phe-Gl
11-^1a-LeuCII*5 Z-P'h
e-Gly-Leu-Lcu(:ll*Compound Z-Ph
When incorporated into agar adducts at a concentration of 50 μg/ff+1, e -Gly Al a -Leu CHl reduced plaques harboring poliovirus type 2 and leivirus type IA by more than 90% compared to controls. Ta. In assays for this compound, no cytotoxicity was observed at concentrations up to 1 mg/ml.

Comparison - Examples 1 to 30: Nijidai in Virus Kiritsuguyodo
As stated above, the range of compounds representing the present invention is defined by expectations based on the observed antiviral activity of similar chloromethyl ketone derivatives. The results of a series of Twill cut VfI assays that evaluated the protease inhibitory activity of chloromethyl ketones and F ribeptide are shown in Table 1. The results are reported as follows.

-No activity 10/-Low activity+ 100 deer/mβ and 10+ activity 50 μtl/a+j! Active with +++
Activity +++ 1 at 10 cycles/111β <5 cycles/Activity 1 at ml, Z-Phe-Gly-ΔIa-LeuCII2CI
++ +2.1lPhe-Gly-81a
-LeuCl12Cl +/
-3, ^c-Phe-GIS'-^1a-LeuCI
I2CI ++ +4.5ue-Phc-Gl
y-81a-LeuCIIzC1++ 15, MeO
Suc-Pbc-Gly-8In-LeuCI
12CI + 10+6, DNS-Phe-Gly-
^1a-LcuCII2CI +++7, D
NP-Pbe-Gly-ΔIa-LeuCH2C
I + + 18, mouth oc-Gly-^1a-
LeuCHzCl ++9.2-8la-
nily-^1a-LeuCIIzC1+/-10, Z-
Leu-Gly-^1a-LeuCI (zcl
+ Master +11, Z - G I n - G l
y-^Ia-LeuCIlzCl +1
2, Z-Phe-C: 1y-Gly-LeuCt12C
l + 10+13.2-Phe-Gly-^1
a-LcuCI12Br +++14.2-P
he-Gly-^1a-ValCII2Cl
+ +15.2-Phe-Gly-Leu-Le
uC112C1 Shijuju+16.2-Pl+e-Lsu-
^1a-LeuCI12CI ++17.2-
Phe-C1y-PI+e-LcuCIIzCl
++18, Z-Phe-Phe-81a-LeuC
l12Cl + /-19,2
-Phe-Gly-5er-LeuC1i□C!
±20.2-Phe-Ser-Δ1a-LeuC
112C1+21.2-Pbe, -Gly-Lys-L
euCIlzCl +22.2-Phe
-Lys-^la LeuCII2'Cl
+23.2-Phe-Gly-Pro-LeuC
H2CI + 124.1-Phe-Pro layer n-LcuCII2Cl 25, 8c-
Phe-Gly-Glu-LeuCl□CI
+/-26, ^c-Phe-Glu(OEt)-
^1n-LeuCllzC1+ /-27, MeOSu
c-ΔIa-Ile-Pbe-LeuCII2Cl
+++28, MeOSuc-Phe-Gly-1,e
u- to 1u (OCH:+) CII2C1 10++ 129, MeOSuc-Δ1a-11e-Pbe-Glu
(OCII*) CIl□CI Officer

Claims (1)

[Claims] 1. The following formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ In the formula, A^1 is Ala, Val, Leu, Ile, Phe, T
is an amino acid residue selected from the group consisting of yr, Gly, Pro, Ser and Thr; A^2 is an amino acid residue selected from the group consisting of Ala, Val, Leu, He and Gly; 3 is Ala, Val, Leu, Ile, Phe, T
An amino acid residue selected from the group consisting of yr and Gly, R^1 is an N-terminal protecting group, and R^2 is methyl, isopropyl, isobutyl, 4-hydroxybenzyl, or ▲ where the mathematical formula, chemical formula, table, etc. ▼, where R^3 is amino, methoxy, ethoxy, benzyloxy, or alkyl having 1 to 6 carbon atoms, provided that A^1 is Ala, A^2 is Gly, A^3 is P
When he and R^2 are isobutyl, R^1 is Boc
or a physiologically acceptable salt thereof. 2, A^1 is Ala, Val, Leu, Ile, Phe
, Gly, Pro, and Ser, A^2 is an amino acid residue selected from the group consisting of Ala, Val, Leu, He, and Gly, and A^3 is Ala. , Val, Leu, He and Phe, and R^1 is Z, Ac, Boc, MeOSuc, Suc, D
The compound according to claim 1, which is NS or DNP, and R^2 is isopropyl or isobutyl. 3.A^1 is Ala, Val, Leu, Gly, Pro
or Ser, A^2 is Ala, Val, Leu or Gly, A^3 is Ala, Val, Leu or Phe, R^1 is Z, MeOSuc or Suc, R^2 is Isopropyl or isobutyl, the compound according to claim 2. 4, A^1 is Ala, Val, Leu or Gly, A^2 is Leu or Gly, A^3 is Leu or Phe, R^1 is Z or MeOSuc, R^2 is The compound according to claim 3, which is isopropyl or isobutyl. 5. A^1 is Ala or Leu, A^2 is Gly, A^3 is Phe, R^1 is Z or MeOSuc, and R^2 is isobutyl. Compound according to item 4. 6. The compound according to claim 5, wherein A^1 is Ala and R^1 is Z. 7. The compound according to claim 5, wherein A^1 is Leu and R^1 is Z. 8. A^1 is Gly, A^2 is Gly, A^3 is Phe, R^1 is Z, and R^2 is isobutyl according to claim 4. Compound. 9. A^1 is Ala, A^2 is Leu, A^3 is Phe, R^1 is Z, and R^2 is isobutyl according to claim 4. Compound. 10. Treatment of picornavirus infections in mammals, comprising a compound according to claim 1 in an amount effective to inhibit viral protease activity and a physiologically acceptable carrier or diluent. Composition for. 11. Treatment of picornavirus infections in mammals, comprising a compound according to claim 2 in an amount effective to inhibit viral protease activity and a physiologically acceptable carrier or diluent. Composition for. 12. Treatment of picornavirus infections in mammals, comprising a compound according to claim 3 in an amount effective to inhibit viral protease activity and a physiologically acceptable carrier or diluent. Composition for. 13. Treatment of picornavirus infections in mammals, comprising a compound according to claim 4 in an amount effective to inhibit viral protease activity and a physiologically acceptable carrier or diluent. Composition for. 14. Treatment of picornavirus infections in mammals, comprising a compound according to claim 5 in an amount effective to inhibit viral protease activity and a physiologically acceptable carrier or diluent. Composition for. 15. Treatment of picornavirus infections in mammals, comprising a compound according to claim 6 in an amount effective to inhibit viral protease activity and a physiologically acceptable carrier or diluent. Composition for. 16. Treatment of Picornavirus infections in mammals, comprising a compound according to claim 7 in an amount effective to inhibit viral protease activity and a physiologically acceptable carrier or diluent. Composition for. 17. Treatment of picornavirus infections in mammals, comprising a compound according to claim 8 in an amount effective to inhibit viral protease activity and a physiologically acceptable carrier or diluent. Composition for. 18. Treatment of picornavirus infections in mammals, comprising a compound according to claim 9 in an amount effective to inhibit viral protease activity and a physiologically acceptable carrier or diluent. Composition for. 19. A method for treating picornavirus infection in a mammal, which comprises administering the composition according to claim 10 to the mammal. 20. A method for treating picornavirus infection in a mammal, which comprises administering the composition according to claim 11 to the mammal. 21. A method for treating picornavirus infection in a mammal, which comprises administering the composition according to claim 12 to the mammal. 22. A method for treating picornavirus infection in a mammal, which comprises administering the composition according to claim 13 to the mammal. 23. A method for treating picornavirus infection in a mammal, which comprises administering the composition according to claim 14 to the mammal. 24. A method for treating picornavirus infection in a mammal, which comprises administering the composition according to claim 15 to the mammal. 25. A method for treating picornavirus infection in a mammal, which comprises administering the composition according to claim 16 to the mammal. 26. A method for treating picornavirus infection in a mammal, which comprises administering the composition according to claim 17 to the mammal. 27. A method for treating picornavirus infection in a mammal, which comprises administering the composition according to claim 18 to the mammal.