Inhibition of viral protease activity by peptide halomethyl ketones

Selected tripeptide and tetrapeptide halomethyl ketones are employed in processes for treating viral infection in mammals. These compounds inhibit picornavirus protease activity.

BACKGROUND OF THE INVENTION 
This invention relates generally to inhibition of viral proteases, and more 
particularly, to use of certain peptide halomethyl ketones as specific 
inhibitors of picornavirus protease activity. 
Proteases are enzymes which cleave proteins at specific peptide bonds. In 
living systems, highly specific proteases and complementary protease 
inhibitors mediate or control a broad spectrum of biological functions. 
For example, proteases cleave precursors to form active proteins in 
post-translational processing of polypeptides, provide mechanisms for 
zymogen activation cascade reactions such as blood coagulation, 
fibrinolysis, and certain immunological reactions, and mediate transport 
of selected proteins across biological membranes. Accordingly, proteases 
represent potential targets for therapeutic agents designed to function as 
specific inhibitors of protease activity. 
Proteases encoded by viral genomes play a critical role in viral 
reproduction. Viral proteases cleave large precursor polypeptides produced 
by infected cells into smaller protein components, or subunits, which are 
subsequently assembled to form functional virus structures. Lozitskii et 
al., Usp. Sovrem. Biol. 93:352-362 (1982) have reviewed the role of 
proteolysis in reproduction of avian and mammalian viruses, and have 
surveyed part of the literature relating to viral protease inhibitors. 
Picornaviruses represent a significant class of viral pathogens in humans 
and other mammals. Included within this class are polioviruses, 
rhinoviruses, and the viruses which are the etiologic agents of hepatitis 
A and hoof-and-mouth disease. During picornavirus replication, viral mRNA 
is translated in a continuous passage of ribosomes along a viral mRNA 
molecule, producing a linear protein product which is cleaved at selected 
sites by virus-specified proteases prior to dissociation of a 
protein/ribosome complex. 
A number of workers have sought specific inhibitors of picornavirus 
protease activity. Korant, J. Virol. 10:751-759 (1972), discloses 
inhibition of poliovirus and echovirus-12 protein processing by 
chloromethyl ketone derivatives of simple amino acids. Specifically, 
Korant discloses inhibition by tolylsulfonylphenylalanyl chloromethyl 
ketone (TPCK) and tolylsulfonyllysyl chloromethyl ketone (TLCK). Summers 
et al., J. Virol. 10:880-884 (1972), similarly disclose inhibition of 
protease cleavage of large poliovirus-specific polypeptides by TPCK, TLCK, 
and D- and L-isomers of carbobenzyloxyphenylalanyl chloromethyl ketone 
(ZPCK). In a subsequent report, Korant et al., Proc. Natl. Acad. Sci. USA 
76:2992-2995 (1979), describe inhibition of poliovirus protein processing 
by carbobenzyloxyleucyl chloromethyl ketone (ZLCK). 
Various peptide derivatives with capacity to inhibit protease activity are 
known. Powers, "Haloketone Inhibitors of Proteolytic Enzymes", in 
Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, 
Weinstein, ed. (Marcel Dekker, New York, 1977), pp. 65-178, has surveyed 
the literature reporting inhibition of protease activity by haloketone 
derivatives of amino acids and peptides. Powers et al., Biochem. Biophys. 
Acta 480:246-261 (1977), disclose inhibition of subtilisin BPN', a 
bacterial protease, by a series of peptide chloromethyl ketones. Of the 
compounds tested, acetyl-L-phenylalanyl-L-glycyl-L-alanyl-L-leucyl 
chloromethyl ketone (Ac-Phe-Gly-Ala-leuCH.sub.2 Cl) was the fastest 
inhibitor. A related compound, 
methoxy-succinyl-L-phenylalanyl-L-glycyl-L-alanyl-L-leucyl chloromethyl 
ketone (MeOSuc-Phe-Gly-Ala-LeuCH.sub.2 Cl) is disclosed by Enzyme Systems 
Products in a November 1981 product bulletin. 
Ito et al., Biochem. Biophys. Res. Commun. 49:343-349 (1972), describe 
experiments involving inhibition of chymotrypsin, a digestive protease, by 
certain peptide aldehydes. Ito et al. also tested for inhibition of 
chymotrypsin by Ac-Leu-Leu-PheCH.sub.3, a tripeptidyl methyl ketone. 
However, no inhibition was observed at an inhibitor concentration of 600 
.mu.g/mL. 
Finally, Fittkau et al., "Synthesis and Properties of Peptide Ketones", in 
Peptides 1982, Blaha et al., eds., (de Gruyter, New York, 1983) pp. 
617-622, disclose inhibition of thermitase, a thermostable serine protease 
of Thermoactinomyces vulgaris, by certain peptide methyl ketones. 
It has now been found that picornavirus protease activity can be inhibited 
by certain peptide halomethyl ketones. These compounds can potentially be 
employed in treatment of viral infection in mammals. 
SUMMARY OF THE INVENTION 
The present invention provides processes for treating picornavirus 
infection in a mammal, comprising administering to a mammal an effective 
antiviral amount of a compound of the formula 
##STR1## 
or a physiologically acceptable salt thereof, wherein 
A.sup.1 is an amino acid residue selected from the group consisting of Ala, 
Val, Leu, Ile, Phe, Tyr, Gly, Pro, Ser and Thr; 
A.sup.2 is an amino acid residue selected from the group consisting of Ala, 
Val, Leu, Ile, and Gly; 
A.sup.3 is an amino acid residue selected from the group consisting of Ala, 
Val, Leu, Ile, Phe, Tyr and Gly; 
R.sup.1 is an N-terminal protecting group; and 
R.sup.2 is methyl, isopropyl, isobutyl, 4-hydroxybenzyl, or 
##STR2## 
where R.sup.3 is amino, methoxyl, ethoxyl, benzyloxy or alkyl of 1 to 6 
carbon atoms; 
X is Cl or Br; and 
n is 0 or 1; with the proviso that both A.sup.3 and A.sup.1 are not 
simultaneously Ala. 
The present invention also provides compounds of the formula 
##STR3## 
or physiologically acceptable salts thereof, wherein 
A.sup.1, A.sup.2, A.sup.3, R.sup.1, X and n are as defined above, and 
R.sup.2 is 
##STR4## 
where R.sup.3 is methoxyl or ethoxyl.

DETAILED DESCRIPTION OF THE INVENTION 
The compounds employed in processes of the present invention are halomethyl 
ketone derivatives of selected tripeptides and tetrapeptides. These 
compounds inhibit processing of picornavirus capsid proteins by 
virus-encoded proteases. 
As used throughout the specification, the following abbreviations for amino 
acid residues or amino acids apply: 
Ala: L-alanine 
Gly: glycine 
Gln: L-glutamine 
Glu: L-glutamic acid 
Leu: L-leucine 
Ile: L-isoleucine 
Lys: L-lysine 
Phe: L-phenylalanine 
Pro: L-proline 
Ser: L-serine 
Thr: L-threonine 
Tyr: L-tyrosine 
Val: L-valine 
As used throughout the specification, "N-terminal protecting group" means 
an arylcarbonyl, alkylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, 
aralkoxycarbonyl, arylsulfonyl, alkylsulfonyl, or arylsulfonyl peptide 
protecting group, or other equivalents known to those skilled in the art 
of peptide synthesis. Gross and Meienhofer, eds., The Peptides, Vol. 3, 
(Academic Press, New York, 1981) pp. 3-81, the disclosure of which is 
hereby incorporated by reference, describe numerous suitable amine 
protecting groups. As used herein, either individually or as part of a 
larger group, "alkyl" means a linear, cyclic, or branched-chain aliphatic 
moiety of 1 to 10 carbon atoms; "aryl" means an aromatic moiety, e.g., 
phenyl, of 6 to 18 carbon atoms, unsubstituted or substituted with one or 
more alkyl, nitro, alkoxy, or halo groups; and "aralkoxy" means an aryl 
moiety of 7 to 19 carbons having an aliphatic substituent, and, 
optionally, other substituents such as one or more alkyl, alkoxy, nitro or 
halo groups. As used herein, "halo" means Cl or Br. 
Examples of suitable values for N-terminal protecting group R.sup.1 include 
formyl, acetyl, trifluoroacetyl, benzyloxycarbonyl (carbobenzoxy), 
substituted benzyloxycarbonyl, tert-butyloxycarbonyl, 
isopropyloxycarbonyl, allyloxycarbonyl, phthaloyl, benzoyl, acetoacetyl, 
chloroacetyl, phenoxycarbonyl, methoxysuccinyl, succinyl, 
2,4-dinitrophenyl, dansyl, p-methoxybenzenesulfonyl, p-toluenesulfonyl, 
methanesulfonyl and phenylthio. Particularly convenient values for R.sup.1 
are carbobenzoxy (Z), tert-butyloxycarbonyl (Boc) and acetyl (Ac). 
Certain values for N-terminal protecting group R.sup.1 are abbreviated as 
follows throughout the specification: 
Z: Carbobenzoxy 
Boc: t-Butyloxycarbonyl 
Ac: Acetyl 
Et: Ethyl 
Suc: Succinyl 
MeOSuc: Methoxysuccinyl 
DNS: Dansyl 
DNP: 2,4-Dinitrophenyl 
In naming compounds useful in the process of the invention, C-terminal 
amino acid moiety 
##STR5## 
is assigned the name of a corresponding amino acid. Thus, a compound of 
formula I, above, wherein R.sup.1 is Z, A.sup.1 is Leu, A.sup.2 is Gly, 
A.sup.3 is Phe, R.sup.2 is isobutyl, and X is Cl is conventionally named 
N-carbobenzoxy-L-phenylalanyl-L-glycyl-L-leucyl-L-leucine chloromethyl 
ketone; this compound is abbreviated herein as Z-Phe-Gly-Leu-LeuCH.sub.2 
Cl. 
Contemplated classes of compounds of formula I preferred for use in various 
embodiments of the invention include the following. A first class includes 
compounds wherein R.sup.1, R.sup.2, A.sup.1, A.sup.2, A.sup.3, and X are 
as previously defined and n is 0. A second class includes compounds 
wherein R.sup.1, R.sup.2 and X are previously defined, n is 1, A.sup.1 is 
selected from the group consisting of Phe, Gly, Ala, Pro, Leu, and Ser; 
A.sup.2 is selected from the group consisting of Ala, Val, Leu, Ile, and 
Gly; and A.sup.3 is selected from the group consisting of Ala, Val, Leu, 
Ile, Phe, Tyr and Gly. 
On the basis of superior effectiveness in inhibiting viral replication, 
those compounds of formula I wherein n is 1; R.sup.2 is isobutyl or 
methoxycarbonylethyl; R.sup.1 is Z or MeOSuc; A.sup.1 is Ala, Gly or Leu; 
A.sup.2 is Ile or Gly; and A.sup.3 is Phe, Ala or Leu are particularly 
preferred for use in the processes of the present invention. 
The compounds of the present invention are compounds of the foregoing 
formula I wherein R.sup.2 is methoxycarbonylethyl or ethoxycarbonylethyl. 
Contemplated classes of compounds within this genus include classes 
corresponding in scope to those previously defined for the compounds 
employed in the processes of the invention. Preferred compounds of the 
invention are those wherein n is 1; R.sup.1 is Z or MeOSuc; R.sup.2 is 
methoxycarbonylethyl; A.sup.1 is Ala or Leu; A.sup.2 is Gly; and A.sup.3 
is Phe or Ala. Particularly preferred compounds are those wherein A.sup.1 
is Leu; A.sup.3 is Phe; and R.sup.1 is MeOSuc. 
Specific examples of compounds useful in various embodiments of the 
invention include 
Z-Phe-Gly-Leu-LeuCH.sub.2 Cl; 
Z-Phe-Gly-Ala-LeuCH.sub.2 Cl; 
Ac-Phe-Gly-Ala LeuCH.sub.2 Cl; 
Suc-Phe-Gly-Ala-LeuCH.sub.2 Cl; 
MeOSuc-Phe-Gly-Ala-LeuCH.sub.2 Cl; 
DNS-Phe-Gly-Ala-LeuCH.sub.2 Cl; 
DNP-Phe-Gly-Ala-LeuCH.sub.2 Cl; 
Boc-Gly-Ala-LeuCH.sub.2 Cl; 
Z-Leu-Gly-Ala-LeuCH.sub.2 Cl; 
Z-Phe-Gly-Gly-LeuCH.sub.2 Cl; 
Z-Phe-Leu-Ala-LeuCH.sub.2 Cl; 
Z-Phe-Gly-Phe-LeuCH.sub.2 Cl; 
Z-Phe-Gly-Ser-LeuCH.sub.2 Cl; 
Z-Phe-Gly-Pro-LeuCH.sub.2 Cl; 
Z-Phe-Gly-Ala-LeuCH.sub.2 Br; 
MeOSuc-Ala-Ile-Phe-LeuCH.sub.2 Cl; 
MeOSuc-Phe-Gly-Leu-Glu(OCH.sub.3)CH.sub.2 Cl; 
MeOSuc-Ala-Ile-Phe-Glu(OCH.sub.3)CH.sub.2 Cl; 
Z-Phe-Gly-Ala-ValCH.sub.2 Cl; and 
Z-Phe-Gly-Ala-TyrCH.sub.2 Cl. 
Physiologically acceptable salts of compounds of formula I include acid 
addition salts of free base, if present, wherein the acid can be organic 
or inorganic, e.g., hydrochloric, phosphoric, maleic, acetic, citric, 
succinic, etc. Alternatively, salts of free peptidic acids, including 
sodium, potassium, and ammonium salts, are included within the scope of 
compounds useful in the present invention. 
In practicing the process of the invention, the foregoing compounds can be 
employed alone, in combination with one another, in combination with other 
therapeutic agents, or in combination with various inert pharmaceutically 
acceptable carriers in a variety of dosage forms, orally or parenterally. 
Dose requirements will vary with the compound and dosage form employed and 
the animal being treated. Typically, therapy is initiated at lower dosages 
and dosage is increased until the desired inhibiting effect is achieved. 
The compounds employed in the invention can be prepared by techniques 
generally corresponding to those disclosed by Kettner et al., [Arch. 
Biochem. Biophys. 162:56 (974). 
First, N-protected peptides or amino acids are reacted with about one 
equivalent of N-methylmorpholine and one equivalent of isobutyl 
chloroformate at about -20.degree. C., generating a mixed 
peptide-isobutyric acid anhydride. This standard technique is described by 
Anderson et al., J. Amer. Chem. Soc., 89:5012 (1967). Second, the 
resulting mixed anhydride is treated with about one equivalent of 
diazomethane in tetrahydrofuran or other suitable inert, aprotic solvent 
at 0.degree. C., generating an N-protected peptide or amino acid 
diazomethyl ketone. Third, the latter compound is treated with a solution 
of HCl or HBr in anhydrous ethanol or ether at 0.degree. C., producing an 
N-protected halomethyl ketone. 
Larger peptide halomethyl ketones can be assembled by repetitively coupling 
a deprotected halomethyl ketone to mixed anhydrides of other N-protected 
peptides or amino acids generated according to the foregoing procedure. 
Deprotection of N-terminal amino groups can be accomplished by treatment 
with trifluoroacetic acid, anhydrous HF, anhydrous HCl, or by other 
methods known to those skilled in the art. 
Procedures suitable for producing specific compounds employed in processes 
within the scope of the present invention are described in paragraphs 
preceding the Examples set forth below. In the preparative procedures and 
Examples, all parts and percentages are by weight, and all degrees are 
Celsius, unless otherwise noted. 
GENERAL SYNTHETIC PROCEDURES 
1. Mixed Anhydride Coupling Procedure 
Approximately 1 g of an N-protected amino acid or peptide is dissolved in 
20 mL of tetrahydrofuran (THF), and the resulting solution is cooled to 
-20.degree.. N-methylmorpholine (1 eq) and isobutyl chloroformate (1 eq) 
are added and after 5 minutes, an additional 10 mL of cold THF and an 
equivalent of triethylamine are added. The resulting mixture is 
immediately added to an equivalent of an amine hydrochloride or 
trifluoroacetate dissolved in 5 mL of dimethylformamide (DMF). The ensuing 
reaction is allowed to stir 1 hour at -20.degree. and then 2 hours at 
about 23.degree.. The resulting mixture is filtered and the filtrate 
thereby obtained is then concentrated to approximately 5 mL by 
evaporation. The resulting concentrate or residue is dissolved in ethyl 
acetate and washed sequentially with 0.2N-hydrochloric acid, 5% sodium 
bicarbonate solution, and saturated aqueous sodium chloride. The resulting 
organic solution is then dried briefly over sodium sulfate, filtered, and 
finally evaporated to leave a crude peptide product. 
2. N-Hydroxysuccinimide (OSu) Coupling Procedure 
N-hydroxysuccinimide esters of N-protected amino acids and peptides can be 
prepared by procedures substantially similar to those disclosed by 
Anderson et al., J. Am. Chem. Soc., 86:1839 (1964). An OSu ester is 
dissolved in a minimial volume of dioxane, and the resulting solution is 
added to an equal volume of an aqueous solution consisting of 1.5 eq of 
triethylamine and either 1.5 eq of an amino acid or 1.1 eq of a peptide, 
forming a reaction mixture. After about 5 minutes, if a complete solution 
is not obtained, a small test sample of the reaction mixture can be 
diluted with water and another sample diluted with dioxane. On the basis 
of the results obtained, the reaction mixture is then diluted with the 
indicated solvent (either water or dioxane) until completely dissolved. 
After the reaction has proceeded to completion, the reaction mixture is 
acidified with hydrochloric acid and the resulting product extracted into 
ethyl acetate. The resulting extract is then washed with 0.2N hydrochloric 
acid followed by 0.2N hydrochloric acid in saturated sodium chloride. The 
washed extract is then dried over sodium sulfate, filtered, and finally 
evaporated to dryness to leave a crude peptide. 
3. Other Coupling Procedures 
Dansyl, 2,4-dinitrophenyl, and methoxysuccinyl derivatives of peptides are 
prepared by reacting a selected chloride, fluoride, or 
N-hydroxysuccinimide ester with an appropriate peptide. Acetyl and 
succinyl derivatives can be prepared from the corresponding anhydrides. A 
peptide hydrochloride or trifluoroacetate salt is dissolved in 50% aqueous 
dioxane at a level of 0.25 mmol/mL and the resulting solution is cooled to 
0.degree.. A selected coupling agent (1.0-1.2 eq) is dissolved in dioxane 
and added along with 2 eq of sodium bicarbonate. The resulting reaction is 
monitored by following the disappearance of ninhydrin positive material. 
4. Saponification of Methyl Esters 
An N-protected methyl ester is dissolved in dioxane (1 mL/mmole), and an 
equal volume of 1.00N sodium hydroxide is added over a period of 30 
minutes. Disappearance of starting material is monitored by thin-layer 
chromatography. After the resulting reaction has proceeded to completion, 
an equivalent of 1.00N hydrochloric acid is added, and the solution is 
diluted to 100 mL with water. The product is then extracted into ethyl 
acetate, and the resulting organic phase washed with 0.2N hydrochloric 
acid followed by 0.2N hydrochloric acid in saturated sodium chloride. 
Solvent is then removed by evaporation to leave a crude carboxylic acid. 
5. Hydrolysis of the Boc Group 
Boc protecting groups are removed from peptides by dissolving a selected 
peptide in trifluoroacetic acid and stirring the soution for 5 minutes at 
room temperature. Cold ether is then added. If a precipitate is obtained 
upon addition of ether, it is triturated with ether and isolated. If no 
precipitate is obtained, the ether is evaporated and toluene is added to 
precipitate deprotected peptide as a trifluoroacetic acid salt. 
Alternatively, a Boc-protected peptide can be dissolved in ethanolic 
hydrochloric acid (2.0-3.5N) and the resulting solution stirred at about 
23.degree. for about 30 minutes, followed by evaporation of solvent. In 
all cases, peptide hydrochloride or trifluoroacetate salts are dried 
overnight under vacuum in the presence of solid potassium hydroxide and 
phosphorus pentoxide. 
6. Hydrolysis of t-Butyl Esters (Bu) 
t-Butyl peptide esters are dissolved in trifluoroacetic acid, and the 
resulting solution is stirred for 1 hour at room temperature. Solvent is 
then evaporated, the resulting residue is redissolved in toluene. 
Following a second toluene evaporation step, the remaining residue is 
dried under vacuum with solid potassium hydroxide. Crude product is 
crystallized from an appropriate solvent, e.g., toluene or ethyl acetate. 
7. Thin Layer Chromatography (TLC) Procedures 
TLCs are run on 5.times.10 cm silica gel plates, using a fluorescent 
indicator. Spots are visualized by conventional techniques, using either a 
UV light or an iodine jar. Peptides having free amino groups protected by 
Boc groups are exposed to HCl vapors, and then stained with ninhydrin. The 
following solvent systems are useful for chromatography: 
methanol:chloroform (1:9) 
butanol:acetic acid:water (4:1:1) 
ethyl acetate:hexane (8:2) 
SYNTHESIS OF PEPTIDE HALOMETHYL KETONES 
Representative peptide halomethyl ketones were prepared by the indicated 
literature procedure or as described below: 
A. Ac-Phe-Gly-Ala-LeuCH.sub.2 CL 
Ac-Phe-Gly-Ala-LeuCH.sub.2 Cl was prepared by condensation of 
N-acetyl-L-phenylalanylglycyl-L-alanine with leucine chloromethyl ketone 
hydrochloride using a mixed anhydride procedure employing isobutyl 
chloroformate, Powers et al., Biochem. Biophys. Acta. 480:246-261 (1977), 
describes an analogous procedure. Crude product was recrystallized from 
ethyl acetate to provide a white crystalline solid, m.p. 
152.4.degree.-153.8.degree. (reported m.p. 167.degree.-168.degree.). 
B. Z-Phe-Gly-Ala-LeuCH.sub.2 Cl 
A solution of 4.00 g (9.4 mmol) of Z-Phe-Gly-Ala-OH and 0.95 g of 
N-methylmorpholine in 100 mL of THF was cooled to -20.degree. under 
nitrogen, and then 1.28 g of isobutyl chloroformate was added. After 5 
minutes a solution of 1.14 g of leucine chloromethyl ketone hydrochloride 
in dry acetone was added, followed by 0.95 g of N-methylmorpholine. The 
resulting reaction mixture was stirred and allowed to warm to about 
25.degree. over a period of 2 hours. Solvent was evaporated, and the 
resulting residue was dissolved in ethyl acetate. This solution was washed 
with ice water, followed by cold 5% citric acid. The resulting solution 
was dried and then solvent was evaporated to leave 5.63 g of a frothy 
solid. This material was triturated overnight with 250 mL of ether. The 
resulting gel-like product was filtered from the ether and washed with 
additional ether, providing 3.10 g of crude product as a fine white 
powder. A 2.56 g portion was dissolved in 25 mL of warm ethyl acetate, 
forming a cloudy solution. This solution was filtered through carbon 
(Celite) and an additional 200 mL of ether was added. After seeding, 1.15 
g of fine needle-like crystals of Z-Phe-Gly-Ala-LeuCH.sub.2 Cl 
precipitated and were collected. These crystals melted at 
138.degree.-141.8.degree.; .delta..sub.D.sup.25 -59.degree. (C=0.45 g/100 
mL in acetone). 
Anal: Calcd. for C.sub.29 H.sub.37 ClN.sub.4 O.sub.6 : C, 60.78; H, 6.51; 
N, 9.78; Found: C, 60.45; H, 6.44; N, 9.69. 
C. Z-Phe-Gly-Leu-LeuCH.sub.2 Cl 
As a preliminary step in synthesis of the foregoing compound, 
Z-Phe-Gly-Leu-OH was prepared by the following procedure. 
The N-hydroxysuccinimide ester of Z-Phe-OH (19.15 g, 48.3 mmol) was 
dissolved in 50 mL of dioxane and the resulting solution was filtered into 
a solution consisting of H-Gly-Leu-OH (10.0 g, 53.1 mmol), triethylamine 
(10.1 mL, 72.4 mmol), and water (50 mL). This reaction mixture was stirred 
overnight at about 23.degree., and then concentrated approximately 60% by 
evaporation. The resulting concentrated solution was diluted with 1.0N 
hydrochloric acid until acidic. Product Z-Phe-Gly-Leu-OH was extracted 
into ethyl acetate, and the resulting extract was washed with 0.2N 
hydrochloric acid, followed by saturated aqueous sodium chloride adjusted 
to 0.10N hydrochloric acid. After washing, the ethyl acetate solution was 
dried over anhydrous sodium sulfate and filtered. The ethyl acetate was 
then evaporated to yield 22.5 g of a white, foamy product, which was 
subsequently crystallized from ethyl acetate to yield 16.7 g of 
Z-Phe-Gly-Leu-OH (m.p. 147.degree.-148.degree.). 
Anal: Calcd. for C.sub.25 H.sub.31 N.sub.3 O.sub.6 : C, 63.94; H, 6.67; N, 
8.95; Found: C, 64.19; H, 6.65; N, 8.90. 
H-LeuCH.sub.2 Cl.HCl was prepared substantially according to the procedure 
disclosed by Kettner et al., Arch. Biochem. Biophys. 165:739-743 (1974). 
A mixed anhydride was prepared by dissolving Z-Phe-Gly-Leu-OH (1.05 g, 2.24 
mmol) in 10 mL of tetrahydrofuran, cooling the resulting solution to 
-20.degree., adding N-methylmorpholine (0.25 mL, 2.24 mmol) and isobutyl 
chloroformate (0.29 mL, 2.24 mmol). The resulting mixture was stirred for 
5 minutes at -20.degree., and then 20 mL of cold tetrahydrofuran and 
triethylamine (0.31 mL, 2.24 mmol) were added. This mixture was added to a 
solution of H-LeuCH.sub.2 Cl.HCl (0.48 g, 2.24 mmol) in 5 mL cold 
N,N-dimethylformamide. The resulting reaction mixture was stirred for 
about 1 hour at -20.degree., and then for 2 hours at about 23.degree.. The 
mixture was then filtered, and tetrahydrofuran evaporated from the 
filtrate. The resulting residue was dissolved in 100 mL of ethyl acetate. 
This solution was then sequentially washed with 0.2N hydrochloric acid, 5% 
aqueous sodium bicarbonate, and saturated aqueous sodium chloride. The 
washed extract was then dried over anhydrous sodium sulfate and then 
solvent was evaporated to yield 1.2 g of a foam. This product was 
crystallized from ethyl acetate to yield 0.94 g (m.p. 
160.degree.-161.5.degree.) of Z-Phe-Gly-Leu-LeuCH.sub.2 Cl. 
Anal: Calcd. for C.sub.32 H.sub.43 N.sub.4 O.sub.6 Cl: C, 62.47; H, 7.06; 
N, 9.11; Found: C, 62.32; H, 6.90; N, 9.14. 
D Z-Phe-Gly-Ala-LeuCH.sub.2 Br 
A mixed anhydride of Z-Phe-Gly-ALa-OH (0.41 g, 0.95 mmol) was prepared and 
coupled to H-LeuCH.sub.2 Br.HBr substantially according to the procedure 
described for preparation of Z-Phe-Gly-Leu-CH.sub.2 Cl, above. The 
resulting product was crystallized from ethyl acetate:hexane to yield 0.30 
g. Product was recrystallized from the same solvents to yield 0.07 g, m.p. 
135.5.degree.-138.degree. (dec.). 
Anal: Calcd. for C.sub.29 H.sub.37 N.sub.4 O.sub.6 Br: C, 56.39; H, 6.05; 
N, 9.07; Found: C, 56.70; H, 6.26; N, 8.97. 
E. Z-Phe-Gly-Ala-ValCH.sub.2 Cl 
First, precursor H-ValCH.sub.2 Cl.HCl was prepared by dissolving Boc-Val-OH 
(6.5 g, 30 mmol) in 10 mL of THF and treating it with N-methylmorpholine 
(3.3 mL, 30 mmol) and isobutyl chloroformate (3.9 mL, 30 mmol) for 10 
minutes at -20.degree.. The resulting mixture was filtered and the 
retained material was washed with 40 mL of cold THF. The combined 
filtrates were added to 200 mL of diazomethane:ether. The resulting 
solution was stirred for 2 hours at 0.degree., and then solvent was 
removed by evaporation to yield an oil. The oil was dissolved in ethyl 
acetate, washed with 5% sodium bicarbonate followed by saturated aqueous 
sodium chloride, and then dried over sodium sulfate. Evaporation of 
solvent left 6.4 g of an oil. The oil was dissolved in 100 mL of ether, 
and the resulting solution was treated with a 5% excess of ethanolic HCl 
for 15 minutes at 0.degree.. This solution was washed with cold water 
followed by saturated aqueous sodium chloride, and then dried over sodium 
sulfate. After evaporation of solvent, product was washed with hexane to 
yield 2.7 g of Boc-ValCH.sub.2 Cl (m.p. 70.degree.-73.degree.). 
Boc-ValCH.sub.2 Cl (1.0 g) was deblocked by stirring with 5 mL of 3N 
ethanolic HCl for 30 minutes at about 23.degree.. Solvent was evaporated 
and the resulting residue was triturated with ether to yield 0.69 g of 
H-ValCH.sub.2 Cl.HCl. 
Z-Phe-Gly-Ala-OH (1.60 g, 3.73 mmol) was then coupled to H-ValCH.sub.2 
Cl.HCl by a procedure substantially similar to that described for the 
preparation of Z-Phe-Gly-Leu-LeuCH.sub.2 Cl, above. The resulting product 
was crystallized from ethyl acetate:ether to yield 1.29 g of 
Z-Phe-Gly-Ala-ValCH.sub.2 Cl (m.p. 143.degree.-144.degree.). 
Anal. Calcd. for C.sub.28 H.sub.35 N.sub.4 O.sub.6 Cl: C, 60.14; H, 6.32; 
N, 10.02; Found: C, 59.92; H, 6.25; N, 10.05. 
F. Z-Phe-Gly-Ala-TyrCH.sub.2 Cl 
Boc-TyrCHN.sub.2 was prepared by dissolving Boc-Tyr-OH (10 g, 35.5 mmol) in 
30 mL of tetrahydrofuran and then reacting it with N-methylmorpholine 
(3.91 mL, 35.5 mmol) and isobutyl chloroformate (4.62 mL, 35.5 mmol) for 5 
minutes at -20.degree.. The resulting reaction mixture was filtered, and 
the material retained by the filter was washed with 50 mL of cold 
tetrahydrofuran. The resulting filtrates were collected and combined. The 
combined filtrates were added to 150 mL of diazomethane:ether (.about.40 
mmol) and the resulting reaction mixture was stirred for 15 minutes at 
0.degree.. Solvent was then evaporated with a stream of nitrogen. The 
remaining residue was dissolved in ethyl acetate and the resulting 
solution was washed with water followed by saturated aqueous sodium 
chloride. The washed solution was then dried over sodium sulfate, and then 
solvent was evaporated to yield a crude product. This material was 
chromatographed on a 4 cm column containing 75 g of silica gel, using 
chloroform as a solvent, to yield 3.67 g of Boc-TyrCHN.sub.2. This product 
crystallized from ether to yield 1.86 g (m.p. 136.degree.-137.degree.) in 
a first crop, and 0.58 g (m.p. 133.5.degree.-134.5.degree.) in a second 
crop. NMR in CDCl.sub.3 indicated a diazo proton at .delta. 5.27. 
Anal: Calcd. for C.sub.15 H.sub.19 N.sub.3 O.sub.4 : C, 58.99; H, 6.28; N, 
13.76; Found: C, 59.08; H, 6.16; N, 13.65. 
Boc-TyrCHN.sub.2 (1.81 g, 5.92 mmol) was dissolved in 30 mL of 
tetrahydrofuran, and the resulting solution wwas treated with 3.45N 
ethanolic:HCl (1.72 mL, 5.92 mmol) for 5 minutes at 0.degree.. Solvent was 
removed by evaporation on a rotary evaporator without temperature 
regulation. The remaining residue was dissolved in ethyl acetate, and the 
resulting solution was washed with 0.2N hydrochloric acid followed by 
saturated aqueous sodium chloride. The washed solution was dried over 
sodium sulfate, and then solvent was evaporated to yield crystalline 
Boc-TyrCH.sub.2 Cl. Crystals were isolated and washed with cold ether to 
yield 0.45 g (m.p. 110.degree.-112.degree.) in a first crop, and 0.90 g 
(m.p. 110.degree.-112.degree.) in a second crop. NMR spectra in CDCl.sub.3 
corresponded to that expected, except that methylene protons of 
--COCH.sub.2 Cl appears as a doublet at .delta.4.1. 
Anal: Calcd. for C.sub.15 H.sub.20 NO.sub.4 Cl: C, 57.41, H, 6.44; N, 4.46; 
Found: C, 57.66; H, 6.66; N, 4.52. 
Boc-TyrCH.sub.2 Cl (0.5 g) was deblocked by treatment with 20 mL of 3.5N 
ethanolic HCl for 30 minutes at about 23.degree.. Solvent was evaporated 
and the resulting product, H-TyrCH.sub.2 Cl.HCl (0.45 g), was dried in 
vacuo over solid potassium hydroxide and phosphorus pentoxide. 
Z-Phe-Gly-Ala-OH (0.77 g, 1.80 mmol) was coupled to H-TyrCH.sub.2 Cl.HCl by 
a mixed anhydride coupling procedure substantially similar to that 
described for preparation of Z-Phe-Gly-Leu-LeuCH.sub.2 Cl in Procedure E, 
above. Product was crystallized from ethyl acetate to yield 0.73 g of 
Z-Phe-Gly-Ala-TyrCH.sub.2 Cl. Product slowly decomposed at 
140.degree.-160.degree. and melted with complete decomposition at 
160.degree.-160.5.degree.. 
Anal: Calcd. for C.sub.32 H.sub.35 N.sub.4 O.sub.7 Cl: C, 61.67; H, 5.67; 
N, 8.99; Found: C, 61.60; H, 5.92; N, 8.69. 
G. MeOSuc-Phe-Gly-Leu-LeuCH.sub.2 Cl 
MeOSuc-Phe-Gly-Leu-OH was prepared from Z-Phe-Gly-Leu-OH, as described 
above in Procedure C. Z-Phe-Gly-Leu-OH (4.00 g, 8.52 mmol) was dissolved 
in 100 mL of methanol and hydrogenated in the presence of 1.0 eq of 
anhydrous HCl and 0.50 g of 10% Pd on carbon overnight in a Parr 
apparatus. The resulting reaction mixture was filtered and solvent was 
evaporated to yield 3.1 g of H-Phe-Gly-Leu-OH as a foam. 
This product was dissolved in 5 mL of N,N-dimethylformamide, to which 
N-hydroxysuccinimide ester of methoxysuccinic acid (1.9 g, 8.4 mmol) and 
triethylamine (1.2 mL, 8.4 mmol) were added. This reaction mixture was 
stirred for 0.50 hr at ambient temperature, then additional triethylamine 
(0.58 mL, 4.2 mmol) was added. The reaction mixture was then stirred 
overnight. At this point, 5 mL of a 5% solution of aqueous sodium 
bicarbonate was added. After 5 min, the reaction mixture was diluted to 50 
mL with 5% aqueous sodium bicarbonate to yield a precipitate, which was 
discarded. Ethyl acetate was added to the reaction mixture to extract 
product MeOSuc-Phe-Gly-Leu-OH. The aqueous portion of the resulting 
extraction mixture was acidified with hydrochloric acid, and then the 
ethyl acetate phase or extract was separated from the aqueous portion. The 
extract was washed with 0.20N hydrochloric acid, followed by saturated 
sodium chloride prepared in 0.20N hydrochloric acid. The washed solution 
was then dried over sodium sulfate. Solvent was evaporated and the 
resulting residue was crystallized from ethyl acetate, yielding 1.30 g of 
MeOSuc-Phe-Gly-Leu-OH (m.p. 167.5.degree.-168.5.degree.). 
Anal: Calcd. for C.sub.23 H.sub.32 N.sub.3 O.sub.7 : C, 58.91; H, 6.76; N, 
9.37; Found: C, 59.20; H, 6.99; N, 9.06. 
MeOSuc-Phe-Gly-Leu-OH (1.0 g, 2.23 mmol) was coupled to H-LeuCH.sub.2 Cl 
HCl by a procedure substantially similar to that described for preparation 
of Z-Phe-Gly-Leu-LeuCH.sub.2 Cl. After coupling, solvent was evaporated to 
yield a foamy product which crystallized from ethyl acetate, providing 
0.30 g (m.p. 116.degree.-117.degree.) of MeOSuc-Phe-Gly-Leu-LeuCH.sub.2 Cl 
in a first crop and 0.25 g (m.p. 115.degree.-116.degree.) in a second 
crop. A sample of the first crop was analyzed. 
Anal: Calcd. for C.sub.29 H.sub.43 N.sub.4 O.sub.7 Cl: C, 58.51; H, 7.30; 
N, 9.42; Found: C, 58.67; H, 7.20; N, 9.31. 
H. Suc-Phe-Gly-Ala-LeuCH.sub.2 Cl 
Boc-Phe-Gly-Ala-OH was prepared substantially according to the mixed 
anhydride coupling procedure described previously, except amine components 
were added in tetrahydrofuran or tetrahydrofuran containing the minimum 
quantity of water needed to dissolve the amine hydrochlorides. Methyl 
esters were saponified with aqueous sodium hydroxide in methanol. 
Boc-Gly-Ala-OCH.sub.3 was prepared by coupling Boc-Gly-OH (35.0 g, 200 
mmol) to H-Ala-OCH.sub.3.HCl (27.9 g, 200 mmol) using a mixed anhydride 
procedure. The resulting product (43.5 g, 167 mmol) was deblocked with 
anhydrous HCl in dioxane. After deblocking, crystallization from 
dioxane:ether yielded 27.1 g of H-Gly-Ala-OCH.sub.3.HCl. 
Boc-Phe-OH (26.5 g, 100 mmol) was coupled to H-Gly-Ala-OCH.sub.3.HCl (19.6 
g, 100 mmol) using a mixed anhydride procedure. After crystallization from 
ethyl acetate:hexane, 30.2 g of Boc-Phe-Gly-Ala-OCH.sub.3 (m.p. 
126.4.degree.-127.8.degree.) were obtained. 
Anal: Calcd. for C.sub.20 H.sub.29 N.sub.3 O.sub.6 : C, 58.95; H, 7.71; N, 
10.31; Found: C, 59.49; H, 7.05; N, 10.26. 
Boc-Phe-Gly-Ala-OCH.sub.3 (28.4 g, 69.7 mmol) was saponified to yield 22 g 
of solid Boc-Phe-Gly-Ala-OH. It was crystallized from acetone:ether to 
yield 20 g of product (m.p. 105.7.degree.-109.degree.). 
Anal: Calcd. for C.sub.19 H.sub.27 N.sub.3 O.sub.6 : C, 58.00; H, 6.92; N, 
10.68; Found: C, 58.04; H, 6.78; N, 10.66. 
Boc-Phe-Gly-Ala-LeuCH.sub.2 Cl was prepared by coupling Boc-Phe-Gly-Ala-OH 
(11.8 g, 30 mmol) to H-LeuCH.sub.2 Cl.HCl (6.06 g, 30 mmol) using a mixed 
anhydride procedure. A mixed anhydride of Boc-Phe-Gly-Ala-OH was prepared 
in a solution of 300 mL of tetrahydrofuran and 25 mL of acetone, and then 
H-LeuCH.sub.2 Cl HCl was added in acetone. The resulting crude product, 
17.6 g, was dissolved in ether, treated with carbon, and filtered. Product 
Boc-Phe-Gly-Ala-LeuCH.sub.2 Cl crystallized from ether (10.0 g). 
Anal: Calcd. for C.sub.20 H.sub.39 N.sub.4 O.sub.6 Cl: C, 57.93; H, 7.29; 
N, 10.39; Found: C, 57.92; H, 7.53; N, 10.17. 
Boc-Phe-Gly-Ala-LeuCH.sub.2 Cl (0.30 g) was treated with 1 mL of anhydrous 
trifluoroacetic acid for 5 minutes at about 23.degree., forming a 
trifluoroacetate salt, which was precipitated by addition of cold ether 
and then washed with additional cold ether. The salt was then dried over 
solid potassium hydroxide and phosphorus pentoxide to yield 0.31 g (0.55 
mmole) of H-Phe-Gly-Ala-LeuCH.sub.2 Cl.trifluoroacetate. This product was 
dissolved in 2 mL of 50% dioxane:water, and the resulting solution was 
cooled to 0.degree.. Sodium bicarbonate (0.09 g, 1.11 mmol) and succinic 
anhydride (0.066 g, 0.66 mmole) dissolved in 1 mL of dioxane were added. 
After 1 hour, 0.55 mL of 1.0N hydrochloric acid was added, and the 
resulting solution was diluted to 10 mL with water and held overnight at 
40.degree.. During this period product Suc-Phe-Gly-Ala-LeuCH.sub.2 Cl 
crystallized from solution. This product was filtered and washed with 100 
mL of cold 0.10N hydrochloric acid. After drying in vacuo, 0.18 g of 
Suc-Phe-Gly-Ala-LeuCH.sub.2 Cl (m.p. 157.5.degree.-158.degree.) was 
obtained. 
Anal: Calcd. for C.sub.25 H.sub.35 N.sub.4 O.sub.7 Cl: C, 55.70; H, 6.56; 
N, 10.40; Found: C, 55.47; H,6.59; N, 10.48. 
I. MeOSuc-Phe-Gly-Ala-LeuCH.sub.2 Cl 
Suc-Phe-Gly-Ala-LeuCH.sub.2 Cl (0.20 g, 0.37 mole) was dissolved in 10 mL 
of acetone, and the resulting solution was treated with diazomethane:ether 
(10 mL) for 30 minutes at 0.degree.. Solvent was evaporated after the 
solution was warmed to about 23.degree.. The remaining residue was 
dissolved in acetone, and the resulting solution was filtered and 
concentrated by evaporation of solvent. The concentrate was diluted with 
ethyl acetate, and this solution was again concentrated by evaporation to 
remove most of the acetone. A crystalline product separated. This product 
was isolated and washed with cold ethyl acetate to yield 0.11 g of 
MeOSuc-Phe-Gly-Ala-LeuCH.sub.2 Cl (m.p. 128.5.degree.-129.5.degree.). 
Anal: Calcd. for C.sub.26 H.sub.37 N.sub.4 O.sub.7 Cl: C, 56.45; H, 6.76; 
N, 10.13; Found: C, 56.26; H, 6.69; N, 10.01. 
J. Boc-Gly-Ala-LeuCH.sub.2 Cl 
Boc-Gly-Ala-OH was prepared by an N-hydroxysuccinimide coupling procedure. 
Boc-Gly-OSu (7.67 g, 28.2 mmol) was dissolved in 10 mL of 
dimethylsulfoxide, and the resulting solution was added to a solution 
consisting of H-Ala-OH (3.76 g, 42.3 mmol) and triethylamine (5.89 mL, 
42.3 mmol) in 20 mL of water. After stirring overnight, a precipitate was 
removed by filtration. The remaining solution was diluted with water and 
then acidified with hydrochloric acid. An aqueous phase separated from 
which product was extracted by addition of ethyl acetate. The ethyl 
acetate extract was washed with 0.2N hydrochloric acid and then saturated 
sodium chloride prepared in 0.2N hydrochloric acid. The washed ethyl 
acetate solution was then dried over sodium sulfate and filtered. Solvent 
was then evaporated, leaving a foamy product, which was crystallized from 
ethyl acetate:hexane to yield 2.88 g of Boc-Gly-Ala-OH (m.p. 
128.degree.-130.degree.). 
Anal: Calcd. for C.sub.10 H.sub.18 N.sub.2 O.sub.5 : C, 48.76; H, 7.38; N, 
11.38; Found: C, 49.12; H, 7.27; N, 11.28. 
Boc-Gly-Ala-OH (2.5 g, 10.2 mmol) was coupled to H-LeuCH.sub.2 Cl.HCl by a 
procedure substantially similar to that described for the preparation of 
Z-Phe-Gly-Leu-LeuCH.sub.2 Cl. Evaporation of ethyl acetate provided 3.7 g 
of a foam. Crystallization from ethyl acetate:hexane gave 3.1 g of 
Boc-Gly-Ala-LeuCH.sub.2 Cl (m.p. 95.degree.-99.5.degree.). 
Anal: Calcd. for C.sub.17 H.sub.30 N.sub.3 O.sub.5 Cl: C, 52.09; H, 7.73; 
N, 10.72; Found: C, 52.21; H, 7.46; N, 10.63. 
K. Z-Phe-Ser-Ala-LeuCH.sub.2 Cl 
Z-Phe-Ser(OCH.sub.2 C.sub.6 H.sub.5)-Ala-OH was prepared according to the 
following mixed anhydride coupling procedure. 
First, Boc-Ser(OCH.sub.2 C.sub.6 H.sub.5)-OH (4.93 g, 16.7 mmol) was 
coupled to H-Ala-OCH.sub.3.HCl (2.80 g, 20.0 mmol) by a mixed anhydride 
procedure to yield an oil, 6.1 g. Boc-Ser(OCH.sub.2 C.sub.6 
H.sub.5)-Ala-OCH.sub.3 was then deblocked with trifluoroacetic acid, and 
the resulting product, H-Ser(OCH.sub.2 C.sub.6 
H.sub.5)-Ala-OCH.sub.3.trifluoroacetate (6.15 g, 15.6 mmol), was coupled 
to Z-Phe-OH (3.89 g, 13.0 mmol) using a mixed anhydride procedure. This 
product was crystallized from ethyl acetate to yield 5.4 g of 
Z-Phe-Ser(OCH.sub.2 C.sub.6 H.sub.5)-Ala-OCH.sub.3 (m.p. 
158.degree.-159.degree.). 
Anal: Calcd. for C.sub.31 H.sub.35 N.sub.3 O.sub.7 : C, 66.28; H, 6.29; N, 
7.48; Found: C, 65.54; H, 6.24; N, 7.37. 
Z-Phe-Ser(OCH.sub.2 C.sub.6 H.sub.5)-Ala-OCH.sub.3 (5.63 g, 8.24 mmol) was 
saponified substantially as previously described, except that 16 mL of 
dioxane and 8.25 mL of 1.00N sodium hydroxide were used. The desired 
product, Z-Phe-Ser(OCH.sub.2 C.sub.6 H.sub.5)-Ala-OH, crystallized from 
methanol:ethyl acetate to yield 3.5 g (m.p. 171.5.degree.-172.5.degree.). 
Anal: Calcd. for C.sub.30 H.sub.33 N.sub.3 O.sub.7 : C, 65.79; H, 6.08; N, 
7.67; Found: C, 65.50; H, 5.86; N, 7.84. 
Z-Phe-Ser(OCH.sub.2 C.sub.6 H.sub.5)-Ala-LeuCH.sub.2 Cl was prepared by 
coupling Z-Phe-Ser(OCH.sub.2 C.sub.6 H.sub.5)-Ala-OH (1.37 g, 2.5 mmol) to 
H-LeuCH.sub.2 Cl.HCl by a mixed anhydride coupling procedure substantially 
similar to that used for the preparation of Z-Phe-Gly-Leu-LeuCH.sub.2 Cl 
in Procedure C, above. Product was obtained as a crystalline solid, 0.98 g 
(m.p. 167.degree.-168.degree.). Anal: Calcd. for C.sub.37 H.sub.45 N.sub.4 
O.sub.7 : C, 64.09; H, 6.56; N, 8.08; Found: C, 64.22; H, 6.38; N, 8.08. 
Z-Phe-Ser(OCH.sub.2 C.sub.6 H.sub.5)-Ala-LeuCH.sub.2 Cl (0.71 g, 10.2 mmol) 
was treated with a mixture of 15 mL of anhydrous HF and 1 mL of anisole in 
a commercial HF apparatus (Peptide Institute, Inc.) After 70 minutes at 
0.degree., HF was removed by evaporation, leaving a residue which was 
dried in vacuo over potassium hydroxide overnight. 
H-Phe-Ser-Ala-LeuCH.sub.2 Cl.HF, 0.35 g, was obtained after triturating 
the residue with ether. 
This hydrofluoride salt (0.39 g, 0.71 mmole) was dissolved in a mixture of 
2 mL water and 1 mL of dioxane. The resulting solution was cooled to 
0.degree. and carbobenzoxychloride (0.10 mL, 0.71 mmole) and sodium 
bicarbonate (0.12 g, 1.42 mmol) were added. After 30 minutes at 0.degree., 
no ninhydrin positive material could be detected. The resulting reaction 
mixture was diluted with ethyl acetate and the resulting organic layer was 
washed sequentially with 5% sodium bicarbonate solution, 0.2N hydrochloric 
acid, and saturated sodium chloride. The washed solution was dried over 
sodium sulfate and solvent was evaporated, leaving a residue, which was 
diluted by 50% with hexane to yield 0.13 g of Z-Phe-Ser-Ala-LeuCH.sub.2 Cl 
(m.p. 155.degree.-157.degree.). 
Anal: Calcd. for C.sub.30 H.sub.39 N.sub.4 O.sub.7 Cl: C, 59.73; H, 6.53; 
N, 9.29; Found: C, 59.81; H, 6.47; N, 9.13. 
L Z-Phe-Gly-Ser-LeuCH.sub.2 Cl 
Z-Phe-Gly-Ser-LeuCH.sub.2 Cl was prepared by the following procedure. 
First, Z-Phe-Gly-Ser(OCH.sub.2 C.sub.6 H.sub.5)LeuCH.sub.2 Cl (0.98 g, 
14.4 mmol) was deblocked by treatment with 15 mL of anhydrous HF and 1 mL 
of anisole (Procedure K) to yield 0.69 g of H-Phe-Gly-Ser-LeuCH.sub.2 
Cl.HF. This intermediate (0.59 g, 12.4 mmol) was then coupled with 
carbobenzoxychloride substantially according to the procedure of Procedure 
K to yield 0.4 g of Z-Phe-Gly-Ser-LeuCH.sub.2 Cl (m.p. 
146.degree.-146.5.degree.) after crystallization from ethyl 
acetate/hexane. 
Anal: Calcd. for C.sub.29 H.sub.37 N.sub.4 O.sub.7 Cl: C, 59.12; H, 6.34; 
N, 9.51; Found: C, 59.45; H, 6.37; N, 9.46. 
Biological Activity of Selected Peptide Halomethyl Ketones 
Selected compounds useful in the processes of the present invention have 
been shown to inhibit viral protease activity in two assays. One assay, 
known as a viral cleavage assay, involves comparison of patterns of 
protein synthesis (as visualized by incorporation of labeled amino acids) 
in virus-infected HeLa cells grown in the presence and absence of a 
selected test compound. A second assay, known as a plaque inhibition 
assay, involves an assessment of the effects of test compounds upon the 
infectivity of virus in agar-overlaid cell cultures. Toxic effects of a 
test compound, if any, will also be observed during the incubation period 
of the plaque inhibition assay. Both assays are described in greater 
detail below. 
Viral Cleavage Assay 
In this assay, samples of growing HeLa-O cells were exposed to human 
poliomyelitisvirus type 2 or human rhinovirus type 1A at a virus 
concentration of about 10 infectious virus particles per cell. After 
several hours, host cell metabolism was markedly inhibited, and added 
radioactive amino acids were incorporated into viral proteins only. After 
varying concentrations of a test compound were added to cell samples, 
viral proteins were labeled for 60 minutes at the mid-cycle of infection 
(3-5 hours after first exposure of the cells to the virus). Cell samples 
were then solubilized in 0.01M tris(hydroxymethyl)-aminomethane buffer, pH 
6.8, containing 1% (w/v) sodium dodecyl sulfate and 1% (v/v) 
2-mercaptoethanol. The resulting labeled viral proteins were separated on 
a polyacrylamide gel and detected by autoradiography, as previously 
described by Korant et al., Proc. Natl. Acad. Sci. USA 76:2992 (1979). If 
a selected concentration of the test compound disrupted the usual pattern 
of virus protein processing, that compound was scored as active at that 
concentration. Gels corresponding to cell samples labeled in the presence 
of test compounds exhibiting viral protease-inhibiting activity were 
generally distinguishable by appearance of high molecular weight protein 
species not apparent on control gels. 
Plaque Inhibition Assay 
In this assay, cultured HeLa cells were grown to confluency in 60 mm 
plastic petri dishes. Each culture was then infected with approximately 
300 plaque-forming units of virus. Human rhinovirus type 1A was used in 
the test reported below. The virus employed in this experiment was allowed 
to absorb to the cells for 30 minutes at 34.5.degree.. 
The compound tested was dissolved in ethanol at a concentration 100 times 
greater than the highest concentration to be tested. The resulting 
solution was then diluted 1:100 into a solution of McCoy's medium 
containing 5% heat-inactivated fetal calf serum and 0.38% agar. Two-fold 
dilutions were then made into agar medium. 
After virus adsorbed to the cells, excess virus was washed away and each 
culture was overlaid with 5 mL of agar medium containing a pre-selected 
dilution of the compound to be tested. Controls were overlaid with agar 
medium only. Each culture was then incubated at 34.5.degree. to allow 
development of plaques. 
A plaque is a roughly circular region of dead cells in a culture, 
indicating an area where one plaque-forming unit of virus first infected 
one cell. The agar overlay restricts virus mobility, so that viral 
infection is communicated only between contiguous cells. 
When plaques in control cultures were large enough to be observed, yet 
still relatively discrete, all cultures were stained with 1% crystal 
violet. Plaques appeared as clear areas in a deep purple field of 
uninfected cells. Toxic doses of the test compound resulted in visible 
cell detachment in culture dishes. 
EXAMPLES 1-19 AND COMISONS A-J: PROTEASE-INHIBITING ACTIVITY OF PEPTIDE 
HALOMETHYL KETONES IN VIRAL CLEAVAGE ASSAY 
The results obtained in a series of experiments in which representative 
tetrapeptide and tripeptide chloromethyl ketones were tested for 
protease-inhibiting activity in the viral cleavage assay using poliovirus 
are set forth in Tables 1 and 2 below. The results are scored as follows: 
TABLE 1 
______________________________________ 
Protease-Inhibiting Activity of Selected 
Peptide Halomethyl Ketones 
Ex- 
am- 
ple Peptide Analog Activity 
______________________________________ 
1 Z--Phe--Gly--Ala--LeuCH.sub.2 Cl 
+++ 
2 Ac--Phe--Gly--Ala--LeuCH.sub.2 Cl 
+++ 
3 Suc--Phe--Gly--Ala--LeuCH.sub.2 Cl 
+++ 
4 MeOSuc--Phe--Gly--Ala--LeuCH.sub.2 Cl 
+++ 
5 DNS--Phe--Gly--Ala--LeuCH.sub.2 Cl 
+++ 
6 DNP--Phe--Gly--Ala--LeuCH.sub.2 Cl 
+++ 
7 Boc--Gly--Ala--LeuCH.sub.2 Cl 
++ 
8 Z--Leu--Gly--Ala--LeuCH.sub.2 Cl 
+++ 
9 Z--Phe--Gly--Gly--LeuCH.sub.2 Cl 
+++ 
10 Z--Phe--Gly--Ala--LeuCH.sub.2 Br 
+++ 
11 Z--Phe--Gly--Ala--ValCH.sub.2 Cl 
++ 
12 Z--Phe--Gly--Leu--LeuCH.sub.2 Cl 
++++ 
13 Z--Phe--Leu--Ala--LeuCH.sub.2 Cl 
++ 
14 Z--Phe--Gly--Phe--LeuCH.sub.2 Cl 
++ 
15 Z--Phe--Gly--Ser--LeuCH.sub.2 Cl 
++ 
16 Z--Phe--Gly--Pro--LeuCH.sub.2 Cl 
++ 
17 MeOSuc--Ala--Ile--Phe--LeuCH.sub.2 Cl 
+++ 
18 MeOSuc--Phe--Gly--Leu--Glu(OCH.sub.3)CH.sub.2 Cl 
++++ 
19 MeOSuc--Ala--Ile--Phe--Glu(OCH.sub.3)CH.sub.2 Cl 
+++ 
20 Z--Phe--Gly--Ala--TyrCH.sub.2 Cl 
++ 
______________________________________ 
- no activity 
+/- low activity 
+ active at 100 .mu.g/mL 
++ active at 50 .mu.g/mL 
+++ active at 10 .mu.g/mL 
++++ active at &lt;5 .mu.g/mL 
TABLE 2 
______________________________________ 
Comparative Experiments Involving 
Other Peptide Halomethyl Ketones 
Comparison 
Peptide Analog Activity 
______________________________________ 
A H--Phe--Gly--Ala--LeuCH.sub.2 Cl 
+/- 
B Z--Ala--Gly--Ala--LeuCH.sub.2 Cl 
+/- 
C Z--Gln--Gly--Ala--LeuCH.sub.2 Cl 
+ 
D Z--Phe--Phe--Ala--LeuCH.sub.2 Cl 
+/- 
E Z--Phe--Ser--Ala--LeuCH.sub.2 Cl 
+ 
F Z--Phe--Gly--Lys--LeuCH.sub.2 Cl 
+ 
G Z--Phe--Lys--Ala--LeuCH.sub.2 Cl 
+ 
H Z--Phe--Pro--Ala--LeuCH.sub.2 Cl 
+ 
I Ac--Phe--Gly--Glu--LeuCH.sub.2 Cl 
+/- 
J Ac--Phe--Glu(OEt)--Ala--LeuCH.sub.2 Cl 
+/- 
K Ac--Phe--Glu--Ala--LeuCH.sub.2 Cl 
+/- 
______________________________________ 
EXAMPLE 20 
The antiviral activity of Z-Phe-Gly-Leu-LeuCH.sub.2 Cl on rhinovirus type 
1A was evaluated by a plaque inhibition assay as described above. 
Antiviral activity, as scored by 90% plaque reduction, was noted at 1 
.mu.g/mL, whereas cytotoxicity was first detected at a concentration of 
about 15 .mu.g/mL with human HeLa-O cells. 9n