CA2109646C

CA2109646C - Para-nitroanilide peptides

Info

Publication number: CA2109646C
Application number: CA002109646A
Authority: CA
Inventors: Gaston O. Daumy; Lawrence A. Reiter
Original assignee: Pfizer Corp SRL
Current assignee: Pfizer Corp SRL
Priority date: 1992-11-24
Filing date: 1993-11-22
Publication date: 2000-03-07
Anticipated expiration: 2013-11-22
Also published as: CA2109646A1; US5498695A

Abstract

A compound of the formula R1-A2-A1-Asp-p-nitroanilide I
wherein:

A1 is a residue of any of the naturally occurring .alpha.-amino acids or a homolog, analog or derivative of a natural .alpha.-amino acid;
A2 is a residue of a lipophilic .alpha.-amino acid;
R1 is alkylcarbonyl,phenalkylcarbonyl,alkoxycarbonyl,phenalkoxycarbonyl, alkylaminocarbonyl, phenalkylaminocarbonyl or R2-A3 wherein A3 is a residue of a lipophilic .alpha.-amino acid; and R2 is alkylcarbonyl, alkoxycarbonyl or phenylalkoxycarbonyl, and a method of detecting inhibitors of interleukin 1.beta. converting enzyme (ICE) comprising evaluating a test compound's capacity to inhibit the ICE-induced hydrolysis of a compound of the formula I. The greater the ability of a test compound to inhibit such hydrolysis, the greater its expected activity in treating inflammation as well as diseases whose pathogenesis is induced or sustained by interleukin-1.beta..
Also disclosed is the following intermediate, useful for synthesizing the compounds of formula I:
(see above formula)

Description

~149~46 _, _ PARA-NITROANILIDE PEPTIDES
Background of the Invention _..
This invention is concerned with para-nitroanilide peptides and methods of using N
m such peptides to detect inhibitors of interleukin 1 f3 converting enzyme (ICE). Such U
inhibitors are useful in treating inflammatory conditions in mammals, especially man.
Current therapies for arthritis are severely limited by the side effects of available drugs and their ineffectiveness beyond treatment for disease symptoms. The most widely used drugs are agents (the non-steroidal antiinflammatory drugs, NSAIDS) which inhibit the cyclooxygenase pathway of arachidonic acid metabolism. While these compounds are effective in controlling the symptoms of arthritis, they are not disease remittive. Furthermore, cyclooxygenase inhibition is generally associated with the major side-effect of NSAID therapy, gastrointestinal irritation. Steroids are used in the more severe cases of arthritis and are very effective. However, long term therapy using steroids is seldom tolerable. Second line antiinflammatory agents such as gold, penicillamine, chloroquine and methotrexate are also beset with side effect issues which severely limit their general utility.
Interleukin-1 (IL-1 ) has been strongly implicated as a key mediator of tissue damage in osteo-and rheumatoid arthritis. Lowering levels of IL-1 in a diseased joint would be expected to halt continued degeneration and perhaps allow joint repair to take place. One approach to reducing levels of IL-1 is to block the generation of mature IL-1 fi from its biologically inactive precursor, pro-IL-1 fi, by inhibition of the interleukin-1 f3 converting enzyme (ICE). This invention relates to a novel series of compounds which are substrates for ICE. The compounds may be used to detect ICE
inhibitors which are useful for the treatment of diseases characterized by inflammation as well as diseases whose pathogenesis is induced or sustained by interleukin-1 fi.
Such diseases include inflammatory bowel disease, psoriasis, allergic encephalitis, gingivitis, systemic lupus erythematosus, diabetes melitis, gout, septic shock and adult respiratory distress syndrome. It is expected that such inhibitors will not elicit the side effects associated with NSAID therapy (due to cyclooxygenase inhibition), steroids or other treatments currently in use.

Summary of the Invention The present invention relates to a compound of the formula Rl-A2-A1-Asp-p-nitroanilide I
wherein:
A1 is Ala;
A2 is Val or Ala;
R1 is alkylcarbonyl, phenalkylcarbonyl, alkoxycarbonyl, phenalkoxycarbonyl, alkylaminocarbonyl, phenalkylaminocarbonyl or R2-A~3 wherein A3 is Val; and R2 is alkylcarbonyl, alkoxycarbonyrl or phenylalkoxycarbonyl.
The alkylcarbonyl is preferably Ac, the phenalkylcarbonyl is preferably PhCH2CH2C0, the alkoxycarbonyl is preferably t-BOC, the phenalkoxycarbonyl is preferably CBZ and the phenylalkylaminocarbonyl is preferably PhCH2NHC0.
The following are preferred compounds of the invent ion PhCH2CH2COValAlaAsp-p-nitroanilide;
PhCH2NHCOValAlaAsp-p-nitroanilide ;
t-BOCValAlaAsp-p-nitroanlade;
AcValAlaAsp-p-nitroanilide; and CBZValAlaAsp-p-nitroanilide.
The present invention also relates to a method of detecting inhibitors of interleukin lp converting enzyme (ICE) comprising evaluating a compound's capacity to inhibit the ICE-induced hydrolysis of a compound of the formula I.
The greater the ability of a compound to inhibit such hydrolysis, the greater its expected activity in treating inflammation as well as diseases whose pathogenesis is induced or sustained by interleukin 1/3.
The abbreviations used herein to denote amino acids are well known and standard in the art and include the following: Ala, alanine; Pro, proline; His, histidine; Cys, cysteine; Cys(Me), methylcysteine; 1?he, phenylalanine; Val, valine; IIe, isoleucine; Leu, leucine; Tyr, tyrosine; Glu, glutamic acid; Lys, lysine; Asp, aspartic acid; and Val, valine.
Other abbreviations used herein include the following: FMOC, fluorenylmethyloxycarbonyl; CBZ, benzyloxycarbonyl; Ac, acetyl; Ph, phenyl; t-BOC, t-butoxycarbonyl.
Detailed Description of the Invention The compounds of the present invention, having the formula I, as defined above, are readily and generally prepared by the general methods described below.

.__ 2109646 - 3a -Scheme 1 Z-t-butyl -butyl H
HZN ~ \ Rl-AZ-Al-NH N ~ \
O / O

Rl-AZ-AIOH
4s% TFA
2.5% Vila 2.5% ~miieole SO% ~
Rl_AZ_Al_~ ~~ \
O

The most preferred procedure, shown in Scheme 1, is to first couple a di- or tripeptide (R'-A2-A'OH), which can be prepared by standard methods known in the art, with the fi-t-butyl ester of aspartyl-p-nitroaniilide (the product of Preparation 1 ). This coupling can be induced by any number of methods known in the art such as, but not limited to, those based an dicyclohexylc~arbondiimide, 1-(dimethylaminopropyl)-ethylcarbodiimide hydrochloride (DEC ~ HC;I) (the method used herein), isobutyl chloroformate, and N,N-bis[2-oxo-3-oxazolidinyl]phophorodiamidic chloride.
Additives such as, but not limited to, N-hydroxysuccinirnide or N-hydroxybenzotriazole, which are typically used in such couplings can be included. The solvent used for this coupling can be any reaction inert solvent such as, but not limited to, DMF
(dimethylformanide), THF (tetrahydrofuran), dioxane, and methylene chloride. The coupling reaction can be performed at from about -20 to about 100°C, with temperatures about 15 to about 30°C being preferred. The second step of the preferred procedure involves cleaving the f3-t-butyl ester of the aspartyl residue which is carried out with strong acids such as, but not limited to, trifluoroacetic acid (TFA), hydrogen chloride, hydrogen bromide, p-toluenesulfonic acid, and methanesulfonic acid. Co-solvents such as, but not limited to, methylene chloride, dioxane and ethyl aceatate can be used. TFA/methylene chloride mixtures are preferred. The reaction can be performed at from about -50 to about +50°C, with about 15 to about 30°C being preferred. Additives such as, but not limited to, anisole and thioanisole can be included to prevent side reactions from occurring during the cleavage of the ester.

Scheme 2 HzN ~ ~ H
0 ~ N
t-BOC-R1-X NO2 t-BOC-R1-NH
0 v \NO
a CO~H
H
1) TFR N
- t-BOC-R~-R1NH
2) t-BOC-R~-X ~
0 v \NO., CO~H
H
1> TFR
' t-BOC-R3-R2-R1PJH N w 2) t-BOC-R3-X

X = e.g. N-hydroxysuccm made, pentaflurophenyl In those cases where R' or RZ is a t-butoxycarbonyl (t-BOC) group the preferred procedure is to first react aspartyl-p-nitroanilide hydrochloride (the product of Example 4, Step B) with a preactivated N-t-BOC-protecaed amino acid derivative in the presence of a base. This will give t-BOC-A'-Asp-p-nitroanilide. Preactivation of the t-BOC-protected amino acid derivative may b~e as, but is not limited to, the N-hydroxysuccinimide or pentafluorophenyl esters. The base can be, but is not limited to, tertiary amine bases such as triethyl aminEa, diisopropyl ethyl amine, pyridine and N-methylmorpholine. The reaction is performed in a reaction inert solvent such as, but not limited to, DMF, THF, dioxane, and m~ethylene chloride. The reaction can be performed at from about -20 to about 100° C with temperatures about 15 to about 30° C
being preferred. In the second step the N-terminal t-BOC group is removed with strong acids such as, but not limited to, trifluoroacetic: acid (TFA), hydrogen chloride, hydrogen bromide, p-toluenesulfonic acid, and methanesulfonic acid. Co-solvents such as, but not limited to, methylene chloride, dioxane and ethyl acetate can be used.
TFA/methylene chloride mixtures are preferred. This reaction can be performed at from about -50 to about +50°C with about 15 to about 30°C being preferred. Co-solvents such as, but not limited to, anisole and thioanisole can be included to prevent side reactions from occurring during removal of the t-BOC group. In a third step, the product of this reaction is coupled as described above with a preactivated N-t-BOC-protected amino acid derivative in the presence of a base which will give t-BOC-~A1-Asp-p-nitroanilide. The two step sequence can be repeated again to give t~-A3-A2-Al-Asp-p-nitroanilide.
The compounds of formula I are substrates for ICE and, in conjunction with ICE
that has been partially to totally purified or more preferably that has been immobilized in an active form by adsorption onto antibody coated protein A functionalized agarose beads, can be used for detecting inhibitors of ICE. Inhibitors of ICE may be used in treating inflammatory diseases in which interl~eukin-1 fi plays a role.
Adsorption of ICE
onto protein A functionalized agarose beads is achieved by coating the beads with a polyclonal antibody specific to the N-terminal i~egion of ICE wherein the F~
region of the antibody binds to the protein A. The Feb portion of the antibody remains free to bind to ICE which it does when the beads area then heated with a partially purified preparation of ICE derived from THP-1 cells. The ICE so bound retains its catalytic activity.

-6a- 210964fi The compound of the formula:

H

O / ~N02 (which may be abbreviated as HAsp(S-t-butyl)-p-nitroanilide) may be produced by coupling FMOC-Asp((3-t-butyl)OH with p-nitroaniline using POC13 in pyridine according to the procedure of Rijkers et al. (Recl. Trav. Chim. Pays-Bas, 110, 347 (1991)), to form FMOC-Asp(S-t-butyl)-p-nitroanilide and then removing the FMOC
group by treating the coupling product with DBU(1,8-diazalicyclo-[5.4.0)undecene-7) or similar strong base.

~~~~s~s _7_ The following Preparations and Examples illustrate the preparation of the compounds of the present invention and their use in detecting ICE inhibitors.
Abbreviations used below are defined either the first time they are used or on pages 2, 4 and 6 above.
Preparation 1 HAsp i(fi-t-butt,)-p-nitroanilide A. FMOCAs~(f3-t-butyl)i-p-nitroanilide Using the procedure of Rijkers et al. Recl. Trav. Chim Pays-Bas, 110, 347 (1991 )) FMOCAsp(f3-t-butyl)OH (10.29 g, 25.0 mmole), p-nitroaniline (3.45 g, 25.0 mmole) and POC13 (4.22 g = 2.56 mL, 27.5 mmole) in pyridine (75 mL) gave, after being passed through a pad of silica gel (40:60 - ethyl acetate:hexane), 12.38 g (93%) of light yellow foam. Recrystallization of a portion from cyclohexane/ethyl acetate gave an analytical sample as a light yellow powder: mp 163-164°C (dec. with gas evol.); 'H
NMR (CDCI3) a 1.47 (s, 9H), 2.68 (dd, J=7.~4, 16.8 Hz, 1 H), 2.97 (dd, J=3.8, 16.8 Hz, 1 H), 4.24 (t, J=6.8 Hz, 1 H), 4.51 (d, J=6.8 t-iz, 2H), 4.6-4.7 (m, 1 H), 6.0-6.15 (m, 1 H), 7.25-7.35 (m, 2H), 7.35-7.45 (m, 2H), 7.58 (d, J=7.4 Hz, 2H), 7.67 (d, J=9.2 Hz, 2H), 7.77 (d, J=7.6 Hz, 2H, 8.21 (d, J=9.2 Hz, 2H), 9.0-9.1 (br s, 1 H); MS (LSIMS) m/e 532 (11, M++1), 476 (29), 179 (100); [a]pz°+39.2° (c=1.0, DMF);
Analysis calculated for Cz9Hz9N30,: C, 65.52; H, 5.50; N, 7.91; found: C, 65.61; H, 5.58; N, 7.80.
B. HAsp(f3-t-butyl-p-nitroanilide FMOCAsp(f3-t-butyl)-p-nitroanilide (2.13 g, 4.0 mmole) and DBU (1,8-diazabicyclo[5.4.0]undecene-7) (609 g, 4.0 mmole) were stirred together in dry dimethylformanide (DMF)(40 mL) for 1 hour. The reaction mixture was then diluted with ether (200 mL) and extracted with 1 N HCI (3 x 20 mL). The combined aqueous extracts were washed with ether and then basified wii:h a calculated amount of KZC03 (8.3 g, 60 mmole). This aqueous solution was extracted with ether (9 x 50 mL). The combined ether extracts were dried over MgS04. Filtr<~tion and concentration gave a yellow oil consisting of the desired product, some DBI.I and DMF. This was taken up in 1:1 ethyl acetate:hexane (50 mL) and poured onto a. pad of silica gel. Elution with 1:1 ethyl acetate:hexane (7 x 50 mL) and ethyl aceltate (3 x 200 mL) completely eluted the product. Concentration of the appropriate fractions gave 955 mg (77%) of light yellow solid. Recrystallization from cyclohexane/ethyl acetate gave an analytical sample as a pale yellow powder:mp 143-144°C; 'H NMR~ (CDCI3) d 1.44 (s, 9H), 2.1-2.3 (brs, 2H), _8_ 2.82 (dd, J=6.7, 16.9 Hz, 1 H), 2.89 (dd, J=~4.4, 16.9 Hz, 1 H), 3.82 (dd, J=4.4, 6.7 Hz, 1 H), 7.77 (d, J=9.2 Hz, 2H), 8.20 (d, J=92 Hz, 2H), 10.0-10.1 (br s, 1 H); MS
(LSIMS) m/e 310 (43, M++1 ), 254 (97), 154 (100); ~[a]pzo-4.9° (c=1.0, DMF);
HPLC ret. time:
2.07 min (40%), 5.56 min (50%); Analysis calculated for C,4H,9N3O5: C,54.36;
H, 6.19;
N, 13.59; found C, 54.69; H, 6.22; N, 13.37..
Preparation 2 CBZVaIAIaOCH3 CBZVaI N-hydroxysuccinimid~e ester (8.71 g, 25.0 mmole), alanine methyl ester hydrochloride (3.49 g, 25.0 mmole), DIEA (diisopropylethylamine) (3.23 g. 25.0 mmole) were combined in CHZCIz (250 mll) and stirred at room temperature for hours. The reaction mixture was washed twice, each time first with saturated NaHC03 and then with 1 N HCI, and was then dried over MgS04, filtered, and concentrated giving a white solid. This was recrystallized from ethylacetate to give 5.49 g (65%) of fine white needles. A second crop of 1.56 c~ (18%) of fine white needles was obtained from the mother liquors: mp 163-164°C;'H INMR (CDCI3) a 0.93 (d, J=6.8 Hz, 3H), 0.98 (d, J=6.8 Hz, 3H), 1.40 (d, J=7.2 Hz, 3H), 2.11 (hept, J=6.7 Hz, 1 H), 3.74 (s, 3H), 4.01 (br t, 1 H), 4.58 (pent, J=7.2 Hz, 1 H), 5.11 (s, 2H), 5.38 (b d, 1 H), 6.38 (br d, 1 H), 7.3-7.4 (m, 5H); MS (LSIMS) m/e 337 (100, M++1), 255(66);[a]ozo-46.0°
(c=1.0, methanol);
Analysis calculated for C"Hz4N205: C, 60.70; H, 7.19; N, 8.33; found: C, 60.70; H, 7.14;
N, 8.33.
Exam le a 1 AcTyrValAlaAs~-p-nitroanilide A. CBZTyr(O-t-butyl)ValAIaOCH3 CBZVaIAIaOCH3 (6.67 g, 19.8 mmole) was hydrogenated at 3 atm. over 10% Pd on carbon (700 mg) in CH30H (100 mL) at room temperature. After 1 hour, the catalyst was removed by filtration through a nylon ifilter. The filtrate was evaporated in vacuo giving a white solid which was dissolved in a 1:1 mixture of CH2CIz and DMF
(200 mL).
To this solution was added CBZTyr(O-t-butyl) N-hydroxysuccinimide ester (9.28 g, 19.8 mmole). After being stirred at room ternperature for 18 hours, the mixture was concentrated in vacuo to remove the CHzCIz and then water (300 mL) was added to precipitate the product. The solid was collected, washed with water and dissolved in ethyl acetate (500 mL). This solution was washed twice with saturated NaHC03 and twice with 1 N HCI and dried over MgS04. Filtration and evaporation in vacuo gave a _g_ white solid which was recrystallized from cyclohexane (100 mL) ethyl acetate (70 mL) yielding 7.10 g (65~°) of white fluffy solid. A second crop of 1.25 g (11 ~o) was obtained from the mother liquors: mp 189-190°C;'H NMR (DMSO-de) d 0.85 (d, J=6.8 Hz, 3H), 0.88 (d, J=7.0 Hz, 3H), 1.2-1.35 (m, 12H), 1.85-2.05 (m, 1 H), 2.83 (dd, J=10.9, 13.8 Hz, 1 H), 2.92 (dd, J=3.7, 13.8 Hz, 1 H), 3.3-3-4 (m, 2 (partially obscured by H20 absorption)), 3.60 (s, 3H), 4.2-4.4 (m, 3H), 4.94 (s, 2H), 6.85 (d, J=8.4 Hz, 2H), 7.17 (d, J=8.4 Hz, 2h), 7.2-7.35 (m, 5H), 7.52 (d, J==8.7 Hz, 1 H), 7.83 (d, J=9.2 Hz, 1 H), 8.45 (d, J=6.5 Hz, 1 H); MS (LSIMS) m/e 556 (100, M++1 ), 453 (31 ); [a]o~°-35.1 ° (c=1.0 methanol); Analysis calculated for C3°H4,N3O,: C, 64.84; H, 7.44; N, 7.56; found C, 64.96; H, 7.35; N, 7.52.
B. AcTyr(O-t-butyl)ValAIaOCH3 CBZTyr(O-t-butyrl)ValAIaOCH3 (5.55 g, 10.0 mmole) was hydrogenated at 3 atm.
over 10~° Pd on carbon (500 mg) in CH30H (100 mL) at room temperature.
After 1 hour, the catalyst was removed by filtration through a nylon filter. The filtrate was evaporated in vacuo giving an oil which was dissolved in THF (100 mL). To this solution was added DIEA (1.55 g, 12 mmole~) and acetyl chloride (942 mg, 12 mmole).
After being stirred at room temperature overnight, the reaction mixture was concentrated in vacuo and the residue dissolved in CHCI3. This solution was washed with 1 N HCI, and then with saturated NaHC03 and then dried over MgS04.
Filtration and evaporation in vacuo gave a gel-like solid which was recrystallized from ethyl acetate/CH30H to give 2.86 g (620) of a gel-like solid which was dried under high vacuum. A second crop of 1.42 g (31 ~°) was obtained from the mother liquors: mp 209-211 ° C; ' H NMR (DMSO-de) d 0.82 (d, J=6.8 Hz, 3H), 0.86 (d, J=6.8 Hz, 3H), 1.24 (s, 9H), 1.27 (d, J=7.3 Hz, 3H), 1.73 (s, ~~H), 1.94 (hept, J=6.8 Hz, 1 H), 2.66 (dd, J=10.0, 14.0 Hz, 1 H), 2.90 (dd, J=4.3, 14.0 IHz, 1 H), 3.59 (s, 3H), 4.15-4.3 (m, 2H), 4.5-4.6 (m, 1 H), 6.82 (d, J=8.4 Hz, 2H), 7.12 (d, J=8.4 Hz, 2H), 7.79 (d, J=9.0 Hz, 1 H), 8.06 (d, J=8.4 Hz, 1 H) 8.39 (d, J=6.6 Hz, 1 H); MS (LSIMS) m/e 464 (100, M++1 ); [a]p 17.0° (c=1.0, DMF); Analysis calculated for Cz4H3,N3O6: C, 62.18; H, 8.05; N, 9.06;
found: C, 62.27; H, 8.18; N, 9.00.
C. AcTyr(O-t-butyl)ValAIaOH
AcTyr(O-t-butyl)ValAIaOCH3 (2.32 g, 5.0 mmole) was slurried in 10% aqueous CH30H (50 mL) and treated with LiOH ~ HZO (1.05 g, 25.0 mmole) in one portion.
The reaction mixture was stirred at room temperature for 2 hours and the reaction was then 2109fi46 quenched by the addition of an excess of sulfonic acid ion exchange resin (56 g, 125 meq of H+). After being stirred for 15 minutes, the mixture was filtered and the resin washed thoroughly with CH30H. The filtrates was concentrated in vacuo to give a white solid which was recrystallized from ethyl acetate/CH30H yielding, after drying under high vacuum, 1.96 g (87~) of a white powdesr: mp 191-192°C (dec. with gas evolution);
' H NMR (DMSO-de) 30.82 (d, J=6.8 Hz, 3H;1, 0.86 (d, J=6.8 Hz, 3H), 1.25 (s, 9H), 1.27 (d, J=7.3 Hz, 3H, partially obscured), 1.74 ('s, 3H), 1.95 (hept, J=6.8 HZ, 1 H), 2.66 (dd, J=10.1, 13.9 Hz, 1 H), 2.92 (dd, J=4.2, 13.9' Hz, 1 H), 4.1-4.25 (m, 2H), 4.5-4.6 (m, 1 H), 6.82 (d, J=8.4 Hz, 2H), 7.12 (d, J=8.4 Hz, .?H), 7.79 (d, J=9.0 Hz, 1 H), 8.07 (d, J=8.4 Hz, 1 H), 8.23 (d, J=6.9 Hz, 1 H); MS (LSIMS;I m/e 450 (53, M++1 ), 189(100);
[a]p2°-8.7°
(c=1, DMF); Analysis calculated for calculated for C23H35N3O6: C, 61.45; H, 7.85; N, 9.35; found C, 61.18; H, 8.05; N, 9.26.
D. AcTyr(O-t-buty~ValAlaAsp(f3-t-butylp-nitroanilide.
AcTyr(O-t-butyl)ValAIaOH (687 mg, 1.53 mmole), HAsp(fi-t-butyl)-p-nitroanilide (473 mg, 1.53 mmole), N-hydroxysuccinimicle (264 mg, 2.29 mmole) and DEC ~ HCI
(352 mg, 1.84 mmole) were combined in dry DMF (15 mL) and the resulting pale yellow solution stirred at room temperature for 44 hours. The reaction mixture was diluted with 1 N HCI and the resulting precipitated solidl triturated to break all chunks of solid into a finely dispersed solid. This was then collected and washed with 1 N HCI. The solid was resuspended in aqueous NaHC03, trii:urated for 15 minutes and collected.
After washing with water and drying under high vacuum 875 mg (77°~) of a white powder was obtained. A portion of this was recrystallized from ethyl acetate/CH30H to give an analytical sample: mp 234-235°C (dec. witlh gas evolution);'H NMR (DMSO-ds) a0.82 (t,J=7.1 Hz, 3H), 1.21 (d, J=7.1 Hz, 3H), 1.24 (s, 9H), 1.34 (s, 9H), 1.74 (s, 3H), 2.57 (dd, J=7.6, 15.9 Hz, 1 H), 2.63-2.72 (m, 1 I-I), 2.76 (dd, J=6.8, 15.9 Hz, 1 H), 2.92 (dd, J=0.6, 10.1 Hz, 1 H), 4.1-4.2 (m, 1 H), 4.2-4..3 (m, 1 H), 4.5-4.6 (m, 1 H), 4.6-4.7 (m, 1 H), 6.82 (d, J=8.4 Hz, 2H), 7.13 (d, J=8.4 Hz, 2H), 7.88 (d, J=9.3 Hz, 2H), 8.08 (d, J=8.4 Hz, 1 H), 8.14 (d, J=6.7 Hz, 1 H), 8.22 (d, J==9.3 Hz, 1 H), 8.38 (d, J=7.5 Hz, 1 H), 10.52 (s, 1 H); MS(LSIMS) m/e 741 (32, M++1 ), 7'10 (34), 432 (53), 361 (75), 305 (100); [a]pzo-16.4° (c=1, DMF); Analysis calculated for C3~H5zN6O,°: C, 59.98;
H, 7.08; N, 11.35;
found: C, 59.78; H, 6.90; N, 11.20.

2~~964~

E. AcTyrValAlaAsp-p-nitroanilide:
A slurry of AcTyr(O-t-butyl)ValAlaA;sp(f3-t-butyl)-p-nitroanilide (148 mg, 0.20 mmole) in CHZCIZ (10 mL) at 0°C was treated with 10 mL of a prechilled mixture of TFA:anisoleahioanisole (90:5.5). The resulting solution was stirred at 0°C for 30 minutes and at room temperature for 4 hours. The mixture was concentrated in vacuo.
CHZCIZ was added to the concentrate and the solvent evaporated in vacuo. The residue was then triturated with ether for a few hours. The solid was collected, washed thoroughly with ether and dried under vacuum to give 109 mg (870) of a white powder;
mp 205-206 ° C (dec.); ' H NMR (DMSO(dime~thylsulfoxide)-ds) d 0.82 (t, J=7.3 Hz, 6H), 1.22 (d, J=7.2 Hz, 3H), 1.74 (s, 3H), 1.9-2.0 (m, 1 H), 2.5-2.7 (m, 3H), 2.86 (dd, J=0.6, 10.2 Hz, 1 H), 4.1-4.2 (m, 1 H), 4.2-4.3 (m, 1 H), 4.45-4.55 (m, 1 H), 4.6-4.7 (m, 1 H), 6.61 (d, J=8.4 Hz, 2H), 7.01 (d, J=8.4 Hz, 2H), 7.84 (d, J=9.2 Hz, 2H), 7.88 (d, 1 H, partially obscured by adjacent peak), 8.06 (d, J=8.3~ Hz, 1 H), 8.12 (d, J=7.0 Hz, 1 H), 8.20 (d, J=9.2 Hz, 2H) 8.31 (d, J=7.5 Hz, 1 H), 11.2-11.4 (br s, 1 H); MS (LSIMS) m/e 651 (5, M++Na), 629 (3, M++1), 491 (7), 424, (3), 376 (18), 305 (47), 178 (136), 136 (100);
[a]p2°-18.8°(c=1.0, DMF); Analysis calculated for CZ9H36N60,°: C, 55.40; H, 5.77; N, 13.37: found C, 55:60; H, 6.24; N, 13.49.
Exam~~le 2 PhCHZCHzCOValAlau4sp-p-nitroanilide A. PhCH2CH2COValAIaOCH3 CBZVaIAIaOCH3 (1.35 g, 4.00 mmole) was hydrogenated at 3 atm. over 10~ Pd on carbon (150 mg) in CH30H (40mL) at room temperature. After 1 hour, the catalyst was removed by filtration through a nylon filter. The filtrate was evaporated in vacuo giving a white solid which was slurred in CHC13(40 mL) and treated with DIEA
(620 mg, 4.8 mmole) and hydrocinnamoyl chloride (741 mg, 4.4 mmole). After 1 hour at room temperature, the reaction mixture was washed with 1 N HCI, dried with MgS04 filtered and concentrated in vacuo to a white solid. This was recrystallized from ethylacetate to give 617 mg (48%) of white powder: mp 207-208°C; 'H NMR (DMSO-de) a 0.79 (d, J=6.8 Hz, 3H), 0.84 (d, J=6.7 Hz, 3H), 1.28(d, J=7.3 Hz, 3H), 1.85-2.0 (m, 1 H), 2.35-2.5 (m, 2H, partially obscured by the DMSO-d5 peak), 2.75-2.85 (m, 2H), 3.61 (s, 3H), 4.15-4.3 (m, 2H), 7.1-7.3 (m, 5H), 7.90 (d, J:=9.1 Hz, 1 H), 8.43 (d, J=6.7 Hz, 1 H); MS
(FAB) m/e 335 (88, M++1), 232 (100), 204 (5~t); [a]p2°-71.7°(c=1.0, methanol); Analysis calculated for C,8HZ6N204: C, 64.65; H, 7.84; N, 8.38; found: C, 64.85; H, 7.62; N, 8.05.

21~9~46 B. PhCHzCHZCOVaIAIaOH
By the same procedure used to prepare AcTyr(O-t-butyl)ValAIaOH, PhCHZCH2COValAIaOCH3 (508 mg, 1.52 mmole) and LiOH ~ OH (319 mg, 7.6 mmole) in 1096 aqueous CH30H (15 mL) gave, after quenching with sulfonic acid ion exchange resin (17.0 g, 38 meq), 511 mg (1000 of pure product as a white powder.
Recyrstallization of a portion from ethylace~tate gave an analytical sample:
mp 205-206°C;'H NMR (DMSO-de) a 0.76 (d, J=6.'7 Hz, 3H), 0.81 (d, J=6.8 Hz, 3H), 1.24 (d, J=7.3 Hz, 3H), 1.8-1.95 (m, 1 H), 2.35-2.55 (m, 2H, partially obscured by the DMSO-d5 peak), 2.75-2.85 (m, 2H), 4.1-4.25 (m, 2H), 7.1-7.3 (m, 5H), 7.84 (d, J=9.1 Hz, 1 H), 8.23 (d, J=6.9 Hz, 1 H); MS (LSIMS) m/e 321 (45, M++1 ), 232 (29), 204(11 ), 157(100); [a]p2o-2.0° (c=1.0, DMF); Analysis calculated for C"H24N204: C, 63.73; H, 7.55; N, 8.75;
found: C, 63.78; H, 7.30; N, 8.60.
C. PhCHZCHZCOVaIAIaAsp(f3-t-k>utY)_p-nitroanilide By the same procedure used to prepare the title compound of Example 1 D, PhCH2CHzCOVaIAIaOH (401 mg, 1.20 mmcde), HAsp(13-t-butyl)-p-nitroanilide (387 mg, 1.20 mmole), N-hydroxysuccinimide (216 mg, 1.5 mmole) and DEC ~ HCI (288 mg, 1.88 mmole) in DMF (12 mL) gave 661 mg (90%.) of a tan powder. This was recrystallized from ethyl acetate to give 459 mg (62%) of a white powder; mp 222-224°C
(dec. with gas evolution); 'H NMR (DMSO-ds) 6 0.77 (t, J=6.9 Hz, 6H), 1.20 (d, J=7.1 Hz, 3H), 1.34 (s, 9H), 1.85-1.95 (m, 1 H), 2.35-2.65 (m, 3, partially obscured by the DMSO-d5 peak), 2.7-2.85 (m, 3H), 4.1-4.15 (m, 1 H), 4.2-4.3 (m, 1 H), 4.6-4.7 (m, 1 H), 7.1-7.3 (m, 5H), 7.85-7.95 (m, 3H), 8.13 (d, J=6.8 Hz, 1 H), 8.21 (d, J=9.3 Hz, 2H), 8.31 (d, J=7.6 Hz, 1 H), 10.56 (s, 1 H); MS (LSIMS) m/e 612 (4, M++1 ), 556 (16), 418 (15), 325(7), 303(26), 232(100), 204(62); [a]oz°-17.6° (c=1.0, DMF); Analysis calculated for C3, H4, N508: C, 60.87; H, 6.76; N, 11.45; found: C, 61.04; H, 6.59; N, 11.23.
D. PhCHZCHZCOVaIAIaAspp-nii~roanilide By the same procedure used to prepare the compound of Example 1 E, PhCHzCHZCOVaIAIaAsp(f3-t-butyl)-p-nitroaniilide (122 mg, 0.2 mmole) gave 104 mg (94~°) of a light tan powder: mp 206-208°C (dec.); ' H NMR (DMSO-ds) d 0.77 (t, J=7.0 Hz, 6H), 1.21 (d, J=7.1 Hz, 3H), 1.85-2.0 (m~, 1 H), 2.35-2.7 (m, 3, partially obscured by the DMSO-d5 peak), 2.7-2.85 (m, 3H), 4.1-4.18 (m, 1 H), 4.18-4.3 (m, 1 H), 4.6-4.7 (m, 1 H), 7.1-7.3 (m, 5H), 7.85-7.95 (m, 3H), 8.12 (d, J=6.7 Hz, 1 H), 8.21 (d, J=9.3 Hz, 2H), 8.29 (d, J=7.6 Hz, 1 H), 10.55 (s, 1 H); MS (t_SIMS) m/e 578 (10, M++Na), 418 (4), 303 (26), 232 (100), 204 (89); [a]o °-21.6° (c=1.0, DMF); Analysis calculated for CZ~H33N5O8:
C, 58.37; H, 5.99; N, 12.61; found: C, 58.29; H, 5.84; N, 12.43.
Exami~le 3 PhCH2NHCOValAl2u4sp-p-nitroanilide A. PhCH2NHCOVaIAIaOCH, CBZVaIAIaOCH3 (1.35 g, 4.00 mmole) was hydrogenated at 3 atm. over 10~ Pd on carbon (150 mg) in CH30H (40 mL) at room temperature. After 1 hour, the catalyst was removed by filtration through a nylon filter. The filtrate was evaporated in vacuo giving a white solid which was slurried in CHCI3 (40 mL) and treated with benzyl isocyanate (586 mg, 4.4 mmole). After 1 hour at room temperature, the reaction mixture was washed three times with 'I N HCI, dried over MgSO4,filtered and concentrated in vacuo giving 791 ~mg (61 %) of the desired product as a white powder.
Recrystallization of a portion from ethyl acetate/CH30H gave an analytical sample: mp 227-228°C; 'H NMR (DMSO-ds) a 0.80 (d, J=6.8 Hz, 3H), 0.86 (d, J=6.7 Hz, 3H), 1.26(d, J=7.3 Hz, 3H), 1.85-1.95 (m, 1 H), 3.59 (s, 3H), 4.10 (dd, J=5.9, 9.2 Hz, 1 H), 4.15-4.3 (m, 3H), 6.08 (d, J=9.3 Hz, 1 H), 6.53 (t, J=6.0 Hz, 1 H), 7.15-7.35 (m, 5H), 8.39 (d, J=6.7 Hz, 1H); MS (LSIMS) m/e 336 (100, M++1), 233 (62), 203 (76);
[a]p2°+6.1 (c=1.0, DMF); Analysis calculated for C"H,5N3O4; C, 60.88; H, 7.51; N, 12.53;
found:
C, 60.98; H, 7.30; N, 12.34.
B. PhCHZNHCOVaIAIaOH
By the same procedure used to prepare the title compound of Example 1 C, PhCH2NHCOVaIAIaOCH3 (671 mg, 2.00 mmole) and LiOH ~ OH (168 mg, 4.00 mmole) in 10°~ aqueous CH30H (20 mL) gave after quenching with sulfonic acid ion exchange resin (18.0 g, 40 meq) 658 mg (100%) of pure product as an off-white flaky solid.
Recyrstallization of a portion from ethyl ace~tate/CH30H gave an analytical sample as very fine white crystals: mp 205-206°C; 'H INMR (DMSO-de) a 0.79 (d, J=6.8 Hz, 3H), 0.86 (d, J=6.7 Hz, 3H), 1.25 (d, J=7.3 Hz, 31H), 1.85-2.0 (m, 1 H), 4.05-4.3 (m, 4H), 6.09 (d, J=9.3 Hz, 1 H), 6.54 (t, J=6.0 Hz, 1 H), 7.'I 5-7.35 (m, 5H), 824 (d, J=7.0 Hz, 1 H); MS
(LSIMS) m/e 344 (22, M++Na), 322 (83, M++1), 233 (70), 205(18), 189 (100);
[a]o2°+15.3° (c=1.0, DMF); Analysis calculated for C,6H23N3O4:
C, 59.79; H, 7.21; N, 13.08; found: C, 59.87; H, 7.30; N, 12.80.

C. PhCH2NHCOVaIAIaAspi(f3-t-butyl)-p-nitroanilide By the same procedure used to prepare the title compound of Example ID, PhCHZNHCOVaIAIaOH (553 mg, 1.72 mmolle), HAsp(f3-t-butyl)-p-nitroanilide (532 mg, 1.72 mmole), N-hydroxysuccinimide (247 mgt, 2.15 mmole) and DEC ~ HCI (515 mg, 2.69 mmole) in DMF (17 mL) gave 1.04 g (99W) of a light yellow powder. This was recrystallized from ethyl acetate/CH30H to give 667 mg (63~) of a white powder: mp 209-210°C (dec. with gas evolution);'H NMI~ (DMSO-de) a 0.79 (d, J=6.8 Hz, 3H), 0.84 (d, J=6.7 Hz, 3H), 1.21 (d, J=7.1 Hz, 3H}, 1.34 (s, 9H), 1.85-2.0 (m, 1 H), 2.55 (dd, J=7.7, 16.0 Hz, 1 H), 2.74 (dd, J=6.7, 16.0 Hz, 1 H), 4.02 (dd, J=5.8, 8.3 Hz, 1 H), 4.15-4.3 (m, 3H), 4.6-4.7 (m, 1 H), 6.13 (d, J=8.4 Hz, 1 H), 6.56 (t, J=6.0 Hz, 1 H), 7.15-7.35 (m, 5H), 7.88 (d, J=9.3 Hz, 2H), 8.15-8.25 (m, 3H), 8.33 (d, J=7.6 Hz, 1 H), 10.50 (s, 1H); MS (LSIMS) m/e 635 (1, M++Na), 613 (3, M++1), 557 (13), 424(12), 419 (12), 304 (13), 286 (19), 233 (100), 205 (29), 171 (27); (a)oZ°-2.8°
(c=1.0, DMF); Analysis calculated for C3°H4°NeOe: C, 58.81; H, 6.;i8; N,13.72; found:
C, 58.92; H, 6.56; N, 13.64.
D. PhChZNHCOVaIAIaAsp-p-nitroanilide By the same procedure used to prepare the title compound of Example IE, PhCHZNHCOVaIAIaAsp(fi-t-butyl)-p-nitroanilide (123 mg, 0.2 mmole) gave 102 mg (92%) of an off-white powder: mp 207-209°C; 'H IVMR (DMSO-ds) d 0.78 (d, J=6.8 Hz, 3H), 0.84 (d, J=6.7 Hz, 3H), 1.22 (d, J=7.1 Hz, 3H), 1.85-2.0 (m, 1 H), 2.60 (dd, J=7.7, 16.0 Hz, 1 H), 2.78 (dd, J=6.7, 16.0 Hz, 1 H), 4.02 (dd, J=5.8, 8.3 Hz, 1 H), 4.15-4.3 (m, 3H), 4.6-4.7 (m, 1 H), 6.12 (d, J=8.5 Hz, 1 H), 6.5E~ (t, J=6.0 Hz, 1 H), 7.15-7.35 (m, 5H), 7.88 (d, J=9.3 Hz, 2H), 8.15-8.25 (m, 3H), 8.33 (d, J=7.6 Hz, 1 H), 10.47 (s, 1 H), 12.45 (br s, 1 H); MS (LSIMS) m/e 579 (4, M++ Na), 557(15, M++1 ), 304 (14), 286 (20), (100); (a]ozo-3.5° (c=1.0, DMF); Analysis calculated for CZeH32NB°08 ~ 0.5 HZO; C, 55.01;
H, 6.22; N, 14.81; found: C, 55.15, H, 6.06; N, 14.54.
Examt~le 4 t-BOCVaIAIaAs~~-p-nitroanilide A. t-BOCAsp~(f3-t-butyl)-p-nitroanilide Using the procedure of Rijkers et <~I. Recl. Trav. Chim Pays-Bas, 110, 347 (1991 )) t-BOCAsp(fi-t-butyl)OH (7.65 g, 25.0 mmole), p-nitroaniline (3.45 g, 25.0 mmole) and POCI3 (4.22 g = 2.56 mL, 27.5 mmole) in pyridine (75 mL) gave, after being passed through a pad of silica gel (25:75 ethyl acetate:hexane), 8.93 g (87°~) of light 21099:6 yellow foam: 'H NMR (CDCI3) ~' 1.47 (s, 91-I), 1.49 (s, 9H), 2.69 (dd, J=6.7, 17.0 Hz, 1 H), 2.91 (dd, J=4.3, 17.0 Hz, 1 H), 4.59 (m, 1 H), 5.90 (br d, 1 H), 7.69 (d, J=9.2 Hz, 2H), 8.20 (d, J=9.2 Hz, 2H), 9.20 (br s, 1 H); MS (LSIMS) m/e 410 (22), 394 (9), 354 (18), 298 (100), 282 (17), 254 (26); [a]p2°-30.0° (c=1.0, methanol); Analysis calculated for C,9HZ,N30,: C, 55.73; H,6.65; N, 10.26; found: C, 55.50; H, 6.41; N, 10.22.
B. HAsp-p-nitroanilide hydrochlc>ride t-BOC-Asp(f3-t-butyl)-p-nitroanilide (8.68 g, 21.2 mmole) was dissolved in a mixture of dioxane (200 mL) and ethyl acetate (50 mL) and cooled to O°C. The solution was then saturated with HCI gas and stirred for one hour at O°C. The HCI
was then purged from the reaction with a stream of NZ and the reaction mixture concentrated to a yellow glass. This was triturated with ether, collected and dried under high vacuum to give 6.79 g (yield greater than 100%; NMR indicated some ether still present) of a light yellow powder: 'H NMR (DMSO-d6) a 2.93 (dd, J=7.3, 17.5 Hz, 1 H), 3.01 (dd, J=5.2, 17.5 Hz, 1 H), 4.32 (m, '1 H), 7.18 (d, J=9.1 Hz, 2H), 8.26 (d, J=9.1 Hz, 2H); MS (LSIMS) m/e 254 (54, M++1), 239 (92), 221 (100), 197 (94), 195 (76).
C. t-BOCAIaAspJ~-nitroanilide HAsp-p-nitroanilide hydrochloride (2.90 g, 10.0 mmole), t-BOCAIa N-hydroxysuccinimide ester (2.86 g, 10.0 mmole) and DIEA (1.29 g, 10.0 mmole) were combined in CHzCl2 (100 mL) and stirred for 24 hours at room temperature. The resulting turbid solution was washed twice vuith 0.1 N HCI and then dried over MgS04.
Filtration and concentration in vacuo gave <~ yellow foam that was chromatographed (5:30:65 - acetic acid: ethyl acetate:hexane to 5:35:60 - acetic acid:ethylacetate:hexane) to give 3.03 g (71%) of a light yellow foam: mp 74-80°C; 'H NMR (CDC13) ~' 1.39 (s, 9H), 1.44 (d, J=7.2 Hz, 3H), 2.77 (dd, J=4.6, 17.6 Hz, 1 H), 3.42 (dd, J=2.5, 17.6 Hz, 1 H), 4.1-4.2 (m, 1 H), 4.95-5.1 (m, 2H), 7.62 (br d, J=9.2 Hz, 1 H), 7.93 (br d, J=9.0 Hz, 2H), 8.18 (d, J=9.0 Hz, 2H), 9.28 (br s, 11-I); MS (LSIMS) m/e 425 (46, M++1), (100); [a]o~°-11.8° (c=1.0, DMF); Analysis calculated for C,eHz4N408: C, 50.94; H, 5.70;
N13.20; found: C, 50.86; H, 5.68; N, 12.85.
D. t-BOCVaIAIaAsp~-nitroanilide:
t-BOCAIaAsp-p-nitroanilide (424 mg, 't .00 mmole) was dissolved in neat TFA
(10 mL) and stirred at room temperature for 1 hour. The solvent was then evaporated in vacuo and the residue was then dissolved in 10 mL of CHzCIz which was then evaporated in vacuo. This dissolution with CHZCIz and subsequent evaporation was repeated two more times giving a light yellow foam. This foam was suspended in CHZCIZ (10 mL) and treated with DIEA (129 rng, 1.00 mmole). To this suspension was added t-BOCVaI N-hydroxysuccinimide ester (314 mg, 1.00 mmole), DMF (10 mL) and sufficient additional DIEA to make the mixtuire neutral. After the mixture was stirred at room temperature for 24 hours, the solvents 'were removed in vacuo (high vacuum) and the residue triturated with 1 N HCI. The resulting light yellow solid was collected, washed with water and dried. Chromatography (5:35:60 - acetic acid: ethyl acetate:hexane to 5:55:40 - acetic acid:ethyl acetate:hexane) gave 348 mg (66~) of a light yellow glass that was recrystallized firom ethyl acetate to give 128 mg of an amorphous solid: mp 200-202°C; 'H NMR (DMSO-ds) d 0.78 (d, J=6.7 Hz, 3H), 0.81 (d, J=6.8 Hz, 3H), 1.20 (d, J=8.1 Hz, 3H), 1.37 (s, 9H), 1.85-1.95 (m, 1 H), 2.60 (dd, J=7.5, 16.6 Hz, 1 H), 2.76 (dd, J=6.3, 16.6 Hz, 1 H), 3.75-3.85 (m, 1 H), 4.2-4.3 (m, 1 H), 4.63 (m, 1 H), 6.77 (d, J=8.6 Hz, 1 H), 7.88 (d, J=9.2 Hz, 2H), 8.02 (d, J=7.7 Hz, 1 H), 8.21 (d, J=9.2 Hz, 2H), 8.41 (d, J=7.0 Hz, 1 H), 10.53 (brs 1 H); MS (LSIMS) m/e 546 (21, M++Na), 524 (22, M++1), 507 (12), 458 (45), 424 (57), 330 (35), 286 (34), (100); [a]p2°-27.7° (c=1.0, DMF); Analysis calculated for C23HssNsOs: C, 52.76; H, 6.35;
N, 13.38; found: C, 52.45; H, 6.57; N, 12.76~.
Ex- am~~le 5 AcValAlaAsp-~~-nitroanilide t-BOCVaIAIaAsp-p-nitroanilide (174 mg, 0.33 mmole) was dissolved in neat TFA
(5 mL) and stirred at room temperature for 1 hour. The TFA was removed in vacuo and the residue was then dissolved in 5 mL of ChizCl2 which was then evaporated in vacuo.
This dissolution with CHZCIZ and subsequent evaporation repeated two more times.
The residue was dissolved in dioxane/water (5 mL, 4:1 ) and treated with acetic acid N-hydroxysuccinimide ester (63 mg, 0.40 mmole) and NaHC03 (139 mg, 1.65 mmole).
After 18 hours, the mixture was diluted with 1 N HCI (25 mL) and extracted three times with ethyl acetate. The combined extracts were dried with MgS04, filtered and concentrated to a yellow solid which was r~acrystallized from ethyl acetate/ethanol to give 33 mg (21%) of light yellow powder: mp 196-200°C (dec.);'H NMR
(DMSO-de) d 0.80 (d, J=6.6 Hz, 3H), 0.82 (d, J=5.1 Hz, 3H), 1.20 (d, J=7.2 Hz, 3H), 1.85 (s, 3H), 1.85-2.0 (m, 1 H), 2.60 (dd, J=7.5, 16.6 Hz, 'I H), 2.75 (dd, J=6.2, 16.6 Hz, 1 H), 4.1-4.2 (m, 1 H), 4.2-4.3 (m, 1 H), 4.6-4.7 (m, 1 H), 7.8-7.9 (m, 3H), 8.11 (d, J=6.8 Hz, 1 H), 8.21 (d, J=9.3 Hz, 2H), 8.27 (d, J=7.2 Hz, 1 H), 10.51 (s, 1 H), 12.45 (br s, 1 H);
MS (LSIMS) m/e 488 (6, M++Na), 466 (23, M++1 ), 449 (7) 328 (15), 213 (44), 142 (100);
[aJpz°-24.9° (c=1.0, DMF); Analysis calculated for Cz°HZ,N508: C, 51.60; H, 5.85; N, 15.05; found: C, 50.16; H, 5.79; N, 14.06.
Examc~le 6 CBZVaIAIaAsa-~a-nitroanilide A. CBZAsp(f3-t-butyl)-p-(t-BOC amino anilide CBZAsp(f3-t-butyl)OH dicyclohexylannine salt (2.52 g, 5.00 mmole), 4-(t-BOC
amino)aniline (1.04 g, 5.00 mmole), DEC ~ HCI (1.44 g, 7.5 mmole), N
hydroxybenzotriazole hydrate (675 mg, 5.0 mmole) and DIEA (323 mg, 2.5 mmole) were combined in dry DMF (50 mL) and stirred at room temperature for 24 hours. The mixture was diluted with ether (150 mL) and washed twice with 1 N HCI, twice with saturated NaHC03, and once with 1 N HCII. After drying over MgS04 filtration and concentration in vacuo, an off-white solid was obtained which was recrystallized from cyclohexane/ethyl acetate to give 2.17 g (84'~%) of tan solid. An analytical sample was prepared by recrystallization from hexane/ethylacetate:mp 131-133°C
(softens 120°C);
' H NMR (CDC13) a 1.43 (s, 9H), 1.51 (s, 9H;1, 2.67 (dd, J=7.1, 17.2 Hz, 1 H), 2.97 (dd, J=4.1, 17.2 Hz, 1 H), 4.6-4.7 (m, 1 H), 5.16 (s., 2H), 6.10 (br d, 1 H), 6.45 (br s, 1 H), 7.30 (d, J=9.1 Hz, 2H), 7.35-7.45 (m, 7H), 8.41 (b s, 1 H); MS (LSIMS) m/e 513 (46, M+), 457 (40), 402 (38), 358 (40), 243 (32), 178 (31), 152 (100); [a]o2°-19.2° (c=1.0, methanol);
HPLC retention time: 4.32 minutes (30%), 1 T.17 minutes (40%); Analysis calculated for CZ,H35N3O,: C, 63.14; H, 6.87; N, 8.18; found: C, 63.24; H, 6.94; N, 8.05.
B. CBZAIaAsp(f3-t-butyl pit-BOC aminoJwanilide CBZAsp(f3-t-butyl)-p-(t-BOC amino)anilide (1.17 g, 2.28 mmole) was hydrogenated over 10% Pd-C (120 mg) at 3 atm. in CH30H (20 mL) at room temperature for 1 hour. The reaction mixtur~a was filtered through a nylon filter and the filtrate concentrated to an oil. This was dissolved in CH2CI2 (23 mL) and CBZAIa N-hydroxysuccinimide ester (803 mg, 2.51 mm~ole) was added. After being stirred at room temperature for 24 hours, the reaction mixture was washed twice with 1 N HCI
and twice with saturated NaHC03 and then dried ovs~r MgS04. Filtration and concentration in vacuo gave a white solid which was recrystallized from cyclohexane/ethyl acetate to give 750 mg (56%) of white powder: mp '183-185°C (with gas evolution);
'H NMR
(DMSO-ds) ~ 1.19 (d, J=7.2 Hz, 3H), 1.35 (s, 9H), 1.45 (s, 9H), 2.54 (dd, J=7.7, 15.6 21Q9fi46 Hz, 1 H), 2.72 (dd, J=6.4, 15.6 Hz, 1 H), 4.0-4.1 (m, 1 H), 4.6-4.7 (m, 1 H), 4.99 (d, J=12.5 Hz, 1 H), 5.03 (d, J=12.5 Hz, 1 H), 7.25-7.4 (im, 7H), 7.48 (d, J=8.9 Hz, 2H), 7.58 (br d, J=6.5 Hz, 1 H), 8.23 (br d, J=8.1 Hz, 1 H), 9.26 (br s, 1 H), 9.68 (br s, 1 H); MS (LSIMS) m/e 585 (34, M++1 ), 584 (44, M+), 529 (4'I ), 528 (29), 473 (50), 321 (94) 243 (100);
[a]p2o-28.9° (c=1.0, methanol); Analysis calculated for C3°H40N408' C. 61.63; H, 6.90;
N, 9.58; found: C, 61.66; H, 7.15; N, 9.52.
C. CBZVaIAIaAsp(f3-t-butyl,-p-(t-BOC aminoyanilide CBZAIaAsp(fi-t-butyl)-p-(t-BOC amino)anilide (1.97 g, 3.37 mmole) was hydrogenated over 10% Pd on carbon (200 mg) at 3 atm. in CH30H (50 mL) at room temperature for 1 hour. The reaction mixtur~a was filtered through a nylon filter and the filtrate concentrated to a white gummy solid. This solid was suspended in DMF
(12 mL) and CBZVaI N-hydroxysuccinimide ester (1.;?9 g, 3.71 mmole) was added. After being stirred at room temperature for 24 hours, the reaction mixture was diluted with a saturated solution of NaHC03~and stirred for' 15 minutes. The precipitated product was collected, washed with water and dried under high vacuum to give 2.16 g (94%) of a fine white powder: mp 232-233°C (with gays evolution); 'NMR (DMSO-ds) d 0.81 (d, J=8.7 Hz, 3H), 0.84 (d, J=6.9 Hz, 3H), 1.1'3 (d, J=7.0 Hz, 3H), 1.33 (s, 9H), 1.85-2.0 (m, 1 H), 2.52 (dd, J=7.6, 15.8 Hz, 1 H), 2.69 (dd, J=6.4, 15.8 Hz, 1 H), 3.8-3.9 (m, 1 H), 4.2-4.3 (m, 1 H), 4.6-4.7 (m, 1 H), 5.01 (d, J=12.8 Hz, 1 H), 5.03 (d, J=12.8 Hz, 1 H), 7.25-7.4 (m, 8H), 7.47 (d, J=9.0 Hz, 2H), 8.08 (br d, J=6.9 Hz, 1 H,) 8.21 (br d, J=7.9 Hz, 1 H), 9.25 (br s, 1 H), 9.79 (br s, 1 H); MS (LSI MS) m/e 684 (51, M+), 420 (40), 119 (100);
[a]p2°-16.5° (c=1.0, DMF); Analysis calculated for C35H49N5O9:
C, 61.47; H, 7.22; N, 10.24; found: C, 61.29; H, 6.93; N, 10.20.
D. CBZVaIAIaAsp p-nitroanilide CBZVaIAIaAsp(f3-t-butyl)-p-(t-BOC arnino)anilide (900 mg, 1.32 mmole) was dissolved in cold TFA (13 mL) and stirred at 0°C for 4 hours. The TFA
was removed in vacuo and acetic acid (26 mL) added to the residue. To the resulting suspension was added NaB03 (2.03 g, 13.2 mmole) and the mixture stirred for 18 hours at room temperature. The reddish-orange reaction mixture was concentrated. Water and ethyl acetate (200 mL) were added to the residue and a small amount of 1 N HCI added to bring the pH to about 1. The separated ethyl acetate layer was twice washed with 1 N
HCL and was then dried with MgS04, filtered and concentrated to a light brown solid.
This was absorbed onto silica gel and charged onto a column. Elution (2.5:2.5:95 -_19_ acetic acid:methanoI:CH2C12) gave 474 mg (64%) of product. Further purification was achieved by preparative thin layer chromatography (5:5:95 - acetic acid:methanoI:CH2C12): mp 204-206°C (dsac., softens 188°C); 'H
NMR (DMSO-de) a 0.80 (d, J=8.1 Hz, 3H), 0.83 (d, J=7.0 Hz, 3H), 1.21 (d, J=7.1 Hz, 3H), 1.85-2.0 (m, 1 H), 2.61 (dd, J=7.7, 16.6 Hz, 1 H), 2.77 (dd, J=6.0, 16.6 Hz, 1 H), 3.8-3.9 (m, 1 H), 4.2-4.3 (m, 1 H), 4.6-4.7 (m, 1 H), 5.02 (s, 2H), 7.25-7.4 (m, 6H), 7.88 (d, J=9.3 Hz, 2H), 8.12 (d, J=6.6 Hz, 1 H), 8.21 (d, J=9.3 Hz, 2H), 8.38 (d, J=7.3 Hz, 1 H), 10.48 (s, 1 H), 12.40 (br s, 1 H); MS (LSIMS) m/e 580 (M++Na, 17) 558 (M++1, 71 ), 541 (19), 527(32), 420 (51 ), 305 (100); [a]p2°-23.9° (c=1.0, DMF); Analysis calculated for CZSH3, N5O9 ~ 0.25 HZO:C, 55.56; H, 5.65 N, 12.46; found: C, Ei5.33; H, 5.56; N, 12.09.
Example 7 Asst The use of the compounds of this invention in identifying inhibitors of interleukin 1 f3 converting enzyme (ICE) and, consequently, demonstrating the latter compounds' effectiveness for treating inflammatory diseases is disclosed by the following in vitro assay. Other procedures for purification an<i assaying ICE are disclosed in Black et al., FEBS Letters, 247, 386-390 (1989), and Thornberry et al., Nature, 356, 768-774 (1992).
Cell Culture And Lysates.
Human monocy~te cell line, THP-1 (,ATCC-TIB 202) was grown in RPMI media 1640 (Gibco BRL Gaithersburg, MD 20877) with 10% fetal bovine serum, harvested by centrifugation, washed twice in Dulbecco's PBS dithiothreitol without Ca++, and resuspended in 10 mM Tris-HCI pH 8 buffer containing 5 mM DTT
(dithiothreitol), 1 mM
EDTA (ethylene diamine tetraacetic acid), 1 mM PMSF (phenylmethyl sulfonylfluoride), 1 Ng/ml pepstatin, and 1 ,ug/ml leupeptin at 1-3 x 108 cells per ml. Cells were frozen at -70°C until use and then lysed by thawing. Lysates were cleared by centrifugation at 20,000 x g for 1 hour followed by 120,000 x g for 1 hour.
Partial Purification Of ICE Activity By Ion-E~:chancte Chromatography ICE activity was purified from THP-1 cell lysates by three chromatographic steps:
(A) Thp-1 cell lysate (1.5L) was desalted by G25 column chromatography (Pharmacia LKB Biotechnology) (B) The protein fraction was then subjected to ion-exchange chromatography on 4-Sepharose Fast Flow (Pharmacia LKB Biotechnology) in buffer A (20 mM Tris pH 7.8 containing 5 mM EDTA, 1 mM PMSF, 1 ,ug/ml pepstatin, and ,ug/ml leupeptin). ICE activity was eluted ~nrith a gradient of NaCI in buffer A. (C) The 21~96~6 active fractions from B were desalted, concentrated and subjected to MonoQ
(trademark) (Pharmacia LKB Biotechnology) column chromatography. ICE activity was then eluted in a NaCI gradient. Active ICE ifractions from C were pooled and used to bind immunoaffinity beads containing covalently linked antibodies raised against the first 11 N-terminal residues of ICE (NH2-Asp-Pro-Ala-Met-Pro-Thr-Ser-Ser-Val-Lys-Leu-Cys-CONHZ).
Immobilization Of ICE
Immobilization of ICE to immunoaffinity beads was done following standard protocols. Briefly, IgG fractions were covaleintly linked to protein A beads as described by the manufacturers (Antibody Orientation Kit Protein A Agarose supplied by Affinica (trademark) (Product manufactured by Schleicher and Schuell)). Protein A beads were pelleted by centrifugation and washed with 5 times their volume with Affinica (trademark) supplied "binding buffer". IgG, appropriately diluted in "binding buffer," was then bound to the beads. The beads were then washed and the bound IgG was covalently linked with dimethyl suberimidate. After the reaction was stopped with the Nquenching buffer" supplied by Affinica (trademark), the immunoaffinity beads were washed and stored in PBS buffer containing 0.02% NaN3. To bind the immunoaffinity beads with MonoQ purified ICE preparations, the beads were washed in 10 mM
Tris HCL buffer pH 7.8 containing 5 mM DDT, 1 mM EDTA-NA, 1 ug/ml peptstatin, 1 ug/ml leupeptin and 10% glycerol ("washing buffer"). After the wash, the beads were mixed with ICE in the presence of 10% DMSO final concentration. The suspension was rotated slowly for 1 hour at room temperature. Subsequently, the beads were thoroughly washed with the "washing buffer" before they were used and resuspended in an equal volume of this buffer in the enzymatic assay.
Assay Procedure The enzymatic reaction was carried out at 25°C in 96 microtiter wells (100 N) final volume) with active enzyme immobilized to anti-ICE antibodies covalently linked to protein A beads as described above. 1'he incubation mixtures for the enzymatic assays contained 0.01 to 1.5 mM of the pNA substrates and was made up in 8.35 mM
MES, 4.17mM Tris, 4.17 mM acetic acid, 4.E. M DMSO, 0.8mM EDTA and 4.17mM DTT
(final concentrations) adjusted to a final pH of 7.
The enzymatic reaction was monitored spectrophotometrically at 405 nM. The increase in absorbance at this wave length resulted from the release of pNA

chromophore after hydrolysis by ICE of the peptidic-pNA substrate. The release of the chormophore was linear with reaction time and the rates observed were proportional to ICE and substrate concentration. The spectrophotometric assay greatly facilitated the quantitative determination of kinetic constants and assessment of the enzyme specificity (Vmax/Km). In addition, compari:>on of inhibitory compounds was possible.
These compounds could easily be introduced in the assay and compared as to the type (competitive, uncompetitive, mixed) and degree (IG) of inhibition they can effect on catalysis (See A. Cornish-Bowden, Fundamentals of Enzyme Kinetics, Butterworth and Co., Ltd., London (1979).
The rates, kinetic constants and relative specificity for the pNA substrates for ICE
are presented in Table 1. The data are consistent with the interpretation that tripeptides and tetrapeptides are substrates for this enzyme, compounds smaller than tripeptides are not. In addition two known peptidic compounds (prepared by a solid phase peptide synthesizer) known to be hydrolyzed by ICE, p70 (H-Asn-Glu-Ala-Tyr-Val-Hls-Asp-Ala-Pro-Val-Arg-Ser-Leu-Asn and p48 (Ac-Tyr-Val-His-Asp-Ala-NH2), were alsofound to behave as competitive inhibitors of the hydrolysis of CBZ-Val-Ala-Asp-pNA
by affecting Km but not Vmax (Ki(mM) was found to be 0.46 mM and 1.6 mM
respectively).
This demonstrated the ability of the assay to quantitatively evaluate potential inhibitors of ICE.

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Claims

1. A compound of the formula R1-A2-A1-Asp-p-nitroanilide I
wherein A1 is Ala;
A2 is Val or Ala;
R1 is alkylcarbonyl, phenyalkylcarbonyl, alkoxycarbonyl, phenalkoxycarbonyl, alkylaminocarbonyl, phenalkylaminocarbonyl or R2-A3 wherein A3 is Val; and R2 is alkylcarbonyl, alkoxycarbonyl or phenylalkoxycarbonyl,

2. A compound according to claim 1, wherein R1 is Ac, PhCH2CH2CPO, t-BOC, PhCH2NHCO, CBZ or R2-A3, and R2 is Ac, t-BOC or CHZ.

3. PhCH2CH2COValAlaAsp-p-nitroanilide, according to claim 1.

4. PhCH2NHCOValAlaAsp-p-nitroanilide, according to claim 1.

5. t-BOCValAlaAsp-p-nitroanalide, according to claim 1.

6. AcValAlaAsp-p-nitroanilide, according to claim 1.

7. CBZValAlaAsp-p-nitroanilide, according to claim 1.

8. A method of detecting inhibitors of interleukin 1.beta.
converting enzyme (ICE) comprising evaluating a compound's capacity to inhibit the ICE-induced hydrolysis of a compound according to any one of claims 1 to 7.

9. A use of a compound according to any one of claims 1 to 7 for detecting inhibitors of interleukin 1.beta. converting enzyme.

10. A process for producing a compound of the formula I
as defined in claim 1, which comprises:
(i) coupling a di- or tripeptide of the formula:

(wherein R1, A2 and A1 are as defined in claim 1) with .beta.-t-butyl ester of aspartyl-p-nitroanilide of the formula:
in the presence of a coupling agent selected from the group consisting of dicyclohexylcarbodiimido, 1-(dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride, isobutyl, chloroformate and N,N-bis[2-oxo-3-oxazolidinyl]
phosphorodiamidic chloride in an inert solvent at a temperature of -20°C to +100°C, to form a compound of the formula:
, and (wherein R1, A2 and A1 are as defined in claim 1) (ii) cleaving the t-butyl group from the compound of the immediately above formula by a strong acid in a co-solvent at a temperature of -50°C to +50°C.

11. A process for producing a compound of the formula I
as defined in claim 1, which comprises:
(i) reacting aspartyl-p-nitroanilide of the formula or hydrochloride thereof, with a preactivated N-t-BOC-protected amino acid derivative of the formula:
t-BOC-A1-X
(wherein t-BOC is t-butoxycarbonyl, A1 is as defined in claim 1, and X is a preactivated carboxyl group) in the presence of a base in a reaction-inert solvent at a temperature of from about -20°C to about +100°C, to form a t-BOC-A1-aspartyl-p-nitroanilide of the formula (ii) removing the t-BOC group from the t-BOC-A1-aspartyl-p-nitroanilide with a strong acid in a co-solvent at a temperature of from about -50°C to about +50°C, to form a A1-aspartyl-p-nitroanilide, (iii) repeating the steps (i) and (ii) except that t-HOC-A2-X is used in place of t-HOC-A1-X, to form a compound of the formula:
, and (iv) where required, repeating the steps (i) and (ii) except that t-BOC-A3-X is used in place of t-BOC-A1-X, to form a compound of the formula: