interleukin-1β converting enzyme : synthesis of hydroxyethyl dipeptide surrogate-containing...

Jnt. J. Peptide Protein Res. 41. 1993. 476-483 Printed in Belgium - all rights reserL,ed

Copyright 0 Munksgaard 1993

INTERNATIONAL JOURNAL OF PEPTIDE & PROTEIN RESEARCH

ISSN 0367-8377

Interleukin-l/? converting enzyme Synthesis of hydroxyethyl dipeptide surrogate-containing compounds as potential

ICE inhibitors

LAWRENCE A. REITER* and JOHN J . MARTIN

P h e r Inc., Central Reseurch Division, Department of Medicinal Chemistry, Groton, Connecticut, USA

Received 4 August, accepted for publication 4 October 1992

A series of compounds containing a hydroxyethyl-based dipeptide surrogate have been prepared as probes to evaluate the possibility of ICE being an aspartic protease. The aldehyde t-BocAsp(8-t-butyl)H reacted with the organochromium species derived from phenethyl bromide and CrClz to give the expected addition product. Lactonization, reprotection of the amine and oxidation with RuC13 gave the two protected dipeptide surrogates 7a and 7b. These were incorporated into tetra-, penta- and hexapeptide-like molecules and evaluated as inhibitors of the enzyme. The failure of these compounds to inhibit ICE indicated that this enzyme was very unlikely to be an aspartic protease.

Kej' w0rd.r: hydroxyethyl dipeptide surrogates; interleukin- lp converting enzyme (ICE); organochromium species; peptido- mimetics; ruthenium oxidation

The inhibition of proteases as a therapeutic approach has received increasing attention during the past de- cade. This has been spurred by advances in molecular biology as well as by the notable success of inhibitors of angiotensin-converting enzyme (1). The ongoing efforts to develop inhibitors of the aspartic proteases renin ( 2 ) and HIV-protease (3) are further examples of such approaches. Proteases important in inflammatory diseases (e.g. collagenase, stromelysin, cathepsins B and L, and elastase) have also received some attention in this regard (4). Interleukin- lj? converting enzyme (ICE), a recently described protease (5-8), represents another target whose inhibition may lead to therapeutic benefits in inflammatory diseases (9). This enzyme is believed to be responsible for the conversion of in- active, 33 kDa pro-IL-lj? into its active 17.5 kDa form. Inhibition of ICE, therefore, could lead to a reduction in the levels of active interleukin-1 (IL-I), a major me- diator of inflammation (10). Owing to our interest in modulating the effects of IL-1 by a variety of mecha- nisms, we initiated a program aimed ultimately at in- hibiting ICE in a therapeutically useful manner.

At the start of our work, available evidence indicated that ICE was either an aspartic or a cysteine protease. The inhibition of ICE by N-ethylmaleimide and iodoacetic acid ( 5 ) , while suggesting it to be a cysteine protease, did not eliminate the possibility of it being an aspartic protease. Inhibition by these non-specific thiol- reactive reagents could be due to their reaction with a

476

non-active site thiol(s). Such has been observed with HIV-protease (1 1). Direct evidence regarding the possibility of ICE being an aspartic protease consisted only of the ineffectiveness of pepstatin as an inhibitor (5). However, in the light of the extraordinarily high specificity of ICE for an aspartic acid at PI (6-8), the failure of pepstatin, which has a leucine-like side chain at PI , to inhibit ICE is not surprising.

Our strategy for pursuing the inhibition of ICE began with the synthesis of substrate-based probe compounds that would aid in identifying the class of enzyme to which ICE belongs and which would, if active as inhibitors, provide logical starting points for medicinal chemistry pursuit. The hexapeptide-like compounds la/b, which span from P3 to P i (Fig. 1) and which contain the hydroxyethyl-based dipeptide surrogate Asp" [ CH(OH)CH2]Gly in place of the scissile bond

Pro-IL-lP Tyr113toArg 120 ... TyrValHisHN

0 CH,

- t-BocValPheHN Hozc),p,COProValNH2

OH FIGURE 1

Asp" [ CH(OH)CH2]Gly dipeptide surrogate

residues Asp and Ala, were selected as targets to test the possibility that ICE may be an aspartic protease (12). The highly effective inhibition of renin (2) and HIV-protease (3) by compounds containing hydroxyethyl-based dipeptide surrogates gave us confidence that these targets would, if ICE were an aspartic protease, display an easily detected level of inhibition. We chose to pursue the Asp" [ CH(0 H)CH2]Gly dipeptide surrogate rather than the Asp" [ CH(OH)CHz]Ala dipeptide surrogate for synthetic convenience, and we were confident that the exchange of Gly for Ala would not dramatically affect the results of our study, since we knew that peptidic substrates containing a Gly at Pi were as good as the corresponding substrates containing Ala (6,8).

In both the renin and HIV-protease areas, compounds containing a hydroxyethyl dipeptide surrogate with an absolute S-configuration display the more po- tent inhibition (2,3). Nevertheless, for our purposes we felt that the synthesis of both isomers would be prudent, and we therefore sought a synthetic method that would give us both isomers in a reasonable ratio and which would be compatible with the "aspartic acid" functionality at the PI site. Of the many published syntheses of hydroxyethyl dipeptide surrogates (13), we were at- tracted to a method described by Holladay et al. in which the skeleton of the dipeptide surrogate was con- structed by treating an amino acid aldehyde with an appropriate Grignard reagent (14). Their method offers both stereoisomers in a reasonable ratio and, in addition, their papers describe subsequent chemical con- versions of some relevance to our problem. We were concerned, however, that Grignard reagents would not be compatible with the P-carboxylate of the aspartic acid residue. Since organochromium species add selec- tively to aldehydes in the presence of esters (13, the use of an appropriate organochromium species as the nu- cleophile in place of a Grignard reagent appeared to offer a solution to this potential problem.

RESULTS AND DISCUSSION

In their preparation of the Leu" [ CH(OH)CH2]Ala dipeptide surrogate, Holladay et al. protected the hydroxy group of the initial adduct, deprotected a latent carboxy group at the C-terminus and oxidized this functionality, yielding the carboxylate (14). We felt that a variation of this strategy would serve our purposes. Thus, we anticipated that the hydroxy group of our initial adduct could be protected internally through lactone formation with the P-carboxylate function of the aspartic acid residue (Scheme 1). This lactone would serve to protect both of these key functionalities during the entire synthesis and would allow their simultaneous release in the final step.

With this plan in mind, we reduced t-BocAsp(P-t- buty1)OH with diborane (16), and the resulting alcohol was oxidized using Swern conditions (17) to give alde-

CH2C02fbutyl y; CH,CO,-t-butyl zrlm t-BOCNHAC02H * t-BOCNHACH20H

2 6 H

I X 38 R CH20-t-bulyl 3b S CH20-t-bUlyI 4 1 R P h 4 6 S P h

NH-CBOC RuCWNa104

r x ac810wHz0

NH-t-Boc

0

NH-CBOC RuCWNa104

k x ac810wHz0

NH-t-Boc

I ? ( 5a R C Y O H 7 8 R 5b S CHzOH 7b S 68 R Ph sb S Ph

SCHEME 1

hyde 2. This was directly coupled with two organochromium species, either of which could provide the "glycine" portion of the dipeptide surrogate. The first of these, derived from 3-bromo- 1-tert-butoxypropane (18), closely paralleled the Grignard reagent used by Holladay et al. and led to a product which would yield the C-terminal carboxy group after deprotection and oxidation. The second organochromium species was derived from phenethyl bromide, and the adduct from this would require a more vigorous oxidation to release the C-terminal carboxyl group. A ruthenium-mediated oxidation of the phenyl group to a carboxy group did, however, appear to be a viable option, since a ruthenium oxidation has been successfully achieved with similarly substituted peptide-like compounds (19). We found that both organochromium species reacted with 2 as desired, each giving a mixture of two diastereomers, 3a/b and 4a/b (20). Subsequent treatment of these mixtures with neat TFA led to cleavage of the tert-butyl esters and t-Boc groups, lactonization, and, in the case of 3a and b, cleavage of the C-terminal tert- butyl ethers. In both series, reprotection of the N-terminal m i n e with (Boc)zO provided easily sepa- rable mixtures of the expected lactones, 5a/b and 6a/b. In initial experiments the overall yield of lactones from 2 was 20 and 29% for 3-bromo-1-tert-butoxypropane and phenethyl bromide, respectively. In both cases the ratio of the two diastereomers was about 2:1, and the major product was found to have R absolute stereo- chemistry at the newly formed stereocenter. This was established by single-crystal X-ray analysis of lactone 6a (21) and subsequent correlation by comparison of NMR spectra.

Ruthenium-mediated oxidation of the phenyl groups in lactones 6a/b proceeded in relatively good yield (81 and 62%, respectively) (22). This, together with the better overall yield obtained with phenethyl bromide in the organochromium addition/lactonization sequence

411

L.A. Reiter and J.J. Martin

(which was improved to 45 o/o upon scale-up), led us to abandon the pursuit of the series derived from 3-bromo- 1-tert-butoxypropane.

Lactones 7a and 7b were each coupled with reasonable efficiency to HProValNHz using the BOP-Cl method (Scheme 2) (23). Extension of the N-terminus, first with t-BocPheOSucc (24) and then with t-BocValOSucc, proceeded as expected, although in both cases the yield in the final coupling step was quite low. Each of the resulting six lactones, Sa/b-lOa/b, was then treated with an excess of sodium hydroxide in cold methanol to open the lactone. The resulting hydroxycarboxylic acids, l la/b, 12a/b and la/b, were isolated by neutralization of the excess base with sulfonic acid ion-exchange resin, filtration and concentration. These compounds proved to be stable in the solid state but relactonized to a significant extent over the course of a few hours in solution in the presence of acid.

Evaluation of l l a /b , 12a/b and la/b as inhibitors of ICE revealed that none of these displayed inhibition at 1 0 0 ~ ~ (25). The failure of these Asp” [ CH(OH)CH2]Gly dipeptide surrogate-containing compounds to inhibit ICE at this concentration strongly suggested that the enzyme is not an aspartic protease. This result and our related work (12) led us to conclude that ICE is a cysteine protease. Subsequent to the completion of this work, our conclusion was confirmed by two other studies. That ICE is not an aspartic protease was indicated by its 1 ’ sequence (26) from which the characteristic aspartic protease sequence of AspThrGIy (27) is absent. That ICE is a cysteine protease was indicated through both its inhibition by compounds designed specifically to inhibit it (28, 29) and an examination of the kinetics of its inhibition by iodoacetic acid (29).

While the hydroxyethyl-based dipeptide surrogates reported herein did not inhibit ICE and do not in them- selves provide starting points for medicinal chemistry pursuit, the synthetic route described will be of value in designing syntheses of related compounds as potential inhibitors of both ICE and other enzymes that have a specificity for an acidic group at PI .

7a/b-1 Oa/b l la /b , 12alb and la ib

7a 7b 8a - 1 l a 8b - 1 l b 9a -12a 9b -12b 10a - l a 10b -1b

SCHEME 2

478

: B 91 R t-BOC OH s t-aoc OH R i-BOc PTOVaINH2 s t-BOC ProValNH2 R t-BocPhe ProVaINH2 S 1-BocPhe ProValNHz R t-BocValPhe ProValNHz S 1-BocValPhe ProValNH2

EXPERIMENTAL PROCEDURES

General procedures NMR spectra were obtained on a Bruker AM-300 spectrometer. EI mass spectra were obtained on a Finnigan EI-C1 mass spectrometer and LSIMS mass spectra on a Kratos Concept mass spectrometer using a 4: 1 mixture of dithiothreitol and dithioerythritol as a matrix. Melting points are uncorrected and were deter- mined in open capillaries. Elemental analyses were performed by the Analytical Department in the Central Research Division of Pfizer Inc. Optical rotations were obtained on a Perkin Elmer 241 MC polarimeter. HPLC analyses were performed on a Waters Nova- Pak Ct8 column (3.9 mm x 150 mm) and were run iso- cratically with a flow rate of 1 mL/min with mixtures of CH3OH and water (both containing 0.1 % TFA); the percentage of water is noted in each experiment. Eluted materials were detected by UV monitoring at 220 nm. The DMSO, DMF and CH2C12 used in reaction mixtures were of Sure-Seal@ grade from Aldrich. Chro- mous chloride (99.9%) was obtained from the AESAR Group of Johnson Matthey. Vitamin BIZ was obtained from Schweizerhall. 2-Phenethyl bromide was distilled before use. Diisopropylethylamine (DIEA) and other solvents and reagents used were of standard grades and were used as obtained from commercial sources. Chro- matography was performed with 40 VM Flash “Baker” Silica Gel from J.T. Baker, Inc.

(S)- tert- ButyI-3 -(tert - butoxycarbonylamino)-4 - hydroxy- butanoate. N-x-t-Boc-L-Aspartic acid P-tert-butyl ester dicyclohexylamine salt (31.06 g, 66.0 mmol) and sulfonic acid ion-exchange resin (150 g, 2.23 meq/g) were stirred together for 15 min in methanol (500 mL). The resin was removed by filtration and washed well with methanol. The filtrate was concentrated in vacuo to an oil. This oil was dissolved in dry T H F (60inL) and added slowly to an ice cold solution of BH3.THF (200 mL, 1.0 M). After 3 h the reaction was carefully quenched with 10% acetic acid in methanol (67 mL). The reaction mixture was then concentrated in vacuo. The resultingoil was dissolved in ethyl acetate (500 mL), and this solution washed with 1 N hydrochloric acid (2 x ) and brine. After drying, filtration and concentration, the residual oil was chromatographed (45:55 ethyl acetate:hexane), yielding 14.86 g (82%) of a colorless oil: ‘H NMR (CDCI3) 6 1.43 (s, 9H), 1.44 (s, 9H), 2.51 (m, 2H), 2.58 (br s, lH), 3.67 (br t, 2H), 3.94 (m, lH), 5.22 (br s, 1H); 13C NMR (CDC13) 6 28.0, 28.4, 37.4, 49.7, 64.8, 79.8, 81.3, 155.9, 171.1; MS (EI) m/z 244 (8), 188 (25), 146 (60), 132 (47), 88 (93), 57 (100).

(S)-tert-Butyl-3-(tert-butoxycarbonylamino)-4 -0xobutun- oate(2). Tooxalylchloride(l8.2 g = 12.5 mL, 143 mmol) in CHzClz (420mL) at -70°C was added dimethyl sulfoxide (15.7 g = 14.3 mL, 201 mmol) in CHZC12 (70 mL). Ten min after complete addition, (S)-tert-


2.85 (m, 2H), 2.91 (dd, J = 8.0, 18.0 Hz, lH), 4.18 (m, lH), 4.23 (m, lH), 4.69 (br d, lH), 7.15-7.3 (m, 5H);

84.8, 126.3, 128.5, 128.6, 140.4, 154.9, 174.3; IR (KBr) 3365, 1782, 1680cm-'; MS (EI) mjz 249 (36, M +

(100); [a];' + 33.4" (c = 1.0, MeOH); Analysis calcd. for C17H23N04: C, 66.86; H, 7.59; N, 4.59; found: C, 66.75; H, 7.51; N, 4.60. The more polar product was recrystallized from cyclohexane/EtOAc giving 3.20 g (1 5 %) of (2S,3S)-3-(tert-butoxycarbonylamino)-5-oxo- 2-(2-phenyIethyl)tetrahydrofuran (6b): m.p. 11 5- 116°C; 'H NMR (CDC13) 6 1.42 (s, 9H), 1.9-2.1 (m, lH), 2.46 (dd, J=2.4, 17.8 Hz, IH), 2.7-2.95 (m, 3H), 4.4-4.55 (m, 1H),4.7-4.8 (br d, lH), 7.3-7.45 (m, 5H);

82.4, 126.3, 128.5, 128.6, 139.7, 140.5, 174.9; IR (KBr) 3375, 1768, 1682 cm- l; MS (EI) m/z 249 (39, M +

(100); [a];' -87.6" (c = 1.0, MeOH); Analysis calcd. for C17H23N04: C, 66.86; H, 7.59; N, 4.59; found: C, 66.83; H, 7.64; N, 4.74.

13C NMR (CDC13) 6 28.2, 31.4, 35.2, 35.3, 51.9, 80.7,

-56), 188 (27), 150 (50), 132 (76), 126 (45), 91 (73), 57

13C NMR (CDC13) 6 28.3, 30.9, 31.6, 36.9, 50.0, 80.5,

-56), 232 ( l l ) , 188 (29), 150 (41), 132 (65), 91 (62), 57

butyl-3-(tert-butoxycarbonylamino)-4- hydroxybutano - ate (19.8g, 71.8 mmol) in CH2Cl2 (140mL) was added, keeping the reaction temperature below -70 " C. Fifteen minutes after complete addition, DIEA (51.9 g = 70.0 mL, 402 mmol) in CH2Cl2 (70 mL) was added, keeping the reaction temperature below -70 " C. After 2 h at -7O"C, the mixture was allowed to warm to -10°C and was quenched with water (50 mL). The mixture was then washed with 0.1 M H2S04 (3 x 100 mL) and saturated NH4CI (100 mL), dried with MgS04, filtered and concentrated in vucuo without heating yielding an oil (> loo"/,,) which was used directly in the subsequent reaction: lH NMR (CDCl3) 6 1.42 ( s , 9H), 1.43 ( s , 9H), 2.72 (dd, J = 5.0, 17.2 Hz, IH), 2.89(dd,J=4.3, 17.2 Hz, IH), 4.31 (m, lH), 5.59 (br d, lH), 9.64 (s, 1H).

tert - Butyl-(3S,4R/S) -3 -(tert - buroxycurbonylumino)-4 - hydroxy-6-phenylhexanoate (4a/ bl. To N2-purged DMF (580 mL) containing chromous chloride (35.28 g, 287.0 mmol) and vitamin B12 (3.89 g, 2.87 mmol) was added, through a cannula, a solution of 2-phenethyl bromide (26.56 g, 143.5 mmol) and crude 2 (171.8mmol) in N2 purged DMF (140mL). The reaction mixture was stirred for 16 h at 30°C. The cooled reaction mixture was diluted with Et20 (1.5 L) and 1 N HCI (700mL). The separated aqueous layer was extracted with Et2O (3 x 250 mL). All EtzO fractions were combined and washed with 1 NHCI (3 x 250 mL) and dried over MgS04. Filtration and concentration gave 25.20 g (> 100%) of a yellow-green oil which was a mixture of diastereomeric alcohols: 'H NMR (CDCL) 6 1.42 (s, 9H), 1.57 (s, 9H), 1.7-1.8 (m, 2H), 2.45-2.55 (m, 2H), 2.6-2.9 (m, 2H), 3.6-3.7 (m, lH), 3.8-3.9 (m, lH), 5.1-5.2 (m, lH), 7.1-7.3 (m, 5H); MS (EI) m/z 380 (M +, 4), 324 (3), 323 (2), 132 (100).

(2R/S,3S) -3 -(tert - Butoxycurbon,vlumino) -5 - 0x0 -2 -(2 - pheny1ethyl)tetruhydrofuran (6alb). Prechilled TFA (360mL) was added to the mixture of 4a/b and the resulting solution allowed to stand overnight at 0°C. The TFA was removed in vucuo and chased with CCL (3 x 100 mL). The brown oily residue was dissolved in CH2Cl2 (300mL) and the pH adjusted to 7 with DIEA. Additional DIEA (1 1.13 g, 86.1 mmol) was then added followed by di-tert-butyl dicarbonate (18.79 g, 86.1 mmol). After stirring for 4 h at room temperature, the reaction mixture was extracted with 1 N HC1. The separated aqueous layer was extracted with CH2CL (250 mL). The combined CHzC12 layers were washed with 1 N HCI (500 mL), dried over MgS04, filtered and concentrated in vacuo giving a brown oil. This was chromatographed (25:75 Et0Ac:hexane). Theless polar product was recrystallized froni cyclohexane/EtOAc, giving 6.66 g (30%) of (2R,3S)-3-(tert-butoxycarbonylamino)-5-oxo-2-(2-phenylethyl)tetrahydrofuran (6a): m.p. 142-143°C; IH NMR (CDC13) 6 1.42 (s, 9H), 1.9-2.1 (m, 2H), 2.42 (dd, J = 5.9, 18.0 Hz, IH), 2.65-

(2R/S,3S)-3-(tert-Butoxycarbonylumino)-5-oxo-2-(3- hydroxypropyl) tetruhydrofuran (5a/ b). To N2-purged DMF (20 mL) containing chromous chloride (1.22 g, 10.0 mmol) and vitamin B I Z (135 mg, 0.1 mmol) was added, via a syringe, a solution of 1-bromo-3-tert- butoxypropane (976 mg, 5.0 mmol) and crude 2 (I 2.5 mmol) in N2-purged DMF (20 mL). The reaction mixture was stirred for 18 h at 34°C. The cooled reaction mixture was diluted with Et20 (100 mL), washed with 1 N HCl (2 x 25 mL) and dried over MgS04. Filtration, concentration and chromatography (25:75 Et0Ac:hexane) gave 384 mg (39%) of colorless oil. To this was added prechilled TFA (10 mL), and the resulting solution was stirred for 4 h at 0°C. The TFA was removed in vucuo and chased with CCL (10 mL). The residue was dissolved in CHzCl2 (10 mL) and the pH adjusted to 7 with DIEA. Additional DIEA (145 mg, 1.12 mmol) was then added followed by di-tert-butyl dicarbonate (245 mg, 1.12 mmol). After stirring for 1.5 h at room temperature, the reaction mixture was washed with 1 N HCl(2 x ), dried over MgS04, filtered and concentrated in vucuo, giving an oil. This was chromatographed (30:70 Et0Ac:hexane) giving 83 mg (13 % ) of (2R,3S)-3-(tert-butoxycarbonylamino)-5-oxo- 2-(3-hydroxypropyl)tetrahydrofuran (5a): 'H NMR (CDC13) 6 1.44 (s, 9H), 1.65-1.9 (m, 4H), 2.43 (dd, J = 7.7, 18.0 Hz, lH), 2.97 (dd, J = 8.6, 18.0 Hz, lH), 3.35-3.5 (m, 2), 4.0-4.1 (m, lH), 4.3-4.4 (m, lH), 5.17 (br s, 1H); and 43 mg (7%) of (2S,3S)-3-(tevt-butoxycarbonylamino)-5-oxo-2-( 3-hydroxypropy1)tetrahydrofuran (5b): lH NMR (CDC13) 6 1.45 (s, 9H), 1.65-1.8 (m, 4H), 2.48 (dd, J=2.4, 17.1 Hz, lH), 2.87 (dd, J = 7.3, 17.1 Hz, lH), 3.3-3.45 (m, 2H), 4.45-4.55 (m, 2H), 4.75-4.85 (br d, 1H).

479


3-/(2R,3 S)-3-(tert-Butoxycarbonylumino)-5-oxo-2 - tetra- hydrofuranyljpropionic acid (7a). 6a (5.29 g, 17.3 mmol) was dissolved in acetone (200 mL). Water (140 mL) was added followed by RuC13 (359 mg, 1.73 mmol) and NaI04 (37.0 g, 173 mmol). After 30 min a slight exo- therm was controlled by cooling with an ice-water bath. The mixture was then stirred at room temperature. Ad- ditional RuCL (180 mg) and NaI04 (17.5 g) were added after 18 h, 26 h and 42 h. After another 24 h the reaction was quenched with 2-propanol(lOO mL), and after 30 min the solids were removed by filtration through diatomite and washed thoroughly with acetone. The filtrate was concentrated to remove most of the organic solvents. The resulting dark-brown mixture was acidi- fied with 1 N HCI (100 mL) and extracted with ethyl acetate (250 mL). The organic layer was separated and washed with 1 N HCI (100 mL) and saturated NH4CI (100 mL), dried with MgS04, filtered and concentrated to a yellow foam. This was chromatographed (5:35:60 HOAc:EtOAc:hexane), yielding 3.84 g (81 %) of 7a as a yellow gum which solidified upon standing. Also isolated were 167 mg (3 %) of recovered 6a and 160 mg (3%) of (2R,3S)-3-(tert-butoxycarbonylamino)-5-oxo- 2-(2-phenyl-2-oxoethyl)tetrahydrofuran. An analytical sample of the title compound was prepared by recrys- tallization from cyclohexane/ethyl acetate: m.p. 86- 89°C; 'H NMR (acetone-d6) 6 1.40 (s, 9H), 1.9-2.0 (m,lH),2.0-2.l(m,lH),2.5(m,2H),2.55(dd,J=6.6, 17.7 Hz, lH), 2.90 (dd ,J= 8.5, 17.7 Hz, lH), 4.15 (m, lH), 4.35 (m, lH), 6.60 (br d, 1H); I3C NMR (CDCl3) 6 28.4, 28.6, 29.7, 35.1, 52.0, 80.8, 84.6, 155.5, 174.8, 177.1; IR (KBr) 3370, 2981, 2955, 2933, 1779, 1718, 1678 cm- l; MS (EI) mjz 274 (< 1, M' + l), 218 (17), 200 (35), 182 (52), 173 (30), 156 (52), 143 (43), 114 (58), 87 (55 ) , 59 (99), 57 (100); [a]? + 16.9" (c= 1.0, MeOH); Analysis calcd. for C I ~ H I ~ N O ~ : C, 52.74; H, 7.01; N, 5.13; found: C, 52.45; H, 7.12; N, 4.90.

3 -[(2 S .3 S) -3-(tert - Butoxycarbonylamino) -5 -ox0 -2-tetrahydrofuranyl]propionic acid (76). By the same procedure used to prepare 7a, 6b (2.90 g, 9.50 mmol) gave after chromatography (5:35:60 H0Ac:EtOAc:hexane) 1.60 g (62%) of a foam. A portion of this was crystallized from EtOAc/hexane: m.p. 160-162°C; 'H NMR (DMSO-d6) 6 1.38 (s, 9H), 1.77 (m, 2H), 2.29 (m, lH), 2.33 (m, 2H), 2.93 (dd, J = 8.5, 17.7 Hz, lH), 4.31 (m, lH),4.50(m, lH),7.52(brd,J=8.5Hz, lH), 12.19fbr s, 1H); MS (LSIMS) mjz 274 (16, M' + l), 267 (13), 255 (4), 240 (4), 218 (loo), 200 (16), 182 ( l l ) , 174 (14); [a]:' -91.3" (c = 0.9, MeOH); Analysis calcd. for C I Z H I ~ N O ~ : C, 52.74; H, 7.01; N, 5.13; found: C, 52.56; H, 7.17; N, 5.07.

1 -[3- f(2 R,3 S)-3~tert-Butoxycarbonylamino)-5-oxo-2-tetra- hydrofuranyll-1 -oxopropylj-L-prolyl-L- valinamide (Sa). l a (2.14 g, 7.85 mmol) was dissolved in CHzC12 (80 mL) and cooled to 0°C. To this solution was added BOP-CI (3.00 g, 11.8 mmol), DIEA (5.07 g, 39.25 mmol) and

480

HProValNH2.HCl (30) (2.94 g, 11.8 mmol). The mixture was allowed to come to room temperature and was stirred for 72 h. The reaction mixture was then washed with 1 N HCl (2 x ) and saturated NaHC03, and dried over MgS04. The residue obtained after filtration and concentration was chromatographed (5:95 CH30H:CH2C12) to give 2.58 g (68%) of a white solid: m.p. 108-110°C; 'H NMR (CDC13) 6 0.83 (d, 3H), 0.87 (d, 3H), 1.43 (s, 9H), 1.7-1.85 (m, 2H), 1.9-2.2 (m, 5H), 2.2-2.35 (m, lH), 2.52 (dd, J = 6.9, 17.9 Hz, lH), 2.55-2.7 (m, IH), 2.85 (dd ,J= 7.9, 17.9 Hz, lH), 3.4-3.55 (m, lH), 3.65-3.75 (m, lH), 4.0-4.1 (m, lH), 4.15-4.25 (m, lH), 4.3-4.4 (m, lH), 4.4-4.5 (m, lH), 5.71 (br s, lH), 5.97 (br d, lH), 6.36 (br s, lH), 6.80 (br d, 1H); MS (LSIMS) mjz 469 (73, M + + l), 453 (30), 413 (50), 396 (75), 369 (24), 352 (24), 297 (91), 269 (100); [ x]2' + 44.3" (c = 1.0, MeOH), HPLC ret. time: 6.24 min (60%); Analysis calcd. for C22H36N407.HzO: C, 54.30; H, 7.87; N, 11.52; found: C, 54.33; H, 7.64; N, 11.30.

I - f3 f(2 S ,3 S)-3~tert-Butoxycarbonylamino)-5-oxo-2-tetru- hydrofuranylj-1 -oxopropyl]-~-prolyl-~-valinamide (Sb). By the same procedure used to prepare Sa, 7b (1.5 g, 5.5 mmol), BOP-C1 (1.68 g, 6.6 mmol), DIEA (2.84 g, 22 mmol) and HProValNHz.HC1 (2.05 g, 8.25 mmol) gave after chromatography (5:95 MeOH:CH2C12) 1.20g (47%) of a white solid: m.p. 198-200°C; 'H NMR (CDC13) 6 0.88 (d, J=8 .2 Hz, 3H), 0.91 (d, J = 8.2 Hz, 3H), 1.41 (s, 9H), 2.23-2.64 (m, 3H), 2.89 (dd, J = 7.3, 17.1 Hz, lH), 3.47 (m, lH), 3.64 (m, lH), 4.21 (dd, J = 7.3, 8.2 Hz, lH), 4.35-4.62 (m, 3H), 5.81 (br d, J = 8.5 Hz, lH), 6.01 (br s, lH), 6.50 (br s, lH), 7.06 (br d, J = 8.2 Hz, 1H); MS (LSIMS) m/z 469 (22, M + + l), 413 (14), 391 (16), 341 (22), 281 (53), 267 (31), 207 (54), 155 (100); HPLC ret. time: 4.38min (60 yo); exact mass calcd. for C2zH36N407: 469.2678, found: 469.2628.

1 -/3-/(2R,3S)-3-//N - tert-(Butoxycarbony1)-L -phenylalanyl]amino]-5 -oxo-2-tetrahydrofuranyl]-l -oxopropyI]- L-prolyl-L-valinamide (9a). 8a (1 17 mg, 0.25 mmol) was added to chilled (ice/water bath) TFA ( 5 mL) and stirred at that temperature for 1 h. The TFA was removed in vucuo and the residue dissolved in CH2CL (10 mL). DIEA was added until the mixture was basic. tBocPhe N-hydroxysuccinimide ester (109 mg, 0.30 mmol) was then added and the mixture was stirred at room temperature for 4 h. The reaction mixture was washed with 1 N HCI (2 x ) and saturated NaHCO3 (2 x ) and dried over MgS04. Filtration and concentration gave a white solid that was chromatographed (5:95 CH30H:CH2C12) to give 127 mg (82%) of a white powder. A portion was recrystallized from EtOAc: m.p. 164-169°C; 'H NMR (CDCL) 6 0.89 (t, J=6.4Hz,6H),1.34(~,9H),l.7-1.8(m,2H),1.95-2.6 (m, 7H), 2.6-2.8 (m, lH), 2.8-3.0 (m, 2H), 3.0-3.2 (m, lH), 3.45-3.6 (m, lH), 3.7-3.85 (ni, lH), 4.1-4.5 (m,

Asp'P[ CH(OH)CH2]Gly dipeptide surrogate

(m, lH), 4.68-4.89 (m, 3H), 5.99 (br s, lH), 6.39 (br s, IH), 6.47 (br d, J = 8.6 Hz, lH), 6.96 (br d , J = 8.6 Hz, IH), 7.13 (m, 2H), 7.30 (m, 4H), 7.60 (br d, J = 8.6 Hz, 1H); MS (LSIMS) m/z 715 (100, M' + I), 699 (21), 615 (loo), 543 (lo), 471 (14), 391 ( 5 ) ; HPLC ret. time: 17.53 rnin (40%).

4H), 4.55-4.7 (m, lH), 5.19 (br d, IH), 6.00 (br s, lH), 6.54 (br s, lH), 6.76 (br d, lH), 7.1-7.3 (m, 5H), 7.85 (br d, 1H); MS (LSIMS) m/z 616 (80, M + + I), 516 (loo), 471 (31), 444 (37), 416 (42), 372 (75); HPLC ret. time: 7.31 min (50%), 27.19 min (60%); Analysis calcd. for C ~ I H ~ ~ N ~ O S . H ~ O : C, 57.30, H, 7.29; N, 10.78; found: C, 57.97; H, 6.93; N, 10.75.

I -/3-/(2S,3S)-3-//N- tert-(Butoxycarb0nyl)-L-phenylalanyl]amino]-S-oxo-2-tetrahydr~~furanyl]- 1 -oxopropyl]- L-prolyl-L-valinamide (9b). By thc same procedure used to prepare 9a, 8b (970 mg, 2.07 mmol) and t-BocPhe N-hydroxysuccinimide ester (903 mg, 2.48 mmol) gave after chromatography (595 MeOH:CHK12) 832 mg of a foam (66%): 'H NMR (CDC13) 6 0.9 (m, 6H), 1.38 (s, 9H), 1.80-2.60 (m, 9H), 2.80-3.12 (m, 3H), 3.42- 3.79 (m, 3H), 4.26 (m, IH), 4.43 (m, IH), 4.49 (m, lH), 4.57 (m, lH), 4.76 (m, lH), 5.11 (m, lH), 6.11 (br s, lH), 6.30 (br s, lH), 6.88 (m, IH), 7.14-7.31 (m, 5H), 7.59 (m, 1H); MS (LSIMS) m/z 638 (35, M + + Na+ ), 616 (30, M + + l), 5 16 (loo), 47 1 (28), 372 (32); HPLC ret. time: 7.31 rnin (50%).

1 -/3 -/(2R,3 S)-3-//N -(N-tert-(Butoxycarbonyl)-~- valy1,l- ~-phenylalanyl]amino]-5-oxo-2-tetrahydrofurunyl]-l -oxopropyl]-~-prolyl-~-valinamide ( IOU). By the same procedure used to prepare 9a, with the exception that THF was used in place of CH2C12, Ya (308 mg, 0.5 mmol) and tBocVal N-hydroxysuccinimide ester (189 mg, 0.6 mmol) gave a precipitate which was collected, washed with EtOAc and dried under high vacuum. This white solid was chromatographed (5:95 CH30H:CHCL), yielding 126 mg* (35%) of a white foam: 'H NMR (DMSO-d6) 6 0.7-0.8 (m, 6H), 0.8- 0.9 (m, 6H), 1.38 (s, 9H), 1.7-2.1 (m, 8H), 2.15-2.45 (m, 4H), 2.8-3.0 (m, 2H), 3.4-3.6 (m, 2H), 3.73 (br t, J = 7.7 Hz, lH), 3.9-4.25 (m, 3H), 4.4-4.6 (m, 2H), 6.73(brd,J=8.6Hz, lH),7.05(brs, lH),7.15-7.3(m, 5H), 7.35 (br s, lH), 7.72 (br d, J = 9.4 Hz, lH), 7.99 (brd,J= 8.1 Hz, lH), 8.45-8.55 (m, 1H); MS (LSIMS) m/z 715 (33, M + + l), 699 (27), 615 (100); HPLC ret. time: 17.41 min (40%).

1 - / 3 -/(2 S ,3 S)-3 - //N-/N-tert-(Btrtoxycarbonyl)-~-vulyl]- ~-phenylalanyl]amino]-S-oxo-2-tetrahydrofuranyl]-l -oxopropyl]-~-prolyl-~-valinarnide (lob). By the same procedure used to prepare 9a, 9b and t-BocVal N-hydroxysuccinimide ester (33 1 mg, 1.05 mmol) gave, after chromatography (5:95 MeOH:CHK&), 61 mg (10%) of a foam: 'H NMR (CDC13) 6 0.79 (d, J = 8.2 Hz, 3H), 0.92-1.01 (m, 9H), 1.35 (s, 9H), 1.82 (m, IH), 1.91-2.61 (m, lOH), 2.83 (m, 2H), 3.10 (m, 2H), 3.50 (m, IH), 3.69 (m, lH), 3.78 (t, J=4 .3 Hz, lH), 4.33 (dd,J=7.7, 9.4Hz, IH), 4.50 (m, lH), 4.62

* 'H NMR and HPLC showed that this sample contained 1.1 molar equivalents of N-hydroxysuccinimide.

1 -/(4R,S S)- 6- Carboxy-S -(tert- butoxycarbonylamino)-4- hydroxy-I-oxohexyll-~-prolyl-~-valinurnide ( l lu) . 8a (47 mg, 0.10 mmol) was dissolved in CH3OH (5 mL) and treated with 1 N NaOH (0.5 mL) in one portion. After 2 h the reaction was quenched by adding sulfonic acid ion exchange resin (1.12 g, 2.5 meq H + ). After stirring with the resin for 15 min, the resin was removed by filtration and washed thoroughly with CH30H. The filtrate was concentrated in vucuo and chased with EtOH to remove water. This yielded 42 mg (86%) of a white solid: 'H NMR (CD30D) 6 0.96 (d, J = 2.8 Hz, 3H),0.98(d,J=2.7Hz,3H), 1.41(s,9H), 1.55-1.7(m, IH), 1.85-2.2 (m, 6H), 2.41 (dd ,J= 8.6, 16.4 Hz, lH), 2.5-2.6 (m, 2H), 2.66 (dd,J=4.1, 16.4 Hz, lH), 3.45- 3.75 (m, 3H), 3.8-3.9 (m, lH), 4.18 (d , J= 6.7 Hz, lH), 4.47 (dd, J = 3.3, 8.2 Hz, 1H); MS (LSIMS) m/z 487 (46, M + + l), 387 (85), 119 (100); HPLC ret. time: 4.18 rnin (60%).

1 -/(4S,SS)-6- Carboxy-5-(tert-butoxycarbonylumino) - 4 - hydroxy-1 -oxohexyl]-~-prolyl-~-valinamide ( l lb ) . By the same procedure used to prepare lla, 8b (117mg, 0.25 mmol), gave 86 mg (70%) of a glass: 'H NMR (DMSO-d6)60.84(m,6H), 1.38(s,9H), 1.70-2.05(m, 6H), 2.15-2.45 (m, 7H), 3.47 (m, lH), 3.82 (m, lH), 4.10 (m, lH), 4.35-4.50 (m, lH), 6.47 (m, lH), 7.05 (m, IH), 7.31 (m, IH), 7.69 (m, 1H); MS (LSIMS) m/z 487 (27, M' + I), 387 (43), 214 (14), 135 (27), 119 (100); HPLC ret. time 4.20 rnin (60%).

I -/(4R,5S)-6- Carboxy-5-//N -(tert-butoxycnrbonyI)-L - phenylalanyl]amino]-4 -hydroxy- I -oxohexyl]-L -prolyl-L- valinamide (12a). By the same procedure used to prepare lla, 9a (149 mg, 0.24 mmol) gave 151 mg (99%) of a white powder: 'H NMR (DMSO-d6) 6 0.75-0.9 (m, 6H), 1.2-1.3 (m, 9H), 1.4-1.5 (m, lH), 1.6-1.7 (m, lH), 1.7-1.8 (m, lH), 1.8-2.0 (m, 4H), 2.2-2.4 (m, 4H), 2.6-2.75 (m, lH), 2.9-3.0 (m, lH), 3.3-3.6 (m, 3H), 3.9-4.2 (m, 3H), 4.35-4.45 (m, lH), 6.8-6.85 (m, lH), 7.0-7.1 (m, IH), 7.15-7.35 (m, 6H), 7.66 (br d, J = 8.9 Hz, IH), 7.75-7.9 (m, 1H); MS (LSIMS) m / z 656 (26, M + + l), 634 (64), 534 (100); HPLC ret. time: 6.77 rnin (50%), 25.03 rnin (60%).

1 -/(4S,5S)-6- Carboxy-5-/m-(tert-butoxycarbonyl)-L - phenylalanyl]amino]-4-hydroxy- 1 - oxohexyl]-~ -prolyl- L - valinamide (126). By the same procedure used to prepare lla, 9b (154 mg, 0.25 mmol) gave after trituration with EtOAc/hexanes (10 mL, 50:50) and filtration 120mg (76%) of an amorphous solid: 'H NMR (DMSO-d6) 6 0.82 (m, 6H), 1.30 (s, 9H), 1.45 (m, 2H),

48 1


1.77 (m, lH), 1.81-2.04 (m, 5H), 2.30 (t, J = 8.1 Hz, 2H), 2.71 (m, lH), 2.94 (m, lH), 3.39-3.55 (m, 5H), 4.01-4.20 (m, 3H), 4.39 (m, lH), 6.91 (m, lH), 7.04 (in, lH), 7.19 (m, lH), 7.25-7.34 (m, 5H), 7.66 (d, J = 8.6 Hz, 1H); MS (LSIMS) m / z 656 (8, M + + l) , 634 (74), 534 (loo), 321 (32), 214 (45); HPLC ret. time: 5.91 min (50%).

I -[(4R,5 S)-6- Carboxy-5 -/[N-/Nftert-butoxycurboi?~~l)- L - vaLvl]- L -phenylalanyl]amino]-4 -hydroxy- 1 -oxohe.q*l]- L-prolyl-L-vulinamide (la). By the same procedure used to prepare l l a , 10a (1 15 mg, 0.16 mmol) gave 100 mg* (85%) of a white powder: 'H NMR (DMSO-d6) d 0.6-0.7 (m, 6H), 0.75-0.9 (m, 6H), 1.38 (s, 9H), 1.5- 1.7 (m, 2H), 1.7-2.1 (m, 6H), 2.1-2.4 (m, 4H), 2.65- 2.8 (m, 2H), 3.4-3.5 (m, l H , partially obscured by HzO peak), 3.5-3.6 (m, lH), 3.65-3.75 (m, IH), 3.9-4.0 (m, lH), 4.0-4.1 (m, lH), 4.1-4.2 (m, lH), 4.35-4.45 (m, IH), 4.45-4.6 (m, lH), 6.67 (br d , J = 8.6 Hz, lH), 7.02 (br s, lH), 7.1-7.35 (m, 5H), 7.33 (br s, lH), 7.68 (br d, J = 8.6 Hz, IH), 7.87 (br d, J = 8.6 Hz, IH), 7.9-8.0 (m, 1H); MS (LSIMS) m / z 755 (56, M + + N a + ), 733 (100, M + + l), 717 (27), 633 (83); HPLC ret. time: 16.58 min (40%).

1 -114 S ,5 S) -6- Carboxy- 5 -//N-[Nitei~-buto.u).carboti?,l)- L - vuly1J- L -phenylalunyl]amino J - 4-hydro.xj.- 1 -oxoheq-lJ- r-prolyl-L-valinamide (lb). By the same procedure used to prepare l l a , 10b (57 mg, 0.08 mmol) gave 51 mg (86%) of a foam: 'H NMR (DMSO-d6) 6 0.67 (m, 6H), 0.82 (m, 6H), 1.20-1.32 (m, 3H), 1.39 (s, 9H), 1.70-2.05 (m, 6H), 2.27 (m, 3H), 2.72 (m, lH), 2.97 (m, lH), 3.18 (d, J = 6.8 Hz, lH), 3.38-3.52 (m, 2H), 3.70 (m, lH), 4.00-4.21 (m, 2H), 4.39 (m, lH), 4.61 (m, lH), 6.65 (br d, J = 8.6 Hz, IH), 7.03 (m, lH), 7.10- 7.23 (m, 5H), 7.31 (m, lH), 7.67 (br d , J = 8.6 Hz, lH), 7.75-8.01 (m, 2H); MS (LSIMS)m/z 733 (14, M + + l), 633 (7), 275 (4), 214 (7), 119 (100); HPLC ret. time: 16.80 min (40%).

ACKNOWLEDGMENTS

We thank Drs. D.J. Hoover and R.P. Robinson for helpful discus- sions.

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2. Grccnlee, W.J. (1990) Med. Res. Rev. 10, 173-236: Ocain, T.D. & Abou-Gharbia, M. (1991) Drirgs Fur. 16, 37-51

3 . Norbeck, D.W. & Kempf, D.J. (1991) in Anriuul Reports iri Me- dicinal Chemistry (Bristol, J.A. ed.), vol. 26, pp. 141-150. Aca- demic Press. Neu York

4. Rich. D.H. (1990) in Comprehensive Medicinal Chemisrr).

* 'H NMR and HPLC showed that this sample containcd 1.2 molar equivalcnts of ,~-hydroxysuccinimidc.

(Sammes. P.G. ed.), vol. 2, pp. 391-440, Pcrgamon Press, Lon- don; for more specific reviews on: collagenase inhibition, see

5 .

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18

Hendcrson, B., Docherty, A.J.P. & Beeley, N.R.A. (199O)Dmgs Fur. 15. 495-508; Wahl. R.C., Dunlap, R.P. & Morgan, B.A. (1990) in Ariiiiral Reports in Medicinal Chemistry (Bristol, J.A., ed.), vol. 25, pp. 177-184, Academic Press, New York; Johnson, W.H.. Roberts. N.A. & Borkakoti, N. (1987) J . Enzyme Inhibi- tion 2. 1-22; cathepsin B and L inhibition, see Shaw, E. (1990) in Advurices iri Eiizyrnology (Meister, A. ed.), vol. 63, pp. 271- 347, John Wiley & Sons, New York Black, R.A., Kronheim, S.R. & Sleath, P.R. (1989) FEBS Lett.

Sleath. P.R., Hendrickson, R.C., Kronheim, S.R., March, C.J. & Black, R.A. (1990) J. Biol. Chenz. 265, 14526-14528 Kostura, M.J., Tocci, M.J., Limjuco, G., Chin, J., Cameron, P., Hillman. A.G., Chartrain, N.A. & Schmidt, J.A. (1989) Proc. h r l . Acad. Sci. U S A 86, 5227-5231 Howard, A D . , Kostura, M.J., Thornberry, N., Ding, G.J.F., Linijuco, G.. Weidner, J., Salley, J.P., Hogquist, K.A., Chaplin, D.D., Mumford, R.A., Schmidt, J.A., & Tocci, M.J. (1991) 1. Irii i i i ioiol . 147, 2964-2969 For a review on the therapeutic potential of cytokine manipula- tion sec: Henderson, B. & Blake, S. (1992) Trends Pharnuzcoi. Sci. 13. 145-152 For a revie\\ on interleukin-1 sce: Dinarello, C.A. (1991) Blood 77, 1627-1652 Meek. T.D., Dayton, B.D., Metcalf, B.W., Dreyer, G.B., Strick- ler. J.E., Gorniak, J.G., Rosenberg, M., Moore, M.L., Magaard, V.W. & Debouck, C. (1989) Proc. Natl. Acad. Sci. USA 86, 1841-1845 Our efforts concerning probes to examine thc possibility of ICE being a cysteinc protease included: peptidic nitriles, Robinson, R.P. unpublished results, and peptidyl difluoroketones, Robin- son, R.P. & Donahuc, K.M. (1992)J. Org. Chem. 57,7309-7314 Askin, D.. Wallace, M.A., Vacca, J.P., Reamer, R.A., Volante, R.P. & Shinkai, I. (1992) J . Org. Chem. 57, 2771-2773; Poss, M . A . & Reid, J.A. (1992) Tetrahedron Lett. 33, 1411-1414; Ghosh, A.K., McKee, S.P. & Thompson, W.J. (1991) J . Org. Cheni. 56, 6500-6503; Decamp, A.E., Kawaguchi, A.T., Vol- ante, R.P. & Shinkai, I. (1991) Tetrahedron Lett. 32, 1867-1870; Boyd, S.A. , Mantei, R.A., Hsiao, C.-N. & Baker, W.R. (1991) J . Org. Cheni. 56, 438-442; and references therein Holladay, M.W., Salituro, F.G. & Rich, D.H. (1987) J . Med. Chein. 30, 374-383; Holladay, M.W. & Rich, D.H. (1983) Tet- rohedrori Lett. 24, 4401 -4404 Takai. K . . Nitta, K., Fujimura, 0. & Utimoto, K. (1989)J. Org. Cheni. 54. 4732-4734 Stanfield. C.F., Parker, J.E. & Kanellis, P. (1981)J. Ovg. Chem. 46. 4799-4800; Wilk, S. & Thurston, L.S. (1990) Neuropeptides

Luly, J.R., Dellaria, J.F., Plattner, J.J. ,Soderquist, J.L. &Yi, N. (1987) J . Org. Cheni. 52, 1487-1492 Baker, R. & Kecn, R.B. (1985)J. Organomet. Chem. 285, 419- 427

247, 386-390

16, 163-168

19. Harbcson. S.L. & Rich, D.H. (1989) J . Med. Cherrr. 32, 1378- 1392

20. In considcring the results of Holladay e ta / . (14), as well as those of Jin et a/ . (31) in which alkenylchromium reagents were added to a chiral aldehyde, we felt that significant epimeriration of the r-center of 2 (i.e. C-3) was unlikely, and we thcrefore did not directly address this point. Lactone derived from cpimerized aldehyde may have been rcmoved during the recrystallizations of 6a and 6b. During subsequcnt couplings of 7a and 7b with the dipeptidc HProValNHz and the amino acid derivatives t-

46L


Cannizzaro, L.A., Huebner, K. & Black, R.A. (1992) Scierzre

27. Pearl, L.H. & Taylor, W.R. (1987) Nuture(London) 329.35 1-354 28. Chapman, K.T. (1992) Bioorg. Med. Chem. Lelr. 2, 613-618 29. Thornberry, N.A., Bull, H.G., Calaycay, J.R., Chapman, K.T.,

Howard, A.D., Kostura, M.J., Miller, D.K., MoIineaux, S.M., Weidner, J.R., Aunins, J., Elliston, K.O., Ayala, J.M., Casano, F.J., Chin, J., Ding, G.J.-F., Egger, L.A., Gaffney, E.P., Lim- juco, G., Palyha, O.C., Raju, S.M., Rolando, A.M., Salley, J.P., Yamin,T.-T., Lee,T.D., Shively, J.E., MacCross, M., Mumford, R.A., Schmidt, J.A. & Tocci, M.J. (1992) Nature (London) 356,

30. Eberle, A,, Fauchere, J.-L., Tesser, (3.1. & Schwyzer, R. (1975)

31. Jin, H., Uenishi, J.-I., Christ, W.J. & Kishi, Y. (1986) J . Am.

256, 97-100

768-774

Helv. Chim. Acta 58, 2106-2129

Chem. SOC. 108, 5644-5646

BocPheOSu and tBocValOSu, we observed no evidence indi- cating the presence of other diastereomers

21. X-Ray analysis of 6a performed by Dr. J. Bordner of Pfizer Inc, Central Research Division, Eastern Point Rd., Groton, CT 06340, USA

22. Garner, P. & Park, J.M. (1990) 1. Org. Chem. 55, 3772-3787; Carlsen, P.H.J., Katsuki, T., Martin, V.S. & Sharpless, K.B. (1981) J . Org. Chem. 46, 3936-3938

23. Colucci, W.J., Tung, R.D., Petri, J . S . & Rich, D.H. (1990) J . Org. Chem. 55, 2895-2903; and references therein

24. Work with related series of inhibitors and peptidic substrates indicate that Phe at Pz is an adequate replacement for the nat- urally occurring His: Andrews, G.C., Arriola, M.W., Carty, T.J., Contillo, L.G., Cronin, B.J., Danley, D.E., Daumy, G.O., Donahue, K.M., Downs, J.T., LaLiberte, R.E., McColl, A.S., Otterness, I.G., Reiter, L.A., Robinson, R.P., Singleton, D.H. & Wilder, C., unpublished work from Pfizer Inc; also see Thorn- berry et al. (29)

25. Evaluated using a preparation of ICE derived from THP-1 cells (7) and pro-IL-lD that had been metabolically labeled with 35S- methionine: Arriola, M.W., Carty, T.J., Danley, D.E., Daumy, G.O., Downs, J.T., LaLiberte, R.E., McColl, A.S., Otterness, I.G. & Wilder, C. , unpublished work from Pfizer Inc

26. Cerretti, D.P., Kozlosky, C.J., Mosley, B., Nelson, N., Van Ness, K., Greenstreet, T.A., March, C.J., Kronheim, S.R., Druck, T.,

Address:

Lawrence A . Reiter Central Research Division Department of Medicinal Chemistry Eastern Point Road Groton, CT 06340 USA

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interleukin-1β converting enzyme : synthesis of hydroxyethyl dipeptide surrogate-containing...

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