In!. J. Peptide Protein Res. 40, 1992, 233-242

Synthesis and biological activity of new conformationally restricted analogues of pepstatin” ZBIGNIEW SZEWCZUK, KAREN L. REBHOLZ and DANIEL H. RICH*

School of Pharmacy, University of Wisconsin-Madison,Madison, WI, USA

Received 2 January, accepted for publication 28 March 1992 A new statine derivative, 3-hydroxy-4-amino-5-mercaptopentanoic acid; cysteinylstatine (CySta), was synthesized and used to prepare a series of conformationally restricted analogues of pepstatin (Iva-Val-Val-StaAla-Sta) in which the conformational constraint was introduced via a bis-sulfide connecting the appropriately substituted residues in the PI and the P3 inhibitor side chains. The precursor peptide, Iva-Cys-Val-CyStaAla-Iaa, was synthesized and alkylated with a series of dibromoalkanes and alkenes to produce the cyclic structures. This strategy permitted the carbon atom spacing between the PI and the P3 inhibitor side chains to be systematically varied so as to produce inhibitors with 15-, 16-, and 17-membered ring systems. Additional non-cyclic analogues were synthesized as controls by alkylating the bisthiol intermediates with methyl iodide. The inhibitory potency of the analogues were determined against porcine pepsin and penicillopepsin by using standard enzyme kinetic assays. The cyclic inhibitor were found to be potent inhibitors of both aspartic proteases; inhibitor that contained a trans-2-butene link between the two sulfur atoms was found to be the most potent inhibitor with a Ki less than 1 nM against pepsin and 3.94 nM against penicillopepsin. This series of compounds illustrates a new type of conformational restriction that can be used to probe for the bioactive conformation of peptides. Key words: aspartic protease; conformation; cystatine; enzyme inhibitor; pepsin; pepstatin; restricted; statine

Dedicated to Professor Bruce Merrifield on the occasion of his 70th birthday Pepstatin, the pentapeptide (1) discovered by Umezawa and co-workers in 1970 as part of their efforts to identify novel inhibitors of therapeutically important enzymes (l), remains the most potent naturally occurring inhibitor of the aspartic proteinases. The remarkable affinity of 1 for most aspartic proteinases depends on the unique amino acid, statine (2), which mimics a dipeptidyl tetrahedral intermediate for amide bond hy-

drolysis of this class of enzyme (2, 3). Detailed study of the mechanism of action of pepstatin led to the development of selective inhibitors of aspartic proteinases by modifying the non-statine amino acids to more closely mimic the ancillary residues in substrate;; for targeted proteases. Thus, replacement of the dipeptidyl cleavage unit in substrates by statine or related mimetics produced tight-binding inhibitors of renin (4, 9, cathepsin D (6), and penicillopepsin (7, 8). Pepstatin and derived analogues have also played important roles Abbreviations used follow the IUPAC-IUB Commission on Bio- in efforts to determine the catalytic mechanism of aschemical Nomenclature recommendations. Additional abbreviations: partic proteinases (2, 3). Boc, tert.-butyloxycarbonyl; DCC, dicyclohexylcarbodiimide; Iaa, The availability of a well defined X-ray crystal strucisoamylamine; Iva, isovaleryl; CySta, (3S,4R)-S-mercapto-4-amino- ture of the complex of pepstatin bound to the aspartic 3-hydroxypentanoic acid; Sta, (3S,4S)-6-methyl-4-amino-3-hydroxyproteinase, Rhizopus chinensis pepsin (9) and the cryshexanoic acid. tal structures of other inhibitors bound to penicillopep* A preliminary report of this work, “Conformational restrictions of sin (lo), and endothia pepsin (1 l), led us to consider pepstatin via amino acid side chain groups in a study of the biologically active conformation of pepstatin” by D.H. Rich and Z. Sze- if new classes of inhibitors might be designed by analwczuk was presented at the Polish Peptide Conference “Peptydy 85” ysis of the conformation of the bound inhibitor. Many renin inhibitors have been reported recently as a result in 1985. 233

Z. Szewczuk et al. TABLE 1 Inhibition of pepsin and penicillopepsin by CyStu-derived inhibitors

No.

Compound

Enzyme

Iva-Xxx-Val-CySta-Ala-Iaa

I

I Y

12 14 15 16 17 18 19 20 21 22

Porcine pepsin

Penicellopepsin

Ki (nM)

Ki (nM)

25.05 3.4 0.23 0.89 2.1 3.8 3.52 Sta-Ala-laa 14 Boc-CySta(Bz1)-OH (4), obtained by saponification of the corresponding ethyl ester, was converted to the bis-S-benzyl precursor (12) by classical solution methods (Scheme 2). Conventional deprotection procedures CMe / and standard coupling methods (DCC/HOBt or sym15 laa-Val-Val-CySta-AIa-Iaa metrical anhydrides) were used (16). Reasonable yields SCHEME 3 were obtained for each coupling step, and the overall yield for the synthesis shown in Scheme 2 was 3 1%. Subsequent oxidation afforded aldehyde (7) in 47% Additional linear analogs of pepstatin 13-15 were preoverall yield. To check for epimerization during the pared as shown in Scheme 3. To prepare the cyclic analogs of pepstatin (17-22), preparation of aldehyde (7), a portion of the aldehyde

/B” Boc-Val-CySta-Ala-laa

i

I

/

A

6H

=

12

Na I NH3 Br-X-Br

CH31 Na I NH3

16

22,

-CHzaC 52%

SCHEME 4

235

Z. Szewczuk et al. both sulfhydryl groups in the linear tetrapeptide 12 were tors could be formed by connecting the side chains of deprotected simultaneously by reaction with sodium in amino acids known to project toward the interior of an liquid ammonia (3 1). The bis-thiolate intermediate was enzyme binding pocket rather than toward solvent, and converted in situ into linear and cyclic derivatives. Re- the wealth of information about the pepstatin-aspartic action with methyl iodide gave the bis-S-methyl deriv- proteinase system suggested that this would be a parative 16. Reaction with a series of dibromoalkanes gave ticularly good system on which to test the concept. the cyclic structures 17-22 (Scheme 4). The cyclic in- Aspartic proteinase inhibitors are known to adopt an hibitors 17-22 are relatively insoluble in cold methanol extended beta-structure when bound in the active site so that the cyclic products were easily purified by crys- of the enzyme (9-1 l), and in this conformation, side tallization from methanol. To avoid polymerization of chains of alternating residues (e.g. P1,P3; P2,P4, etc.) the reactive bis-thiol intermediate, the cyclization reac- [nomenclature that of Schechter & Berger (29)] lie adtions were carried out under highly dilute solutions jacent to each other. In the case of pepsin and renin (0.15 mmol/L). Analysis of the final products by mass inhibitors, two of these side-chains (P1,P3) are particspectrometry established that the purified products ob- ularly critical for binding and enzyme inhibition (3). Thus, cyclic inhibitors of aspartic proteinases prepared tained were monomers. by joining the PIP3 side chains seemed structurally plausible and might lead to novel types of low molecEnzyme kinetics Inhibition of porcine pepsin was determined by previ- ular weight enzyme inhibitors. ously described methods (17, 18) that use Phe-Gly-HisWe began the design of the cyclic structures by exPhe(N02)-Phe-Ala-Phe-OMe as the substrate. The amining the X-ray crystal structure of pepstatin bound penicillopepsin catalyzed reaction was monitored by to Rhizopus chinensis pepsin (Fig. 1A) (9). The close use of Ac-Ala-Ala-Lys-Phe(N02)-Ala-AlaNH2 as the proximity of the isobutyl side chain of statine (PI ressubstrate (18). The inhibitors were dissolved in a small idue) to the isopropyl side chain of valine (P3 residue) amount of methanol and then diluted in 0.02 M sodium is highlighted by the use of a dot surface to indicate the acetate (pH 5.50) to give a stock solution of known Van der Waal’s radii of the fully protonated side chains concentration. The reaction rate at 25 O was followed by (Fig. 1B). The fact that these two side chains clearly are monitoring the decrease in absorbance at 3 10 nm. Ini- in contact with each other supported the idea that it tial velocity versus inhibitor concentration data were fit should be possible to form a cyclic structure by judiby using non-linear regression (19) to the equation for ciously connecting the P I and P3 side chains. For syntight-binding inhibition of Williams & Morrison (20), thetic ease, we elected to begin these studies by using and K, values were calculated by use of the method of cysteinyl side chains. However as shown in Fig. lC, the Cha eta[. (21). The results of the inhibition studies are distances between the cystejnyl sulfur atoms in the P1,P3 positions are too great (4A) to form a disulfide bond shown in Table 1. without distorting the peptide chain conformation. Instead, the molecular modeling suggested that additional DISCUSSION atoms would be required in order to connect the sulfur Cyclic analogues of highly flexible linear peptides are atoms without distorting the peptide chain conformafrequently synthesized in order to characterize the “bio- tion found in the pepstatin-Rhizopus chinensis pepsin active conformation” of the parent peptide (22-25). complex. To quickly determine the optimal ring size For synthetic ease, cyclic structures often have been needed to obtain undistorted cyclic inhibitors, we degenerated by joining the side chains of amino acids via vised a general and rapid method for generating a sedisulfide bonds or lactams (22-26). These approaches ries of cyclic structures by connecting the sulfur atoms restrict the conformational space available to the re- with a series of carbon bridges. The number of carbon , sulting products and, in some cases, have produced atoms needed to close the ring system without distortremarkably active or metabolically stable peptide ana- ing the peptide backbone conformation of the enzyme logues (24). However, side chain cyclization can suc- bound inhibitor appeared to be about 4-6. Fig. 1D ilceed only when the modified positions do not interfere lustrates one cyclic inhibitor (20) formed by connecting with the binding of the analogue to the receptor or the sulfur atoms in the P I P3 side chains with trans-2enzyme. Not surprisingly, most initial attempts to eval- butene. Additional molecular modeling studies carried uate the feasibility of conformational restriction pro- out with analogue 20 bound in the active site of R. chinduced cyclic structures in which the modified side ensis pepsin (data not shown), suggested that 20 could chains of the hormone or enzyme inhibitor were found adopt a variety of conformations without encountering to project away from the receptor or enzyme into what obvious steric interactions between the trans-2-butene is likely to be solvent. In recent years, several additional atoms and the enzyme. Extensive molecular modeling methods for introducing conformationally restricting and energy minimization studies of the cyclic structures cyclic structures into peptides have been reported (27- were not carried out, but in view of the number of tight-binding inhibitors found here, such studies would 30). We wished to explore whether tight-binding inhibi- be warranted in the future. 236

Pepstatin analogues

B

A

FIGURE 1 A. Relaxed stereo projection of the P1 and P3 side chains in pepstatin bound to R. chinensis pepsin; B. Same projection as in Fig. 1A except that van der Waal‘s surfaces are shown about the valine and statine side chains; C. Same projection as in Fig. 1A and 1B except that the valine and statine side chains have been changed to Cys and CySta side chains (Iva-Cys-Val-CySta-Ala-Sta); D. Structure 20 formed by connecting the two sulfur atoms in Fig. 1C with trans-2-butene.

dibromoalkanes to form cyclic structures 17-22, or were reacted with methyl iodide to give the corresponddures shown in Scheme 1. Cysteine was converted to ing bis-thiomethyl ether 16, which served as the stanthe N-protected S-benzyl cysteinol6 by standard meth- dard for comparing the potencies of cyclic vs. linear ods and oxidized to the aldehyde 7, which was reacted inhibitors. The products were isolated by evapor ting with lithio ethyl acetate (14). The two diastereomers the ammonia, and purified by recrystallization rom obtained were separated by column chromatography methanol. Analogues 14 and 15 were synthesized by over silica gel as described for the parent compound variations of the above methods to provide additional (14), and the correct diastereomer (3S4R-CySta examples of linear analogues with valine in the P3 po(Bzl)OH, 4) was used to synthesize the protected sition. Our procedure, which uses sodium in liquid amCySta-containing peptides shown in Schemes 2 and 3. monia (3 1) to remove the benzyl protecting groups and The inhibitors were synthesized by coupling Boc- generate the bis-thiolate anions, gave higher yields of CySta(Bz1)-OH to the isoamyl amide derivative of ala- the desired products than the procedure described by nine (Ala-Iaa), followed by stepwise elaboration of the Mosberg & Orinass (13). This may reflect the results linear inhibitors by use of established procedures (16). obtained for different synthetic targets, or it could reThe formation of the cyclic structures was achieved flect a more efficient formation of the intermediate bisby first reacting the bis-benzyl derivative 12 with so- thiolate anions. The inhibition constants (Ki) for each analogue were dium in liquid ammonia, a modification of the procedure of du Vigneaud (31). The bis-thiolate derivatives determined by using standard methods (Table 1) (17, formed were reacted in situ immediately with a series of 18). Remarkably good inhibitors of pepsin and penicilDerivatives ofthe cysteinyl analogue of statine, CySta

(3), were synthesized by following the standard proce-

!

231

.

Z. Szewczuk et al. lopepsin were obtained. The bis-S-methyl inhibitor 16 established that cysteinyl side chains are tolerated in the S1 and S3 binding pockets of either enzyme. The cyclic analogues, which used four-, five- and six-carbons to bridge the two sulfur atoms in the PIP3 positions, were excellent inhibitors of these two enzymes. The best inhibitor appeared to be 20, which was formed from the four carbon bridge, trans-2-butene 20. However, a comparison of the linear analogue 16 with the trans-2-butene analogue 20 indicated that the cyclic structure was no more potent than the linear analogue. Thus, the entropic gain expected from a conformationally restricted enzyme inhibitor did not appear to be realized and this suggested that some unfavorable steric or electrostatic interaction between the trans-2-butene unit and the enzyme probably exists in the complex formed between enzyme and inhibitor 20. The nature of this unfavorable interaction has not been identified. We were somewhat surprised that the much more sterically demanding ortho-xylyland meta-xylyl derivatives 21 and 22 were also very good inhibitors of porcine pepsin (Kj = 4.2 and 5.3 nM respectively), because the molecular modeling studies suggested that these groups might be too large to be accommodated by the enzyme. The kinetic results indicate that the active sites of both enzymes are able to accommodate many atoms in or between the S1 and S3 subsites. Our results establish that tight-binding, cyclic inhibitors of aspartic proteases can be obtained by forming cyclic bis-thio-ether derivatives between the P I - P ~ side chains. This strategy may prove useful for designing inhibitors of other proteases, which are known to bind inhibitors in conformations that approximate an antiparallel 8-sheet (3, 32). In addition, the use of the bis sulfide inhibitors may provide an additional method for determining the “bioactive” conformation of peptides. EXPERIMENTAL PROCEDURES Materials and methods Boc-amino acids were purchased from Sigma; other chemicals were obtained from Aldrich. All solvents were freshly distilled from proper drying materials before use. Melting points were determined on a Thomas Hoover Capillary melting Apparatus and are uncorrected. Optical rotations were measured on a PerkinElmer Model 241 automatic polarimeter. NMR spectra were recorded on a Varian EM-390 (90 MHz) or a Bruker WH-270 spectrometer, in CDC4 with tetramethylsilane as internal standard (chemical shifts in ppm). Low resolution mass spectra were recorded on a Finnigan Model 1015 mass spectrometer. Elemental analyses were determined by Galbraith Laboratories, Inc., Knoxville, TN. TLC was performed on silica gel “POLYGRAM Sil. G/UV254” plates obtained from SYBRON/Brinkmann. The following TLC solvent systems were used: A, 10% methanol in chloroform; B, 238

4% methanol in chloroform; C, 2.5% methanol in chloroform; D, 10% methanol in ethyl acetate; E, dichloromethane. Column chromatography was carried out with Merck silica gel 60 (70-230 mesh) by using 100 g of silica gel in a gravity column per 1-2 g of chromatographed material. All compounds used in the kinetic studies appeared as a single spot on TLC and were analytically pure. The synthesized cyclic compounds were identified as monomers by mass spectrometry. General synthetic procedures Procedure A : removal of the tert.-butoxycarbonyl group. The Boc-peptide (1 mmol) in a solution of 4 N HCI in dioxane (2 mL) was stirred at room temperature and the reaction monitored by TLC. Complete reaction was generally achieved in less than 1 h. Excess reagent was removed under reduced pressure and the residue triturated with dry ethyl ether. The solid product was filtered, washed with ethyl ether and dried in vacuo for several hours. The resulting hydrochlorides were used without further purification. Procedure B: coupling reactions using dicyclohexylcarbodiimidell -hydroxybenzotriazole. A solution of peptide hydrochloride (1 mmol) in methylene chloride (5 mL) was cooled in an ice bath and neutralized with N-ethylmorpholine (1 mmol). Boc-amino acid (1 mmol) and 1-hydroxybenzotriazole (1 mmol) were added followed by DCC (1.1 mmol). The reaction mixture was allowed to stir at 0” for 2 h and at room temperature overnight. DCU was filtered off and the solution was concentrated in vacuo. The residue was dissolved in ethyl acetate and washed with cold 0.5 N HCI, saturated NaHC03, and brine, and dried over MgS04. The peptide was purified by silica gel chromatography and crystallized. Procedure C: preparation of symmetrical anhydrides. Boc-amino acid (2 mmol) and DCC (1 mmol) in methylene chloride (1 mL) were stirred at 0” for 40 min. The reaction mixture was cooled for 20-30 min on dry ice and filtered to remove DCU. The filtrate was used immediately without further purification. I Procedure D: coupling reactions via symmetrical anhydrides. A solution of peptide hydrochloride (1 mmol) in DMF (1 mL) was cooled in an ice bath and neutralized with triethylamine (1 mmol). After addition of symmetrical anhydride (1.2-1.8 mmol), stirring was continued at 0” for 4 h and at room temperature overnight. The solution was concentrated in vacuo and the residue triturated with 0.1 N NaOH (150 mL). The solid product was filtered, washed with water, purified by silica gel chromatography and crystallized. Procedure E: removal of benzyl groups with sodium in liquid ammonia and alkylation of the resulting thiolgroups. Protected peptide (0.03 mmol) was dissolved in dry

Pepstatin analogues liquid ammonia (200 niL) and sodium was added until a light blue color persisted for a few minutes; then either a suitable dibromide (0.033 mmol) or methyl iodide (0.12 nimol) was added. The solution was allowed to reflux for 1 h. The ammonia was removed with a stream of nitrogen, and water was added to the residue. The solid product was filtered, washed with water followed by Skclly B, and purified either by silica gel chromatography or by crystallization from methanol.

the precipitate was filtered and used immediately in the next reaction. Yield: 64""; m.p. 66-68"; TLC RXA) 0.80; RXD) 0.58; NMR (CDCIx) (90 MHz) 6: 1.4 (s, 9H); 2.8 (d, 2H, 5 Hz); 3.7 (s, 2H); 4.3 (m, 1H); 5.3 (m, 1H); 7.3 (s, 5H); 9.5 (s, 1H). Anal. calc. for C I ~ H ~ I N OC~60.99, S : H 7.17, N 4.74, S 10.85. Found: C 61.11, H 7.34, N 4.60, S 11.05.

Nqtert. -but~~~o~~carboriy~-S-(beri~j~~-4(R~-aniitio-3(S)h~drox~~-5-mercaptopeiitu11oiiic acid(Boc-CyStn(Bz1)-OH) N-(tert.-hutj~lor,~carbori~l~-S-(beti~~~l)-~-c~~steitze methyl (4). To dry freshly distilled T H F (20 mL) cooled to ester (5). To a solution of Boc-~-Cys(Bzl)-OH(15.6 g, -20" was added diisopropylamine (2.1 mL, 15 mmol) 50 mniol) in ethyl ether was added etheral diazomethane under a nitrogen atmosphere, followed by a solution of in small portions. The addition was stopped when a 1.55 M n-butyl-lithium in hexane (9.68 mL, 15 niniol). permanent yellowish color persisted. Excess diaz- After 1 h the temperature was lowered to -78", and omethane was destroyed by the dropwise addition of freshly distilled ethyl acetate (1.47 mL, 15 niniol) was acetic acid. The solution was washed with 0.5 N HC1, added and stirred for 15 min. The solution of comsaturated NaHCOI, and brine, and dried over MgSO4. pound 7 (2.95 g, 10 mmol) in T H F (10 mL; cooled to The solvent was evaporated to dryness, and the residue -78") was added, and the reaction mixture was stirred was dissolved in hexane and kept in a freezer overnight. for 10 min before 1 N NaHS04 (350 mL) was added. The resulting crystals were collected in 94% yield: The product was extracted with ethyl acetate, washed n1.p. 50-5 1 ";TLC RAA) 0.86; RAE) 0.17; [ C X -39.1 ] ~ ' with brine, dried over MgS04 and evaporated to dry(c 1, methanol); NMR (CDC13) (90 MHz) 6: 1.4 (s, ness. The resulting oil was chroniatographed on 300 g 9H); 2.8 (d, J = 5 Hz, 2H); 3.7 (d, 5Hj; 4.5 (m, 1H); 5.2 silica gel eluting with 0.75",; of ethanol in chloroform. (d, 1H); 7.3 (s, 5H). The chromatography allowed the resulting estcr diasAnal. calc. for C ~ ~ H ~ J N CO 59.06, ~ S : H 7.12, N 4.31, tereomers to be separated: higher R, (BocS 9.84. Found: C 58.88, H 7.29, N 4.38, S 9.61. (3S,4R)CySta(Bzl)-OEt; yield: 29.90,b; oil; TLC RXC) 0.53; NMR (CDCIj) (90 MHz) 6 1.2 (t, 3H; J = 7 Hz); 1.4 (s, 9H); 2.3-2.8 (m, 4H, includes d, J = 7 Hz); 3.4 Nftert. -butj*lo~~~cnrbori~~l)-Sfberizj~~-2(R)-nmino-3mercaptopropari-I-ol(6). Compound 5 (8.92 g, 30 mmol) (d, IH, J = 3 Hz); 3.5-3.8 (ni, 3H); 4.0-4.4 (m, 3H was dissolved in a mixture of ethanol (200 mL) and includes q, J = 7 Hz); 5.0 (d, l H , J = 9 Hz); 7.3 (s, 5H)); T H F (80 mL) and stirred at room temperature under lower Rf (Boc-(3R,4R)CySta(BzI)-OEt; yield: 8.3 ;,o ; argon. Then 2 M LiBH4 in T H F (36 mL) was added. oil; TLC RXC) 0.46; NMR (CDC13) (90 MHz) 6 1.2 (t, The mixture was stirred for 5 h at room temperature, 3H; J = 7 Hz); 1.4 (s, 9H); 2.4-2.6 (ni, 2H); 2.7 (d, 2H, diluted with cold water and acidified with 5% citric J = 6 Hz); 3.5 (d, l H , J = 4 Hz); 3.6-4.3 (m, 6H inacid. The precipitate was filtered and washed with cludes q, J = 7 Hz); 4.9 (d, l H , J = 9 Hz); 7.3 (s, 5H). water. The crude product was purified by crystalliza- Dry Boc-(3S,4R)CySta(Bzl)-OEt (74 mg, 0.193 mmol) tion from ethyl acetate-hexane. Yield: 78%; m.p. 73- was dissolved in methanol (1.4 mL) and 4 N NaOH (C 1, (0.075 mL, 3 mmol) was added. The reaction mixture 74"; TLC RXA) 0.70; RXD) 0.82; [ ~ ] g -18.2" methanol); NMR (CDCL3) (90 MHz) b: 1.4 (s, 9H); was allowed to stir at room temperature overnight. After 2.4-2.7 (m, 3H); 3.6-3.9 (m, 5H); 5.0 (d, 1H); 7.3 (s, evaporation of volatile components under reduced pres5H). sure the dry residue was dissolved in water (15 mL), Anal. calc. for C15H~iN07S:C 60.58, H 7.79, N 4.71, washed with ethyl ether, acidified with 0.5 N HC! and extracted with ethyl ether. The ether extract was washed S 10.78. Found: C 60.67, H 7.75, N 4.61, S 11.07. with brine, dried (MgS04) and evaporated to give chroN-(tert.-butj-lo~jrarborijll)-S-(betizj~~-2(Ri-amino-3- matographically pure oily product. Overall yield 19;'. mercaptopropnri-1-a1(7). Compound 6 (2.97 g, 10 mmol) TLC RLA) 0.40; RXD) 0.32; NMR: (CDCI3) (90 MHz) was dissolved in DMSO (3 mL) and TEA (6.75 mL, 6: 1.4 (s, 9H); 2.5-2.7 (m, 4H); 3.5-3.9 (m, 4H); 4.3 50 nimol) was added. After cooling, in an ice bath, (m, 1H); 4.9-5.8 (m, 2H); 7.3 (s, 5H). while vigorously stirring, SO3-pyridine complex Analysis for DCHA salt of compound 4: [ x ] E (50 mmol) in DMSO (20 mLj was added. After the -21.6' (c, 0.5, methanol), m.p. 168-170". solution was stirred for 10 min at room temperature, Anal. calc. for C29H48N205S: [ BOC-CySta(Bz1)-OHwater (800 inL) was added, and the mixture was acid- DCHA]: C 64.89, H 9.01, N 5.22, S 5.97. Found: C ified with KHSOJ. The product was extracted with 64.90, H 9.06, N 5.15, S 6.18. ethyl ether. The combined extracts were washed with 0.5 N HCI, and brine, and dried over MgSO4. The sol- 2-Oxazolidone derivative of S-lben~?,.l)-4(R)-nrni,zo-3/S/acid (8). The Boc-group of vent was evaporated in vacuo and the residue was crys- hydrox~~-5-mercaptopentarioic tallized from 17-pcntane. After 3 h of cooling at -12", compound 4 was reniovcd according to general procc239

Z. Szewczuk et al. dure A. The resulting amino acid (20 mg) was dissolved in 1 M potassium hydroxide (6 mL) and was cooled to 5 " . A solution of 10% phosgene in toluene (10 mL) was added. After stirring for 3 h the aqueous layer was separated, and the toluene layer was washed with 1 N KOH. The combined aqueous layers were acidified by hydrochloric acid and extracted with ethyl acetate. The organic extracts were dried (MgS04), and the solvent was removed in vacuo. The residue (TLC RAA) 0.40; RAD) 0.24) was dissolved in CDC13 (0.5 mL), one drop of D20 was added, and the organic layer was analyzed by NMR. NMR: (CDC13 + D20) (90 MHz) 6: 2.1-2.9 (m,4H); 3.6-3.8 (m, 1H); 3.8 (s, 2H);4.4-4.6 (m, IH); 7.4 (s, 5H); 53.4 = 5.4 Hz (established after decoupling the resonance at 2.6 ppm).

Iva-Cys(Bzl)- Val-CySta(Bzl)-Ala-Iaa(l2). Compound 11 (316 mg, 0.394 mmol) was deprotected according to general procedure A. The resulting hydrochloride was coupled with isovaleric anhydride (0.5 mmol) according to general procedure D. The crude product was purified by column chromatography eluting with 30: 1 chloroform/methanol (v/v) and by crystallization from ethyl acetate. Yield: 67%, m.p. 228-230"; TLC RAA) 0.56, Rf(D) 0.33; [a]'," -52.4" (c 0.2, methanol); NMR (CDCL;CD3OD 1:l v/v)) (90 MHz) 6: 0.8-1.15 (m, 18H); 1.3-2.9(m, 13H); 3.1-4.5 (m, 14H); 7.3 (s, 10H). Anal. calc. for C ~ O H ~ ~ N ~cO 62.23, ~SZ H :7.96, N 9.07, S 8.31. Found: C 62.12, H 8.01, N 8.96, S 9.11.

Boc- Val- Val-CySta(Bzl)-Ala-Iaa (13). Compound 7 was deprotected according to general procedure A. The reBoc-CySta(Bz1)-Ala-Iaa (9). The title compound was sulting hydrochloride was coupled with Boc-valine anprepared from HCl H-Ala-Iva (16) and compound 4 by hydride according to general procedure D. (The Bocgeneral procedure B. The product was purified by sil- valine anhydride was obtained according to general ica gel chromatography by eluting with 10% ethyl ac- procedure C). The crude product was purified by silica etate in Skelly B. Yield: 72%; m.p. 92" (soft. 78") gel chromatography eluting with 2 4 1 chloroform/ (methanol-water); TLC RAA) 0.56, RXD) 0.42; [ a ] g methanol (v/v). Yield: 81 %; m.p. 208-209" (chloro-21.1" (c 1, chloroform); NMR (CDC13) (90 MHz) 6: form-Skelly B); TLC RAA) 0.56, RAD) 0.36, [ c c ] ~ 0.9 (d, J = 6 Hz, 6H); 1.1-2.7 (m, 19H); 3.1-4.7 (m, -49.3" (c 2, chloroform); NMR (CDCl3) (90 MHz) 6: 8H); 5.1 (m, IH); 6.5 (m, 1H); 6.9 (m, 1H); 7.3 (s, 5H). 0.8-1.2 (m, 18H); 1.3-2.8 (m, 21H); 3.1-5.5 (m, IlH); Anal. calc. for C ~ ~ H ~ I N C~ 60.59, O ~ SH: 8.34, N 8.48, 6.6-7.4 (m, 9H). Anal. calc. for C ~ ~ H ~ ~ NCS60.58, O ~ SH:8.57, N 10.09, S 6.47. Found: C 60.72, H 8.36, N 8.28, S 6.89. S 4.62. Found: C 60.31, H 8.47, N 9.94, S 4.81. Boc- Val-CySta(Bzl)-Ala-Iaa (10). Compound 9 (61 mg, 123 pmol) was deprotected according to general pro- Iva- Val-Val-CySta(Bzl)-Ala-Iaa (14). Compound 13 was cedure A. The resulting hydrochloride was coupled with deprotected according to general procedure A. The reBoc-valine anhydride (221 pmol) according to general sulting hydrochloride was coupled with isovaleric anprocedure E. The Boc-valine anhydride was obtained hydride according to general procedure D. The crude according to general procedure C . The crude product product was purified by column chromatography elutwas purified by silica gel chromatography eluting with ing with 20: 1 chloroform/methanol (v/v). Yield: 82%; 24:l chloroform/methanol (v/v). Yield: 7 8 2 , m.p. 184- m.p. 260-261"; TLC RAA) 0.47, RAD) 0.21; [ a ] g 185" (chloroform-Skelly B); TLC RLA) 0.59, RAD) -84.6" (c 0.34, methanol). O ~ SH: 8.77, N 10.33, 0.43; [u]'," -48.9" (c 1, chloroform); NMR (CDCl3) Anal. calc. for C ~ ~ H ~ ~ NCS62.01, (90 MHz) 6: 0.8-1.15 (m, 12H); 1.2-2.8 (m, 20H); S 4.73. Found: C 62.08, H 8.50, N 10.42, S 5.07. Mass spectrum m/e (rel. intensity) 679 (0.40), 678 3.1-5.3(m, 10H);6.9(m, lH);7.3(~,5H);7.6(m,2H). Anal. calc. for C ~ O H ~ O N C ~O 60.58, ~ S ;H 8.47; N 9.42, (0.06), 677 (M , not observed), 587 (0.40), 586 (0.27), S 5.39. Found: C 60.73, H 8.64, N 9.25, S 5.69. 563 (M + -CO-Iaa, 0.02), 536 (0.7), 520 (M + -Ala-Iaa, 0.02), 499 (0.24), 481 (0.21), 447 (0.32), 403 (0.23), 380 Boc-Cys(Bzl)-Val-CySta(Bz1)-Ala-Iaa (11). Compound (0.32), 337 (0.59), 326 (0.78), 304 (0.62), 300 (0.70), 283 10 (286 mg, 0.581 mmol) was deprotected according to (4.8),255(1.4),241(3.2),240(2.3),200(2.3), 184(2.1), general procedure A. The resulting hydrochloride was 159 (7.7), 156 (14.5), 143 (7.0), 124 (7.3), 117 (6.8), 91 coupled with the Boc-S-benzylcysteine anhydride (50), 72 (loo), 44 (33). (0.72 mmol) according to general procedure D. The above anhydride was obtained from Boc-Cys(Bz1)-OH Iva-Val-Val-CySt4Me)-Ala-Iaa (15). The title comaccording to procedure C. The crude product was pu- pound was prepared by general procedure E from 14 rified by silica gel chromatography eluting with 30:l and methyl iodide. The product was purified by silica chloroform/methanol(v/v).Yield: 82%; m.p. 185-187" gel chromatography eluting with 30: 1 chloroform/ (chloroform-Skelly B); TLC RAB) 0.13, Rf(D) 0.49; [ a methanol (v/v). Yield: 62% m.p. 260" (decomp.); TLC ]g -49.8" (c 1, methanol); NMR (CDCL3) (90 MHz) RAA) 0.46. 6: 0.8-1.15 (m, 12H); 1.3-2.9 (m, 22H); 3.1-4.6 (m, Anal. calc. for C29H55N506S:c 57.88, H 9.21, N 11.63, S 5.32. Found: C 57.98, H 9.07, N 11.59, S 5.63. 12H); 5.4 (d, 1H); 6.6-7.4 (m, 14H). Mass spectrum m/e (rel. intensity): 603 (0.48), 602 Anal. calc. for C40H61N507S2: c 60.97, H 7.80, N 8.89, (0.13), 601 ( M + , not observed), 515 (0.10, M+-Iaa), S 8.14. Found: C 60.71 H 7.59, N 8.74, S 8.28. +

240

Pepstatin analogues 487 (0.33, M -CO-Iaa), 469 (0.07), 444 (M -Ala-Iaa. +

+

Mass spectrum mje (rel. intensity) 660 (0.34), 659

Oslo),326 (1.87), 304 (1.02), 300 (l.O), 283 (4.4), 261 (M +,0.14), 642 (M + -OH, 0.07), 573 (M -Iaa, 0.12), +

(2.6), 255 (6.Q 241 (6.7), 184 (16), 159 (8.6), 156 (9.7), 143 (9.3), 117 (lo), 88 (36), 72 (loo), 44 (24).

545 (M + -CO-Iaa, 0.82), 502 (M -Ala-Iaa, 0.64), 430 (0.35), 389 (0.23), 304 (6.31), 255 (14.14), 241 (6.16), 167 (4.29), 159 (12.26), 88 (41.97), 72 (92.38), 44 (100). +

Iva-Cy$Me)- Val-CySta(Me)-Ala-Iaa(16). The title compound was prepared by general procedure E from compound 12 (23.16 mg, 30 pmol) and methyl iodide (7.5 pL, 120 pmol). The product was purified by silica gel chromatography, eluting with 25: 1 chloroform/ ethanol (vlv). Yield: 68%; m.p. 236-239" (decomp.); TLC RAA) 0.47 [ a ] g -37.5' (c 0.56, DMSO); NMR ( D M s 0 - d ~(270 ) MHz) 6: 0.8-0.95 (m, 18H); 1.1-1.6 (m, 7H); 1.9-2.85 (m, 16H); 3.0-3.1 (m, 2H); 3.8-3.9 (m, 1H); 4.05-4.25 (m, 3H); 4.5-4.6 (m, 1H); 7.55 (d, 1H); 7.8-8 (m, 3H); 8.15 (d, IH). h i d . Cdc. for C2&3N50&2: c 54.27, H 8.62, N 11.30. Found: C 53.58, H 8.41, N 11.10. Mass spectrum mle (rel. intensity) 620 (0.01), 619 (M +, not observed), 602 (M +-OH, < 0.01), 554 (0.05), 533 (M -Iaa, O.Ol), 505 (M + -CO-Iaa, 0.04), 487 (0.05), 370 (0.25), 301 (1.85), 255 (7.14), 241 (4.38), 183 (3.92), 159 (8.0), 88 (40.3), 72 (70.48), 44 (100). +

(CH2)4

I

\

Iva-Cys-Val-CySta-Ala-Iaa(17). The title compound was prepared by general procedure E from compound 12 (23.16 mg, 30 pmol) and 1,4-dibromobutane (33 pmol). The product was purified by crystallization from methanol. Yield: 59%; m.p. >310"; TLC RAA) 0.52 [ ~(]g -23.9' (C 0.26, DMSO); NMR (DMSO-d6) (270MHz) 6: 0.8-0.95 (m, 18H); 1.1-1.6 (m, 11H); 1.9-2.95 (m, 14H); 3.0-3.1 (m, 2H); 3.9-4.4 (m, 4H); 4.55-4.65 (m, 1H); 7.7-7.85 (m,2H); 7.9-8.0(m,2H); 8.1-8.2 (d, 1H). Anal. calc. for C ~ O H ~ S N S C ~ G55.79, S ~ : H 8.57, N 10.84, S 9.92. Found: C 55.43, H 8.38, N 10.62, S 10.02. Mass spectrum mje (rel. intensity) 646 (M , 0.17), 628 (M+-OH, 0.88), 559 (M+-Iaa, 0.15), 531 ( M + CO-Iaa, 0.94), 488 (M -Ala-Iaa, 0.5 l), 389 (0.3), 274 (2.63), 255 (7.57), 241 (6.31), 159 (11.32), 88 (43.22), 72 (92.21), 44 (100). +

+

(CH2)5

I

\

(CH2)6

I

\

ha-Cys-Val-CySta-Ala-Iaa(19). The title compound was prepared by general procedure E from compound 12 (23.16 mg, 30 pmol) and 1,4-dibromohexane (33 pmol). The product was puried by crystallization from methanol. Yield: 65%; m.p. 270-275 ' (decomp.); TLC RAA) 0.53; [ a ] g -33.8" (c 0.61, DMSO); NMR (DMSO-d6) (270 MHz) 6: 0.8-0.95 (m, 18H); 1.1-1.6 (m, 15H); 1.9-2.85 (m, 14H); 3.0-3.1 (m, 2H); 3.8-3.9 (m, 1H); 4.0-4.1 (m, 1H); 4.15-4.3 (m, 2H); 4.5-4.6 (m, 1H); 7.75-7.85 (m, 2H); 7.9-8.0 (m, 3H). Anal. calc. for C32H59N~06S2:C 57.03, H 8.82, N 10.39, S 9.51. Found: C 56.85, H 8.70, N 10.19, S 9.52. Mass spectrum m/e (rel. intensity) 674 (0.42), 673 ( M + , 0.14), 656 (M+-OH, 0.07), 587 (M+-Iaa, O.l), 559 (M + -CO-Iaa, 0.49), 516 (M -Ala-Iaa, 0.36), 444 (0.28), 303 (3.7), 255 (7.1), 241 (4.2), 167 (5.8), 159 (lO.l), 88 (33), 72 (90), 44 (100). +

CH2-CHSH-CH2

\

I

Iva-Cys-Val-CySta-Ala-Iaa(20). The title compound was prepared by general procedure E from compound 12 (23.16 mg, 30 pmol) and 1,4-dibromo-2-butene (33 pmol). The product was purified by crystallization from methanol. Yield: 56%; m.p. -280' (decomp.); TLC RAA) 0.45. H 8.30, N Anal. calc. for C ~ O H ~ ~ N S ~C. &55.96, : 10.88, S 9.96. Found: C 54.72, H 8.27, N 10.58, S 10.47. Mass spectrum m/e (rel. intensity) 644 (OS), 643 ( M + , not observed, 626 (M+-OH, 0.01), 529 ( M + CO-Iaa, O.Ol), 486 (M+-Ala-Iaa, 0.03), 387 (0.03), 356 (0.08), 341 (0.09), 339 (0.09), 302 (0.08), 255 (1.6), 253 (1.2), 239 (0.97), 159 (4.5), 88 (13), 72 (48), 44 (100).

Q

Iva-Cys-Val-CySta-Ala-Iaa (18). The title compound CH2 CHz was prepared by general procedure E from compound 12 (23.16 mg, 30 pmol) and 1,4-dibromopentane Iv~-dys-Val-CySta-Ala-laa \ (21). The title compound (33 pmol). The product was purified by crystallization was prepared by general procedure E from compound from methanol. Yield: 64%; m.p. 295-300" (decomp.); 12 (23.16 mg, 30 pmol) and a-a'-dibromo-o-ksylene TLC RAA) 0.53; [N]',"-26.6' (C 0.45, DMSO); NMR (33 pmol). The product was purified by crystallization (DMSO-d6) (270 MHz) 6: 0.8-0.95 (m, 18H); 1.1-1.6 from methanol. Yield: 61 % ; m.p. 280" (decomp.); (m, 13H); 1.9-2.9 (m, 14H); 3.0-3.1 (m, 2H); 3.9-4.05 TLC RdA) 0.47). (m, 2H); 4.1-4.35 (m, 2H); 4.5-4.6 (m, 1H); 7.75-8.0 Anal. calc. for C ~ ~ H ~ S N S CO 58.85, ~ S ~ : H 7.99, N 10.09, S 9.24. Found: C 58.58, H 7.91, N 9.88, S 9.27. (m, 5H). Anal. calc. for C31H57N506S2: C 56.42, H 8.71, N Mass spectrum m/e (rel. intensity) 694 (0.08), 693 10.61, S 9.72. Found: C 56.08, H 8.67, N 10.37, S 9.59. ( M + , 0.05), 676 (M+-OH, ~ 0 . 0 1 )356 (0.04), 324

-

24 1

.

Z. Szewczuk et al, 1 1 . Blundell, T., Sibanda, B.L. & Pearl, L. (1983) Nature 304,273275; Foundling, S.I., Cooper, J., Watson, F.E., Cleasby, A., Pearl, L.H., Sibanda, B.L., Hemmings, A,, Wood, S.P., Blundell, T.L., Valler, M.J., Norey, C.G., Kay, J., Boger, J., Dunn, B.M., Leckie, B.J., Jones, D.M., Atrash, B., Hallett, A. & Szelke, M. (1987) Nature 327, 349-352 12. Boger, J. (1985) in Aspartic Proteinuses & Their Inhibitors (KOCH2 CH2 stka, V., ed.), pp. 401-420, de Gruyter, Berlin 13. Mosberg, H.I. & Orinass, J.R. (1985) J . Am. Chem. SOC.107, Iva-dys- Val-&fa-Ma-Iaa (22). The title compound 2986-2987 was prepared by general procedure E from compound 14 Rich, D.H., Sun, E.T. & Boparai, A.S. (1978)J. Org. Chem. 43, 3624-3626 12 (23.16 mg, 30 pmol) and a-a'-dibromo-m-ksylene (33 pmol). The product was purified by crystallization 15. Futagawa, S., Inui, T. & Shiba, T. (1973) Bull. Chem. SOC.Jpn. 46, 3308 from methanol. Yield: 52 % ; m.p. > 3 10O ; TLC RAA) 16 Rich, D.H., Sun, E.T.O. & Ulm, E. (1980)J. Med. Chem. 23, 0.45. 27-33 Anal. calc. for C34HxjNsO&: C 58.85, H 7.99, N 17 Rich, D.H. & Sun, E.T.O. (1980) Biochem. Pharmacol. 29,220510.09, S 9.24. Found: C 58.47, H 8.07, N 9.98, S 9.74. 2212; Fruton, J.S. (1970) Adv. Enzymol. 33, 401-442 Mass spectrum m/e (rel. intensity) 694 (0.27), 693 18 Hofmann, T. & Hodges, R.S. (1982) Biochem. J . 203, 603-610 (M+ 0.09), 676 (M+-OH, 0.45), 607 (M+-Iaa, 0.03), 19 Duggleby, R.G. (1984) Comput. Biol. Med. 14,447-455 579 (M -CO-Iaa, 0.04), 563 (0.10), 536 (M -Ma-Iaa, 20 Williams, J.W. & Morison, J.F. (1979) Methods Enzymol. 63A, 0.12), 494 (0.33), 440 (0.26), 423 (0.30), 407 (0.23), 356 437-467 (0.27), 324 (l.O), 285 (IS), 253 (2.0), 212 (2.2), 201 21 Cha, S., Agarwal, R.P. & Parks, R.E., Jr. (1975) Biochem. Pharmacol. 24, 2187-2197 (4.9, 183 (4.0), 169 (4.5), 159 (5.0), 137 (17), 87 (31), 22 Veber, D.F. (1979) In Peptides: Structure and Biological Function, 72 (47), 44 (100). (Gross E. & Meienhofer, J., eds.), pp. 409-449, Pierce Chem. Co., Rockford, IL ACKNOWLEDGMENTS 23 Veber, D.F., Freidinger, R.M., Schwenk Perlow, D., Paleveda, W.J., Holly, F.W., Strachan, R.G., Nutt, R.F., Anson, B.H., We thank Professor The0 Hofmann for a generous sample of peniHomnick, C., Randall, W.C., Glitzkr, M.S., Saperstein, R. & cillopepsin and Drs. N. Agarwal, D. Kalvin, and J. Maibaum for Hirschmann., R. (19811 . _Nature fLondonl 292, 55-58 samples of pepsin substrates and inhibitors. We thank Professor D. 24. Sawyer, T.K., Cody, W.L., Knittel, J.J., Hruby, V.J., Hadley, Northrop for a computer program used to analyze the inhibition data, M.E., Hirsch, M.D. & ODonohue, T.L. (1984) in Peptides: and Drs. K. Miller and A.A. Pathiaseril for molecular modeling of Structure and Function, Proceedings of the Eighth American Pepthe cyclic products. Partial financial support from the National Intide Symposium, (Hruby,V.J. & Rich, D.H., eds.), pp. 323-331, stitutes of Health (AM20 100) is gratefully acknowledged. Pierce Chem. Co., Rockford, IL 25. Kessler, H. (1982) Angew. Chem. Int. Ed. Engl. 21, 512 26. Freidinger, R.M., Veber, D.F., Perlow, D.S., Brooks, J.R. & REFERENCES Saperstein, R. (1980) Science 210, 656-658 1. Umezawa, H., Aoyagi, T., Morishima, H., Matusaki, M., Ha- 27. Weber, A.E., Halgren, T.A., Doyle, J.J., Lynch, R.J., Siegl, mada, H. & Takeuchi, T. (1970) J. Antibiot. 23,259-262; Aoyagi, P.K.S., Parsons, W.H., Greenlee, W.J. & Patchett, A.A. (1991) T., Morishima, H., Nishizawa, R., Kunimoto, S., Takeuchi, T., J. Med. Chem. 34, 2692-2701 Umezawa, H. & Ikezawa, H. (1972) J. Antibiot. 25, 689-694 28. Sham,H.L.,Bolis,G., Stein,H.H., Fesik, S.W., Marcotte, P.A., 2. Rich, D.H. (1985)J. Med. Chem. 28,263-273 Plattner, J.J., Rempel, C.A. & Greer, J. (1988) J. Med. Chem. 31, 3. Rich, D.H. (1990) in Comprehensive Medicinal Chemistry, (Sam284-295 uels, P.J., ed.), Vol. 2, pp. 391-441, Pergamon Press, Oxford 29. Schechter, I. & Berger, A. (1967) Biochem. Biaphys. Res. Commun. 4. Boger, J., Lohr, N.S., Ulm, E.H., Poe, M., Blaine, E.H., Fanelli, 27, 157-162 G.M., Lin, T.-Y., Payne, L.S. Schorn,T.W., Lamont, B.I.,Vas30. Szewczuk, Z . , Kubik, A., Siemion, I.Z., Wieczorek, Z., Spiegel! sil, T.C., Stabilito, I.I., Veber, D.F., Rich, D.H. & Boparai, A.S. K., Zimecki, M., Janusz, M. & Lisowski, J. (1988) Int. J. Peptide (1983) Nature 303, 81-84; Boger, J., Payne, L.S., Perlow, D.S., Protein Res. 32, 98-103 Lohr, N.S., Poe, M., Blaine, E.H., Ulm, E.H., Scorn, T.W., 31. du Vigneaud, V., Gish, D.T., Katsoyannis, P.G. & Hess, G.P. LaMont, B.I., Liu, T.-Y., Kawai, M., Rich, D.H. &Veber, D.F. (1958) J. Am. Chem. SOC.80, 3355 (1985) J . Med. Chem. 28, 1779-1790 32. Rich, D.H. (1986) in Proteinase Inhibitors, (Barrett, A.J. & Sal5. Greenlee, W.J. (1990) Med. Res. Rev. 10, 173-236 verson, G., eds.), pp. 179-217, Elsevier, New York 6. Agarwal, N. & Rich, D.H. (1983) Anal. Biochem. 1J0, 158-165 7. Salituro, F.G., Agarwal, N., Hofmann, T. & Rich, D.H. (1987) J. Med. Chem. 30,286-295 Address: 8. Maibaum, J. & Rich, D.H. (1988) J. Med. Chem. 31, 625-629 9. Bott, R., Subramanian, E. & Davies, D.R. (1982) Biochemistry Dr. Daniel H . Rich 21,6956-6962; Suguna, K., Padlan, E.A., Smith, C.W., Carlson, Professor of Medicinal Chemistry W.D. & Davies, D.R. (1987) Proc. Natl. Acad. Sci. USA. 84, University of Wisconsin-Madison 425 North Charter Street 7009-7013 10. James, M.N.G., Sielecki, A. Salituro, F., Rich, D.H. & Hof- Madison, Wisconsin 53706 USA mann, T. (1982) Proc. Natl. Acad. Sci. USA. 79, 6137-6141

(0.07), 322 (0.04), 287 (0.62), 255 (1.9), 253 (1.4), 225 (1.1), 159 (4.4), 135 (12), 88 (15), 72 (52), 44 (100).

+

242

+