peptidic p-nitroanilide substrates of interleukin-1β-converting enzyme

Inr. ,/ Pepride PrtiieinReJ. 43, 1YY4. 87-96 Prinred m Bdgium - all righr.s m e w e d

Copprighi 0 Murrhigoord 1YY4

INTERNATIONAL JOURNAL OF PFPTIDt. & PROTEIN RFSE.WCH

IS% 0367-8377

Peptidic p-nitroanilide substrates of interleukin-1B-converting enzyme

LAWRENCE A. REITER

Department of Medicinal Chemistry, Centrul Research Division, P$zer Inc, Groton, Connecticut. USA

Received 29 January, accepted for publication 15 June 1993

Peptidic p-nitroanilides are useful colorimetric substrates for enzymes. With the aim of developing a convenient, quantitative assay for inhibitors of interleukin-lg-converting enzyme (ICE), we have explored three approaches to the synthesis of peptidic p-nitroanilides relevant to this enzyme. The first approach involved a late stage oxidation of a p-aminoanilide such as CbzValAlaAsp(g-tert-butyl)-p-(t-Boc-amino)anilide. The second and third approaches used the preformed amino acid p-nitroanilides HAsp-p-nitroanilide hydrochloride and HAsp(g-tert-buty1)-p-nitroanilide which were coupled iteratively with preactivated amino acid derivatives or with an appropriate peptide, respectively. While each approach had it merits and limitations, all three produced p-nitroanilides that were substrates for ICE. 0 Munksgaard 1994.

Key words: ICE; interleukin-1 P-converting enzyme; p-nitroanilide; sodium perborate

Interleukin-1P-converting enzyme (ICE) is believed to be responsible for the conversion of pro-interleukin- lp (pro-IL- 1p) into the active, mature cytokine IL- l j (1). Inhibition of this enzyme may form the basis for a novel approach towards the treatment of inflammatory dis- eases. With the aim of developing a convenient, quantitative assay for evaluating potential inhibitors of ICE, we sought to prepare a small peptidic substrate for ICE. The P’ residues (2) do not appear to be important for recognition of small peptidic substrates by the enzyme (1, 3); substrates based on P residues alone are recog- nized by the enzyme. We therefore anticipated that appropriate peptidic p-nitroanilides, which would allow the colorimetric detection of enzyme activity, would be suitable substrates for our purposes. Herein we report three approaches to the synthesis of such peptidic p-nitroanilides.

Studies of the specificity of ICE (1, 3-5) have re- vealed that the L-aspartic acid group at P1 is critical for recognition by the enzyme. The histidine residue at P2 is not required for recognition, and a wide variety of amino acids are tolerated at this position. Little is

116 117 ... Tyr Val His Asp Ala Pro Val Arg ...

p4 p1 P1‘ P4’

FIGURE 1 Residues 113-120 of human pro-IL-1/3.

known about the importance of the P j valine. The P4 residue, though, appears to be another important residue for recognition, and peptides devoid of the P4 tyrosine are not efficiently processed (1, 3). We therefore sought to prepare the tetrapeptidic p-nitroanilide 1. Since the side chain of the tyrosine might be mimicked by the N-terminal protecting group of a tripeptide derivative, we also synthesized and examined a number of tripeptide p-nitroanilides as well.

RESULTS AND DISCUSSION

In the first approach the p-nitroanilide functionality was introduced in a late-stage sodium perborate- mediated oxidation of a peptidic p-aminoanilide (6). This approach has been applied to the synthesis of p-nitroanilides containing basic amino acids (7). CbzAsp(B-tert-butyl)OH was coupled to p-t-Boc- aminoaniline using a standard carbodiimide coupling procedure (Scheme 1). Deprotection of the p-t-Boc- aminoaniline with TFA, which also led to concomitant

CO2H

0

1 “.“o NO2

Ac Tyr Val Ala NH

FIGURE 2

L.A. Reiter

DEC HOB1

2 steps

NH-1-BOC \ ,COz-t-butyl

CBZVal AlaNH

3

NH-t-BOc

CBZTyr(0-1-buty1)ValAlaNH 1) TFA 2) NaB4 / HOAc

4 I

CBZryrValAlaNH 0 @o NO2

CBNal AlaNH

5

SCHEME 1

cleavage of the P-tert-butyl ester, followed by treatment with sodium perborate in acetic acid, gave the expected p-nitroanilide 2. With the compatibility of the oxidation with the P I aspartic acid group established, we pro- ceeded to extend the n-terminus using standard Cbz chemistry, yielding tripeptide 3 and tetrapeptide 4. Deprotection of 3 with TFA and oxidation gave the p-nitroanilide 5 in good yield. Similar treatment of 4, however, gave only a very low yield of the expected product. Apparently the tyrosine side chain was not completely compatible with the sodium perborate oxidation.

In the second and third approaches a pre-formed aspartic acid p-nitroanilide derivative was utilized. These approaches are similar to that of Yoshida el (7).

(8), with the exception that in our second approach the peptide chain was built sequentially from the starting aspartic acid p-nitroanilide derivative, while in our third

approach the P-carboxylic acid remained protected as an ester until the final step of the sequence.

Aspartic acid p-nitroanilide 6 was prepared in two steps from t-BocAsp(fl-tert-buty1)OH using first, the high yielding phosphorus oxychloride coupling procedure of Rijkers et al. (9a) and second, hydrogen chloride in dioxane (Scheme 2 ) . N-Terminal extension of 6 was carried out using standard Boc chemistry and N-hydroxysuccinimide esters. While this approach led to the preparation of p-nitroanilide derivatives 7-10, the purification of these compounds was complicated by their low solubility in organic solvents, a property which derives in part from the presence of the free carboxyiic acid. The third approach avoided this prob- lem.

Aspartic acid (P-tert-butyl ester) p-nitroanilide, 11, was prepared in two steps from FmocAsp(B-tert- buty1)OH using first the procedure of Rijkers, and second DBU in DMF to cleave the Fmoc group (10) (Scheme 3)t. Compound 11 was coupled to the tri-

+ While the ovcr oxidation of anilines containing a phenolic hydroxy group with sodium perborate has been observed (6 ) . in an initial control experiment, treatment of AcTyrOEt with sodiuni perborate in acetic acid did not lead to any significant reaction. The ineompat- ibility of 4 to the reaction conditions was therefore unexpected.

88

Piperidine in DMF also removed the FMOC group without af- fecting the p-nitroanilide functionality; however, we did not fully investigate this procedure, since we anticipated that separating excess

PNA substrates of ICE

1) p-nllroanlllne

* H 2 N Q L POCIS I pyrldlne

2) HCVdioxane

Ni 0 0

87% HCI

t-BOCNH

0 2 6

Go/ 1)TFA 2) AcOSu - DlEA

t-BOCAlaNH 28% AcAlaNH o /

7 NO2 N 0,

- N 0,

DlEA

t-BOCAlaNH 28% AcAlaNH

7 8

NO2

DlEA

21 % AcValAlaNH

10

1-BOCVal AlaNH

9

SCHEME 2

COz-t-butyl 1) p-nltroanlline POCIS I pyrldine

G O H "%

0 77% FMOC NH 2) DBU / DYF

NO2 11

CO2-t-bUtyl

0

13 "."a NO2

AcTyr(O-t-butyl)VaiAlaOH (1 2)

DECl HOSu I DMF - Ac Tyr(O-t-butyl) Val Ala NH

77%

TFA:CH2C12 50:40 anloole:thloanlsole 5:5

t 1 87%

SCHEME 3

peptide 12, and the product 13 was easily purified by recrystallization. If tetrapeptide 13 was purified to analytical purity, then the subsequent deprotection led without any further purification to analytically pure 1.

Each of these approaches has its merits and limitations. The oxidative approach allows the rapid con- struction of the desired peptidic intermediates, and these intermediates are easily handled and purified. However, the final oxidation step is not quantitative,

piperidine and the piperidine-containing side products from 11 would be more complicated than the simple work-up employed after the DBU reaction.

and purification of the desired product can be complicated by its low solubility in organic solvents. In addition, functionality sensitive to oxidation is not compatible. The iterative approach starting from aspartic acid p-nitroanilide (6) allows the preparation of peptide substrates with a wide range of functionality; however, the intermediates, as well as the final products, all contain a free carboxylic acid group which again imparts low solubility in organic solvents. The third approach is probably the most versatile in that the key aspartic acid derivative 11 can be coupled to any desired peptide to give an easily handled and purified intermediate. The final step, treatment with strong acid in the presence of appropriate scavengers, proceeds sufficiently cleanly

89

L.A. Reiter

such that purification of the final carboxylic acid- containing compounds is not necessary. However, an acid-sensitive functionality such as an "terminal t-Boc group is not compatibles.

Because of the high specificity that ICE has for an L-aspartic acid residue in the P I position (1, 3-5), the presence of D-aspartic acid-containing impurities in ICE substrates would complicate the kinetic analysis of the cleavage of these substrates by ICE. While no evi- dence of epimerization at the rx-carbon of the PI L-aspartic acid residue was observed by HPLC or NMR in any of the p-nitroanilide substrates prepared herein, as a final point, the presence of such impurities was directly assessed by preparing three p-nitroanilides containing a D-aspartic acid residue at P I . In order to allow comparison with products derived from each of the three synthetic paths, the three compounds synthesized were CbzValAla-D-Asp-p-nitroanilide (14), AcValAla-D-Asp-p-nitroanilide (15) and AcTyrValAla- D-Asp-p-nitroanilide (16). All three of these were prepared by coupling D-aspartic acid (P-iert-butyl ester) p-nitroanilide (17) to the appropriate peptides, CbzV- alAlaOH, AcValAlaOH or 12, followed by deprotection with TFA. The D-aspartic acid-containing peptides 14 and 15 were sufficiently differentiated from the corresponding L-aspartic acid-containing compounds 5 and 10 by HPLC on a C-18 column such that the presence of 100~ of the D-isomer in the L-isomer \vas readily detected. Thc separation of 16 from 1 was not readily achieved under these conditions; however, the precursors to 16 and 1, i.e. AcTyr(0-tert-buty1)ValAla-D and L-Asp@-tert-buty1)-p-nitroanilides, were separable, and again the presence of 10"~o of the D-isomer in the L-isomer was readily apparent. Since epimerization of the aspartic acid residue during the removal of the rert- butyl groups in the latter case is unlikely, we conclude that no significant amounts of D-aspartic acid- containing substrates are produced by any of the three methods.

In summary, we have described three approaches to the synthesis of p-nitroanilides containing a PI aspartic acid group. These approaches have led to a set of tetrapeptide and tripeptide p-nitroanilides 1, 5, 9 and 10 which are substrates for ICE and which can be used in a colorimetric assay for evaluating inhibitors of ICE (3). In addition, the syntheses described, and particularlj that which utilized the key intermediate 11, augment the methods already available for the preparation of peptidic p-nitroanilides containing an aspartic acid residue at P1 (8). The oxidative method for the preparation of p-nitroanilides that has been exemplified might also prove to be useful in the preparation of other peptidic p-nitroanilides.

S. However, an .Werrninal t-Boc group could probably be reintro- duced after the TFA treatment. This possibilit! was not investigated.

90

EXPERIMENTAL PROCEDURES

Geriernl procedures HPLC Analyses were performed on a Waters Nova- Pak C I X column (3.9 mm x 150 mm) and were run iso- cratically with a flow rate of 1 mL/min with mixtures of CH30H and water (both containing 0.1 % TFA); the percentage ofwater is noted for each experiment. Eluted materials were detected by UV monitoring at 220 nm. The D M F and CHzClz used in reaction mixtures were of Sure-Seal@ grade from Aldrich. Reaction mixtures and extracts were concentrated in V ~ C U O on a rotary evaporator; all extracts were dried with MgS04. Ab- breviations: diisopropylethylamine, DIEA; 1-(3- dimethylaniinopropyl)-3-ethylcarbodiimide hydrochloride. DEC.

CbzAssp(P-tert-buty1)-pii-Boc-amino)-anilide. CbzAsp- (P-tert-but] 1)OH dicyclohexylamine salt (2.52 g, 5.00 mmol), 4-(t-Boc-amino)aniline (1.04 g, 5.00 mmol), DEC (1.44 g, 7.5 mmol), N-hydroxybenzotriazole hy- drate (675 mg, 5.0 mmol) and DIEA (323 ing, 2.5 mmol) were combined in dry D M F (50 mL) and stirred at room temperature for 24 h. The mixture was diluted with ether (150 mL) and washed with 1 N HCI (2 x ), satd. NaHCO3 (2 x ), and 1 N HC1. After drying, filtration and concentration, an off-white solid was obtained which was recrystallized from cyclohexane/ EtOAc to give 2.17 g (84%) of tan solid. An analytical sample was prepared by recrystallization from hexane/ EtOAc: m.p. 131-133 " C (softens 120 "C); IH NMR (CDC13) 6 1.43 (s, 9H), 1.51 (s, 9H), 2.67 (dd, J=7 .1 , 17.2 Hz, lH), 2.97 (dd, J = 4.1, 17.2 Hz, lH), 4.6-4.7 (m, lH), 5.16 (s, 2H), 6.10 (br d, lH), 6.45 (br s, lH), 7.30 (d, J = 9.1 Hz, 2H), 7.35-7.45 (m, 7H), 8.41 (br s, 1H); MS (LSIMS) tnjz 513 (46, M + ) , 457 (40), 402 (38); 358 (40), 243 (32), 178 (31), 152 (100); [a]20,, -19.2 ' (c = 1.0, CH3OH); HPLC ret. time: 4.32 min (30",), 17.17 min (40%); Analysis: calcd. for C~H35N307: C, 63.14; H, 6.87; N, 8.18; found: C, 63.24; H, 6.94; N, 8.05.

Cb_Asl~-p-iiitroariilide, 2. CbzAsp(p-tert-butyl)-p-(t- Boc-amino)anilide (1.03 g, 2.00 mmol) in TFA (10 mL) was stirred at 0 " C for 30 min. The solution was then concentrated and the residue dissolved in HOAc (20 mL). NaBO3.4H20 (3.08 g, 20 mmol) was added and the mixture was stirred for 16 h at room temperature. The mixture was filtered and the solids washed with HOAc. The filtrate was concentrated to an orange gum which was taken up in EtOAc. After washing with water (3 x ), drying, filtration and concentration an orange oil was obtained which was chromatographed (5:40:55 H0Ac:EtOAc:hexane) to give 482 mg (62%) of pale yellow solid: m.p. 164-165 "C; IH NMR

J = 5.4, 16.7 Hz, lH), 4.5-4.6 (m, lH), 5.03 (s, 2H), 7.25-7.4 (m, SH), 7.8-79 (m, 3H), 8.21 (d, J = 9.2 Hz,

(DMSO-dti) 6 2.58 (dd,.J= 8.6, 16.7 Hz, lH), 2.74 (dd,


2H), 10.73 (s, lH), 12.42 (br s, 1H); MS (EI) m/z 387 (2, M + ), 224 (20), 178 (19), 108 (37), 91 (100); Analysis calcd. for C18H17N307: C, 55.81; H, 4.42; N, 10.85; found: C, 55.12; H, 4.21; N, 10.50.

CbzAluAsp(B-tert-butyl)-pit-Boc-umino)unilide. CbzAsp(P-tert-buty1)-p-(t-Boc-amino)anilide (1.17 g, 2.28 minol) was hydrogenatedover 10% Pd-C (120 mg) at 40 p.s.i. in CH3OH (20 mL) at room temperature for 1 h. The reaction mixture was filtered through a nylon filter and the filtrate concentrated to an oil. This was dissolved in CH2C12 (23 mL), and CbzAla-N- hydroxysuccinimide ester (803 mg, 2.51 mmol) was added. After being stirred at room temperature for 24 h, the reaction mixture was washed with 1 N HC1 (2 x ) and satd. NaHCO3 (2 x ) and dried. Filtration and concentration gave a white solid which was recrystallized from cyclohexane/EtOAc to give 750 mg (56%) of white powder: m.p. 183-185 "C (with gas evol.); 'H NMR

1.45 (s, 9H), 2.54 (dd, J = 7.7, 15.6 Hz, lH), 2.72 (dd, J=6.4, 15.6 Hz, lH), 4.0-4.1 (m, lH), 4.6-4.7 (m, lH), 4.99 (d, J = 12.5 Hz, lH), 5.03 (d, J = 12.5 Hz, lH), 7.25-7.4 (m, 7H), 7.48 (d, J = 8.9 Hz, 2H), 7.58 (br d, J = 6.5 Hz, lH), 8.23 (br d, J = 8.1 Hz, lH), 9.26 (br s, lH), 9.68 (br s, 1H); MS (LSIMS) m/z 585 (34, M + + l), 584 (44, M + ), 529 (41), 528 (29), 473 (50), 321

HPLC ret. time: 4.08 min (30%), 16.37 min (40%); Analysis calcd. for C30H40N408: C, 61.63; H, 6.90; N, 9.58; found: C, 61.66; H, 7.15; N, 9.52.

(DMSO-dh) 6 1.19 (d, J = 7 . 2 Hz, 3H), 1.35 (s, 9H),

(94), 243 (100); [C(I2'~ -28.9" ( c = 1.0, CH3OH);

Cbz VulA luAs~~-tert-buty~-p-(t-Boc-umino)unilide, 3. By the same procedure used to prepare CbzAlaAsp(P- tert-buty1)-p-(t-Boc-amino)anilide, CbzAlaAsp(b-tert- buty1)-p-(t-Boc-amino)anilide (1.97 g, 3.37 mmol) was hydrogenated in CH30H (50 mL) and coupled to CbzVal N-hydroxysuccinimide ester (1.29 g, 3.7 1 mmol) in DMF (12 mL). The reaction mixture was worked-up by dilution with satd. NaHCO3 and stirring for 15 min. The precipitated product was collected, washed with water and dried under high vacuum to give 2.16 g (94%) of a fine white powder. Recrystallization from EtOAc/ CH30H gave 937 mg (41%) of fine white powder: m.p. 232-233 "C (with gas evol.); 'H NMR (DMSO- ds )60 .81 (d , J=8 .7H~,3H) ,0 .84 (d , J=6 .9H~,3H) , 1.19 (d, J = 7.0 Hz, 3H), 1.33 (s, 9H), 1.45 (s, 9H), 1.85-2.0 (m, lH), 2.52 (dd, J = 7.6, 15.8 Hz, lH), 2.69 (dd,J=6.4, 15.8 Hz, lH),3.8-3.9(m, lH),4.2-4.3(m, IH), 4.6-4.7 (m, lH), 5.01 (d, J = 12.8 Hz, lH), 5.03 (d, J = 12.8 Hz, lH), 7.25-7.4 (m, 8H), 7.47 (d, J = 9.0 Hz, 2H), 8.08 (br d, J = 6.9 Hz, lH), 8.21 (br d, J = 7 . 9 Hz, lH), 9.25 (br s, lH), 9.79 (br s, 1H); MS (LSIMS)m/z684(5l,M+), 420(40), 119(100); [xI2O, -16.5 O ( c = 1.0, DMF); HPLC ret. time: 3.99min (30%), 31.12 min (40%); Analysis calcd. for C35H49N509: C, 61.47; H, 7.22; N, 10.24; found: C, 61.29; H, 6.93; N, 10.20.

CbzTyifO-tert-butyl) VulA luAsdfl-tert-butyl)-p-(t-Boc- amino)anilide, 4. By the same procedure used to prepare CbzAlaAsp(P-tert-buty1)-p-(t-Boc-amino)anilide, 3 (819 mg, 1.20 mmol) was hydrogenated in CH30H (70 mL)/DMF (15 mL) and coupled to CbzTyr(0-tert- butyl) N-hydroxysuccinimide ester (562 mg, 1.20 mmol) in the D M F remaining after concentration of the filtrate from the hydrogenation. The reaction mixture was worked up by dilution with satd. NaHC03 and stirring for 15 min. The precipitate was collected, washed with satd. NaHC03 and water and dried under high vacuum giving 976 mg (90%) of a white powder. Recrystalliza- tion from EtOAc/CH3OH gave an analytical sample: m.p. 224-225 "C; 'H NMR (DMSO-dh) 6 0.83 (d, J=6 .5 Hz, 3H), 0.86 (d, J=5.7 Hz, 3H), 1.21 (d, J = 6.8 Hz, 3H), 1.25 (s, 9H), 1.34 (s, 9H), 1.46 (s, 9H), 1.9-2.15 (m, lH), 2.5-2.6 (m, lH), 2.6-2.7 (m, 2H), 2.9-3.0 (m, lH), 4.15-4.4 (m, 3H), 4.6-4.7 (m, lH), 4.94 (s, 2H), 6.84 (d,J= 8.4 Hz, 2H), 7.18 (d,J= 8.4 Hz, 2H), 7.2-7.4 (m, 7H), 7.49 (d, J = 9.1 Hz, 2H), 7.53 (d, J = 8 . 7 Hz, lH), 7.93 (d, J = 9 . 0 Hz, lH), 8.16 (d, J = 7.0 Hz, lH), 8.24 (d, J = 8.0 Hz, lH), 9.27 (s, lH), 9.83 (s, 1H); MS (FAB) m/z 903 (23,M' + l), 639 (21), 583 (54), 524 (42), 469 (62), 453 (68), 108 (38), 91 (100); [ct]'OD -18.3 O ( c= 1.0, DMF); HPLC ret. time: 9.86 min (30%); Analysis calcd. for C4~H66Nh01 I : C, 63.84; H, 7.37; N, 9.31; found: C, 63.46; H, 6.96; N, 8.96.

CbzValAluAsp-p-nitrounilide, 5. By the same procedure used to prepare 2, 3 (900 mg, 1.32 mmol) was depro- tected with cold TFA (13 mL) for 4 h and oxidized with NaBO3 (2.03 g, 13.2 mmol) in HOAc (26 mL). After 18 h at room temperature, the reddish-orange reaction mixture was concentrated. Water and EtOAc (200 mL) were added to the residue and a small amount of 1 N HC1 added to bring the pH to about 1. The separated EtOAc layer was washed with 1 N HCI (2 x ), dried, filtered and concentrated to a light-brown solid. This was absorbed onto silica gel and charged onto a chromatography column. Elution (2.5:2.5:95 HOAc: CH30H:CH2C12) gave 474 mg (64%) of product. Fur- ther purification was achieved by preparative thin-layer chromatography (5:5:95 HOAC:CH~OH:CH~CI~): m.p. 204-206 "C (dec., softens 188 "C); IH N M R

J = 7.0 Hz, 3H), 1.21 (d, J = 7.1 Hz, 3H), 1.85-2.0 (m,

16.6 Hz, lH), 3.8-3.9 (m, lH), 4.2-4.3 (m, IH), 4.6- 4.7 (m, lH), 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, lH), 8.21 (d, J = 9.3 Hz, 2H), 8.38 (d , J= 7.3 Hz, lH), 10.48 (s, IH), 12.40 (br s, 1H); MS (LSIMS) m/z 580 (17, M + + Na), 558 (71, M + + l), 541 (19), 527 (32), 420 (51), 305 (100); [ c1I2OD -23.9 O (c = 1.0, DMF); HPLC ret. time: 9.02 min (40%); Analysis calcd. for C'bH31N509.0.25 H20: C, 55.56; H, 5.65; N, 12.46; found: C, 55.33; H, 5.56; N, 12.09.

(DMSO-d6) 6 0.80 (d, J z 8 . 1 Hz, 3H), 0.83 (d,

lH), 2.61 (dd, J = 7.7, 16.6 Hz, lH), 2.77 (dd, J = 6 . 0 ,

91

L.A. Reiter

t-BocAsp(8-tert-butyl)-p-nitroanilide. Using the procedure of Rijkers et al. (9a) t-BocAsp-(b-rert-butyl)OH (7.65 g, 25.0 mmol), p-nitroaniline (3.45 g, 25.0 mmol) and POCIS (4.22 g = 2.56 mL, 27.5 mmol) in pyridine (75 mL) gave, after being passed through a pad of silica gel (25:75 EtOAc:hexane), 8.93 g (87"") of light yellow foam: 'H NMR (CDC13) 6 1.47 (s, 9H), 1.49 (s, 9H), 2.69 (dd, J=6.7, 17.0 Hz, lH), 2.91 (dd, J=4 .3 , 17.0Hz, lH), 4.59 (m, lH), 5.90 (br d, lH), 7.69 (d, J = 9 . 2 Hz, 2H), 8.20 (d, J = 9 . 2 Hz, 2H), 9.20 (br s, 1H); MS (LSIMS) m/z 410 (22), 394 (9). 354 (18), 298

CH3OH) [lit. (9b) [xl2OD -19.8 ' (c = 1.0, CH3OH)I; HPLC ret. time: 4.28 min (30°/,), 14.10 min (40",,); Analysis calcd. for C19H~N307: C, 55.73; H, 6.65; N. 10.26; found: C, 55.50; H, 6.41; N, 10.22.

(loo), 282 (17), 254 (26); [ x I z o D -30.0" ( c = 1.0,

HAsp-p-nitroanilide hjidrochloride, 6. t-Boc-Asp(b-tert- buty1)-p-nitroanilide (8.68 g, 2 1.2 mmol) was dissolved in a mixture of dioxane (200 mL) and EtOAc (50 mL) and cooled to 0 ° C . The solution was then saturated with HC1 gas and stirred for 1 h at 0 " C . The HC1 was then purged from the reaction with a stream of N2 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 (> 1000,, some ether still present by NMR) of a light yellow powder: 'H NMR

J = 5.2, 17.5 Hz, lH), 4.32 (m, lH), 7.18 (d, J = 9.1 Hz,

(54, M i + l), 239 (92), 221 (loo), 197 (94), 195 (76).

(DMSO-d6)62.93(dd,J=7.3, 17.5Hz, lH),3.01(dd,

2H), 8.26 (d, J = 9.1 Hz, 2H); MS (LSIMS) i n / z 254

t-BocAlaAsp-p-nitroanilide, 7. 6 (2.90 g, 10.0 mmol), t-BocAla N-hydroxysuccinimide ester (2.86 g, 10.0 mmol) and DIEA (1.29 g, 10.0 mmol) in CHzClz (100 mL) were stirred for 24 h at room temperature. The turbid solution was washed with 0.1 N HCl (2 x ) and dried. Filtration and concentration gave a yellow foam that was chromatographed (5:30:65 H0Ac:Et- 0Ac:hexane to 5:35:60 H0Ac:EtOAc:hexane) to give 3.03 g (71 %) of a light yellow foam: m.p. 74-80 'C; 'H NMR (CDC13) 6 1.39 (s, 9H), 1.44 (d, J = 7.2 Hz, 3H), 2.77 (dd, J=4 .6 , 17.6 Hz, lH), 3.42 (dd, J=2 .5 , 17.6 Hz, lH), 4.1-4.2 (m, lH), 4.95-5.1 (m, 2H), 7.62 (br d, J = 9.2 Hz, lH), 7.93 (br d, J = 9.0 Hz, 2H), 8.18 (d, J = 9.0 Hz, 2H), 9.28 (br s, 1H); MS (LSIMS) m/z

DMF); HPLC ret. time: 3.40 rnin (40°0), 7.64min (50%); Analysis calcd. for ClsH24N408: C, 50.94; H, 5.70; N, 13.20; found: C, 50.86; H, 5.68; N, 12.85.

425 (46, M' + l), 369 (100); [2I2OD -11.8' ( c = 1.0,

AcAlaAsp-p-nitroanilide, 8. 7 (212 mg, 0.50 mmol) was dissolved in neat TFA ( 5 mL) and stirred at room temperature for 1 h. The solvent was removed and chased with CHzCl2 (2 x ). The residual gum was dissolved in dioxane (4mL) and treated with acetic acid N-hydroxysuccinimide ester (94 mg, 0.6 mmol), NaH- CO3 (210 mg, 2.5 mmol) and water (1 mL). This mix-

92

ture was stirred for 20 h and then concentrated. The residue was acidified with 1 N HCl and extracted with EtOAc. The extract was dried, filtered and concentrated to 189 mg (> 100%) of a pale yellow solid which was recrystallized from EtOAc/EtOH to give 51 mg (28",) ofhard tan crystals: m.p. 202-203 "C; 'H NMR

2.62 (dd, J = 7 . 9 , 16.5 Hz, lH), 2.78 (dd, J=5 .8 , 16.5 Hz, lH), 4.1-4.25 (m, lH), 4.6-4.65 (m, lH), 7.89 (d, J = 9 . 2 Hz, 2H), 8.15-8.25 (m, 3H), 8.34 (d, J = 7.5 Hz, 1H); MS (LSIMS) m/z 367 (100, M + + 1); [%]'OD + 17.3 (c = 1.0, DMF); HPLC ret. time: 3.23 min (603,); Analysis calcd. for C15H18N407: C, 49.18; H, 4.95; N, 15.30; found: C, 48.86; H, 4.89; N, 14.68.

(DMSO-db) 6 1.18 (d, J z 7 . 1 Hz, 3H), 1.85 (s, 3H),

t-Boc ValAlnAsp-p-nitroanilide, 9. 7 (424 mg, 1.00 mmol) was dissolved in neat TFA (10 mL) and stirred at room temperature for 1 h. The solvent was then concentrated and chased with CH2C12 (3 x ) giving a light yellow foam. This was suspended in CH2C12 (10 mL) and treated with DIEA (129 mg, 1.00 mmol). To this sus- pension was added t-BocVal N-hydroxysuccinimide ester (314 mg, 1.00 mmol), D M F (10 mL) and suffi- cient additional DIEA to make the mixture neutral. After stirring at room temperature for 24 h, the solvents were removed in vucuo (high vacuum) and the residue triturated with 1 N HC1. The resulting light yellow solid was collected, washed with water and dried. Chroma- tography (5: 3 5: 60 HOAc: Et0Ac:hexane to 5: 55 :40 H0Ac:EtOAc:hexane) gave 348 mg (66%) of a light yellow glass that was recrystallized from EtOAc to give 128 mg of an amorphous solid: m.p. 200-202 "C; lH

J = 6.8 Hz, 3H), 1.20 (d, J = 8.1 Hz, 3H), 1.37 (s, 9H), 1.85-1.95(m, lH),2.60(dd,J=7.5, 16.6Hz, 1H),2.76 (dd, J = 6 . 3 , 16.6 Hz, lH), 3.75-3.85 (m, lH), 4.2-4.3 (m, lH), 4.63 (m, lH), 6.77 ( d , J = 8.6 Hz, lH), 7.88 (d, J = 9 . 2 Hz, 2H), 8.02 (d, J = 7 . 7 Hz, lH), 8.21 (d, J = 9.2 Hz, 2H), 8.41 (d, J = 7.0 Hz, lH), 10.53 (br s, 1H); MS (LSIMS) m/z 546 (21, M + +Na), 524 (22, M + + l), 507 (12), 468 (45), 424 (57), 330 (35), 286 (34), 215 (100); [zlzoD -27.7 O ( c= 1.0, DMF); HPLC ret. time: 5.92 min (40%), 17.84 min (50%); Analysis calcd. for C~H33N509: C, 52.76; H, 6.35; N, 13.38; found: C, 52.45, H, 6.57; N, 12.76.

NMR (DMSO-d6) 6 0.78 (d, J = 6.7 Hz, 3H), 0.81 (d,

AcValAlaAsp-p-nitroanilide, 10. By the same procedure used to prepare 8, 9 (174 mg, 0.33 mmol) was depro- tected in neat TFA ( 5 mL) and coupled with acetic acid N-hydroxysuccinimide ester (63 mg, 0.40 mmol) in the presence of NaHC03 (139 mg, 1.65 mmol) in dioxane/ water (5 mL, 4: 1) giving, after recrystallization from EtOAc/EtOH, 33 mg (21%) of light yellow powder: m.p. 196-200 " C (dec.); ' H NMR (DMSO-d6) 6 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, lH), 2.60 (dd, J = 7.5, 16.6 Hz, lH), 2.75 (dd, J = 6.2, 16.6 Hz,


N3Os: C, 54.36; H, 6.19; N, 13.59; found: C, 54.69; H, 6.22; N, 13.37.

lH), 4.1-4.2 (m, lH), 4.2-4.3 (m, lH), 4.6-4.7 (m, lH),7.8-7.9(m,3H),8.1l(d,J=6.8Hz,lH),8.21(d, J = 9.3 Hz, 2H), 8.27 ( d , J = 7.2 Hz, lH), 10.51 (s, lH), 12.45 (br s, 1H); MS (LSIMS) m/z 488 (6, M + + Na), 466 (23, M + + l), 449 (7), 328 (15), 213 (44), 142 (100); [ciI2"D -24.9' ( c = 1.0, DMF); HPLC ret. time: 9.72 rnin (60%); Analysis calcd. for C20H27N508: C, 51.60; H, 5.85; N, 15.05; found: C, 50.16; H, 5.79; N, 14.06.

FMOCAsdj-tert-butyo-p-nitroanilide. Using the procedure of Rijkers et al. (9a) FMOCAsp(j-tert-buty1)OH (10.29 g, 25.0 mmol), p-nitroaniline (3.45 g, 25.0 mmol) and POC13 (4.22 g = 2.56 mL, 27.5 mmol) in pyridine (75 mL) gave, after being passed through a pad of silica gel (4050 EtOAc:hexane), 12.38 g (93 %) of light-yellow foam. Recrystallization of a portion from cyclohexane/ EtOAc gave an analytical sample as a light-yellow powder: m.p. 163-164°C (dec. with gas evol.); IH NMR (CDCl3) 6 1.47 (s, 9H), 2.68 (dd, J = 7.4, 16.8 Hz, lH), 2.97(dd,J=3.8, 16.8Hz, lH),4.24(t ,J=6.8Hz, lH), 4.51 (d , J= 6.8 Hz, 2H), 4.6-4.7 (m, lH), 6.0-6.15 (m, lH), 7.25-7.35 (m, 2H), 7.35-7.45 (m, 2H), 7.58 (d,

J = 7.6 Hz, 2H), 8.21 (d, J = 9.2 Hz, 2H), 9.0-9.1 (br s, 1H); MS (LSIMS) m/z 532 (11, M + + l), 476 (29), 179 (100); [ XI2OD + 39.2 (c = 1.0, DMF); HPLC ret. time: 3.54 rnin (20%), 13.02 rnin (30%); Analysis calcd. for C29H29N307: C, 65.52; H, 5.50; N, 7.91; found: C, 65.61; H, 5.58; N, 7.80.

J=7 .4Hz, 2H), 7.67 (d, J=9 .2Hz, 2H), 7.77 (d,

HAsp(P-tert-butyo-p-nitroanilide, 11. FMOCAsp(j-tert- buty1)-p-nitroanilide (2.13 g, 4.0 mmol) and DBU (609 mg, 4.0 mmol) were stirred together in dry DMF (40 mL) for 1 h. The reaction mixture was then diluted with ether (200 mL) and extracted with 1 N HC1 (3 x 20 mL). The combined aqueous extracts were washed with ether and then basified with a calculated amount of K2CO3 (8.3 g, 60 mmol). This aqueous solution was extracted with ether (9 x 50 mL). The combined ether extracts were dried. Filtration and concentration gave a yellow oil consisting of the desired product, some DBU and DMF. This was taken up in 1:l Et0Ac:hexane (50 mL) and poured onto a pad of silica gel. Elution with 1:l Et0Ac:hexane (7 x 50 mL) and EtOAc (3 x 200 mL) completely eluted the product. Concentration of the appropriate fractions gave 955 mg (77%) of light yellow solid. Recrystallization from cyclohexane/EtOAc gave an analytical sample as a pale yellow powder: m.p. 143-144°C; 'H NMR (CDCI3) 6 1.44 (s, 9H), 2.1-2.3 (br s, 2H), 2.82 (dd, J=6.7, 16.9Hz, lH), 2.89 (dd,J=4.4, 16.9Hz, lH), 3.82 (dd,J=4.4, 6.7 Hz, IH), 7.77 (d, J = 9.2 Hz, 2H), 8.20 (d, J=9 .2 Hz, 2H), 10.0-10.1 (br s, 1H); MS (LSIMS) m/z 310 (43, M + + l), 254 (97), 154 (100); [ XI2'D -4.9 O (c = 1.0, DMF); HPLC ret. time: 2.07 rnin (40%), 5.56 rnin (50%); Analysis calcd. for C14H19-

CbzValAlaOCH3. CbzVal N-hydroxysuccinimide ester (8.71 g, 25.0 mmol), alanine methyl ester hydrochloride (3.49 g, 25.0 mmol), DIEA (3.23 g, 25.0 mmol) were combined in CH2C12 (250 mL) and stirred at room temperature for 20 h. The reaction mixture was alternately washed with satd. NaHC03 and 1 N HCl(2 x ), dried, filtered and concentrated giving a white solid. This was recrystallized from EtOAc to give 5.49 g (65%) of fine white needles. A second crop of 1.56 g (18%) of fine white needles was obtained from the mother liquors: m.p. 163-164°C [lit. (11) m.p. 162.5-163 "C]; 'H NMR (CDC13) 6 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, lH), 3.74 (s, 3H), 4.01 (br t, lH), 4.58 (pent, J=7 .2Hz , lH), 5.11 (s, 2H), 5.38 (br d, lH), 6.38 (br d, lH), 7.3-7.4 (m, 5H); [c1I2O, -46.0' ( c= 1.0, CH30H) [lit. (12) -49.45 O (c = 1.65, CH3OH)I; HPLC ret. time: 2.45 rnin (40%), 4.33 rnin (50%)).

CbzTyq'O-tert-buty1)ValAlaOCH.j. By the same procedure used to prepare CbzAlaAsp(0-tert-buty1)-p-(t-Boc- amino)anilide, CbzValAlaOCH3 (6.67 g, 19.8 mmol) was hydrogenated in CH30H (100 mL) and coupled with CbzTyr(0-tert-butyl) N-hydroxysuccinimide ester (9.28 g, 19.8 mmol) in a 1:l mixture of CH2C12 and DMF (200 mL). After stirring at room temperature for 18 h the mixture was concentrated to remove the CH2Cl2 and then water (300 mL) was added to precipitate the product. The solid was collected, washed with water and dissolved in EtOAc (500 mL). This solution was washed with satd. NaHC03 (2 x ) and l N HCI (2 x ) and dried. Filtration and concentration gave a white solid which was recrystallized from cyclohexane (100 mL)/EtOAc (70 mL) yielding 7.10 g (65%) of white fluffy solid. A second crop of 1.25 g (1 1%) was obtained from the mother liquors: m.p. 189-190 "C;

(d, J = 7.0 Hz, 3H), 1.2-1.35 (m, 12H), 1.85-2.05 (m, lH), 2.83 (dd ,J= 10.9, 13.8 Hz, lH), 2.92 (dd, J = 3.7, 13.8 Hz, lH), 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, lH), 7.83 (d, J = 9.2 Hz, lH), 8.45 (d , J= 6.5 Hz, 1H); MS (LSIMS)

CH30H); HPLC ret. time: 8.04 rnin (40%), 15.37 rnin (50%); Analysis calcd. for C30HdlN307: C, 64.84; H, 7.44; N, 7.56; found: C, 64.96; H, 7.35; N, 7.52.

'H NMR (DMSO-ds) 6 0.85 (d, J = 6.8 Hz, 3H), 0.88

m/Z 556 (100, M' + l), 453 (3 1); [%]'OD -35.1 (C = 1.0,

AcTyq'O-tert-butyl) ValAlaOCH3. By the same procedure used to prepare CbzAlaAsp(0-tert-buty1)-p-(t-Boc- amino)anilide, CbzTyr(O-tert-butyl)ValAlaOCH3 (5.55 g, 10.0 mmol) was hydrogenated in CH30H (100 mL) and coupled with acetyl chloride (942 mg, 12 mmol) in the presence of DIEA (1.55 g, 12 mmol) in

93

L.A. Reiter

T H F (100 mL). After stirring at room temperature over- night, the reaction mixture was concentrated and the residue dissolved in CHCI?. This solution was washed with 1 N HCI, satd. NaHCO3 and dried. Filtration and concentration gave a gel-like solid which was recrystallized from EtOAc/CHJOH to give 2.86 g (62"") of a gel-like solid which was dried under high vacuum. A second crop of 1.42g (31"") was obtained from the mother liquors: m.p. 209-211 'C; 'H NMR (DMSO- d6) 6 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, 3H), 1.94 (hept,J= 6.8 Hz, lH), 2.66 ( d d , J = 10.0, 14.0 Hz, lH), 2.90 (dd, J=4 .3 , 14.0 Hz, lH), 3.59 (s, 3H), 4.15-4.3 (m, 2H), 4.5-4.6 (m, lH), 6.82 ( d , J = 8.4 Hz, 2H), 7.12 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 9.0 Hz, lH), 8.06 (d, J = 8.4 Hz, lH), 8.39 ( d , J = 6.6 Hz, 1H); MS (LSIMS) mjz 464 (100, M + + 1); [ x]'"D -17.0 ' (C = 1.0, DMF); HPLC ret. time: 2.75 min (40",), 5.62 min (50""); Analysis calcd. for C~H37N306: C, 62.18; H, 8.05; N, 9.06; found: C, 62.27; H, 8.18; N, 9.00.

AcT~~O-tert-but~l)YulAluOH, 12. AcTyr(U-reri-buty1)- ValAlaOCH3 (2.32 g, 5.0 mmol) was slurried in lo", aqueous CH30H (50 mL) and treated with LiOH.H20 (1.05 g, 25.0 mmol) in one portion. After stirring at room temperature for 2 h, the reaction was quenched by the addition of an cxccss of sulfonic acid ion- exchange resin (56 g, 125 mequiv. of H + ). After being stirred for 15 min, the mixture was filtered and the resin washed thoroughly with CH30H. The filtrate was concentrated to give a white solid which was recrystallized from EtOAc/CH3OH yielding, after drying under high vacuum, 1.96g (87%) of a white powder: m.p. 191- 192 "C (dec. with gas evol.); 'H NMR (DMSO-d6) 6 0.82 (d, J = 6 . 8 Hz, 3H), 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, lH), 2.66 (dd, J = 10.1, 13.9Hz, lH), 2.92 (dd ,J=4.2 , 13.9Hz. IH), 4.1-4.25 (m, 2H), 4.5-4.6 (m, lH), 6.82 ( d , J = 8.4 Hz, 2H), 7.12 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 9.0 Hz, lH), 8.07 (d, J = 8.4 Hz, lH), 8.23 (d, J = 6.9 Hz, 1H); MS (LSIMS)m/z450(53,M+ + I), l89(100); [ r I 2 ( ' D -8.7' (c = 1, DMF); HPLC ret. time: 4.03 min (50"") ; Analy- sis calcd. for C ~ ~ H X N @ ~ : C, 61.45; H, 7.85; N, 9.35; found: C, 61.18; H, 8.05; N, 9.26.

AcTyr(O-tert-butylj ValAl~IAs~b-tert-bui~,l~-p- nitroanilide, 23. 12 (687 mg, 1.53 mmol), 11 (473 mg, 1.53 mmol), N-hydroxysucciniinidc (264 nig, 2.29 mmol) and DEC (352 mg, 1.84 mmol) were combined in dry D M F (15 mL), and the resulting pale yellow solution was stirred at room temperature for 44 h. The reaction mixture was diluted with 1 N HCI and the resulting precipitated solid 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 satd. aqueous NaHC03, triturated for 15 min and collected. After washing with water and

94

drying under high vacuum, 875 mg (77%) of a white powder was obtained. A portion of this was recrystallized from EtOAc/CH30H to give an analytical sample: m.p. 234-235 " C (dec. with gas evol.); 'H NMR

J = 7.1 Hz, 3H), 1.24 (s, 9H), 1.34 (s, 9H), 1.74 (s, 3H), 1.9-2.05 (m, lH), 2.57 (dd ,J= 7.6, 15.9 Hz, lH), 2.63- 2.72 (m, lH), 2.76 (dd, J = 6.8, 15.9 Hz, IH), 2.92 (dd, J=O.6, 10.1 Hz, lH), 4.1-4.2 (m, lH), 4.2-4.3 (m, IH), 4.5-4.6 (m, lH), 4.6-4.7 (m, lH), 6.82 (d, J = 8.4 Hz, 2H), 7.13 ( d , J = 8.4 Hz, 2H), 7.85-7.95 (m, 3H), 8.08 (d, J = 8.4 Hz, lH), 8.14 (d, J = 6.7 Hz, IH), 8.22 ( d , J = 9.3 Hz, 2H), 8.38 ( d , J = 7.5 Hz, lH), 10.52

(DMSO-d6) 6 0.82 (t, J = 7 . 1 Hz, 6H), 1.21 (d,

(s, 1H); MS (LSIMS) m/z 741 (32, M + + l), 710 (34),

DMF); HPLC ret. time: 21.45 min (35%); Analysis calcd. for C37H~N6010: C, 59.98; H, 7.08; N, 11.35; found: C, 59.78; H, 6.90; N, 11.20.

432 ( 5 3 ) , 361 (75), 305 (100); [c1I2O, -16.4' (c= 1,

AcTi,rValAIuAsp-p-izitroanilide, I . A slurry of 13 (148 mg, 0.20 mmol) in CH2C12 (10 mL) at 0 " C was treated with a prechilled mixture of TFA:anisole:thio- anisole (90:5:5, 10 mL). The resulting solution was stirred at 0 'C for 30 min and at room temperature for 4 h. The mixture was concentrated, chased once with CH'Cl,, and the residue was triturated with ether for a few hours. The solid was collected, washed thoroughly with ether and dried under vacuum to give 109 mg (87",) of a white powder: m.p. 205-206 " C (dec.); 'H NMR (DMSO-d6) 6 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, lH), 2.5-2.7 (m, 2H), 2.7-2.9 (m, 2H), 4.1-4.2 (m, IH), 4.2-4.3 (m, lH), 4.4-4.5 (m, IH), 4.6-4.7 (m, lH), 6.61 ( d , J = 8 . 4 H z , 2H), 7.01 ( d , J = 8 . 4 H z , 2H), 7.82 (d. l H , partially obscured by adjacent peak), 7.88 (d, J = 9 . 2 Hz, 2H), 8.02 (d, J = 8 . 2 H z , lH), 8.14 (d, J = 6 . 7 Hz, lH), 8.20 (d, J = 9 . 2 H z , 2H), 8.35 (d, J = 7.3 Hz, lH), 9.14 (br s, lH), 10.51 (s, 1H); MS (LSIMS) nzjz 651 ( 5 , M + + Na), 629 (3, M + + l), 491 (7), 424 (3), 376 (18), 305 (47), 178 (136), 136 (100); [rIzoD -18.8' ( c = 1.0, DMF); HPLC ret. time: 4.37 min (509,0), 14.14 min (60%); Analysis calcd. for C N H ~ ~ N ~ O ~ U : C, 55.40; H, 5.77; N, 13.37; found: C, 55.60; H, 6.24; N, 13.49.

Cbz ValAlrOH. By the same procedure used to prepare 12, CbzValAlaOCH3 (673 mg, 2.00 mmol) gave upon drying under high vacuum 623 mg (97%) of a white powder: m.p. 173-174 "C [lit. (12) 172-173 "C].

CbzVa/alAIa-D-As~b-tert-butyl)-p-nitroanilide. By the same procedure used to prepare 13, CbzValAlaOH (322 mg, 1.00 mmol) and 17 (309 mg, 1.00 mmol) gavc after recrystallization from cyclohexane/EtOAc 362 mg (59"") of a white powder: m.p. 212-214 "C; 'H NMR

J = 6.7 Hz, 3H), 1.22 (d, J = 7.0 Hz, 3H), 1.85-2.0 (m, lH), 2.57 (dd, J = 8.6, 16.0 Hz, lH), 2.83 (dd, J = 5.3,

(DMSO-dc,) 6 0.83 (d, J = 6 . 6 H z , 3H), 0.84 (d,


(d, J=6 .7 Hz, 3H), 1.21 (d, J=7 .0Hz, 3H), 1.36 (s, 9H), 1.85 (s, 3H), 1.85-2.0 (m, IH), 2.56 (dd, J=8.4, 16.1 Hz, lH), 2.81 (dd ,J= 5.5, 16.1 Hz, lH), 4.05-4.2 (m, 2H), 4.7-4.85 (m, lH), 7.94 (d, J = 8.2 Hz, lH), 8.02 (d, J = 9.3 Hz, 2H), 8.26 (d, J = 9.3 Hz, 2H), 8.44 (d, J = 5.4 Hz, lH), 8.52 (d, J = 8.3 Hz, lH), 10.23 ( s , 1H); MS (LSIMS) mjz 522 ( M + + 1, 13), 466 (44), 328 (50), 213 (50), 142 (100).

16.0 Hz, lH), 3.8-3.9 (m, lH), 4.1-4.2 (m, lH), 4.7- 4.8 (m, lH), 4.99 (d, J = 12.6H2, lH), 5.02 (d, J = 12.6 Hz, lH),7.25-7.35(m,5H),7.5O(d,J= 8.5 Hz, 1H),8.04 (d, J = 9.3 Hz, 2H), 8.22 (d, J = 9.3 Hz, 2H), 8.47 (d , J= 5.0 Hz, lH), 8.62 (d , J= 8.2 Hz, lH), 10.14 (s, 1H); MS (LSIMS) mjz 614 ( M + + 1, 9), 558 (48), 420 (loo), 305 (78).

CbzVulAlu-D-Asp-p-nitrounilide, 14. By the same procedure used to prepare 1, CbzValAla-D-Asp@-tert-buty1)- p-nitroanilide (61 mg, 0.10 mmol) gave, after trituration with hexane (instead of ether), 46 mg (82%) of an off- white powder: m.p. 201-203 "C; lH NMR (DMSO-

1.8-2.0 (m, lH), 2.63 (dd, J = 8.0, 16.8 Hz, lH), 2.80 (dd,J= 5.1, 16.8 Hz, lH), 3.8-3.9 (m, lH), 4.1-4.2 (m, lH), 4.7-4.8 (m, lH), 4.99 (d, J = 12.6Hz, lH), 5.02 (d , J= 12.6 Hz, lH), 7.2-7.4 (m, 5H), 7.48 (d , J= 8.52,

d6) d 0.83 (d , J= 5.2 Hz, 6H), 1.22 (d,J=7.0 Hz, 3H),

lH), 8.05 (d, J = 9.3 Hz, 2H), 8.22 (d, J = 9.3 Hz, 2H), 8.45 (d, J = 5.1 Hz, lH), 8.56 (d, J = 8.0 Hz, lH), 10.14 (s, lH);MS(LSIMS)m/z580(M+Na,50),558(M+ 1, 64), 420 (47), 305 (36), 155 (100); HPLC ret. time: 8.55 rnin (40%)).

Ac VulAIuOBn. By the same procedure used to prepare 13, AcValOH (2.39 g, 15.0 mmol), HAlaOBn.HOTs (5.00 g, 15.0 mmol) and DEC (3.83 g, 20 mmol) in the presence of HOBt (2.30g, 15.0 mmol) (instead of N-hydroxysuccinimide) and 4-methylmorpholine (1.52 g, 15.0 mmol) in DMF (30 mL) gave a white solid which was taken up in CHC13 and washed with 1 N HC1 and satd. NaHC03. Concentration of the dried extract gave 3.49g (73%) of a white flaky solid; 'H

J=6.7Hz,3H), 1.42(d,J=7.2Hz,3H), 1.95-2.15(m, 4H), 4.2-4.3 (m, lH), 4.55-4.65 (m, lH), 5.15 (d,

J = 9.3 Hz, IH), 6.40 (d, J = 6 . 8 Hz, lH), 7.3-7.4 (m, 5H).

AcVulAluOH. By the same procedure used to prepare CbzAlaAsp(/3-tert-butyl)-p-(t-Boc-amino)anilide, Ac- ValAlaOBn (3.37 g, 10.5 mmol) was hydrogenated to give after recrystallization from EtOAcjCH30H 938 mg (39%) of white fluffy solid: m.p. 234-235 "C. A second recrystallization gave an analytical sample: m.p. 240-

NMR (CDCI3) 6 0.93 (d, J = 6.8 Hz, 3H), 0.94 (d,

J = 12.3 Hz, lH), 5.19 (d, J = 12.3 Hz, lH), 6.13 (d,

242 "C; 'H NMR (DMSO-dh) 6 0.82 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H), 1.25 (d, J = 7.3 Hz, 3H), 1.84 (s, 3H), 1.9-2.0 (m, lH), 4.1-4.2 (m, 2H), 7.85 (d, J = 9.1 Hz, lH), 8.25 (d, J = 7.0 Hz, lH), 12.46 (br s, 1H); Analysis calcd. for CIoH18N204: C, 52.16; H, 7.88; N 12.17; found: C, 52.12; H, 7.86; N, 12.02.

A c VulAlu-D-A&-tert-butylf-p-nitroanilide. By the same procedure used to prepare 13, AcValAlaOH (230 mg, 1.00 mmol) and 17 (309 mg, 1.00 mol) gave after recrystallization from cyclohexane/EtOAc 179 mg (34%) of fine white crystalline solid: m.p. 227-229 "C (dec.); 'H NMR (DMSO-dh) 6 0.82 (d, J = 6.7 Hz, 3H), 0.83

Ac ValAlu-D-Asp-p-nitrounilide, 15. By the same procedure used to prepare 1, AcValAla-D-Asp(fl-tevt-buty1)- p-nitroanilide (52 mg, 0.10 mmol) gave after trituration with hexane (instead of ether) 47 mg (loo',;) of a pale yellow powder: 1n.p. 218-220 "C; 'H NMR (DMSO- d6) 6 0.83 (d , J= 6.3 Hz, 6H), 1.21 (d , J= 7.0 Hz, 3H), 1.85 (s, 3H), 1.85-2.0 (m, lH), 2.64 (dd, J = 8.1, 16.7 Hz, lH), 2.81 (dd,J= 5.4, 16.7 Hz, lH), 4.05-4.2 (m, 2H), 4.7-4.8 (m, lH), 7.93 (d , J= 8.1 Hz, lH), 8.04 (d, J = 9.3 Hz, 2H), 8.25 (d, J = 9.3 Hz, 2H), 8.45 (d, J = 5.4 Hz, lH), 8.50 (d , J= 8.1 Hz, lH), 10.18 (s, 1H); MS (LSIMS) m/z 488 ( M + + Na, 23), 466 ( M + + 1, 30), 328 (38), 213 (48), 142 (100); HPLC ret. time: 10.21 min (60%).

AcTyfO-tert-butyl) ValAla-D-Asyf,&tert-but,vl)-p- nitroanilide. By the same procedure used to prepare 13, 12 (449mg, 1.00 mmol) and 17 (309mg) gave after recrystallization from EtOAc/CH3OH 258 mg (35;,) of fluffy white solid: m.p. 240-242 ' C (dec.); 'H NMR (DMSO-d6) 6 0.85 (t, J = 6.7 Hz, 6H), 1.2-1.25 (m,

2.5-2.7 (m, 2H), 2.8-2.95 (m, 2H), 4.1-4.2 (m, 2H), 4.55-4.65(m, lH),4.7-4.8(m, 1H),6.80(d,J=8.5 Hz,

12H), 1.36 (s, 9H), 1.74 (s, 3H), 1.9-2.05 (m, lH),

2H), 7.12 (d, J = 8.5 Hz, 2H), 7.99 (d, J = 7.2 Hz, IH), 8.05 (d, J = 9.3 Hz, 2H), 8.10 (d, J = 8.4 Hz, lH), 8.29 (d, J = 9.3 Hz, 2H), 8.48 (d, J = 4.9 Hz, lH), 8.65 (d, J = 8.4 Hz, lH), 10.11 (s, 1H); MS (LSIMS) tnjz 763 (M+ + Na, 2), 741 (M+ + 1, 3), 724 (4), 710 (9), 685 (12), 547 (14), 491 (33), 361 (53), 305 (78), 178 (100); HPLC ret. time: 22.40 rnin (35%).

AcTyrVa'alAfu-D-Asp-p-nitrounilide, 16. By the same procedure used to prepare 1, AcTyr(0-tert-buty1)ValAla- D-Asp@-tert-buty1)-p-nitroanilide (74 mg, 0.10 mmol) gave 59 mg (94%) of a white powder; m.p. 204-205 "C (dec.); 'H NMR (DMSO-dc) 6 0.85 (t, J = 6.3 Hz, 6H), 1.22 (d , J= 6.6 Hz, 3H), 1.74(s, 3H), 1.85-2.0 (m, lH), 2.5-2.65 (m, 2H), 2.65-2.9 (m, 2H), 4.1-4.2 (m, 2H), 4.5-4.6 (m, lH), 4.7-4.8 (m, lH), 6.59 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 8.5 Hz, 2H), 7.96 (d, J = 7.2 Hz, lH), 8.0-8.1 (m, 3H), 8.29 (d, J = 9.3 Hz, 2H), 8.46 (d, J=4 .9 Hz, lH), 8.62 (d, J = 8.4 Hz, lH), 9.13 (s, 1H); MS (LSIMS) mjz 651 ( M + +Na, 66), 629 ( M + + 1, 19), 491 (25), 305 (71), 177 (100); HPLC ret. time: 4.38 rnin (50%), 14.22 rnin (60%).

FMOC-D-Asp@-tert-butylf-p-nifrounilide. By the same procedure used to prepare FMOCAsp(P-tert-buty1)-p-

95

L.A. Reiter

nitroanilide, FMOC-D-Asp(P -terr-buty1)OH (4.94 g, 12.0 mmol) gave, after being passed through a pad of silica gel, 5.62 g (88%) of a light-yellow foam whose 'H-NMR was identical to that of FMOCAsp(P-terr- buty1)-p-nitroanilide.

H-D-Asp(P-tert-bur~l)-p-nitroanilide, 17. By the same procedure used to prepare 11, FMOC-D-Asp(P-terr- buty1)-p-nitroanilide (5.31 g, 10.0 mmol) gave, after being passed through a pad of silica gel and triturated with hexane/EtOAc, 2.44 g (79"") of a fine light-yellow solid; m.p. 143-144 " C ; [ x ] ) ~ ~ + 4.4 ' (c = 1.0, DMF).

REFERENCES

1.

2.

3 .

4.

5.

Thornberry, N.A., Bull, H.G.. Calaycay. J.R.. Chapman, K.T.. Howard, A.D., Kostura, M.J., Miller, D.K., Molineaux, 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., Gaffne), E.P.. Lim- juco, G., Palyha, O.C., Raju, S.M.. Rolando, A.M., Salleq-, J.P.. Yamin, T.-T., Lee, T.D., Shivcly, J.E., MacCross, M., Mumford, R.A., Schmidt, J.A. & Tocci, M.J. (1992) Nurure (Loiidon) 356. 768-774 and references therein The PJP',, notation of Schechter and Bcrger is used herein: Schechter. I. & Berger, A. (1967) Biochern. Bioph!.s. Res. Coin- intin. 27, 157-162 Daumy, G.O., Reiter, L.A., Andrew, G.C., Arriola. M.W.. Con- tillo, L.G., McColl, A.S., Robinson, R.P. & Singleton, D.H., submitted Sleath, P.R., Hendrickson. R.C.. Kronheim. S.R.. March, C.J. & Black, R.A. (1990)J. Bid. Chew. 265. 13526-14528 Howard, A.D., Kostura, M.J.. Thornberr). N., Ding. G.J.F., Limjuco, G., Weidner, J., Salley, J.P.. Hogquist, K.A., Chaplin.

D.D., Mumford, R.A., Schmidt, J.A. & Tocci, M.J . (1991) J .

6. McKillop. A. & Tarbin, J.A. (1980) Tetrahedron Lett. 24, 1505- 1508; McKillop, A. & Tarbin, J.A. (1987) Tetrahedron 43, 1753- 1758

7. Burdick, D.J., Struble, M.E. & Burnier, J.P. (1993) Terrahedron Lerr. 34, 389-2592.

8. Yoshida. N., Tsuruyama, S., Nagata, K., Hirayama, K., Noda, K. & Makisumi, S. (1988)J. Biochem. 104,451-456; Noda, K., Imanaga, Y., Takei, K. & Yoshida, N. (1991) Bull. Fac. Home L f e Sci. 22, 19-24

9. (a) Rijkers, D.T.S., Hemker, H.C., Nefkens, G.H.L. & Tesser, (3.1. (1991) Recl. Truv. Chint. Pay.s-Eas 110, 347-348; (b)for another synthesis of aspartic acid p-nitroanilides see: Noda, K., Oda, M., Sato, M. & Yoshida, N. (1990) Int. J . Peptide Protein Res. 36, 197-200

10. Wade,J.D., Bedford, J.. Sheppard, R.C.&Tregcar,G.W. (1991) Pepride Res. 4, 194-199; Chang, D.-D., Waki, M., Ahmad, M., Meienhofer, J., Lundell, E.O. & Huag, J.D. (1980) Int. J. Peptide Proreiii Rrs. 15, 59-66

11. Seu, J.H.. Smeby, R.R. & Bumpus, F.M. (1962) J . Am. Chem. Soc. 84, 4948-4950

12. Klieger, E. & Schroedcr, E. (1963) Justus Liehigs Ann. Chem. 661, 193-201

IiI l l7Jl i i lO/ . 147, 2964-2969

Address:

Lartreiice A . Reirer Department of Medicinal Chemistry Central Research Division Pfizer Inc. Eastcrn Point Road Groton, C T 06340 C'SA

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peptidic p-nitroanilide substrates of interleukin-1β-converting enzyme

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