tert-butyl (2S)-2,4-diamino-4-oxobutanoate

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Fragment I was prepared by stepwise chain elongation using the mixed anhydride procedure at -15° C. with careful temperature control. As seen in the scheme of FIG. 2 mixed anhydride (M.A.) coupling (isobutylchloroformate) of Z-Glu(OtBu)-OH with H-Asn-OtBu provided Z-Glu(OtBu)-Asn-OtBu. Catalytic hydrogenation (10% Pd-C) of this compound cleaved the N-terminal protective group and the dipeptide was coupled to Z-Ala-OH using mixed anhydride coupling to give Z-Ala-Glu (OtBu)-Asn-OtBu. The N-terminal was again deprotected by catalytic hydrogenation and the tripeptide coupled with Z-Glu(OtBu)-OH to give the protected tetrapeptide Z-Glu(OtBu)-Ala-Glu(OtBu)-Asn-OtBu which is deblocked by catalytic hydrogenation to provide the desired protected tetrapeptide (1). Fragment I was prepared by stepwise chain elongation at -15° C.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Fragment I was prepared by stepwise chain elongation using the mixed anhydride procedure at -15° C. with careful temperature control. As seen in the scheme of FIG. 2 mixed anhydride (M.A.) coupling (isobutylchloroformate) of Z-Glu(OtBu)-OH with H-Asn-OtBu provided Z-Glu(OtBu)-Asn-OtBu. Catalytic hydrogenation (10% Pd-C) of this compound cleaved the N-terminal protective group and the dipeptide was coupled to Z-Ala-OH using mixed anhydride coupling to give Z-Ala-Glu (OtBu)-Asn-OtBu. The N-terminal was again deprotected by catalytic hydrogenation and the tripeptide coupled with Z-Glu(OtBu)-OH to give the protected tetrapeptide Z-Glu(OtBu)-Ala-Glu(OtBu)-Asn-OtBu which is deblocked by catalytic hydrogenation to provide the desired protected tetrapeptide (1). Fragment I was prepared by stepwise chain elongation at -15° C.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: N-Benzyloxycarbonyl-γ-t-butyl-L-glutamate (50.7 g, 0.15 mol) in 300 ml freshly distilled THF was placed in a 2-liter 3-neck round bottom flask fitted with a thermometer, mechanical stirrer and dropping funnel and immersed in a dry ice-alcohol bath at -15°. N-Methylmorpholine (16.8 ml, 0.15 mol) was added dropwise. The temperature was maintained at -15° and isobutyl chloroformate (19.7 ml, 0.15 mol) was added dropwise over a 2-minute period. The reaction mixture was stirred an additional 2 minutes at -15° and a precooled solution of L-asparagine t-butyl ester (25.6 g, 0.136 mol) dissolved in 270 ml THF and 30 ml DMF was added dropwise over a 4-minute period while the temperature was maintained at -15°. The reaction mixture was stirred at -15° for 30 minutes and at 25° for 2.5 hours, evaporated in vacuo and the residue dissolved in EtOAc and washed with 10% NaHCO3 (2×300 ml), saturated NaCl solution (30 ml), 1 M citric acid (2×300 ml) and saturated NaCl solution (300 ml). The organic layer was dried over MgSO4, filtered and evaporated in vacuo to yield a thick oil. The oil was crystallized from EtOAc-ether to yield 34.5 g. Trituration of the mother liquid with ether gave an additional 31.1 g. Total yield 65.6 g (95%); mp 139° dec; Rf 0.22 (CHCl3 :CH3OH:AcOH:80-2-0.4). The analytical sample was prepared by recrystallization from methanol-ether; mp 142°-144.5°; [α]D25 -19.14° (c 1, CH3OH). Anal. calcd. for C25H37N3O8 (507.6): C, 59.16; H, 7.35; N, 8.28. Found: C, 59.24; H, 7.34; N, 8.40. fitted with a thermometer, mechanical stirrer immersed in a dry ice-alcohol bath at -15° The temperature was maintained at -15° was maintained at -15° The reaction mixture was stirred at -15° for 30 minutes and at 25° for 2.5 hours evaporated in vacuo the residue dissolved in EtOAc washed with 10% NaHCO3 (2×300 ml), saturated NaCl solution (30 ml), 1 M citric acid (2×300 ml) and saturated NaCl solution (300 ml) The organic layer was dried over MgSO4 filtered evaporated in vacuo to yield a thick oil The oil was crystallized from EtOAc-ether to yield 34.5 g

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Fragment I was prepared by stepwise chain elongation using the mixed anhydride procedure at -15° C. with careful temperature control. As seen in Scheme 1 of FIG. 2 mixed anhydride coupling (isobutylchloroformate) of Z-Glu(OtBu)-OH with H-Asn-OtBu provided Z-Glu(OtBu)-Asn-OtBu. Catalytic hydrogenation (10% Pd-C) of this compound cleaved the N-terminal protective group and the dipeptide was coupled to Z-Ala-OH using mixed anhydride coupling to give Z-Ala-Glu(OtBu)-Asn-OtBu. The N-terminal was again deprotected by catalytic hydrogenation and the tripeptide coupled with Z-Glu(OtBu)-OH to give the protected tetrapeptide Z-Glu(OtBu)-Ala-Glu(OtBu)-Asn-OtBu which is deblocked by catalytic hydrogenation to provide the desired tetrapeptide (I). Fragment I was prepared by stepwise chain elongation at -15° C.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: When in the procedure of Example 1, Step (b), thianthrene-2-sulfonyl chloride is replaced by phenoxathiine-2-sulfonyl chloride and (L)-valine-t-butyl ester is replaced with (L)-asparagine-t-butyl ester, (S)-2-(phenoxathiine-2-sulfonylamino)-succinamic acid is obtained; mp 165-175° C.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 23.6 Grams (73.2 mmols) of Z-Asn-OtBu in 200 ml of ethyl acetate are hydrogenated in the presence of 2 g of palladium carbon (10% Pd) until, after 1 hour, hydrogen is no longer absorbed. The catalyst is filtered off and the filtrate concentrated, whereupon the product, melting at 96°-98° C., crystallizes out. Optical rotation [α]D20 =+2° (c=2.1 in methanol). In the thin-layer chromatogram on silica gel the Rf52 =0.30; Rf45 =0.50. after 1 hour hydrogen is no longer absorbed The catalyst is filtered off the filtrate concentrated, whereupon the product, melting at 96°-98° C. crystallizes out Optical rotation [α]D20 =+2° (c=2.1 in methanol)

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The product from Example 100 (56 mg, 0.1 mmole) and H-Asn-OtBu (38 mg, 0.2 mmole) were processed as in Example 213A-B to provide the title compound. 1H NMR (500 MHz, DMSO-d6) δ12.11-12.80 (br.s, 1 H), 8.94 (s, 1 H), 7.99 (s, 1 H), 7.28 (m, 7 H), 7.18 (m, 3 H), 7.03 (m, 1 H), 6.96 (m, 2 H), 6.87 (m, 5 H), 4.50 (m, 1 H), 4.05 (s, 2 H), 4.01 (s, 2 H), 2.91 (s, 3 H), 2.55 (m, 2 H), 2.44 (m, 2 H), 2.39 (s, 3 H), 2.11 (t, 2 H), 1.76 (m, 2 H); MS (APCI+) m/z 673 (M+H)+.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Analogously to Example 100, 5.45 g (31.4 mmol) of quinaldic acid in 161 ml of THF are reacted with 7.08 g (34.3 mmol) of DCC, 4.63 g (34.3 mmol) of HOBT and 5.38 g (28.6 mmol) of (L)-asparagine tert-butyl ester (Bachem, Bubendorf/Switzerland). Filtration, extraction and column chromatography (SiO2, ethyl acetate/hexane 3:1) yields the pure title compound: TLC Rf (C')=0.15; tRet (V)=12.2 min; FAB-MS (M+H)+ =344. Filtration, extraction and column chromatography (SiO2, ethyl acetate/hexane 3:1)

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Fragment I was prepared by stepwise chain elongation using the mixed anhydride procedure at -15° C. with careful temperature control. As seen in the scheme of FIG. 2 mixed anhydride (M.A.) coupling (isobutylchloroformate) of Z-Glu(OtBu)-OH with H-Asn-OtBu provided Z-Glu(OtBu)-Asn-OtBu. Catalytic hydrogenation (10% Pd-C) of this compound cleaved the N-terminal protective group and the dipeptide was coupled to Z-Ala-OH using mixed anhydride coupling to give Z-Ala-Glu (OtBu)-Asn-OtBu. The N-terminal was again deprotected by catalytic hydrogenation and the tripeptide coupled with Z-Glu(OtBu)-OH to give the protected tetrapeptide Z-Glu(OtBu)-Ala-Glu(OtBu)-Asn-OtBu which is deblocked by catalytic hydrogenation to provide the desired protected tetrapeptide (1). Fragment I was prepared by stepwise chain elongation at -15° C.