Hydrazones of a 6-hydrazinonicotinyl-modified cyclic peptide IIb/IIIa receptor antagonist were prepared in order to protect the hydrazine moiety from reaction with trace aldehyde and ketone impurities encountered during the process of manufacturing and compounding lyophilized kits used in radiolabeling with (99m)Tc. Hydrazones were prepared by either a direct reaction of the 6-hydrazinonicotinyl-modified cyclic peptide with carbonyl compounds or by conjugation of the cyclic peptide with hydrazones of succinimidyl 6-hydrazinonicotinate. Stability of the hydrazones was evaluated by treatment with formaldehyde. Hydrazones derived from simple aliphatic aldehydes underwent an exchange reaction with formaldehyde, while hydrazones of aromatic aldehydes and ketones provided the greatest level of stability when challenged with formaldehyde. We have been successful in protecting 6-hydrazinonicotinyl-modified cyclic peptides from reacting with formaldehyde, while still allowing sufficient reactivity for radiolabeling with (99m)Tc. The hydrazones of succinimidyl 6-hydrazinonicotinate are convenient and general reagents for forming 6-hydrazinonicotinyl conjugates with amino-functionalized bioactive molecules.

Using isoxazoline XR299 (1a) as a starting point for the design of highly potent, long-duration GPIIb/IIIa antagonists, the effect of placing lipophilic substituents at positions alpha and beta to the carboxylate moiety was evaluated. Of the compounds studied, it was found that the n-butyl carbamate 24u exhibited superior potency and duration of ex vivo antiplatelet effects in dogs. Replacement of the benzamidin-4-yl moiety with alternative basic groups, elimination of the isoxazoline stereocenter, and reversal of the orientation of the isoxazoline ring resulted in lowered potency and/or duration of action.

MS (70 eV), m/e (relative intensity) 570 (M+, 62), 70 (100); NMR (CDClg, Me4Si) 0.96 (m, 6 H), 1. 00-2.60 (m, 9 ), 1.45 (s, 9 ), 2.75-4.00 (m, 4 ), 3.80 (s, 3 ), 4.00-4.70 (m, 3 H), 5.00-5.50 (m, 2 ), 5.90 (d, J = 8.5 Hz, 1 ), 6.50-7.05 (m, 4 H), 8.35 (d, «7=11 Hz, 1 H); high-resolution MS, caled for CaoHyNA m/e 570.3054, found m/e 570.3052.Zizyphine A (3). A solution of 60 mg (0.105 mmol) of 27a and 0.1 mL of m-dimethoxybenzene in 5 mL of trifluoroacetic acid was stirred for 0.5 h at room temperature and then evaporated in vacuo. The solution of the residue in CH2C12 was washed with 1 N KHC03, dried, filtered, and evaporated. Recrystallization of the residue from ethyl acetate/petroleum ether afforded 50 mg (100%) of cyclo [7V-[3-[[3-[2-(Z)-aminovinyl]-4-methoxyphenyl]oxy]-jV-isoleucyl-(2S,3S)-prolyl]-(S)-prolyl]. This product was added to a solution of 25 mg of 4-(dimethylamino) pyridine and 100 mg (0.3 mmol) of the pentafluorophenyl ester of N-(dimethylamino)-L-isoleucine in 3 mL of absolute dioxane. The reaction mixture was kept at 80 °C for 7 h and then evaporated, and the residue was chromatographed on silica gel with CH2Cl2/i-PrOH (92.5:7.5) to yield zizyphine A (3): 35 mg (54%);[a]\ -430°(c 0.093, CHC13) (lit.18a [a]\ -464°(c 1, CHCI3); lit.18c [«j^n -411°(c 0.086, CHCI3)). The CD of the synthetic product was identical with the CD of the natural product (Ciba-Geigy):