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A series of peptidyl alpha-ketobenzoxazoles were synthesized and evaluated for their in vitro and in vivo inhibition of human neutrophil elastase (HNE). These compounds inhibit HNE by forming both a covalent bond between the ketone carbonyl carbon atom and the hydroxyl group of Ser-195 and a hydrogen bond between the benzoxazole nitrogen atom and His-57. Appending to the parent benzoxazole ring a variety of substituents which spanned a range of physicochemical properties had only a modest effect on in vitro potency (Ki = 3-0.4 nM). This apparent lack of a significant effect is believed to result from the fact that any increased ketone carbonyl activation by the ring substituent is counter balanced by a corresponding decrease in the hydrogen-bonding ability of the benzoxazole nitrogen atom. In contrast to the results in vitro, maximizing in vivo activity was critically dependent upon the choice of the benzoxazole ring substituent. Several substituted peptidyl alpha-ketobenzoxazoles effectively inhibited HNE-induced lung injury when administered intratracheally 24 h prior to the enzyme.

Previously we had shown that tripeptidyl trifluoromethyl ketones (TFMKs) possessing an N-terminal diarylacylsulfonamide, such as ICI 200,880 and ICI 200,355, displayed unparalleled protection against the lung damage induced by human neutrophil elastase (HNE) when the inhibitors were administered intratracheally. Since the diarylacylsulfonamides were designed specifically to afford a long residence time in the lung, it was not unexpected that inhibitors from this class of TFMKs were not active when administered orally. Upon evaluating a large number of peptidyl TFMKs possessing a variety of N-terminal groups, several compounds were identified which demonstrated oral activity. Compounds were evaluated for their oral activity by measuring their ability to inhibit the increase in lung weight relative to body weight (Lw/Bw), the increase in red blood cells, and the increase in white blood cells induced by intratracheally administered HNE (100 micrograms/hamster). A number of tripeptidyl trifluoromethyl ketones containing neutral N-terminal groups displayed good oral activity, while those containing basic, acidic, or polar groups did not. Compound 50, possessing an N-terminal 4-(CH3O)C6H4CO group, was particularly effective, reducing Lw/Bw by 77%, red cells by 89%, and white cells by 91% when dosed at 37.5 mg/kg orally. Thus, by modifying the N-terminal group of tripeptidyl TFMKs, inhibitors can be designed which are effective in vivo when administered either orally or intratracheally.

The potential energy surface for the reaction of atomic carbon with thiophene has been studied computationally. Intermediates which are energetically viable include the 2- and 3-thienylcarbenes 8 and 11, thiacyclohexa-3,5-dien-2-ylidene, 10, and thiacyclohexa-2,3,5-triene, 6. In accord with experimental data, 6 and 8 are in equilibrium. The lowest-energy pathway for rearrangement of 6 to 8, which is endothermic by 14.5 kcal/mol, involves ring opening to Z-2-penten-4-ynthial which then recloses to carbene 8. A 1,4 addition of C across the diene system in thiophene generates an ylid which rearranges with little or no barrier to cyclopentadienethione, the global minimum on this potential energy surface.

Acetolysis of 1,6-anhydro sugars can be achieved by treatment with acetic anhydride and triethylsilyl trifluoromethanesulphonate at 0 "C for 5-15 minutes, under which conditions a wide variety of protecting groups are unaffected, and even the trisulphonate is cleaved, albeit in six hours. TBDPSO 7c OAc 8c Complex mixture TBDPSO 9 L O A c AGO S O Ac OAc 85 13 14 a Typical experimental procedure. The anhydro sugar is dissolved in Ac20 and cooled to 0°C with stirring under argon. Two drops (-20-30 pl) of triethylsilyl trifluoromethanesulphonate are added to the solution. The reaction is followed by TLC and generally requires 5-15 min for completion. A solution of saturated sodium bicarbonate is then added and after being stirred for 30 min, the aqueous mixture is extracted three times with ethyl acetate. The organic extracts are combined and washed with saturated sodium hydrogen carbonate solution followed by brine. The mixture is then dried over sodium sulphate, filtered and the solvents removed under reduced pressure. Percolation through silica gel removes any trace of acetic anhydride affording clean product. b This reaction required 6 hours for completion; Ts = p-tolylsulphonyl. c p: CY = 3 : 1. d TBDPS = tert-butyldiphenylsilyl.

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