Synthesis of the retro-inverso peptide analogues of N - acetylmuramyl-L-alanyl-D-isoglutamine (MDP)

Two novel retro-inverso analogues of a conformationally restricted carbocyclic muramyl dipeptide (MDP) derivative N-(2-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carbonyl)-L-alanyl-D-isoglutamine were prepared and tested in a immunorestoration test in mice. Both retro-inverso MDP analogues did not enhance non-specific resistance to experimental fungal infection in immunosuppressed animals, suggesting that the intact amide bond is essential for the immunorestorating activity in this type of rigidified carbocyclic MDP analogues.


Introduction
3][4] Recently we have shown that replacement of the polyhydroxy substituted pyranose ring of D-glucosamine in MDP by a benzene ring and partial rigidification of the molecule involving incorporation of the lactoyl moiety and acetamido group into a benzo-fused 3-morpholinone ring results in a new series of potent immunostimulatory compounds of general structure 2 (R 1 =H, Me). 5,6wever, modifications of the L-Ala-D-iGln bond of MDP and analogues have not been described so far although modification of the labile peptide bonds is one of the most frequently applied strategy in the design of peptidomimetics. 7Among them, the retroinverso modification of peptide bonds has evolved into one of the most widely used peptidomimetic approaches for the design of novel bioactive molecules which has been applied to many families of biologically active peptides 7,8 including immunostimulatory tuftsin analogues. 9 report now on preparation and results of biological testing of two novel carbocyclic MDP analogues, represented by a general structure 3, with a retro-inverso modified peptide bond between the L-alanyl and D-glutamate moieties.These compounds were prepared with the aim of obtaining information about the importance of the intact peptide bond for immunostimulating activity in this class of partially rigidifed carbocyclic MDP analogues.

Results and Discussion
Starting from commercially available benzyl methyl malonate 4a (R = H) and 4b (R = CH 3 ) prepared by methylation of 4a, alkylation with methyl 3-bromopropionate in the presence of sodium hydride and subsequent hydrogenolysis afforded carboxylic acids 6a and 6b.The intermediary esters 5a and 5b were purified by a high-vacuum distillation to avoid decarboxylation.Coupling of carboxylic esters 6 with D-alanine benzyl ester 10 in the presence of diphenylphosphoryl azide (DPPA) [11][12][13] followed by hydrogenolytical removal of the benzyl group gave intermediates 8a and 8b which were converted to acyl azides 9a,b.These acyl azides were then subjected to Curtius rearrangement in the presence of benzyl alcohol.The rearrangement of acyl azide to isocyanate could be very successfully followed by IR spectroscopy as a gradual disappearing of the azide absorption band at ca. 2150 cm -1 and appearance of the isocyanate band at ca. 2250 cm -1 .
The yields of carbamates 10a,b were generally low due to a well-known side product formation during addition of alcohols to isocyanates. 14

General procedure for preparation of carboxylic acids 6a and 6b
A solution of benzyl methyl 2-alkyl malonate 5a or 5b (10.0mmol) in methanol (30 mL) was purged with argon and hydrogenated over 10 weight % of 10% palladium on charcoal at room temperature and normal pressure until the reaction was complete (ca 1 h).The solvent was removed in vacuo to give the product as a colorless viscous oil.

Issue in Honor of Prof. Miha Tišler ARKIVOC 2001 (v) 7-20 ISSN 1424-6376 Page 11 ARKAT USA, Inc Experimental Section General Procedures. All
reagents and solvents were of commercial grade and were used