J. Med. Chem. 1980,23,6-8
6
Use of Acetylacetone to Prepare a Prodrug of Cycloserine N. P. Jensen,* J. J. Friedman, Department of Membrane and Arthritis Research, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065
H. Kropp,* and F. M. Kahan Department of Exploratory Microbiology, Merck Institute for Therapeutic Research, Rahway, New Jersey 07065. Received February 26, 1979 Several derivatives of cycloserine (1) were prepared and it was found that (R)-4-[(1-methyl-3-oxo-1-buteny1)amino]-3-isoxazolidinone(1l),the condensation product of acetylacetone and cycloserine ( l ) ,was an efficacious prodrug of increased stability under aqueous conditions.
One of the most common problems of a medicinal chemist is to design a prodrug of an active structure in an attempt to obtain some advantage such as greater chemical stability, better transport, or lower Recently, we needed such a prodrug of the antibiotic cycloserine (1) to use in combination with the recently disclosed synthetic Our primary objective antibiotic 3-fluoro-~-alanine-Z-d.~ in this case was to reduce the known instability of cycloserine with respect to the formation of dimer 2. This dimerization takes place particularly well in concentrated aqueous media and can even take place in the solid state.4 Preventing this dimerization would therefore increase the stability of cycloserine both on the shelf and in physiological media. Since the formation of dimer 2 requires a reaction of the amino group of cycloserine, functionalization of this amino group is the easiest and most direct approach to retarding dimer formation (Scheme I). This approach has been previously attempted, but it was found that “analogues in which the amino group is alkylated or acylated are i n a ~ t i v e ”and , ~ the conclusion has been drawn that a free amino group is required for biological a ~ t i v i t y .The ~ only type of amino modification compatible with activity was found to be Schiff base^,^'^ and their activities are attributed to hydrolysis to cyclo~erine.~ We have prepared a number of new cycloserine analogues in which the amino group is functionalized and have tested them for chemical stability vis a vis cycloserine. Table I exemplifies typical conditions of heat and humidity that were used in these stability studies. Derivatives which had stability superior to cycloserine under such (1) A. A. Sinkula, Ann. Rep. Med. Chem., 10, 306 (1975). (2) N. J. Harper, Progr. Drug Res., 4,221 (1962). (3) (a) F. M. Kahan and H. Kropp, 15th Interscience Conference
(4) (5)
(6) (7)
on Antimicrobial Agents and Chemotherapy, Washington, D.C., Sept. 24-26, 1975, Abstract 100; (b) H. Kropp, F. M. Kahan, and H. B. Woodruff, ibid., Abstract 101; (c) J. Kollonitsch, L. Barash, N. P. Jensen, F. M. Kahan, S. Marburg, L. Perkins, S. M. Miller, and T. Y. Shen, ibid., Abstract 102; (d) F. M. Kahan, H. Kropp, H. R. Onishi, and D. P. Jacobus, ibid., Abstract 103. See F. A. Lassen and C. H. Stammer, J . Org. Chem., 36,2631 (1968), for a discussion of the dimerization. F. C. Neuhaus in “Antibiotics, 1, Mechanism of Action”, D. Gottlieb and P. D. Shaw, Ed., Springer-Verlag,Berlin and New York, 1967, p 55. S. Bianchi, E. Felder, and U. Tiepolo, Farmico, E d . Prat., 20, 366 (1965). See ref 5 , p 56.
Scheme I
1
2
4
HN
0-N H
11
Table I. Comparative Stability of 1 and 11 t o Moisture no.
la
conditions 14 h, 3 5 ” C ,
100% humidity 11 14 h , 3 5 ’ C , 100% humidity la 1 2 h, 3 5 ” C , 100% humidity lC 1 h, 7 0 ° C , 98% humidity 11 1 h, 7 0 ° C , 98% humidity 11 3 days, room temp, stirring in acetyl acetone
assay method
% remaining
optical rotation
74
optical rotation
99
colorime tricb
72
colorimetricb
22
colorimetricb
94
optical rotation
100
a Unpurified commercial cycloserine purchased from Nutritional Biochemical. Particle size appears to have some effect on the rate of decomposition of 1 and 11, but 11 was always more stable to moist conditions. L. R. Jones, Anal. Chem., 28, 3 9 (1956). Cycloserine purified by the method of C. H. Stammer, A. W. Wilson, C. F. Spencer, F. W. Bachelor, F. W. Holly, and K. Folkers, J. Am. Chem. Soc., 79, 3226 (1957).
conditions were then tested for their in vitro antibacterial activity and their ability to liberate cycloserine in vivo, as tabulated and described in Table 11. Our results largely substantiated previously reported work’ on such derivatives of cycloserine. Several derivatives, including various types of Schiff bases, proved of insufficient chemical sta-
0022-2623/80/1&323-0006$01.00/0 0 1979 American Chemical Society
Journal of Medicinal Chemistry, 1980, Vol. 23, No. 1 7
Cycloserine Prodrug Preparation
Table 11. Antibacterial Activity and Urinary Recovery of Cycloserine Analogues
D-4-amino-3-isoxazolidinone D-cycloserine antibacterial act. :‘ zone of inhibn diameter, nm
Rl 3 4 5 6 7
-C( = O)NHCH, -C( = O)CH,Cl -C( =O)CH,Cl -C(=O)OC,H, -C( = O)NHCH,
R2
disposition in the mouse: urinary recovery, % of doseb
E. coli Staph. aureus bioassay
chem assay
-H -C( = O)CH,Cl -H -H -C( = O)NHCH,
22
