J. Med. Chem. 1994,37, zyxwvu 3969-3976 3969 Dipeptide Phosphonates as Inhibitors of Dipeptidyl Peptidase zy IV zyxwvutsrqponmlkjihgfedcbaZYXW Bogdan Boduszek,$Jozef Oleksyszyn,g Chih-Min Kam,$ Joe Selzler,$Robert E. Smith,? and James C. Powers*!$ From the School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, and Enzyme Systems Products, 6491 Sierra zyxwvuts Lane, Dublin, California 94568 Received November 1, 1993@ A series of dipeptides which contained phosphonate analogs of proline and piperidine-2- carboxylic acid (homoproline) have been synthesized and tested as inhibitors of DPP-IV. The rates of inhibition of DPP-IV by these compounds are moderate, but the inhibitors are quite specific. The best inhibitor in the series is Ala-PipP(OPh-4-Cl)2 (131, which has a Kinact of 0.353 s-l and KI of 236 pM. The DPP-IV inhibitors Ala-ProP(OPh)2 zyxw (6), Ala-ProP(OPh-4-Cl)2 (12), and Ala-PipP(OPh-4-Cl)2 (13) do not inhibit trypsin, human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), acetylcholinesterase, papain, and cathepsin B. However, compounds 12 and 13 inhibited chymotrypsin slowly. Most of these dipeptides containing a homoproline phosphonate residue (Pipp) or a Pro phosphonate residue (Prop) at the PI site are stable in a pH 7.8 buffer with half-lives of several hours to several days. DPP-IV inhibited by 6, 7 (Ala- PipP(OPh)2), 12, or 13 is quite stable, and no enzyme activity was recovered after removal of excess inhibitor and incubation in buf'fer for 1 day. Since the phosphonate inhibitors are specific toward DPP-IV and the inhibited enzymes are stable, they should be useful in establishing the biological functions of DPP-IV and may be useful therapeutically in the prevention of the rejection of transplanted tissue. Introduction Dipeptidyl peptidase IV (DPP-IV,lEC 3.4.14.5, CD26) is a post-proline cleaving enzyme which will remove the dipeptides AA-Pro (AA zyxwvutsr = amino acid residue) from the N-terminus of proteins or polypeptides. DPP-IV has been found in a variety of mammalian cells and tissues, including kidney, placenta, and blood plasma, and on the surface of certain T-lymphocyte subsets. Despite extensive studies, the biological role of DPP-IV in mammalian systems have not been established, al- though a number of functions have been postulated. DPP-IV may participate in the metabolism and uptake of proline-containing peptides in the intestine and kidney2and may be involved in fibronectin-mediatedcell movement and adhe~ion.~ DPP-IV may also play a role in the metabolism or catabolism of collagen which has a high frequency of Gly-Pro sequence^.^ DPP-IV in human plasma has been shown to cleave N-terminal Tyr-Ala from growth hormone-releasing factor and cause inactivation of this h~rmone.~ DPP-IV is also involved in T-cell activation and regulation of T-cell proliferation.6 Thus, inhibitors of DPP-IV may have therapeutic utility in the modulation of the rejection of transplanted tissue by the host organism. DPP-IV is a serine protease which has been demon- strated by its complete inhibition by DFP.7 The amino acid sequence of rat liver DPP-IV deduced from cDNA has been established, and it contains the sequence of Gly-Trp-Ser-Tyr-Glycorresponding to the common se- quence Gly-X-Ser-X-Gly found in the active site of various serine proteases.a Recent studies have shown that radiolabeled r3H1DFP is bound to Ser-631 of DPP- * Corresponding author: James C. Powers, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332- 0400. Tel: (404) 894-4038. Fax: (404) 894-7452. +Enzyme Systems Products. * Georgia Institute of Technology. * Current address: OsteoArthritis Sciences, Inc., One Kendall Square, Cambridge, MA 02139. @Abstract published in Advance ACS Abstracts, October 1, 1994. 0022-2623l94/1837-3969$04.50/0 IV, and the residues Gly-629, Ser-631, and Gly-633 are essential for the enzyme activity of DPP-IV.g Interest- ingly, the catalytic triad residues (Ser-624,Asp-702, and His-734) of mouse DPP-IV are arranged in a novel sequential order (e.g., Ser-Asp-His) that is different from that of archetypical serine proteases (His-Asp-Ser).lO A wide variety of inhibitors of serine proteases have been reported;ll however, only a few classes of com- pounds are effective inhibitors of DPP-IV. N-Peptidyl- 0-(4-nitrobenzoyl)hydroxylamines irreversibly inacti- vate DPP-IV, but most of the inhibitor is hydrolyzed during the inhibition process.12 Azapeptides such as Ala-AzaPro-OPh form acyl enzyme derivatives with DPP-IV, but they are not very effective inhibitors, and the acyl enzyme can deacylate regenerating active enzyme.13 The boronic acids Ala-boroPro and Pro- boroPro are specific and potent reversible inhibitors of DPP-IV with KI values in the nanomolar range; how- ever, both inhibitors are unstable in solution at neutral pH.14 Many other types of transition-state inhibitors would be expected to be quite unstable when incorpo- rated into a dipeptide structure with a free N-terminal amino group. For example, dipeptide trifluoromethyl ketones, a-keto acids, or chloromethyl ketones would be expected to cyclize and be unstable in aqueous solu- tion.16 Therefore, new specific and potent inhibitors of DPP-IV are needed. A variety of peptide phosphorus derivatives have been reported to be serine protease inhibitors. For example, DFP analogs such as peptide phosphonyl fluqrides inhibit serine proteases potently, but they are extremely unstable in aqueous solution. l6 Peptide phosphonates which incorporate a tetrahedral phosphorus moiety in the peptide substrate inhibit serine proteases p0or1y.l~ Peptidyl (a-aminoalky1)phosphonate diphenyl esters offer one potential route to effective DPP-IV inhibitors since phosphonate esters are relative unreactive with nitrogen nucleophiles (e.g., the N-terminal amino group). These peptide phosphonate esters are specific and zyxwvutsr 0 1994 American Chemical Society