Design and synthesis of glycolic and mandelic acid derivatives as factor Xa inhibitors.

A series of glycolic and mandelic acid derivatives was synthesized and investigated for their factor Xa inhibitory activity. These analogues are highly potent and selective inhibitors against fXa. In a rabbit deep vein thrombosis model, compound 26 showed significant antithrombotic effects (81% inhibition of thrombus formation) at 1.1 microM plasma concentration following intravenous administration.

Antithrombotic and hemostatic capacity of factor Xa versus thrombin inhibitors in models of venous and arteriovenous thrombosis.

Thrombin plays a central role in venous and arterial thrombosis. We utilized two different rabbit models of in vivo thrombosis to investigate the effect of inhibitors of thrombin generation and thrombin activity. The agents tested were specific inhibitors of factor Xa (fXa) [N2-[(phenylmethyl)sulfonyl]-D-arginyl-N-[(1S)-4-[(aminoiminomethyl++ +)a mino]-1-(2-thiazolylcarbonyl)butyl]-glycinamide (C921-78)] and thrombin [D-phenylalanyl-N-[4-[(aminoiminomethyl)amino]-1-(chloroacetyl)but yl]-L-prolinamide (PPACK)], as well as drugs that affect both thrombin and fXa, unfractionated and low molecular weight (enoxaparin) heparin. The agents administered as constant intravenous infusion were evaluated for antithrombotic efficacy in anesthetized rabbits. All four agents were capable of dose dependent inhibition of thrombosis in venous and arteriovenous thrombosis models. However, due to the more aggressive nature of thrombotic stimulation in the arteriovenous shunt model, complete cessation of thrombus growth was not achieved for any of the agents at the doses tested. Comparison between the agents focused on the differences in extension of coagulation parameters (activated partial thromboplastin time, prothrombin time, thrombin clotting time), changes in hematological parameters, and extension of rabbit cuticle bleeding time at doses required to produce maximum inhibition in the thrombosis models. In the venous thrombosis model at the maximally effective dose, C921-78 had minimal extension of ex vivo clotting parameters, while enoxaparin and unfractionated heparin demonstrated a two to sevenfold increase in activated partial thromboplastin times, and PPACK had a threefold extension of thrombin clotting times. In addition, unlike the other three agents, which exhibited no significant changes in hematological parameters, PPACK demonstrated dose dependent thrombocytopenia. A standardized cuticle bleeding time was used as a measure of perturbation of hemostasis. The agents were evaluated for significant increases in bleeding time at doses up to eight times that needed to completely inhibit venous thrombus formation. Unfractionated heparin displayed a significant bleeding time effect at the dose required to inhibit venous thrombosis (100 u/kg+2 u/kg/min). Enoxaparin and PPACK caused significant bleeding time extensions at four times the fully efficacious venous dose (800 u/kg+8 u/kg/min and 30 microg/kg/min). By contrast, C921-78 did not significantly increase bleeding time even at eight times the maximally effective dose (240 microg/kg+7.2 microg/kg/min). Our results demonstrate that specific inhibition of fXa can be utilized to derive potent antithrombotic activity without disrupting extravascular hemostasis.

Design, synthesis and structure-activity relationship of a series of arginine aldehyde factor Xa inhibitors. Part 1: structures based on the (D)-Arg-Gly-Arg tripeptide sequence.

A series of arginine aldehyde inhibitors was designed as transition state (TS) analogues based on the known factor Xa specific substrate Cbz-D-Arg-Gly-Arg-pNA. BnSO2-(D)Arg-Gly-Arg-H (20) was found to be the most potent and selective inhibitor of factor Xa and prothrombinase activity in this series.

Modification of a receptor-binding surface of epidermal growth factor (EGF): analogs with enhanced receptor affinity at low pH or at neutrality.

Six mutants of human epidermal growth factor (EGF), which carry single point substitutions within a surface patch proposed to juxtapose the bound receptor, were prepared and characterized for receptor affinity and mitogenicity. Receptor affinities relative to EGF are G12Q > H16D > Y13W > Q43A approximately = H16A approximately = EGF >> L15A. Notably, the reduced receptor affinity of mutant L15A indicates that Leu15 probably contributes substantially to receptor binding whereas unaltered receptor affinities observed for analogs H16A and Q43A indicate that neither His16 nor Gln43 contributes significantly to this interaction. On the other hand, the observed enhanced receptor affinities of analogs G12Q, Y13W and H16D highlight surface loci where additional productive receptor-binding contacts can be introduced. Interestingly, at acidic pH analog H16A reveals substantially greater receptor affinity than that of EGF, a property which may offer enhanced therapeutic utility in acidic environments in vivo.

Solid phase organic synthesis (SPOS): a novel route to diketopiperazines and diketomorpholines.

The solid phase synthesis of libraries containing a 1,3,4,6-tetrasubstituted-2,5-diketo-1,4-piperazine scaffold (DKP) or a 3,4,6-trisubstituted-2,5-diketo-1,4-morpholine scaffold (DKM) from alpha-bromocarboxylic acids and amines is described. Using a design strategy which we refer to as divergent library design, both templates were prepared from a common intermediate. The general utility of this synthetic route in creating novel, non-peptidyl chemical libraries is discussed.

Quantitative structure-activity relationships of the inhibition of Pneumocystis carinii dihydrofolate reductase by 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(X-phenyl)-s-triazines.

The inhibitory activities of 60 4,6-diamino-1,2-dihydro-2,2-dimethyl-1- (X-phenyl)-s-triazines versus purified, recombinant Pneumocystis carinii (Pc) dihydrofolate reductase (DHFR) have been determined at pH 7.0. Utilization of these Kiapp values has led to the formulation of appropriate quantitative structure-activity relationships (QSAR's) for both meta- and parasubstituted derivatives. The QSAR's from Pc are compared with other triazine QSAR's derived versus chicken, murine tumor, Escherichia coli, and particularly human DHFR. Selectivity indices indicate that hydrophobic triazines are particularly effective versus Pc DHFR; they have lower Ki values for Pc DHFR than for human DHFR.

Peptoids: a modular approach to drug discovery.

Peptoids, oligomers of N-substituted glycines, are described as a motif for the generation of chemically diverse libraries of novel molecules. Ramachandran-type plots were calculated and indicate a greater diversity of conformational states available for peptoids than for peptides. The monomers incorporate t-butyl-based side-chain and 9-fluorenylmethoxy-carbonyl alpha-amine protection. The controlled oligomerization of the peptoid monomers was performed manually and robotically with in situ activation by either benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate or bromotris(pyrrolidino)phosphonium hexaflurophosphate. Other steps were identical to peptide synthesis using alpha-(9-fluorenylmethoxycarbonyl)amino acids. A total of 15 monomers and 10 oligomers (peptoids) are described. Preliminary data are presented on the stability of a representative oligopeptoid to enzymatic hydrolysis. Peptoid versions of peptide ligands of three biological systems (bovine pancreatic alpha-amylase, hepatitis A virus 3C proteinase, and human immunodeficiency virus transactivator-responsive element RNA) were found with affinities comparable to those of the corresponding peptides. The potential use of libraries of these compounds in receptor- or enzyme-based assays is discussed.

Possible role for water dissociation in the slow binding of phosphorus-containing transition-state-analogue inhibitors of thermolysin.

A number of phosphonamidate and phosphonate tripeptide analogues have been studied as transition-state-analogue inhibitors of the zinc endopeptidase thermolysin. Those with the form Cbz-GlyP(Y)Leu-X [ZGP(Y)LX, X = NH2 or amino acid, Y = NH or O linkage] are potent (Ki = 9-760 nM for X = NH, 9-660 microM for X = O) but otherwise ordinary in their binding behavior, with second-order rate constants for association (kon) greater than 10(5) M-1 s-1. Those with the form Cbz-XP(Y)-Leu-Ala [ZXP(Y)LA,XP = alpha-substituted phosphorus amino acid analogue] are similarly potent (Ki for ZFPLA = 68 pM) but slow binding (kon less than or equal to 1300 M-1 s-1). Several kinetic mechanisms for slow binding behavior are considered, including two-step processes and those that require prior isomerization of inhibitor or enzyme to a rare form. The association rates of ZFPLA and ZFP(O)LA are first order in inhibitor concentration up to 1-2 mM, indicating that any loose complex along the binding pathway must have a dissociation constant above this value. The crystallographic investigation described in the preceding paper [Holden, H. M., Tronrud, D. E., Monzingo, A. F., Weaver, L. H., & Matthews, B. W. (1987) Biochemistry (preceding paper in this issue)] identifies a specific water molecule in the active site that may hinder binding of the alpha-substituted inhibitors. The implication of this observation for a mechanism for slow binding is discussed.

Phosphonamidate inhibitors of human neutrophil collagenase.

A series of phosphonamidates has been synthesized and shown to inhibit human neutrophil collagenase. The compounds all have sequences patterned after the cleavage site in the alpha 1(I) chain of type I collagen, except that the carbonyl group of the Gly residue in subsite P1 has been replaced by a P(= O)(OH) group (abbreviated GlyP). As the central GlyP-Leu unit is lengthened in the N- and C-terminal directions, in accordance with the cleavage sequence found in collagen, inhibition is systematically improved. The best inhibitor is Cbz-GlyP-Leu-Ala-Gly, which inhibits competitively with a KI value of 14 microM. These phosphonamidates are thought to be acting as transition-state analogues.

Evaluation of intrinsic binding energy from a hydrogen bonding group in an enzyme inhibitor.

This and two accompanying reports describe the intrinsic binding energy derived from a single hydrogen bond between an inhibitor and an enzyme. The results were obtained by comparing matched pairs of inhibitors of the zinc endopeptidase thermolysin that bind to the enzyme in an essentially identical manner but differ in the presence or absence of a specific hydrogen bond. This report describes five phosphorus-containing analogs of the peptides carbobenzoxy-Gly-Leu-X, in which the Gly-Leu peptide linkage is replaced with a phosphonate ester (-PO2(-)-O-). Values for the inhibition constants of these inhibitors show a direct relation with those of the corresponding phosphonamidate analogs (-PO2(-)-NH- in place of the Gly-Leu peptide moiety), which have been characterized previously as transition state analogs. However, each phosphonate ester is bound about 840 times more weakly than the analogous phosphonamidate, reflecting the loss of 4.0 +/- 0.1 kilocalories per mole in binding energy. From these results and the crystallographic analysis in the next report, it can be inferred that the value of 4.0 kilocalories per mole represents the intrinsic binding energy arising from a highly specific hydrogen binding interaction.

Phosphonamidates as transition-state analogue inhibitors of thermolysin.

Six phosphorus-containing peptide analogues of the form Cbz-NHCH2PO2--L-Leu-Y (Y = D-Ala, NH2, Gly, L-Phe, L-Ala, L-Leu) have been prepared and evaluated as inhibitors of thermolysin. The Ki values for these compounds range from 1.7 microM to 9.1 nM and correlate well with the Km/kcat values for the corresponding peptide substrates [Morihara, K., & Tsuzuki, H. (1970) Eur. J. Biochem. 15, 374-380] but not with the Km values alone. The correlation noted between inhibitor Ki and substrate Km/kcat is the most extensive one of this type, providing strong evidence that the phosphonamidates are transition-state analogues and not simply multisubstrate ground-state analogues. Cbz-NH2CH2PO2--L-Leu-L-Leu (Ki = 9.1 nM) is the most potent inhibitor yet reported for thermolysin.