Synthesis and biological activity of ketomethylene pseudopeptide analogues as thrombin inhibitors.

Ketomethylene pseudopeptide analogues Aa-Pro-Arg psi (COCH2) Gly-pip, 1, where Aa are D- or L-amino acids (Dpa, beta, beta-diphenylalanine; alpha Nal, alpha-naphthylalanine; beta Nal, beta-naphthylalanine; Fgl, fluorenylglycine) with highly lipophilic side chains and psi (COCH2) is a ketomethylene pseudopeptide bond, have been synthesized through a modified Dakin-West reaction under very mild conditions with a high yield using tripeptide 4 with a labile functional group directly on the side chain. Their enzymatic assay of thrombin inhibition has been carried out. The structure-activity relationship study indicated that a lipophilic side chain on the amino acid in the P3 position is very important for binding to the apolar site of thrombin. Compound 1a with D-Dpa at the P3 position has a Ki of 0.2 microM and it doubles thrombin clotting time at only 3 times higher concentration. These values are about 7 times better than those of the corresponding D-Phe analogues. Furthermore, 1a shows poor inhibitory activity against plasmin, factor Xa, urokinase, and kallikrein. Preliminary in vivo testing (3-4-kg rabbit as the animal model) shows no observable side effect (change of blood pressure and accumulation of blood platelet in lungs) at a dose of 1 mg/kg.

Characterization of a class of peptide boronates with neutral P1 side chains as highly selective inhibitors of thrombin.

Z-D-Phe-Pro-boroMpg-OPin (9a)1,2 has been shown previously to be a highly specific inhibitor of thrombin in spite of lacking an arginine-like guanidino group at the P1 site. A range of compounds have been synthesized based upon this lead compound, varying the neutral side chain at the P1 site. Of the 20 examples based upon the structures at P2 and P3 of Z-D-X-Pro (X being Phe or beta,beta-diphenylalanine), all were found to be effective inhibitors of thrombin (Ki's between 10 and 100 nM). Furthermore all exhibited a high specificity toward thrombin having values for a Ki(trypsin)/Ki(thrombin) ratio of between 10- and 100-fold. High ratio values were found for a number of the compounds tested against a range of serine proteinases (plasmin, factor Xa, kallikrein, urokinase, protein Ca, chymotrypsin, elastase, and cathepsin G). As far as potency toward thrombin, compounds containing the methoxypropyl group at P1 were favored over those with a methoxy grouping on a shorter alkyl chain (8) or without the methoxy group (1-5). The compounds display potent anticoagulant activity with values for 18 in thrombin time of 0.63 microM and in activated partial thromboplastin time of 2.0 microM. 11B NMR has been used to confirm interaction of the boron atom with the active site. From the high specificity shown with all the compounds we propose that the compounds, constitute a new class of thrombin inhibitors.

Synthetic peptides and peptidomimetics as substrates and inhibitors of thrombin and other proteases in the blood coagulation system.

The synthesis of peptides as imitations of the thrombin cleavage site of fibrinogen has led to sequences with affinity for the enzyme. These peptides were first developed as chromogenic and fluorogenic substrates for thrombin. The same idea was also used to generate peptide substrates for other serine proteases in blood coagulation and fibrinolysis. Amidolytic methods based upon the substrates have revolutionized assays of proenzymes, enzymes, cofactors and inhibitors in research as well as in clinical laboratories. Like peptidomimetics based only on Arg (P1 of the natural substrate) or Arg analogues, these amino acid sequences also have been developed as active site directed inhibitors of thrombin and of factor Xa. A further interesting development is the synthesis of bivalent thrombin inhibitors which, like hirudin, bind to the thrombin active centre as well as to its anionic exosite. Recently, also, it has been shown that the positively charged side chain of P1 Arg is not an absolute necessity for binding a peptide to the active site of thrombin. Several of the new thrombin inhibitors show interesting properties for pharmaceutical development and some of them are on clinical trials.

Non-chromogenic peptide substrates for detection of enzymes by means of capacitance changes at metal electrodes.

A new type of substrate for enzyme detection has been developed. The substrate is non-chromogenic and is used in an assay method based on electrode adsorption. The rate of change in the electric capacitance of the electrode is monitored and taken as a measure of the substrate adsorption. Substrate adsorption is in turn proportional to substrate bulk concentration and thus subject to changes by enzymes. The new substrate introduces a new concept in enzyme detection: as it is non-chromogenic it may contain appropriate amino acids on both sides of the bond subject to enzymatic cleavage.

The electrode adsorption method for determination of enzyme activity: a study of substrate requirements.

The electrode adsorption method for the determination of enzyme activity requires substrates that, besides having good kinetics constants for the enzyme, also show good adsorption/desorption kinetics to the electrode surface and adsorb in such a way that they change the double-layer capacitance of the electrode. A series of peptide substrates containing one to three aromatic groups has been synthesized. Our results show that the aromatic groups are of crucial importance for the capacitance change caused by the adsorbing/desorbing substrate. Thus, the tripeptide substrate, Bz-Phe(NO2)-Val-Arg-pNA, with three aromatic groups is superior to the other synthesized substrates containing only one or two aromatic groups. Our desorption experiments show that several factors determine the rate of capacitance increase observed when thrombin is added to a substrate solution in equilibrium with a substrate-covered electrode. The kinetic constants of the substrate determine how the substrate concentration in the solution decreases and, consequently, determine the spontaneous desorption measured as capacitance increase. Thrombin does not seem to split adsorbed substrate molecules but it adsorbs to the substrate-covered surface and in that way causes a capacitance decrease counteracting the change caused by desorption of substrate.

Determination of prekallikrein in plasma by means of a chromogenic tripeptide substrate for plasma kallikrein.

A method for plasma prekallikrein determination utilizing a chromogenic tripeptic substrate is presented. The method has a good reproducibility and can easily be automized. Several parameters have been optimized. By using mixtures of deficient plasmas and pooled normal plasma or purified factors it was proved that prekallikrein was the factor determined and that more than 10% (of normal plasma concentration) of FXII and HMW kininogen were essential for the activation of prekallikrein in our method. Further experiments showed that the method was fairly selective and was not influenced by inhibitors present in normal plasma. The later finding was attributed to the high dilution of plasma made possible by using a potent activator and a sensitive substrate.

Methods for the determination of glandular kallikrein by means of a chromogenic tripeptide substrate.

A chromogenic peptide substrate H-D-Val-Leu-Arg-pNA (S-2266) has been used for the determination of glandular kallikrein derived from pancreas, urine and saliva. The conditions used have been optimized. The methods developed are simple and shown to have good reproducibility.

Inhibition of the contractile action of bradykinin on isolated smooth muscle preparations by derivatives of low molecular weight peptides.

The carbonyl terminal tripeptide sequence of bradykinin (Pro-Phe-Arg) is molecularly manipulated to obtain agents with potent antagonistic activity towards the smooth muscle contractile activity of bradykinin. Screening of various peptide derivatives revealed that heptyl amides or esters of H-D-Pro-Phe-Arg, and H-D-Phe-Phe-Arg possessed relatively stronger antibradykinin activity on the isolated smooth muscle preparation. The parent tripeptides, H-D-Pro-Phe-Arg-OH, and H-D-Phe-Phe-Arg-OH, and their amino acid components, i.e. D-Proline, D-Phenylalanine, L-Phenylalanine and Arginine, did not possess any antibradykinin activity in concentrations of up to 10(-4) M. When the heptyl derivatives of these peptides were incubated with either heparinized or citrated whole blood or plasma, the antibradykinin activity was not lost. Incubation of these peptide derivatives with either carboxypeptidase A or B did not result in any loss of the pharmacological effect. However, pancreatic protease extract produced a significant loss of the anti-oxytocic action on the isolated rat uterus preparation. H-D-Pro-Phe-Arg-NH-lauryl derivative also blocked the action of bradykinin and this effect sustained for a longer period of time comparative to the blockade with H-D-Pro-Phe-Arg-NH-heptyl derivative. In concentrations of 10(-7) M and 10(-8) M and 1 min incubation, which blocked the contractile action of bradykinin (1 nmole) on the isolated guinea pig ileum, these peptide derivatives did not block the action of acetylcholine, histamine, and serotonin. However, in concentrations of about 10(-6) M and higher with 5 min. incubation histamin is also blocked. On the isolated rat uterus preparation the contractile action of acetylcholine, angiotensin, oxytocin and vasopressin was blocked at concentrations of 10(-6) M. These findings warrant a differential pharmacological evaluation and in vivo testing of these peptide derivatives to investigate their therapeutic potential.

Structure/function aspects of neutral P1 residue peptide inhibitors of thrombin.

Control of thrombin by its inhibition in indications such as myocardial infarction, unstable angina or stroke has been demonstrated to be therapeutically valuable. However restoration of hemostasis by targeting thrombin while avoiding its fellow serine proteinases, (e.g. plasmin, trypsin), remains a challenge of medicinal chemistry. Tripeptide-boronates and -phosphonates with neutral P1 side chains meet these criteria. Development of novel, high yielding chemical routes furnishes a wide range of un-natural P1 functionalities, demonstrating that this indeed is a class effect with selectivity conferred by the uncharged P1 residue. For example N-benzyloxycarbonyl-D-phenylalanylprolyl-1- (3-methoxypropyl) boroglycine ester (1) has a Ki value for thrombin of 7 nM and greater than two order of magnitude higher with all other serine proteinases tested. The ester group determines the kinetics of inhibition by tripeptide phosphonates, with diphenylphosphonates being slow tight binding inhibitors, showing 50% reversibility of inhibition. Therefore this design of inhibitors offers a facile strategic approach to development as thrombin specific pharmaceutical agents.

Methods for determination of prekallikrein in plasma, glandular kallikrein and urokinase.

New chromogenic tripeptide substrates have been used for the determination of kallikreins and urokinase. The conditions have been optimized. It is possible to determine prekallikrein in plasma after activation with Cephotest. No significant loss in activity caused by plasma kallikrein inhibitors is observed at the dilutions used.

Methods for determination of plasmin, antiplasmin and plasminogen by means of substrate S-2251.

The chromogenic substrate S-2251 (H-D-Val-Leu-Lys-pNA), a selective and sensitive substrate for plasmin activity, has made it possible to develop simple and reproducible methods for the determination of antiplasmin and plasminogen in human plasma. These methods have been optimized and studied in detail and found to be very specific for the respective factors.

Designing of peptide substrates. Different approaches exemplified by new chromogenic substrates for kallikreins and urokinase.

Chromogenic peptide substrates for serine proteases have been designed by using two approaches: (1) by using the natural substrate as a model and (2) by structure-activity correlations obtained through screening of a large number of tripeptides. Some recent examples, substrates for kallikreins and urokinase are given.