Orally active thrombin inhibitors. Part 2: optimization of the P2-moiety

Synthesis and SAR of orally active thrombin inhibitors of the d-Phe-Pro-Arg type with focus on the P2-moiety are described. The unexpected increase in in vitro potency, oral bioavailability, and in vivo activity of inhibitors with dehydroproline as P2-isostere is discussed. Over a period of 24h the antithrombin activity of the most active inhibitors with IC(50)s in the nanomolar range was determined in dogs demonstrating high thrombin inhibitory activity in plasma and an appropriate duration of action after oral administration.     

Protease inhibitors. Part 2. Weakly basic thrombin inhibitors incorporating sulfonyl-aminoguanidine moieties as S1 anchoring groups: synthesis and structure-activity correlations

Two series of derivatives have been prepared and assayed as inhibitors of two physiologically relevant serine proteases, human thrombin and human trypsin. The first series includes alkyl-/ aralkyl-/aryl- and hetarylsulfonyl-aminoguanidines. It was thus observed that sulfanilyl-aminoguanidine possesses moderate but intrinsically selective thrombin inhibitory properties, with KI values around 90 and 1400 nM against thrombin and trypsin respectively. Further elaboration of this molecule afforded compounds that inhibited thrombin with KI values in the range 10-50 nM, whereas affinity for trypsin remained relatively low. Such compounds were obtained either by attaching benzyloxycarbonyl- or 4-toluenesulfonylureido-protected amino acids (such as D-Phe, L-Pro) or dipeptides (such as Phe-Pro, Gly His, beta-Ala-His or Pro-Gly) to the N-4 atom of the lead molecule, sulfanilyl-aminoguanidine, or by attaching substituted-pyridinium propylcarboxamido moieties to this lead. Thus, this study brings novel insights regarding a novel non-basic S1 anchoring moiety (i.e., SO2NHNHC(=NH)NH2), and new types of peptidomimetic scaffolds obtained by incorporating tosylureido-amino acids/pyridinium-substituted-GABA moieties in the hydrophobic binding site(s). Structure-activity correlations of the new serine protease inhibitors are also discussed based on a QSAR model described previously for a large series of structurally-related derivatives (Supuran et al. (1999) J. Med. Chem., in press).     

Novel thrombin inhibitors incorporating weakly basic heterobicyclic P1-arginine mimetics: optimization via modification of P1 and P3 moieties

Optimization of lead compounds 1 and 2 resulted in novel, selective, and potent thrombin inhibitors incorporating weakly basic heterobicyclic P(1)-arginine mimetics. The design, synthesis, and biological activity of racemic thrombin inhibitors 17-29 and enantiomerically pure thrombin inhibitors 30-33 are described. The arginine side-chain mimetics used in this study are 4,5,6,7-tetrahydro-1,3-benzothiazol-2-amine, 4,5,6,7-tetrahydro-2H-indazole, and 2-imino-4,5,6,7-tetrahydro-1,3-benzothiazol-3(2H)-ylamine.     

Protease inhibitors: Part 4. Synthesis of weakly basic thrombin inhibitors incorporating pyridinium-sulfanilylaminoguanidine moieties

Three series of derivatives have been prepared by reaction of sulfanilylaminoguanidine with pyrylium salts, with the pyridinium derivatives of glycine and with the pyridinium derivatives of beta-alanine, respectively. The new compounds were assayed as inhibitors of two serine proteases, thrombin and trypsin. The study showed that in contrast to the leads, possessing KI's around 100-300 nM against thrombin, and 450-1420 nM against trypsin, respectively, the new derivatives showed inhibition constants in the range of 15-50 nM against thrombin, whereas their affinity for trypsin remained relatively low. Derivatives of beta-alanine were more active than the corresponding glycine derivatives, which in turn were more inhibitory than the pyridinium derivatives of sulfanilylaminoguanidine possessing the same substitution pattern at the pyridinium ring. Thus, the present study proposes two novel approaches for the preparation of high affinity, specific thrombin inhibitors: a novel S1 anchoring moiety in the already large family of arginine/amidine-based inhibitors, i.e., the SO2NHNHC(=NH)NH2 group, and novel non-peptidomimetic scaffolds obtained by incorporating alkyl-/aryl-substituted-pyridinium moieties in the hydrophobic binding site(s). The first one is important for obtaining bioavailable thrombin inhibitors, devoid of the high basicity of the commonly used arginine/amidine-based inhibitors, whereas the second one may lead to improved water solubility of such compounds.     

Effect of low-molecular peptides on the transformation of fibrinogen into fibrin

The influence of synthetic peptides on fibrinogen transformation to fibrin under the action of thrombin and fibrin-monomer polymerization was investigated. Peptides Gly-Pro-Arg-Pro; Gly-Pro-Arg-Pro-Lys; Gly-Pro-Arg-Pro-Lys-Boc; Gly-Pro-Arg-Pro-Arg are specific inhibitors of fibrin formation. These peptides interfere with the hydrolysing effect of thrombin due to binding to the central domain of fibrinogen. The interaction of peptides with peripheral D-domains of fibrin-monomer may account for polymerization inhibition. The latter peptide has the largest anticoagulation activity. It is likely that arginine in the fifth position stabilizes the structure of the peptides, with the additional epsilon NH2-group activating its interaction with protein.     

Selective 3-amino-2-pyridinone acetamide thrombin inhibitors incorporating weakly basic partially saturated heterobicyclic P1-arginine mimetics

Novel, highly selective and potent thrombin inhibitors were identified as a result of combing the 3-benzylsulfonylamino-2-pyridinone acetamide P(2)-P(3) surrogate with weakly basic partially saturated heterobicyclic P(1)-arginine mimetics 1-8. The design, synthesis, biological activity, and the binding modes of non-covalent thrombin inhibitors featuring P(1)-4,5,6,7-tetrahydroindazole, 5,6,7,8-tetrahydroquinazoline, and 4,5,6,7-tetrahydrobenzothiazole moieties are described.     

Reciprocal Modulation of Astrocyte Stellation by Thrombin and Protease Nexin-1

When cultured astroglia are treated with agents that elevate intracellular cyclic AMP, they become process-bearing stellate cells and resemble differentiated astrocytes in vivo. Thrombin rapidly reversed the stellation induced by dibutyryl cyclic AMP, forskolin, or isoproterenol in cultured rat astrocytes; half-maximal and maximal effects occurred at 0.5 and 8 pM, respectively. The proteolytic activity of thrombin was required for stellation reversal, as thrombin derivatized at its catalytic site serine with a diisopropylphospho group was inactive. Two thrombin inhibitors, protease nexin-1 and hirudin, blocked and reversed the effect of thrombin. The stellation reversal effect of thrombin was specific, as 300-1,000-fold higher concentrations of other serine proteinases, including plasmin, urokinase, trypsin, and T cell serine proteinase-1, were ineffective. Thrombin is a mitogen for astrocytes at concentrations in excess of 30 pM. Thrombin increased both cell number and ornithine decarboxylase activity, an early marker for mitogenic stimulation, in astrocyte cultures. The lowest thrombin concentrations that completely reversed astrocyte stellation, however, did not increase ornithine decarboxylase activity. Moreover, several other mitogens for astrocytes did not reverse dibutyryl cyclic AMP-induced stellation. Thus, the stellation reversal effect of thrombin is distinct from the mitogenic response.     

Reactivity of Lys(NH2)-containing peptides toward endopeptidases.

Lys(NH2)-containing peptides were subjected to various proteolytic enzymes which were selected for their well-documented specificity for arginyl and/or lysyl peptide bonds. Lys(NH2)-containing peptides were cleaved more rapidly by clostripain than the corresponding lysyl peptides. On the other hand, they proved to be resistant to Achromobacter protease I hydrolysis. The modified peptides synthesized in this study were more stable than the arginyl and lysyl analogues when incubated with trypsin or thrombin. The same tendency was observed when Lys(NH2)-containing peptides were incubated in diluted human serum, suggesting that the replacement of Arg or Lys by Lys(NH2) could be used to increase the stability of peptides in vivo.     

On the activation of human leuserpin-2, a thrombin inhibitor, by glycosaminoglycans

Human leuserpin-2 (hLS2) cDNA variants generated by site-directed mutagenesis were expressed in a transient COS cell system. Functional analysis of the mutants revealed two regions in the NH2-terminal half of hLS2 which are essential for glycosaminoglycan-enhanced thrombin inhibition by hLS2. One of these regions, which encompasses a dimeric structure enriched in basic amino acids, is required for both glycosaminoglycan binding and glycosaminoglycan-mediated acceleration of thrombin inhibition. Deletion of another dimeric region, which spans a sequence with a high negative charge density, resulted in a strong reduction in the glycosaminoglycan-enhanced activity of hLS2. This polyanionic region displays structural and functional similarities to the COOH-terminal end of hirudin, another potent thrombin inhibitor, indicating that both inhibitors may have a common binding site on thrombin. Based on our observations we propose a model for the activation of hLS2 by glycosaminoglycans. The key feature of this model is the suggestion that the glycosaminoglycan-enhanced reaction between hLS2 and thrombin is mediated by at least two regions of contact, involving both the reactive center region and the acidic domain of hLS2. Binding of glycosaminoglycans to hLS2 is suggested to result first in the release of the acidic region from intramolecular interactions. Then, amino acid sequences in thrombin are proposed to interact with the acidic dimer of hLS2.     

Pharmacology of benzamidine-type thrombin inhibitors

Findings on the pharmacology of derivatives of benzamidine characterized as potent competitive inhibitors of the clotting enzyme thrombin were presented including data on their toxicity. Bis-benzamidines and amidinophenylalanine amides were shown to exert pharmacodynamic effects in intact animals and isolated preparations (hypotension; influence on smooth muscle reactions to serotonin and histamine). Studies with 14C-4-amidinophenylpyruvic acid and 3H-N alpha-tosyl-(3-amidino)-phenylalanine piperidide on the pharmacokinetics in rabbits showed that benzamidine derivatives are suited, in principle, for use as anticoagulants in vivo. Pharmacokinetic properties of individual thrombin inhibitors have to be taken into account in order to reach and maintain adequate plasma levels. The antithrombotic effect of benzamidine-type thrombin inhibitors in animals experiments is directly related to their antithrombin activity.     

Development of an oxazolopyridine series of dual thrombin/factor Xa inhibitors via structure-guided lead optimization

Thrombin-inhibitor X-ray crystal structures, in combination with the installation of binding elements optimized within the pyrazinone series of thrombin inhibitors, were utilized to transform a weak triazolopyrimidine lead into a series of potent oxazolopyridines. A modification intended to attenuate plasma protein binding (i.e., conversion of the P3 pyridine to a piperidine) conferred significant factor Xa activity to this series. Ultimately, these dual thrombin/factor Xa inhibitors demonstrated excellent in vitro and in vivo anticoagulant efficacy.     

Retro-binding thrombin active site inhibitors: identification of an orally active inhibitor of thrombin catalytic activity

A series of retro-binding inhibitors of human alpha-thrombin was prepared to elucidate structure-activity relationships (SAR) and optimize in vivo performance. Compounds 9 and 11, orally active inhibitors of thrombin catalytic activity, were identified to be efficacious in a thrombin-induced lethality model in mice.     

Protease Inhibitors: Part 4. Synthesis of Weakly Basic Thrombin Inhibitors Incorporating Pyridinium-Sulfanilylaminoguanidine Moieties

Three series of derivatives have been prepared by reaction of sulfanilylaminoguanidine with pyrylium salts, with the pyridinium derivatives of glycine and with the pyridinium derivatives of β-alanine, respectively. The new compounds were assayed as inhibitors of two serine proteases, thrombin and trypsin. The study showed that in contrast to the leads, possessing K₁'s around 100–300nM against thrombin, and 450–1420nM against trypsin, respectively, the new derivdtives showed inhibition constants in the range of 15–50nM against thrombin, whereas their affinity for trypsin remained relatively low. Derivatives of β-alanine were more active than the corresponding glycine derivatives, which in turn were more inhibitory than the pyridinium derivatives of sulfanilylaminoguanidine possessing the same substitution pattern at the pyridinium ring. Thus, the present study proposes two novel approaches for the preparation of high affinity, specific thrombin inhibitors: a novel S1 anchoring moiety in the already large family of arginine/amidine-based inhibitors, i.e., the SO₂NHNHC(=NH)NH₂ group, and novel non-peptidomimetic scaffolds obtained by incorporating alkyl-/aryl-substituted-pyridinium moieties in the hydrophobic binding site(s). The first one is important for obtaining bioavailable thrombin inhibitors, devoid of the high basicity of the commonly used arginine/amidine-based inhibitors, whereas the second one may lead to improved water solubility of such compounds.     

Synergistic effect of aptamers that inhibit exosites 1 and 2 on thrombin

Thrombin is a multifunctional protease that plays a key role in hemostasis, thrombosis, and inflammation. Most thrombin inhibitors currently used as antithrombotic agents target thrombin''s active site and inhibit all of its myriad of activities. Exosites 1 and 2 are distinct regions on the surface of thrombin that provide specificity to its proteolytic activity by mediating binding to substrates, receptors, and cofactors. Exosite 1 mediates binding and cleavage of fibrinogen, proteolytically activated receptors, and some coagulation factors, while exosite 2 mediates binding to heparin and to platelet receptor GPIb-IX-V. The crystal structures of two nucleic acid ligands bound to thrombin have been solved. Previously Padmanabhan and colleagues solved the structure of a DNA aptamer bound to exosite 1 and we reported the structure of an RNA aptamer bound to exosite 2 on thrombin. Based upon these structural studies we speculated that the two aptamers would not compete for binding to thrombin. We observe that simultaneously blocking both exosites with the aptamers leads to synergistic inhibition of thrombin-dependent platelet activation and procoagulant activity. This combination of exosite 1 and exosite 2 inhibitors may provide a particularly effective antithrombotic approach.     

High density fermentation and activity of a recombinant lumbrokinase (PI239) from Pichia pastoris

A system for the expression of recombinant lumbrokinase (rPI239) was developed in the yeast Pichia pastoris. A total supernatant protein content of 0.174 g/L of high density fermentation broth was obtained. The rPI239 exhibited in vitro fibrinolytic activity. The in vivo activity of rPI239 was measured by prothrombin time, kaolin part thrombin time, thrombin time, and fibrinolytic activity. This work presents the high-density fermentation of rPI239 from P. pastoris and shows that the recombinant protein has similar fibrinolytic activity both in vivo and in vitro.     

The design of potent, selective, non-covalent, peptide thrombin inhibitors utilizing imidazole as a S1 binding element.

Modeling of neutral or mildly basic functional groups in the S1 site of thrombin led to the targeting of imidazole as a S1 binding element and correctly predicted the optimal chain length for connecting this group with the S2 and S3 binding elements. Derivatives of 4-(3-aminopropyl)-imidazole can be selective inhibitors of thrombin demonstrating potent anticoagulant activity.     

Synthesis and characterization of extended and deleted recombinant analogues of parathyroid hormone-(1-84): correlation of peptide structure with function

Recombinant analogues of human parathyroid hormone [hPTH-(1-84)] were expressed in Escherichia coli harboring plasmids containing synthetic genes under the control of the lac promoter. The level of expression of the gene encoding the truncated analogue, hPTH-(3-84), was greater than that of the gene encoding full-length hPTH-(1-84) but less than that of the gene encoding proparathyroid hormone (hProPTH). This may be due in part to the relative efficiency of translation of the mRNA as suggested by secondary structure analysis and in part because of enhanced stability of the extended peptide. Formylmethionyl derivatives of hProPTH and of hPTH-(3-84) and underivatized hPTH-(3-84) were purified by HPLC, and their identity was confirmed by NH2-terminal sequencing and amino acid analysis. The bioactivity of these recombinant peptides was then tested in skeletal and renal adenylate cyclase assays in vitro and in assays examining effects on plasma and urine calcium and phosphate levels and on urine cyclic AMP levels in vivo. The NH2-terminally extended analogue fMet-hProPTH displayed 10% of the in vitro activity of hPTH-(1-84) and was a partial agonist in vivo. The peptides hPTH-(3-84) and fMet-hPTH-(3-84) were inert in vitro and were very weak in vitro antagonists when compared to the NH2-terminal analogue bovine [Nle8,18Tyr34]PTH-(3-34)-NH2. In vivo, hPTH-(3-84) and the bPTH-(3-34) analogue, when assayed at a 10:1 molar ratio relative to bPTH-(1-84), were each inert, and neither demonstrated antagonist activity at these concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation of l-proline based aeruginosin 298-A analogs: Optimization of the P1-moiety

Aeruginosins are a family of naturally occurring oligopeptides that share a common bicyclic amino acid core structure. Many compounds in the family are inhibitors of serine proteases, such as thrombin and trypsin. Thrombin is an important enzyme in the blood coagulation cascade, and is a promising target for anticoagulant drug development. In order to understand the structure–activity relationship (SAR) and to find selective thrombin inhibitors, we synthesized a series of aeruginosin 298-A analogs, in which the P₂ bicyclic amino acid was replaced by a l-proline residue. The structure optimization was focused on modification of the P₁ position. In choosing the P₁ group, an effort was made to avoid using the highly basic guanidine groups present in nearly all naturally occurring aeruginosins. The synthesis and enzyme assays of these aeruginosin analogs against thrombin and trypsin are reported. We found that several compounds with neutral P₁ groups exhibit excellent selectivity over trypsin and good potency against thrombin. The SAR data of the P₁ groups obtained here can be used in preparing other thrombin inhibitors with better selectivity against trypsin.     

Anticoagulant activity of synthetic hirudin peptides

Synthetic peptides based on the COOH-terminal 21 residues of hirudin were prepared in order to 1) evaluate the role of this segment in hirudin action toward thrombin, 2) define the shortest peptide derivative with anticoagulant activity, and 3) investigate the role of tyrosine sulfation in the peptides' inhibitory activities. A hirudin derivative of 20 amino acids, Hir45-64 (derived from residues 45-64 of the hirudin polypeptide), was found to effect a dose-dependent increase in the activated partial thromboplastin time (APTT) of normal human plasma but to have no measurable inhibitory activity toward thrombin cleavage of a tripeptidyl p-nitroanilide substrate. Anticoagulant activity in hirudin derivatives was comparable in peptides of 20, 16, and 12 residues truncated from the NH2 terminus. Additional truncated peptides prepared by synthesis and carboxypeptidase treatment reveal that the minimal sequence of a hirudin peptide fragment with maximal anticoagulant activity is contained within the sequence: NH2-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-COOH. The 12-residue derivative thus identified was reacted with dicyclohexylcarbodiimide in the presence of sulfuric acid to yield a Tyr-sulfated peptide, S-Hir53-64. By comparison to unsulfated peptide, S-Hir53-64 was found to contain a specific inhibitory activity enhanced by one order of magnitude toward increase in APTT and to effect a dose-dependent increase in thrombin time of normal human plasma to yield a 4-fold increase in thrombin time with 2.5 micrograms/ml peptide using 0.8 units/ml alpha-thrombin. Comparison of S-Hir53-64 to hirudin in thrombin time and APTT assays reveals a 50-fold difference in molar specific activities toward inhibition of thrombin. Comparison of antithrombin activities of S-Hir53-64 using a variety of animal thrombins demonstrates greatest inhibitory activity toward murine, rat, and human enzymes and a 10-fold reduced activity toward bovine thrombin.     

Protease Inhibitors. Part 2. Weakly Basic Thrombin Inhibitors Incorporating Sulfonyl-Aminoguanidine Moieties as S1 Anchoring Groups: Synthesis and Structure-Activity Correlations

Abstract

Two series of derivatives have been prepared and assayed as inhibitors of two physiologically relevant serine proteases, human thrombin and human trypsin. The first series includes alkyl-/ aralkyl-/aryl- and hetarylsulfonyl-aminoguanidines. It was thus observed that sulfanilyl-aminoguanidine possesses moderate but intrinsically selective thrombin inhibitory properties, with K_i values around 90 and 1400 nM against thrombin and trypsin respectively. Further elaboration of this molecule afforded compounds that inhibited thrombin with K_i values in the range 10–50 nM, whereas affinity for trypsin remained relatively low. Such compounds were obtained either by attaching benzyloxycarbonyl- or 4-toluenesulfonylureido-protected amino acids (such as D-Phe, L-Pro) or dipeptides (such as Phe-Pro, Gly-His, β-Ala-His or Pro-Gly) to the N-4 atom of the lead molecule, sulfanilyl-aminoguanidine, or by attaching substituted-pyridinium-propylcarboxamido moieties to this lead. Thus, this study brings novel insights regarding a novel non-basic S1 anchoring moiety (i, e., SO₂NHNHC(=NH)NH₂), and new types of peptidomimetic scaffolds obtained by incorporating tosylureido-amino acids/pyridinium-substituted-GABA moieties in the hydrophobic binding site(s). Structure-activity correlations of the new serine protease inhibitors are also discussed based on a QSAR model described previously for a large series of structurally-related derivatives (Supuran et al. (1999) J. Med. Chem., in press).