1,2-Dibenzamidobenzene inhibitors of human factor Xa.

High-throughput screening of a combinatorial library of diamidophenols yielded lead compounds with the ability to inhibit human factor Xa (fXa) at micromolar concentrations (e.g. compound 4, fXa apparent K(ass) = 0.64 x 10(6) L/mol). SAR studies in this novel structural series of fXa inhibitors showed that the phenolic hydroxyl group was not essential for activity. The best activity was found in substituted 1,2-dibenzamidobenzenes in which the phenyl group of one benzoyl group (A-ring) was substituted in the 4-position with relatively small lipophilic or polarizable groups such as methoxy, vinyl, or chloro and the phenyl group of the other benzoyl group (B-ring) was substituted in the 4-position with larger lipophilic groups such as tert-butyl or dimethylamino. The central phenyl ring (C-ring) tolerated a wide variety of substituents, but methoxy, methanesulfonamido, hydroxyl, and carboxyl substitution produced slightly higher levels of activity than other substituents when present in combination with favorable B-ring substitution. Methylation of the amide nitrogen atoms was found to greatly decrease activity. Compound 12 is the highest affinity fXa inhibitor in this group of compounds, having fXa apparent K(ass) = 25.5 x 10(6) L/mol, about 40x more active than the original lead. This lead series does not show potent inhibition of human thrombin. A model for the binding of these ligands to the fXa active site is proposed. The model is consistent with the observed SAR and can serve to guide future SAR studies.

N(2)-Aroylanthranilamide inhibitors of human factor Xa.

Reversal of the A-ring amide link in 1,2-dibenzamidobenzene 1 (fXa K(ass) = 0.81 x 10(6) L/mol) led to a series of human factor Xa (hfXa) inhibitors based on N(2)-aroylanthranilamide 4. Expansion of the SAR around 4 showed that only small planar substituents could be accommodated in the A-ring for binding to the S1 site of hfXa. Bulky groups such as 4-isopropyl, 4-tert-butyl, and 4-dimethylamino were favored in the B-ring to interact with the S4 site of hfXa. The central (C) ring containing a 5-methanesulfonamido group yielded greater activity than carbamoyl groups. Combining the beneficial features from the B- and C-ring SAR, compound 55 represents the most potent hfXa inhibitor in the N(2)-aroylanthranilamide 4 series with hfXa K(ass) = 58 x 10(6) L/mol (K(i) = 11.5 nM).

Structure-based design of potent, amidine-derived inhibitors of factor Xa: evaluation of selectivity, anticoagulant activity, and antithrombotic activity.

To enhance the potency of 1,2-dibenzamidobenzene-derived inhibitors of factor Xa (fXa), an amidine substituent was incorporated on one of the benzoyl side chains to interact with Asp189 in the S1 specificity pocket. Lead molecule 1 was docked into the active site of fXa to facilitate inhibitor design. Subsequently, iterative SAR studies and molecular modeling led to a 1000-fold increase in fXa affinity and a refined model of the new inhibitors in the fXa active site. Strong support for the computational model was achieved through the acquisition of an X-ray crystal structure using thrombin as a surrogate protein. The amidines in this series show high levels of selectivity for the inhibition of fXa relative to other trypsin-like serine proteases. Furthermore, the fXa affinity of compounds in this series (K(ass) = 50-500 x 10(6) L/mol) translates effectively into both anticoagulant activity in vitro and antithrombotic activity in vivo.

A family of arginal thrombin inhibitors related to efegatran.

Three new tripeptide arginal thrombin inhibitors were shown to have potent anticoagulant and antithrombotic activity: D-MePhg-Pro-Arg-H (LY287045), D-1-Tiq-Pro-Arg-H (LY294291), and D-MePhe-Pro-Arg-H (Efegatran). Efegatran and the related arginals differ mechanistically from old and from new anticoagulant agents. As illustrated with x-ray diffraction analysis of crystals of the LY294291 complex with human thrombin, the family of arginals binds thrombin with the P3, P2, and P1 residues interacting with the putative S3, S2, and S1 fibrinogen-binding sites. A hemi-acetal bond at Ser 195 was shown to contribute to the tight-binding reversible competitive thrombin inhibition properties observed with the arginal family. Tight-binding Kass values from thrombin inhibition studies correlated with thrombin clottin inhibition potency. The thrombin time (TT) assay was prolonged twofold with 33 nM Efegatran, which demonstrated an apparent Kass value of 0.8 x 10(8) L/mol (for comparison, 17 nM hirudin was required to prolong the TT assay two-fold). There are empirical anticoagulant selectivity differences between Efegatran and hirudin, manifested by large activated partial thromboplastin time (aPTT)/TT effect ratios (30 to 55) found with the arginals, as compared to the small aPTT/TT effect ratio (2 to 3) found with hirudin. The underlying anticoagulant mechanism differences between the arginals and hirudin appear to be confined to the aPTT pathway and, therefore, might involve different effects toward thrombin feedback activation of factor VIII. The arginals did not substantially inhibit other coagulation factor serine proteases. Antithrombotic effects of Efegatran and the arginal family occur at low infusion doses in dogs and appear to correlate with effects on TT without requiring perturbation of the aPTT. Selectivity properties regarding the fibrinolytic enzymes were shown to be important for successful use of the arginals in vivo as adjunctive agents during tissue plasminogen activator (t-PA) thrombolysis. The data suggest that LY287045, LY294291, and Efegatran should be expected to be useful as antithrombotic adjuncts to thrombolytic therapy with t-PA, urokinase, or streptokinase and should be expected to spare endogenous fibrinolysis. Efegatran has been evaluated in phase I clinical studies and is currently under clinical investigation in phase II protocols as a new cardiovascular anticoagulant.

Comparison of the thrombolytic activity of the novel plasminogen activator, LY210825, to anisoylated plasminogen-streptokinase activator complex in a canine model of coronary artery thrombolysis.

We have compared the thrombolytic efficacy of a novel single-chain, recombinant tissue-type plasminogen activator variant, LY210825, containing the second kringle and serine protease domains of native tissue-type plasminogen activator, with anisoylated plasminogen-streptokinase activator complex (APSAC). Male hounds (16-22 kg) were anesthetized, the left circumflex coronary artery was isolated and an electromagnetic flow probe was placed around the artery proximal to the first main branch for the measurement of coronary blood flow. An occlusive thrombus was formed after electrolytic injury of the intima of the coronary artery. After an occlusion period of 1 hr, either LY210825 (n = 8) or APSAC (n = 6) was administered as a single i.v. injection of 0.45 mg/kg. Blood was drawn (3.8% citrate) for determination of plasma fibrinogen, plasminogen and alpha-2 antiplasmin. Time to reperfusion was significantly faster with LY210825 than with APSAC, 20 +/- 2 vs. 54 +/- 8 min, respectively. The incidence of reocclusion was similar for both agents. APSAC produced significant depletion of alpha-2 antiplasmin, plasminogen and circulating fibrinogen, whereas LY210825 caused only slight consumption of plasminogen and only small decreases in fibrinogen. After a single injection of LY210825, thrombolytic concentrations of plasminogen activator were available immediately, whereas there was a significant delay in lytic concentrations of active streptokinase-plasmin complex. Consequently, LY210825 reperfused the coronary artery faster than did APSAC. In addition, LY210825 spared plasma fibrinogen, plasminogen and alpha-2 antiplasmin and therefore, could potentially minimize the risk of bleeding complications.

Thrombolytic activity of a novel plasminogen activator, LY210825, compared with recombinant tissue-type plasminogen activator in a canine model of coronary artery thrombosis.

LY210825, a recombinant tissue-type plasminogen activator (rt-PA), which contains the kringle-2 and serine protease functional domains of native tissue-type plasminogen activator, was previously produced by site-directed mutagenesis in a Syrian hamster cell line. We studied the thrombolytic potential of this molecule in a canine thrombosis model. Male hounds (16-22 kg) were anesthetized; a 2.0-cm segment of the left circumflex coronary artery (LCX) was isolated proximal to the first main branch, and the dogs were instrumented with an electromagnetic flow probe to measure coronary blood flow. An occlusive thrombus was formed after injury of the intimal surface of the LCX with an electrical current applied by a needle-tipped anode placed distal to the electromagnetic flow probe. After 1 hour of occlusion, either LY210825 or rt-PA was administered intravenously according to the following protocols: 1) a 1-hour infusion of either 0.25 mg/kg LY210825 or 0.4 mg/kg rt-PA, 2) single injections of 0.15-0.6 mg/kg LY210825, and 3) a single injection of 0.45 mg/kg LY210825 and a 3-hour infusion of 1.0 or 1.7 mg/kg rt-PA. Plasma half-lives of LY210825 and rt-PA were 58 +/- 7 and 3.3 +/- 0.3 minutes, respectively. LY210825 produced more rapid reperfusion of the LCX than did rt-PA. In the third study, 90% of the rt-PA-treated vessels reoccluded within 1 hour after cessation of drug, whereas only 25% of the LY210825-treated vessels reoccluded during a 4-hour washout period. There were significant, but relatively small, reductions produced by both plasminogen activators on plasma fibrinogen and plasminogen (25-35% decreases). Because of its longer plasma half-life, LY210825 could be administered intravenously as a single injection. In a canine model of coronary artery thrombosis, LY210825 was a more effective thrombolytic agent than was rt-PA.

Quantification of the skin's topography by skin profilometry.

Synopsis A method of skin profilometry is presented. The data generated using this method are used to (a) uncover sources of variation in skin profilometry, (b) provide information regarding the choice of roughness parameters best suited for characterizing the skin's topography, and (c) determine if skin profilometry is a valuable tool for quantitatively assessing changes in the skin's surface pattern. The data show the roughness parameter values to be dependent on the orientation of the tracings with regard to the major grooves and ridges present in the surface patterns. Large variabilities of roughness parameter values obtained for multiple scans within small areas of replicas are indicative of the nonhomogeneity of the skin's surface. The number of peaks, mean peak size, mean depth of roughness, depth of smoothness, and residual profile length appear to be the most utile roughness parameters for quantifying changes in the skin's topography. The ability of skin profilometry to detect subtle changes in the skin's surface pattern due to hydration indicates the method is a sensitive means of quantifying the skin's topography.