The proprotein convertase furin is a potential target for drug design, especially for the inhibition of furin-dependent virus replication. All effective synthetic furin inhibitors identified thus far are multibasic compounds; the highest potency was found for our previously developed inhibitor 4-(guanidinomethyl)phenylacetyl-Arg-Tle-Arg-4-amidinobenzylamide (MI-1148). An initial study in mice revealed a narrow therapeutic range for this tetrabasic compound, while significantly reduced toxicity was observed for some tribasic analogues. This suggests that the toxicity depends at least to some extent on the overall multibasic character of this inhibitor. Therefore, in a first approach, the C-terminal benzamidine of MI-1148 was replaced by less basic P1 residues. Despite decreased potency, a few compounds still inhibit furin in the low nanomolar range, but display negligible efficacy in cells. In a second approach, the P2 arginine was replaced by lysine; compared to MI-1148, this furin inhibitor has slightly decreased potency, but exhibits similar antiviral activity against West Nile and Dengue virus in cell culture and decreased toxicity in mice. These results provide a promising starting point for the development of efficacious and well-tolerated furin inhibitors.

Novel elongated and shortened derivatives of the peptidomimetic furin inhibitor phenylacteyl-Arg-Val-Arg-4-amidinobenzylamide were synthesized. The most potent compounds, e.g., Nα(carbamidoyl)Arg-Arg-Val-Arg-4-amidinobenzylamide (Ki = 6.2 pM), contain additional basic residues at the N-terminus and inhibit furin in the low picomolar range. Furthermore, to decrease the molecular weight of this inhibitor type, compounds lacking the P5-moiety were prepared. The best inhibitors of this series, 5-(guanidino)-valeroyl-Val-Arg-4-amidinobenzylamide and its P3 tert.leucine analogue, displayed Ki values of 2.50 nM and 1.26 nM, respectively. Selected inhibitors, together with our previously described 4-amidinobenzylamide derivatives as references, were tested in cell culture for their activity against furin-dependent infectious pathogens. The propagation of the alphaviruses Semliki Forest virus and chikungunya was strongly inhibited in the presence of selected derivatives. Moreover, a significant protective effect of the inhibitors against diphtheria toxin was observed. These results confirm that the inhibition of furin should represent a promising approach for a short-term treatment of acute infectious diseases.

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Two bivalent thrombin inhibitors were synthesized, which consist of a benzamidine-based active-site-blocking segment, a fibrinogen recognition exosite inhibitor and a peptidic linker connecting these fragments. BZA-1 hirulog contains an N a -(2-naphthylsulfonyl)-S-3-amidinophenylalanyl-isonipecotic acid residue connected via the carboxyl group to the linker segment. The active-site-directed moiety of BZA-2 hirulog [N a -(2-naphthylsulfonyl-glutamyl)-R-4-amidinophenylalanyl-piperidide] was coupled to the linker via the side chain of the glutamic acid. Both BZA-hirulogs contain almost identical linker-exo site inhibitor parts, except for the substitution of a glycine as the first linker residue in BZA-1 hirulog by a g-amino butyric acid in BZA-2 hirulog, thus increasing flexibility and linker length by two additional atoms. BZA-1 hirulog showed moderate potency (K i = 0.50^0.14 nm), while BZA-2 hirulog was characterized as a slow, tight binding inhibitor of thrombin (K i = 0.29^0.08 pm). The stability in human plasma of both analogs was strongly improved compared with hirulog-1. For BZA-2 hirulog a significantly reduced plasma clearance was observed after intravenous injection in rats compared with BZA-1 hirulog and hirulog-1. The X-ray structure of the BZA-2 hirulog in complex with human a-thrombin was solved and confirmed the expected bivalent binding mode.Keywords: anticoagulants; bivalent inhibitor; enzyme kinetics; hirudin; thrombin.The hirulogs [1,2] are highly potent and specific synthetic thrombin inhibitors derived from the naturally occurring 65-amino acid polypeptide hirudin, which was originally isolated from the medicinal leech [3]. Similar to hirudin, the hirulogs inhibit thrombin by a bivalent binding mode. The C-terminus of the hirulogs is almost identical to the C-terminal residues H53±65 of the desulfated r-hirudin (prefix H used for numbering based on the hirudin sequence), which bind to the fibrinogen recognition exo site (FRE) of thrombin. This FRE-directed inhibitor segment is connected by a peptidic linker to an additional inhibitor moiety, which occupies the active site of thrombin.The prototype, hirulog-1 (dPhe-Pro-Arg-Pro-(Gly) 4 -Asn-GlyAsp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH; K i = 2. [7,8] enhanced the affinity of the inhibitors by 1±2 orders of magnitude. However, the strongest improvements in activity were achieved by incorporation of more potent and proteolytically stable active site inhibitor segments. One strategy was the introduction of transition-state analogs containing different P1-residues, like arginyl methyl ketones [9±11], a-keto-amides [12] or boronic acid derivatives [13].A second strategy [14] was the incorporation of an arylsulfonyl-arginyl-R-pipecolic acid active-site-directed segment derived from the potent thrombin inhibitor argatroban [15], which also eliminated the scissile peptide bond after the P1-arginyl residue. A combination of this active-site-directed moiety with optimized linker and FRE-directed inhibitor segments resulted in compounds with inhibition ...

Several new analogs of the known thrombin inhibitor NAPAP were synthesized, in which the P2 glycine residue was substituted by natural and unnatural amino acids. The thrombin inhibitory potency was comparable to that of NAPAP. Several of the compounds had inhibition constants lower than 10 nM and a very high selectivity compared to trypsin, factor Xa and plasmin. In addition, analogs were prepared by alkylation of the N alpha-atom of the 4-amidinophenylalanine in P1 position, which showed a more than 10-fold lower thrombin inhibition. Furthermore, azaglycine was introduced instead of P2 glycine. For most of the inhibitors similar fast elimination rates were seen in rats after intravenous dosing, as found previously for NAPAP. Only some compounds, which contained a second basic group showed a slightly decreased cumulative biliary clearance.