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Inhibitors of the haemagglutinin cleaving proteases furin and HAT were synthesized and characterized in this thesis. While the proproteinconvertase furin is activating the haemagglutinin (HA) of highly pathogenic influenza viruses of the subtype H5 and H7, the trypsin-like serine protease HAT activates the HA of seasonal influenzaviruses of the subtypes H1, H2 and H3. Therefore, the described inhibitors may be useful tools for the development of new antiviral drugs for the treatment of the flu.
In a first screening a bis-amidinohydrazone-derivate was identified as a lead structure for new low molecular weight furin inhibitors, which was modified in four subsequent synthesis cycles. Commercially available carbonyl compounds were used in a first series. The most potent derivative was derived from terephthalaldehyde and possesses an inhibitory constant of 1,5 µM. Inhibitors with three or four basic residues were obtained from second and third series. The coupling of 4-guanidionbutyric acid to the lead structure in the second series and the dimerization of the initial screening hit with chloroaceticacidchloride led to compounds with a inhibitory potency of 0,5 to 0,6 µM. A known salicylaldehyde benzylether-derived compound was used as a lead structure of the fourth series. However, all inhibitors from this attempt were less potent than the lead structure.
With one exception all amidinohydrazone derived inhibitors from this thesis feature a high selectivity against the related trypsin-like serineproteases thrombin, factor Xa and plasmin (Ki > 30 µM).
Until now only few inhibitors for the trypsin-like serinportease HAT were described. Thus, the first attempt in this field was the screening of available compounds for HAT inhibition. Besides, substrate analogue compounds with a 4-amidinobenyzlamide moiety on the C-terminal part and inhibitors with a 3-amidinophenylalanine of the TAPAP-type were identified as possible starting points for the development of HAT inhibitors.
For all substrate analogue inhibitors a P4-benzylsulfonyl residue and a P1-Amidinobenzylamidine residue were used. In a first series with a proline in P2-position, several d-configurated aminoacids were well-accepted in P3-position. Especially d-Arg and d-homo-Arg were favored as a P3-residue. Nevertheless, hydrophobic derivatives, like a tert.-butyl protected d-Asp or d-Glu, were also well-suited for incorporation. Due to the preference of HAT for basic or hydrophobic amino acids in P3, cyclized amines were coupled to the side chains of d-Asp or d-Glu in a second series. Most potent compound from this attempt was obtained via coupling of 1-(2-pyrimidyl)piperazin to the free side chain of d-Glu (Ki = 17 nM).
Substrate analogue inhibitors with a P2-Proline are common inhibitors of several trypsine-like proteases. Hence, the influence of other amino acid residues replacing the proline was examined keeping d-Arg in P3-position. Small hydrophobic residues like 2-aminobutyric acid (Abu), norvaline (Nva), Ala or Val are a good replacement for proline and the compounds inhibit HAT with Ki-values below 30 nM. An advantage of these inhibitors is their enhanced selectivity against the other tested proteases thrombin, factor Xa and plasmin.
Selected compounds were also tested in cell based assays to examine their ability to suppress viral replication. In these experiments the inhibitors with an Abu or Nva residue in P2-position effectively inhibit the replication of influenza viruses of the subtypes H1 and H3. It should be noted that this effect is not associated with any toxic effects to the cells.
Several substrate analogue compounds which inhibit HAT with inhibition constants < 30 nM are presented in this thesis. Contrary to until now known antiviral drugs, the synthesized compounds are targeting host cell proteases. Therefore, there will be no rising of resistances against these drugs.