APLICACIÓN DE LA TERMINACIÓN ANTICIPADA EN

HOE140 (D-Arg0-Argi-Pro2-Hyp3-Gly4-Thi5.Ser6-D-Tiq7-Oic8-Arg9)39/i04 js a potent long-lasting BK antagonist, whose therapeutic potential has already

been mentioned (Section 1.2.6.)- A p-turn is believed to be induced by the unusual amino acids at positions 7 and 8 (see Figure 25) and, given the examples in the previous section, it might be possible to construct a BZD framework at this location. Selection of an appropriate BZD scaffold would also need to take into account those observations described in Sections 1.3.3.2. and 1.3.3.3.

2.1.2.1 Benzodiazepine Overlay Model I

There are a number of ways of overlaying a generalised BZD core on the p - turn in HOE140*. Ripka and co-workers have overlaid a BZD at the (i + 1) corner of the p-turn in the model cyclic peptide 47 and the cyclic antibiotic 49 as previously described in 2.1.1. In this overlay model, the BZD acts as an internal p-turn mimetic. An internal p-turn mimetic is a molecule in which the mimetic skeleton approximately replaces the space that was occupied by the peptide backbone in the natural p - t u r n ^ T h e substituents on the BZD then radiate along the corresponding peptide side chain trajectories, with the lactam moiety more or less superimposed onto the peptide bond between amino acid residues (/+ 2) and (/ + 3). Figure 25 illustrates the overlay with respect to the peptide template, HOE140, and highlights the amino acid residues chosen to be mimicked, assuming that this conformation is maintained in the receptor bound state.

By utilising the phenyl substituent at C-5 of the BZD framework as the (/+ 1) binding determinant (retaining the “double ring” motif), the subsequent introduction of additional functionality would be immediately facilitated. A C-3 hydrophobic mimic of the ( /4- 2) residue would easily be incorporated by use of an appropriate amino acid, while a mimic of the important Arg^ side chain could conceivably be elaborated from N-1 by alkylation with a suitable electrophile. Alternatively, the (/ + 3) position could comprise of a carboxylic acid function in accordance with peptide structure activity requirements'*

* There are a number of benzodiazepine scaffolds which could have been chosen as the design core (see Section 1.3.4.4.). The 1,4-benzodlazepln-2-one depicted was chosen on the strength of the literature observations detailed In the previous section (2.1.1.).

Figure 25. Overlay model I design rationale Ser® (0 D-Tlq^ (1 + 1) HQ H -(D )-A rg -A rg -P ro -H y p -G ly -T h i^ N H O l d Arg (/+ 3)

H O E 1 4 0 C-term inus |3-turn

(/ + 2)

B A S IC ■ F U N C T IO N L IN K

Mimics Arg'

Projects towards D-Tic^ / in (R)-enantiomer H Y D R O P H O B I Q S ID E C H A IN

I

The seven membered heterocyclic ring of the unsubstituted BZD can flip between two pseudo-chair conformations of equal energy. In compound 51, the group at C-3 can orientate itself either in a pseudo-axial or a pseudo-equatorial position. Ripka^^ states that, when there is a substituent in the C-3 position, the chair form with the substituent in a p s e u d o -e q u a to ria l position is thermodynamically favoured. Although there are a number of literature precedents for this suggestion^\ an independent determination of the

conformation was made, as this has implications in the correct positioning of the C-5 phenyl substituent.

An X-ray crystal structural analysis of BZD 51 was obtained, showing that the benzyl group orientates itself in an pseudo-equatorial position. The crystal structure, obtained on the racemic mixture, shows that the unit cell exist as a dimer, the components of which have similar but not identical conformations (see Figure 26 and appendix).

Figure 26. X-ray crystallographic structure of 51

C(8)

cno) C(i2) CI131

The first overlay model of the BZD core 51 on HOE140 requires the benzyl group to project in the general area previously occupied by the amino acid residue Oic and the phenyl group to project itself in the region previously occupied by the amino acid D-Tiq. In order to accommodate the C-3 substituent in a pseudo-equatorial position, the seven-membered ring is puckered in such a way that the C-5 substituent is projected on one side of the lactam plane in the (S)-enantiomer and on the other side in the antipode. Molecular modelling reveals that only the (R)-BZD enantiomer is capable of maintaining all three binding determinants in the appropriate positions*. Despite the stereochemical

* Note also that in the model system 48 (Figure 20, p 43) a BZD of (S) stereochemistry is employed to mimic an (R) amino acid at the (i + 2) position in 4 7 . This supports our preference for a BZD of (R) stereochemistry to mimic the (S)- phenylalanine residue at (i + 2) in the C-terminal p-turn of BK, or the (2S )-0ic residue at the same position in HOE140.

bias in this paradigm, it was prudent and expedient to evaluate racemic mixtures at the onset of the lead finding process. In this way the (S)- enantiomers would not be disqualified from revealing an antagonistic activity arising from an alternative mode of binding.

2.1.2.2. Benzodiazepine Overlay Model II

In the overlay model of James et al.^^ for Ras farnesyltransferase inhibitors, the benzodiazepine forms an external p-turn mimetic core. When this model is applied to HOE140 the C-3 substituent mimics the hydrophobic amino acid D- Tiq and the benzo-fused ring in turn mimics the amino acid Oic (Figure 27). The C-5 phenyl substituent has no obvious role in this model but was retained because its presence was shown previously not to be a disadvantage. Because the BZD is located at a slightly greater distance from the basic function that mimics Arg^, an extra acetamido function needs to be inserted in the side chain.

Figure 27. Overlay model II design rationale Ser® (4 D-Tlq^ ( i+1) HO. H -(D )-A rg -A rg -P ro -H y p -G ly -T h i^ N H Oic' ( / + 3 )

H O E -1 4 0 C-term inus p-turn

(1 + 2)

Projects towards D-TIc^ In (S)-enantiomer B A S IC F U N C T IO N Mimics Arg® [ L IN K ] -n' Mimics Ole'