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binding constant and also in reduced aqueous solubility. Also, due to the very tight fit of both naphthyl
moieties in the S1 and S1' subsites, subsequent design targeting the S3/S3' subsites proved to be difficult
and synthetically challenging [44]. In the search for a simpler solution, the di-tertiary amides were
designed using the crystal structure of compound II (Table 6) as a starting model. Branching from the
amide nitrogens provided an interesting possibility to access S2–S3/S2'–S3' subsites while
simultaneously increasing the solubility and stability of the compounds. In the first design, the
hydroxyethyl moieties were fused to the amide nitrogens and the hydroxyl groups were intended to form
hydrogen bonds with the amide nitrogens of Asp29/29' (compound III in Table 6). The addition of both
hydroxyethyl groups resulted in a rather significant increase in the binding constant, and the racemic
mixture had the K i of 1.1μM. When the crystal structure of compound III complexed with HIV PR was
solved at 2.2 Å resolution, it was observed that the inhibitor had undergone an inversion in binding
mode relative to the secondary amide series. The phenyl groups of compound III occupied the S2/S2'
subsites, switching positions with the t-butyl groups, which were in turn occupying the S1/S1' pockets
(Figure 6). Due to this change in binding mode, the R enantiomer would be expected to be preferred
relative to S. The final position of the hydroxyethyl moieties was less effected by the change, and both
hydroxyls were within hydrogen-bonding distance from the amide nitrogens of Asp 29/29'. In the S2/S2'
pockets, the phenyl groups occupied only a fraction of subsites, but the interaction was strengthened by
highly ordered water molecules involved in electrostatic interaction with the aromatic rings and by
forming hydrogen bonds to Asp30/30'. Interestingly the position of the hydrogen bonds with respect to
the flap water was significantly disturbed in the new binding mode, and the conserved Wat301 was no
longer tetrahydrally coordinated [43,45].
This unanticipated change in binding mode presented a potential for new avenues of design different
from those of the secondary amides. The ability to design into neighboring subsites depends to a large
extent on the positions of bond vectors suitable for substitution in the bound conformation of a given
inhibitor. These vectors in the crystallographically discovered new binding mode of compound III were
positioned ideally to access unfilled space in the S3/S3' pockets. The discovery of this new conformation
of compound III highlighted the power of crystallographic feedback in the process of inhibitor design
and, without this structural information, further design in this series would have been severely impeded.
Inspection of the crystal structure of compound III bound to the active site of HIV PR revealed
lipophilic cavities extending off the S1/S1' subsites adjacent to the t-butyl groups of the benzamidine
moiety. The cavities are bordered by flexible loops around Pro81/81' and previous crystallographic
studies indicated that both loops can move back by up to 2.5 Å, extending the size and
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