The Discovery of GS-9131, an Amidate Prodrug of a Novel Nucleoside Phosphonate HIV Reverse Transcriptase Inhibitor - Accounts in Drug Discovery Case Studies in Medicinal Chemistry - page 230

center. Overall, the best prodrugs for Cat A cleavage, 29b-e, tended to be the
ones containing natural amino acids. The more lipophilic phenylalanine bis-
amidate prodrugs, 29b and 29c, were the best substrates for Cat A, superior
even to 11, and demonstrated potent antiviral activity below 10 nM. The less
lipophilic alanine prodrugs 29d and 29e were generally poorer Cat A substrates,
with glycine being the least ecient. In the monoamidate series of prodrugs,
30a-i, one of the amino acid groups was replaced with phenol or tri-
fluoroethoxy, leading to diastereomeric mixtures. Similar Cat A cleavage trends
were observed amongst the linear alkyl and branched alkyl esters, with the
more lipophilic phenylalanine prodrugs such as 30a-c demonstrating good Cat
A cleavage rates followed by the less lipophilic alanines 30d-g. Interestingly, the
replacement of phenol by trifluoroethoxy, example 30a, did not affect the Cat
A-mediated cleavage compared to prodrug 30b, but antiviral activity dropped
by almost 20-fold! This result likely reflected differences in the ability to cyclize
following Cat A cleavage of the ester group, as proposed in the breakdown
scheme described in Figure 10.7. The prodrugs 30h and 30i are also distinct in
that the ester group on the amino acid is proximally branched. The rate of Cat
A turnover of the isopropyl analog 30i was surprisingly slow when considering
this is the same prodrug as 11, albeit as a diastereomeric mixture. The parent
nucleoside phosphonate is clearly an important recognition motif for Cat A in
addition to the phosphonate prodrug groups. Constraining the proximal
branched ester into a ring, for example cyclobutyl prodrug 30h, elevated the
Cat A activity substantially, leading to good antiviral activity. In general,
provided the ester group was linear, branched non-proximally, or proximally
branched in a small ring, the natural L -alanine and L -phenylalanine amino acids
provided sucient Cat A cleavage rates to result in antiviral activities below
100 nM, more than two orders of magnitude lower than that of the diacid 22.A
final point of note, but of great significance, is that the Cat A cleavage rates
correlated well with the cleavage rates observed in human PBMC extracts,
providing evidence that Cat A is the dominant enzyme in the initial breakdown
step, at least in the target PBMCs.
10.3.2 Optimization of In vitro ADME Properties
In parallel to the substrate SAR for Cat A, the ability of the prodrugs to survive
intestinal enzymes, first-pass liver metabolism, and plasma exposure was
evaluated (Table 10.4). 25 The prodrugs were profiled in both dog and human
hepatocytes, intestinal S9 fraction, and plasma. Rodents were avoided since it is
well known that they have high esterase activities compared to humans and
would likely provide misleading results in vivo. Hepatocytes rather than
microsomes were also used to more closely profile in vivo properties, where cell
permeability and non-oxidative metabolism could have an important impact.
All the amidate prodrugs of 22 demonstrated favorable stability in both dog
and human plasma, but hepatocyte stability proved to be more challenging
(Table 10.4). Although some prodrugs, especially the bisamidates 29b and 29c,