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was that the central ring could be opened or cleaved and the amino group could be meta or para
substituted on the latter. In the carboline series, the cyclic amino group was either at the β or γ position
of the cyclohexenyl ring and the methylene chain bearing the “C-terminal” carboxylate could be of
variable length. The spirocyclic series was varied by alkyl, cycloalkyl, and aryl substitution on the fivemembered
ring amine nitrogen. The cinnamic acids had two carbon chains that could be of varying
length, one of which had the further possibility of containing a double bond(s).
Rather than perform individual syntheses of all possible combinations of these nonpeptide units,
members of each ring type or scaffold family were pooled in equimolar amounts prior to incorporation
into the sequence DArg-Arg-X-Y-Arg. Since each individual member of a given pool was constructed
on a similar carbocyclic scaffold, the chemical environment of the N-terminal amino group and Cterminal
carboxylate groups were expected to follow similar kinetic and thermodynamic controls during
the attachment of the nonpeptide residue to the growing peptide chain. The use of these smaller, directed
libraries made it readily practical to obtain HPLC and mass spectral data for each and therefore confirm
the composition of the library.
Ultimately, 10 libraries of novel nonpeptidic structures were synthesized following typical solid-phase
methodologies. Each library contained from nine to thirty-six different compounds in approximately
equimolar amounts. Unpurified libraries were tested in a receptor binding assay utilizing membrane
preparations from a stable CHO cell line expressing the human B2 receptor. Each library was tested at
concentrations between 10 nM and 1μM. The ability of each library to inhibit[ 3H]-bradykinin binding
was assessed and the results are presented in Figure 8a. Although this type of screening is highly
qualitative, certain libraries appear in Figure 8a that show higher affinity to the receptor than other
libraries. Library one (of the series DArg-Arg-PH-CN-Arg) was ultimately selected for further
deconvolution. This library was further broken down (decoded) in order to determine which
compound(s) were responsible for the apparent activity. It is important to note that breaking these
libraries down to elucidate the structure of the hit(s) was feasible due to the inherently small size of each
library.
Library one contained 12 different structures (recall that there were originally three different
phenanthridinones and four different cinnamic acids). The first deconvolution step of the approach is
shown in Figure 9. Here, only the CN position was randomized, and the PH moieties were specific. This
led to the preparation of three new libraries of 4 compounds each. Receptor binding was again
performed as before and only one of these three new sublibraries showed affinity for the receptor at 10
μM (Figure 8b). The final step in the process required to elucidate the active component(s) was to
synthesize and purify each of the 4 members of this library as shown in Figure 9. Receptor binding on
these
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