Ralph Hirschmann

Pyrrolinone-Based Peptidomimetics: “Let the Enzyme or Receptor be the Judge”

Peptides and proteins, evolved by nature to perform vital biological functions, would constitute ideal candidates for therapeutic intervention were it not for their generally poor pharmacokinetic profiles. Nonpeptide peptidomimetics have thus been pursued because they might overcome these limitations while maintaining both the potency and selectivity of the parent peptide or protein. Since the late 1980s, we have sought to design, synthesize, and evaluate a novel, proteolytically stable nonpeptide peptidomimetic scaffold consisting of a repeating structural unit amenable to iterative construction; a primary concern is maintaining both the appropriate peptide-like side-chains and requisite hydrogen bonding. In this Account, we detail how efforts in the Smith-Hirschmann laboratories culminated in the identification of the 3,5-linked polypyrrolinone scaffold. We developed effective synthetic protocols, both in solution and on solid supports, for iterative construction of diverse polypyrrolinones that present functionalized peptide-like side-chains. As a result of the rigid nature of the pyrrolinone scaffold, control over the backbone conformation could be exerted by modulation of the stereogenicity of the constituent monomers and the network of intramolecular hydrogen bonding. The extended conformation of the homochiral 3,5-linked polypyrrolinone scaffold proved to be an excellent mimic for β-strands and β-sheets. Application to enzyme inhibitor design and synthesis led not only to modest inhibitors of the aspartic acid protease renin and the matrix metalloprotease class of enzymes, but importantly to bioavailable HIV-1 protease inhibitors with subnanomolar binding constants. The design and synthesis of a competent peptide-pyrrolinone hybrid ligand for the class II major histocompatibility complex (MHC) antigen protein HLA-DR1 further demonstrated the utility of the 3,5-polypyrrolinone motif as a mimic for the extended polyproline type II peptide backbone. Equally important, we sought to define, by synthesis, the additional conformational space accessible to the polypyrrolinone structural motif, with the ultimate goal of accessing pyrrolinone-based turn and helix mimetics. Toward this end, a mono-N-methylated bispyrrolinone was found to adopt an extended helical array in the solid state. Subsequent synthesis of d,l-alternating (heterochiral) tetrapyrrolinones both validated the expected turn conformations in solution and led to a functionally active mimetic of a peptidal β-turn (similar to somatostatin). Finally, the design, synthesis, and structural evaluation of both acyclic and cyclic heterochiral (that is, d,l-alternating) hexapyrrolinones yielded nanotube-like assemblies in the solid state. Taken together, these results illustrate the remarkable potential of the 3,5-linked polypyrrolinone scaffold as β-strand, β-sheet, β-turn, and potentially helical peptidomimetics.

The carboxyl terminus heptapeptide of the R2 subunit of mammalian ribonucleotide reductase inhibits enzyme activity and can be used to purify the R1 subunit

The heptapeptide, FTLDADF, identical in sequence to the last seven amino acid residues of the carboxyl terminus of the R2 subunit of mouse ribonucleotide reductase (RR), and its Nα‐acetyl derivative both inhibit calf thymus RR. The Nα‐acetyl derivative is considerably more potent, displaying a K i of 20 μM. The same K i was found for N‐AcFTLDADF inhibition of a reconstituted ribonucleotide reductase from calf thymus R1 and mouse R2, indicating that the C‐termini of calf R2 and mouse R2 might be identical. Our results, taken together with previous results of others on inhibition of viral RR, suggest that inhibition of RRs by peptides mimicking the C‐terminus of R2 may be a general phenomenon. In addition, we have shown that an affinity column, FTLDADF‐Sepharose 4B, can be used to prepare ~95% pure calf thymus R1, devoid of contamination with R2, in a very simple procedure that should be generally applicable to Rl purification from many sources.

The cortisone era: aspects of its impact. Some contributions of the Merck Laboratories

The announcement in 1949, by Hench at the Mayo Clinic, that cortisone had a dramatic beneficial effect on bed-ridden patients suffering from rheumatoid arthritis ushered in the cortisone era. This medical landmark was made possible by the prior steroid research of distinguished chemists and biologists in several countries. The first partial synthesis of cortisone by Sarett was the culmination of a worldwide chemical effort. This work ultimately enabled the process research department at Merck, under the direction of Max Tishler, to perform the 37-step conversion of deoxycholic acid to cortisone on a scale that made the initial clinical trials possible. In spite of the enormity of the project, and the fact that neither of two closely related analogs of cortisone had shown any interesting biological activity. Merck elected to embark on this synthetically challenging project. The clinical results reported in 1949, combined with the complexity of the partial synthesis, stimulated highly innovative research to discover new routes to cortisone and to cortisol, the active hormone. This research, particularly in the pharmaceutical industry in the United States, Mexico, and Europe, demonstrated, among other things, the value of microbial transformations in synthetic sequences. The recognition that the chronic administration of cortisol produces several unexpected side effects stimulated an intensive effort in many countries to discover an analog with an improved therapeutic index. This led to more novel chemistry and many analogs were discovered that proved to be more potent than cortisol. Prednisolone, discovered at the Schering Corporation, was the first compound that combined a high level of anti-inflammatory activity with reduced salt retention. Derek Barton contributed greatly to steroid research during the 1950s by applying creative structural thinking to systematize a host of seemingly unrelated chemical and biological observations. The cortisone era had a profound impact on drug discovery also, since it led to the logical application of steric and electronic concepts to medicinal chemistry. Last, but not least, the cortisone era taught medicinal chemists many important lessons about drug-receptor interactions.

The versatile steroid nucleus: Design and synthesis of a peptidomimetic employing this novel scaffold.

Abstract The design and synthesis of peptidomimetic 1 employing the novel cyclopentanoperhydrophenanthrene skeleton is described. The large body of steroid literature allows for the introduction of diverse peptidal side chains with precise regio- and stereoselective control. Compound 1 binds to the fibrinogen receptor on blood platelets (GP IIb/IIIa), for which it was designed, with an IC50 of ca. 100 μM.

Design and synthesis of a tetrahydropyran-based inhibitor of mammalian ribonucleotide reductase

A tetrahydropyran-based inhibitor (2) of mammalian ribonucleotide reductase (mRR) has been designed and synthesized based on the heptapeptide, N-AcFTLDADF (1), corresponding to the C-terminus of the R2 subunit of mRR. Inhibition studies revealed that 2 is indeed a competent inhibitor, albeit less potent than 1.

Transport Characteristics of Peptidomimetics. Effect of the Pyrrolinone Bioisostere on Transport Across Caco-2 Cell Monolayers

AbstractPurpose. To compare the permeation characteristics of amide bond-containing HIV-1 protease inhibitors and their pyrrolinone-containing counterparts across Caco-2 cell monolayers, a model of the intestinal mucosa. Methods. Transepithelial transport and cellular uptake of three pairs of amide bond-containing and pyrrolinone-based peptidomimetics were assessed in the presence and absence of cyclosporin A using the Caco-2 cell culture model. The potential of the peptidomimetics to interact with biological membranes was estimated by IAM chromatography. Results. In the absence of cyclosporin A, apical (AP) to basolateral (BL) flux of all compounds studied was less than the flux determined in the opposite direction (i.e., BL-to-AP). The ratio of the apparent permeability coefficients (Papp) calculated for the BL-to-AP and AP-to-BL transport (PBL⇒AP/PAP⇒BL) varied between 1.7 and 36.2. When individual pairs were compared, PBL⇒AP/PAP⇒BL ratios of the pyrrolinone-containing compounds were 1.5 to 11.5 times greater than those determined for the amide bond-containing analogs. Addition of 25 μM cyclosporin A to the transport buffer reduced the PBL⇒AP /PAP⇒BL ratios for all protease inhibitors to a value close to unity. Under these conditions, the amide bond-containing peptidomimetics were at least 1.6 to 2.8 times more able to permeate Caco-2 cell monolayers than were the pyrrolinone-containing compounds. The intrinsic uptake characteristics into Caco-2 cells determined in the presence of 25 μM cyclosporin A were slightly greater for the amide bond-containing protease inhibitors than for the pyrrolinone-containing analogs. These uptake results are consistent with the transepithelial transport results determined across this in vitro model of the intestinal mucosa. Conclusions. The amide bond-containing and pyrrolinone-based peptidomimetics are substrates for apically polarized efflux systems present in Caco-2 cell monolayers. The intrinsic permeabilities of the amide bond-containing protease inhibitors are slightly greater than the intrinsic permeabilities of the pyrrolinone-based analogs through Caco-2 cell monolayers.

An effective synthesis of scalemic 3,5,5-trisubstituted pyrrolin-4-ones

A new two-step method employs the intramolecular cyclization of metalated imino esters for the construction of scalemic 3,5,5-trisubstituted pyrrolin-4-ones (4). The imino esters in turn derive from α-disubstituted amino acids, the latter readily available via a new protocol exploiting the enantioretentive alkylation of oxazolidinones.

The design and synthesis of 2,5-linked pyrrolinones. A potential non-peptide peptidomimetic scaffold.

The de novo design and initial synthetic studies directed toward construction of a novel non-peptide scaffold for beta-strand/sheet and related secondary peptide structural mimics are described. The scaffold, consisting of a repeating array of 2,5,5-trisubstituted pyrrolinone (enaminone) units punctuated with appropriate amino acid side chains, is conceptually related to our previously successful 3,5-linked polypyrrolinone non-peptide peptidomimetic scaffold. Construction of the 2,5,5-trisubstituted pyrrolinone ring system proceeds via intramolecular condensation of an N-protected amino dione. The latter is prepared from a protected alpha-amino ketone and aldehyde via an aldol-oxidation reaction sequence.

MOLECULAR MODELING, SYNTHESIS, AND STRUCTURES OF N-METHYLATED 3,5-LINKED PYRROLIN-4-ONES TOWARD THE CREATION OF A PRIVILEGED NONPEPTIDE SCAFFOLD

The molecular modeling, synthesis, and elucidations of the solid state and solution structures of N-methylated 3,5-linked bispyrrolin-4-ones are described. Prior investigations established that the 3,5-linked pyrrolin-4-one based scaffold can be incorporated into mimics of beta-sheet/beta-strands and into potent, orally bioavailable inhibitors of the HIV-1 protease. To extend the utility of this scaffold beyond that of the initially designed mimics of beta-sheet/beta-strands, we have now explored the structure of N-methylated pyrrolinones. Molecular modeling indicated that N-methylated bispyrrolinones could adopt three low-energy backbone conformations (ca. 165 degrees, 289 degrees, and 320 degrees). Upon their successful synthesis, structural elucidation both in the solid state and in solution revealed the existence of two of the three predicted backbone conformers (ca. 165 degrees and 289 degrees). Two structures were particularly noteworthy and completely unexpected. Mono-N-methyl bispyrrolinone (+)-1 self assembled in the solid state to form a novel helix, while the acetylene-linked dimer of (+)-1, designed to potentiate the observed helical array, instead associated via an intermolecular hydrogen bond in parallel columns. These serendipitous observations led us to speculate that the pyrrolinone moiety may in fact represent a privileged nonpeptide scaffold, able to mimic not only the extended beta-sheet/beta-strand conformation as initially targeted, but also diverse conformations including those analogous to beta-turns and helices. These seemingly unlimited conformations greatly expand the scope of this scaffold for the development of low-molecular weight ligands for biologically important macromolecules.

A versatile, efficient synthesis of (−)-(2S, 3R, 4S)-2-amino-1-cyclohexyl-3,4-dihydroxy-6-methylheptane, the abbott pseudodipeptidyl insert

Abstract An economic construction (6 steps, 36% yield) of a valuable peptide analog subunit, the Abbott pseudodipeplidyl insert (−)-(2 S , 3 R , 4 S )-2-amino-1-cyclohexyl-3,4-dihydroxy-6-methylheptane (1), has been developed.