Timothy Alan Shepherd

A novel targeted delivery system utilizing a cephalosporin-oncolytic prodrug activated by an antibody β-lactamase conjugate for the treatment of cancer

Abstract Cephalosporins substituted at C-3′ with oncolytic agents such as vinca alkaloids have been developed as prodrugs for the treatment of solid tumors. Conversion of prodrug to drug is mediated by an immunoconjugate consisting of a β-lactamase enzyme covalently attached to a monoclonal antibody Fab′ fragment which is pre-localized at the tumor.

Synthesis and evaluation of peptidyl Michael acceptors that inactivate human rhinovirus 3C protease and inhibit virus replication

Human rhinovirus, the chief cause of the common cold, contains a positive-sense strand of RNA which is translated into a large polyprotein in infected cells. Cleavage of the latter to produce the mature viral proteins required for replication is catalyzed in large part by a virally encoded cysteine proteinase (3Cpro) which is highly selective for -Q approximately GP- cleavage sites. We synthesized peptidyl derivatives of vinylogous glutamine or methionine sulfone esters (e.g., Boc-Val-Leu-Phe-vGln-OR: R = Me, 1; R = Et, 2) and evaluated them as inhibitors of HRV-14 3C protease (3Cpro). Compounds 1 and 2 and several related tetra- and pentapeptide analogues rapidly inactivated 3Cpro with submicromolar IC50 values. Electrospray mass spectrometry confirmed the expected 1:1 stoichiometry of 3Cpro inactivation by 1, 2, and several other analogues. Compound 2 also proved to be useful for active site titration of 3Cpro, which has not been possible heretofore because of the lack of a suitable reagent. In contrast to 1, 2, and congeners, peptidyl Michael acceptors lacking a P4 residue have greatly reduced or negligible activity against 3Cpro, consistent with previously established structure-activity relationships for 3Cpro substrates. Hydrolysis of the P1 vinylogous glutamine ester to a carboxylic acid also decreased inhibitory activity considerably, consistent with the decreased reactivity of acrylic acids vs acrylic esters as Michael acceptors. Incorporating a vinylogous methionine sulfone ester in place of the corresponding glutamine derivative in 1 also reduced activity substantially. Compounds 1 and 2 and several of their analogues inhibited HRV replication in cell culture by 50% at low micromolar concentrations while showing little or no evidence of cytotoxicity at 10-fold higher concentrations. Peptidyl Michael acceptors and their analogues may prove useful as therapeutic agents for pathologies involving cysteine proteinase enzymes.

1,3-Dipolar cycloaddition reactions of pyrazolidinium ylides with vinyl sulfones. A regioselective synthesis of bicyclic pyrazolidinone antibacterial agents

Abstract The 1, 3-dipolar cycloaddition reaction of pyrazolidinium ylide 1 with substituted vinyl sulfones 5 was studied. Elimination of benzenesulfinic acid from the resulting cycloadducts gave rise to bicyclic pyrazolidinones 3 . The (E)-olefin isomers were found to undergo cycloaddition in a highly regioselective fashion. These pyrazolidinones 3 represent the nuclei of an exciting new class of potent antibacterial agents that mimic β-lactams.

Small peptidic aldehyde inhibitors of human rhinovirus 3C protease

Abstract Small peptide aldehydes were designed to mimic the preferred substrate requirements for the human rhinovirus 3C protease. Di- and tripeptide aldehydes containing a methionine sulfone as a P 1 surrogate for glutamine show low micromolar enzyme inhibitory and antiviral tissue culture activity. LY338387, obtained in a short and efficient synthesis, appears to validate the protease as a therapeutic target.

POTENT, ORALLY BIOAVAILABLE HIV-1 PROTEASE INHIBITORS CONTAINING NONCODED D-AMINO ACIDS

Abstract Novel noncoded D-amino acids have been combined with decahydroisoquinoline, octahydrothienopyridine, and urea hydroxyethylamine isosteres to provide potent HIV-1 protease inhibitors with excellent HIV-1 antiviral activity. LY314613 shows a promising combination of potency and oral bioavailability. Trends in the SAR and comparisons to other isostere derivatives will be discussed.

Thioaldehydes in cycloaddition reactions. Synthesis of nuclear analogues of pyrazolidinone antibacterial agents

Abstract In situ generated thioaldehydes have been found to undergo 1,3-dipolar cycloadditions with a pyrazolidinium ylide to produce a nuclear analogue of pyrazolidinone antibacterial agents.

D-amino acids as novel P2/P3 ligands for inhibitors of HIV-1 protease

Abstract Noncoded D-amino acids have been synthesized which effectively replace the P2/P3 ligands of the HIV-1 protease inhibitor LY289612. Several analogues are shown to have potent enzyme inhibitory and antiviral activities.

Development of analytical and preparative chromatographic separations of novel growth hormone secretagogue compounds

Chromatographic separations of new growth hormone secretagogue compounds were developed to support structure-activity relationship (SAR) studies in conjunction with lead optimization. These new compounds differed from Mercks MK-677 by having two chiral centers and thus diastereomeric mixtures were generated. Separation of initial compounds in the SAR was achieved on a Kromasil C18 column using an ammonium acetate buffer and acetonitrile. However, additional candidates were not separable on C18 columns and a chiral Kromasil CHI-DMB column was used to resolve the diastereomeric compounds. The Kromasil CHI-DMB packing was also used in a preparative chromatographic system to resolve multigram quantities of secretagogue candidates for testing. Chiral separations of different intermediates were also developed in support of evolution of an asymmetric synthetic route. This report summarizes development of the preparative chromatographic system used to purify diastereomeric mixtures and chiral separations of intermediates in the synthesis.

Discovery of N-(6-Fluoro-1-oxo-1,2-dihydroisoquinolin-7-yl)-5-[(3R)-3-hydroxypyrrolidin-1-yl]thiophene-2-sulfonamide (LSN 3213128), a Potent and Selective Nonclassical Antifolate Aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase (AICARFT) Inhibitor Effective at Tumor Suppression in a Cancer Xenograft Model

A hallmark of cancer is unbridled proliferation that can result in increased demand for de novo synthesis of purine and pyrimidine bases required for DNA and RNA biosynthesis. These synthetic pathways are frequently upregulated in cancer and involve various folate-dependent enzymes. Antifolates have a proven record as clinically used oncolytic agents. Our recent research efforts have produced LSN 3213128 (compound 28a), a novel, selective, nonclassical, orally bioavailable antifolate with potent and specific inhibitory activity for aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT), an enzyme in the purine biosynthetic pathway. Inhibition of AICARFT with compound 28a results in dramatic elevation of 5-aminoimidazole 4-carboxamide ribonucleotide (ZMP) and growth inhibition in NCI-H460 and MDA-MB-231met2 cancer cell lines. Treatment with this inhibitor in a murine based xenograft model of triple negative breast cancer (TNBC) resulted in tumor growth inhibition.

Synthesis and pharmacokinetics of potent carbamate HIV-1 protease inhibitors containing novel high affinity hydroxyethylamine isosteres

Abstract Using the hydroxyethylamine isosteres 1 and 2 containing the novel cis-octahydrothienopyridine moiety, substantial enhancement of binding potencies for HIV-1 protease inhibitors which incorporate carbamate linked heterocyclic P2 ligands has been realized. This increase in binding has led to a very potent antiviral compound (LY326188). The pharmacokinetics of selected derivatives are detailed in this report.