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A series of self-associating foldamers have been designed as heparin reversal agents, as antidotes to prevent bleeding due to this potent antithrombotic agent. The foldamers have a repeating sequence of Lys-Sal, in which Sal is 5-amino-2-methoxy-benzoic acid. These foldamers are designed to self-associate along one face of an extended chain in a β-sheet-like interaction. The methoxy groups were included to form intramolecular hydrogen bonds that preclude the formation of very large amyloid-like aggregates, while the positively charged Lys side chains were introduced to interact electrostatically with the highly anionic heparin polymer. The prototype compound (Lys-Sal)4 carboxamide weakly associates in aqueous solution at physiological salt concentration in a monomer-dimer-hexamer equilibrium. The association is greatly enhanced at either high ionic strength or in the presence of a heparin derivative, which is bound tightly. Variants of this foldamer are active in an antithrombin III-factor Xa assay, showing their potential as heparin reversal agents.

Anti-AIDS drug candidate and non-nucleoside reverse transcriptase inhibitor (NNRTI) TMC125-R165335 (etravirine) caused an initial drop in viral load similar to that observed with a five-drug combination in naïve patients and retains potency in patients infected with NNRTI-resistant HIV-1 variants. TMC125-R165335 and related anti-AIDS drug candidates can bind the enzyme RT in multiple conformations and thereby escape the effects of drug-resistance mutations. Structural studies showed that this inhibitor and other diarylpyrimidine (DAPY) analogues can adapt to changes in the NNRTI-binding pocket in several ways: (1). DAPY analogues can bind in at least two conformationally distinct modes; (2). within a given binding mode, torsional flexibility ("wiggling") of DAPY analogues permits access to numerous conformational variants; and (3). the compact design of the DAPY analogues permits significant repositioning and reorientation (translation and rotation) within the pocket ("jiggling"). Such adaptations appear to be critical for potency against wild-type and a wide range of drug-resistant mutant HIV-1 RTs. Exploitation of favorable components of inhibitor conformational flexibility (such as torsional flexibility about strategically located chemical bonds) can be a powerful drug design concept, especially for designing drugs that will be effective against rapidly mutating targets.

There are several indications that a given compound or a set of related compounds can bind in different modes to a specific binding site of a protein. This is especially evident from X-ray crystallographic structures of ligand-protein complexes. The availability of multiple binding modes of a ligand in a binding site may present an advantage in drug design when simultaneously optimizing several criteria. In the case of the design of anti-HIV compounds we observed that the more active compounds that are also resilient against mutation of the non-nucleoside binding site of HIV1-reverse transcriptase make use of more binding modes than the less active and resilient compounds.

The synthesis and anti-HIV-1 activity of a series of diarylpyrimidines (DAPYs) are described. Several members of this novel class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) are extremely potent against both wild-type and a panel of clinically significant single- and double-mutant strains of HIV-1.

Studies of two interrelated strategies for the synthesis of members of the cephalotaxus alkaloid family have culminated in a concise route for the preparation of the parent member cephalotaxine (1). As part of efforts exploring the use of an SET-promoted photocyclization reaction of aryl-substituted silylallyliminium salts to generate the spirocyclic DE unit of the target, we noted that attempts to generate the pentacyclic amino ketone 23 by deacylation of the enol ester 20 led to production of a mixture of 23 and the macrocyclic amino enone 24. A rapid equilibrium was shown to exist between 23 and 24, favoring the latter ring-opened form. This contrasts with the behavior of desmethylcephalotaxinone (22), a key late intermediate in several earlier cephalotaxine syntheses, which is known to exist in a ring-closed form. These observations led to the design of a second generation strategy which relies on transannular cyclization of the macrocyclic amino enedione 28. In practice, the sequence following this design transforms the known iodopiperonylethanol derivative 4 to 22 in 13 steps and a 12% overall yield and, thus, corresponds to an efficient formal synthesis of cepahalotaxine.

SummaryPlasmodium falciparum pathogenesis is affected by various cell types in the blood, including platelets, which can kill intraerythrocytic malaria parasites. Platelets could mediate these antimalarial effects through human defense peptides (HDPs), which exert antimicrobial effects by permeabilizing membranes. Therefore, we screened a panel of HDPs and determined that human platelet factor 4 (hPF4) kills malaria parasites inside erythrocytes by selectively lysing the parasite digestive vacuole (DV). PF4 rapidly accumulates only within infected erythrocytes and is required for parasite killing in infected erythrocyte-platelet cocultures. To exploit this antimalarial mechanism, we tested a library of small, nonpeptidic mimics of HDPs (smHDPs) and identified compounds that kill P. falciparum by rapidly lysing the parasite DV while sparing the erythrocyte plasma membrane. Lead smHDPs also reduced parasitemia in a murine malaria model. Thus, identifying host molecules that control parasite growth can further the development of related molecules with therapeutic potential.

A synthesis program directed toward improving the stability of imidoyl thiourea based non-nucleoside reverse transcriptase inhibitors (NNRTIs) led to the discovery of diaryltriazines (DATAs), a new class of potent NNRTIs. The synthesis and anti-HIV structure–activity relationship (SAR) studies of a series of DATA derivatives are described.

Abstract 1096 Structural studies of the binding interaction between the negatively charged heparin pentasaccharide binding site and antithrombin III suggested that an amphiphilic structure resembling features designed into compounds prepared for antimicrobial activity might also provide an efficient design basis for the development of heparin antagonists. A key design element of our antimicrobial approach at PolyMedix involves compounds exhibiting polycationic arrays which are appended to a diverse set of rigid backbones. A broad screening of the antimicrobial compound library in an enoxaparin (ENX) anti-factor Xa assay, coupled with subsequent secondary testing, identified compounds with potent and broad neutralizing activity. A lead compound (PMX-60056) was identified in one structural series, the salicylamides, and this compound has successfully completed Phase 1 human clinical studies. Recently, a new series of antagonists identified in the initial screen, the arylamides, was investigated for heparin neutralizing activities. Compounds were first tested for their ability to neutralize the anticoagulant activity of ENX, tinzaparin (TZP) or fondaparinux (FPX) in an in vitro amidolytic assay for factor Xa activity. Activated partial thromboplastin time (aPTT) assays demonstrated that the compounds were also active against unfractionated heparin (UFH) in a plasma-based clotting assay. The lead compound PMX150 was comparable to or better than PMX-60056 versus all anticoagulants tested (EC50s: UFH 0.6 uM, ENX 0.1 uM, TZP 0.1 uM, FPX 0.7 uM). Early compounds have shown an interesting structure activity relationship (SAR) which has identified specific regions of the molecule that are important for activity. Additionally, rotation thromboelastometry (ROTEM) shows that compounds are able to neutralize heparin and ENX in human whole blood, restoring normal coagulation profiles. As an initial test for safety, compounds were evaluated in hemolysis and cytotoxicity assays using isolated human erythrocytes, a transformed human liver cell line (HepG2 cells) and a mouse fibroblast cell line (NIH3T3). PMX150 was not cytotoxic (or hemolytic) at concentrations 180 fold greater than the EC50 concentrations in the anti-coagulation assays, indicating a high selectivity index between toxicity and efficacy. Furthermore, the SAR has identified synthetic strategies that should further improve this safety margin. The in vivo efficacy of PMX150 was evaluated in a rat coagulation model using ENX (2 mg/kg) administered by IV injection followed 3 minutes later by saline, protamine or PMX150. Blood was collected before dosing with ENX, and at 1, 3, 10, and 60 min after dosing, for aPTT and factor Xa analysis. PMX150 was more efficacious than protamine; neutralizing 89–100% of the ENX anti-factor Xa activity over the entire 60 minute time course. The in vivo assays concur with the in vitro studies, showing that compounds have been identified with high efficacy versus low molecular weight heparin (LMWH). Protamine, the heparin antagonist currently used in the clinic, routinely causes a transient decrease in blood pressure upon dosing. Hemodynamic effects with PMX-60056 have also been observed in human subjects in the absence of heparin. As PMX150 represents a new chemical series, investigations into potential adverse hemodynamic effects were performed. Blood pressure and heart rate were measured via arterial catheters in rats administered protamine or PMX150 by a 10 minute IV infusion. As expected, protamine displayed transient and prolonged blood pressure reductions at 8 and 16 mg/kg dosages, respectively. However, PMX150 had little to no effect on blood pressure at 8 mg/kg and only a minor transient reduction at 16 mg/kg. In conclusion, we have identified a new series of fully synthetic small molecule antagonists that display potent in vitro activity against UFH, the LMWHs, and fondaparinux. The lead compound in this series, PMX150 is able to neutralize ENX in vivo with more efficacy than protamine and, furthermore, has an improved hemodynamic profile with respect to protamine. Disclosures: Scott: PolyMedix, Inc.: Employment, Equity Ownership. Costanzo:PolyMedix, Inc.: Employment, Equity Ownership. Freeman:PolyMedix, Inc.: Employment, Equity Ownership. Kavash:PolyMedix, Inc.: Employment, Equity Ownership. Young:PolyMedix, Inc.: Employment, Equity Ownership. DeGrado:PolyMedix, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Jeske:PolyMedix, Inc.: Research Funding.

A route for construction of the pentacyclic skeleton of the harringtonine alkaloids based upon a photo-SET induced spirocyclization process has been developed.

A route for construction of the pentacyclic skeleton of the harringtonine alkaloids based upon a photo-SET induced spirocyclization process has been developed.