Stereoelectronic Features of a Complex Ketene Dimerization Reaction.

The amidation reaction of a tetrahydroisoquinolin-1-one-4-carboxylic acid is a key step in the multi-kilogram-scale preparation of the antimalarial drug SJ733, now in phase 2 clinical trials. In the course of investigating THIQ carboxamidations, we found that propanephosphonic acid anhydride (T3P) is an effective reagent, although the yield and byproducts vary with the nature and quantity of the base. As a control, the T3P reaction of a 3-(2-thienyl) THIQ was performed in the absence of the amine, and the products were characterized: among them are three dimeric allenes and two dimeric lactones. A nucleophile-promoted ketene dimerization process subject to subtle steric and stereoelectronic effects accounts for their formation. Two novel monomeric products, a decarboxylated isoquinolone and a purple, fused aryl ketone, were also isolated, and mechanisms for their formation from the ketene intermediate are proposed.

Evaluation of 1,1-cyclopropylidene as a thioether isostere in the 4-thio-thienopyrimidine (TTP) series of antimalarials.

The 4-(heteroarylthio)thieno[2,3-d]pyrimidine (TTP) series of antimalarials, represented by 1 and 17, potently inhibit proliferation of the 3D7 strain of P. falciparum (EC50 70-100 nM), but suffer from oxidative metabolism. The 1,1-cyclopropylidene isosteres 6 and 16 were designed to obviate this drawback. They were prepared by a short route that features a combined Peterson methylenation / cyclopropanation transformation of, e. g., ketone 7. Isosteres 6 and 16 possess significantly attenuated antimalarial potency relative to parents 1 and 17. This outcome can be rationalized based on the increased out-of-plane steric demands of the latter two. In support of this hypothesis, the relatively flat ketone 7 retains some of the potency of 1, even though it appears to be a comparatively inferior mimic with respect to electronics and bond lengths and angles. We also demonstrate crystallographically and computationally an apparent increase in the strength of the intramolecular sulfur hole interaction of 1 upon protonation.

Formal [4 + 2] cycloaddition of imines with alkoxyisocoumarins.

A new preparation of δ-lactams is reported. In the presence of a Lewis acid promoter, alkoxyisocoumarins engage a range of N-aryl and N-alkyl imines to form δ-lactams with a pendent carboalkoxy substituent. A sulfonamide-thiourea catalyst enables the synthesis of these products in moderate to good enantioselectivities.

Stereochemistry and Reactivity of the HPA-Imine Mannich Intermediate.

Homophthalic anhydride (HPA) typically reacts rapidly with benzalimines to afford the formal [4+2] adduct, a 1,2,3,4-tetrahydroisoquinolin-1-one-4-carboxylic acid. The stereochemical outcome of this reaction is consistent with an open transition state comprising an iminium species and enolized HPA, leading to a short-lived amino-anhydride intermediate. In the case of N-tert-butylbenzalimine, this Mannich-type intermediate, which would normally cyclize at low temperature to a single isomer of the delta-lactam, is intercepted by base treatment to afford beta-lactam products. A pathway featuring ketene formation followed by ring closure is implicated.

(+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium.

Drug discovery for malaria has been transformed in the last 5 years by the discovery of many new lead compounds identified by phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they induce is revealing new targets that regulate key processes in the Plasmodium parasites that cause malaria. We disclose herein that the clinical candidate (+)-SJ733 acts upon one of these targets, ATP4. ATP4 is thought to be a cation-transporting ATPase responsible for maintaining low intracellular Na(+) levels in the parasite. Treatment of parasitized erythrocytes with (+)-SJ733 in vitro caused a rapid perturbation of Na(+) homeostasis in the parasite. This perturbation was followed by profound physical changes in the infected cells, including increased membrane rigidity and externalization of phosphatidylserine, consistent with eryptosis (erythrocyte suicide) or senescence. These changes are proposed to underpin the rapid (+)-SJ733-induced clearance of parasites seen in vivo. Plasmodium falciparum ATPase 4 (pfatp4) mutations that confer resistance to (+)-SJ733 carry a high fitness cost. The speed with which (+)-SJ733 kills parasites and the high fitness cost associated with resistance-conferring mutations appear to slow and suppress the selection of highly drug-resistant mutants in vivo. Together, our data suggest that inhibitors of PfATP4 have highly attractive features for fast-acting antimalarials to be used in the global eradication campaign.

Glycosylation of Nucleosides.

Nucleoside O-glycosylation represents an archetypal problem in chemical selectivity, inasmuch as the nucleobase (an undesired site of reaction) is usually more nucleophilic than the hydroxyl (the desired site of reaction). Optimized reaction conditions have been developed for the efficient O-glycosylation of nucleoside hydroxyls. Both thioglycoside and Schmidt imidate donors (1.5 equiv) have been employed successfully. Interference by the nucleobase is minimized by the use of indium(III) triflate as the donor activating reagent; the In(OTf)3 serves to promote apparent transfer of the donor glycosyl moiety from nucleobase to hydroxyl. Glycosylation of uridine triacetate gives products resulting from O- and N-glycosylation of the pyrimidine ring.

Dimerization and comments on the reactivity of homophthalic anhydride.

Homophthalic anhydride (HPA) dimerizes under the influence of base to provide, sequentially, the (3-4')-C-acyl dimer, a pair of chiral diastereomeric bis(lactones), 3-(2-carboxybenzyl)isocoumarin-4-carboxylic acid, and finally, 3-(2-carboxybenzyl)isocoumarin. The structures of the bis(lactones) were misassigned in 1970 based on the (presumed) cis thermal decarboxylative elimination reaction of the lower melting one. The preferred pathway should be trans-anti, however, and crystallographic analysis of one of the bis(lactones) reverses the earlier assignment. The formal cycloaddition reaction of HPA with imines occurs in preference to HPA dimerization; the mechanistic implications of this reactivity difference are discussed.

N-methylimidazole promotes the reaction of homophthalic anhydride with imines.

The addition of N-methylimidazole (NMI) to the reaction of homophthalic anhydride with imines such as pyridine-3-carboxaldehyde-N-trifluoroethylimine (9) reduces the amount of elimination byproduct and improves the yield of the formal cycloadduct, tetrahydroisoquinolonic carboxylate 10. Carboxanilides of such compounds are of interest as potential antimalarial agents. A mechanism that rationalizes the role of NMI is proposed, and a gram-scale procedure for the synthesis and resolution of 10 is also described.

Optimization of diastereomeric dihydropyridines as antimalarials.

The increase in research funding for the development of antimalarials since 2000 has led to a surge of new chemotypes with potent antimalarial activity. High-throughput screens have delivered several thousand new active compounds in several hundred series, including the 4,7-diphenyl-1,4,5,6,7,8-hexahydroquinolines, hereafter termed dihydropyridines (DHPs). We optimized the DHPs for antimalarial activity. Structure-activity relationship studies focusing on the 2-, 3-, 4-, 6-, and 7-positions of the DHP core led to the identification of compounds potent (EC50 < 10 nM) against all strains of P. falciparum tested, including the drug-resistant parasite strains K1, W2, and TM90-C2B. Evaluation of efficacy of several compounds in vivo identified two compounds that reduced parasitemia by >75 % in mice 6 days post-exposure following a single 50 mg/kg oral dose. Resistance acquisition experiments with a selected dihydropyridine led to the identification of a single mutation conveying resistance in the gene encoding for Plasmodium falciparum multi-drug resistance protein 1 (PfMDR1). The same dihydropyridine possessed transmission blocking activity. The DHPs have the potential for the development of novel antimalarial drug candidates.

Biohybrid -Se-S- coupling reactions of an amino acid derived seleninate.

We describe the synthesis of the N-(2-seleninatoethyl) amide of N-Boc-phenylalanine, serving here as a peptide model, and its reductive coupling reactions under mild conditions with unprotected thiouridine and glutathione. Selenosulfide products such as these comprise reversibly conjugated bio-components, and can potentially find uses as probes of biological function, such as enzyme inhibitors, delivery systems, or structural mimics.

ß-D-Arabinosyl 1-C-sulfonic acid.

A short synthetic route to ß,d-arabinofuranosyl 1-C-sulfonic acid (7), a possible biomimetic for the arabinofuranosyl anomeric phosphate, is described. The furanosyl 1-C-sulfonate was prepared by buffered DMDO oxidation of an S-acetyl-1-thio-ß-arabinofuranose derivative. Deprotection under mild conditions allowed isolation of the free sulfonic acid without desulfonylation.

Incorporation of an Isohexide Subunit Improves the Drug-like Properties of Bioactive Compounds.

An enhanced ability to pre-engineer favorable drug-likeness qualities into bioactive molecules would focus and streamline the drug development process. We find that phenols, carboxylic acids, and a purine react with isosorbide ("GRAS" designated) under Mitsunobu coupling conditions to deliver the isoidide conjugates selectively and efficiently. Such conjugates show improved solubility and permeability properties compared with the bare scaffold compounds themselves, and the purine adduct may have applications as a 2'-deoxyadenosine isostere. We anticipate additional benefits, implied by their structures, in metabolic stability and reduced toxicity of the isoidide conjugates as well.

A Bis(imidazole)-based cysteine labeling tool for metalloprotein assembly.

Precise metal-protein coordination by design remains a considerable challenge. Polydentate, high-metal-affinity protein modifications, both chemical and recombinant, can enable metal localization. However, these constructs are often bulky, conformationally and stereochemically ill-defined, or coordinately saturated. Here, we expand the biomolecular metal-coordination toolbox with the irreversible attachment to cysteine of bis(1-methylimidazol-2-yl)ethene ("BMIE"), which generates a compact imidazole-based metal-coordinating ligand. Conjugate additions of small-molecule thiols (thiocresol and N-Boc-Cys) with BMIE confirm general thiol reactivity. The BMIE adducts are shown to complex the divalent metal ions Cu++ and Zn++ in bidentate (N2) and tridentate (N2S*) coordination geometries. Cysteine-targeted BMIE modification (>90% yield at pH 8.0) of a model protein, the S203C variant of carboxypeptidase G2 (CPG2), measured with ESI-MS, confirms its utility as a site-selective bioconjugation method. ICP-MS analysis confirms mono-metallation of the BMIE-modified CPG2 protein with Zn++, Cu++, and Co++. EPR characterization of the BMIE-modified CPG2 protein reveals the structural details of the site selective 1:1 BMIE-Cu++ coordination and symmetric tetragonal geometry under physiological conditions and in the presence of various competing and exchangeable ligands (H2O/HO-, tris, and phenanthroline). An X-ray protein crystal structure of BMIE-modified CPG2-S203C demonstrates that the BMIE modification is minimally disruptive to the overall protein structure, including the carboxypeptidase active sites, although Zn++ metalation could not be conclusively discerned at the resolution obtained. The carboxypeptidase catalytic activity of BMIE-modified CPG2-S203C was also assayed and found to be minimally affected. These features, combined with ease of attachment, define the new BMIE-based ligation as a versatile metalloprotein design tool, and enable future catalytic and structural applications.

Mechanism of a redox coupling of seleninic acid with thiol.

Equimolar quantities of 2-ethoxyethaneseleninic acid and p-thiocresol react rapidly in dichloromethane solution to give the selenosulfide along with disulfide, diselenide, and two products oxidized at sulfur, the thiosulfonate and the selenosulfonate. The latter two are new for this sort of coupling; their formation may be the result of an early thioseleninate to selenosulfinate isomerization. A radical chain mechanism is proposed to account for all five products, as well as their relative amounts.

OGA inhibition by GlcNAc-selenazoline.

The title compound, which differs from the powerful O-GlcNAcase (OGA) inhibitor GlcNAc-thiazoline only at the chalcogen atom (Se for S), is a much weaker inhibitor in a direct OGA assay. In human cells, however, the selenazoline shows comparable ability to induce hyper-O-GlcNAc-ylation, and the two show similar reduction of insulin-stimulated translocation of glucose transporter 4 in differentiated 3T3 adipocytes.

GlcNAc-Thiazoline conformations.

The title compound, a powerful inhibitor of retaining N-acetylhexosaminidases, can move freely among three pyranose solution conformations of similar energy-two twist boats and the (4)C(1) chair-as revealed by NMR, calculational, and crystallographic studies. It binds in the enzyme active site only in the pseudo-(4)C(1) conformation, however, in which it most closely resembles the hypothetical bound substrate transition state, a (4)E sofa that is approximately trigonal bipyramidal at the anomeric carbon.

Biomimetic seleninates and selenonates.

The synthesis of a variety of pyranose-, nucleoside-, (amino acid)-, and polyhydric-based seleninic and selenonic acids by DMDO oxidation of the corresponding selenoesters is reported, as well as some unusual coupling reactions of the seleninate and selenonate functionality with biological nucleophilic groups (sulfhydryl, indole, phenol, imidazole, carboxamide) that are found in proteins and enzyme active sites.

Seleninate in place of phosphate: irreversible inhibition of protein tyrosine phosphatases.

A homotyrosine based seleninic acid irreversibly inhibits protein tyrosine phosphatases by forming a covalent selenosulfide linkage with the active site cysteine sulfhydryl specifically. The details of the event are revealed by model synthetic studies and by kinetic, mass spectrometric, and crystallographic characterization.

Trans- and cis-stilbene polyphenols induced rapid perinuclear mitochondrial clustering and p53-independent apoptosis in cancer cells but not normal cells.

We previously reported that 3,4,5,4'-tetramethoxy-trans-stilbene (MR-4) induces p53 and perinuclear mitochondrial clustering in cancer cells [Gosslau, A., Chen, M., Ho, C.-T., Chen, K.Y., 2005, A methoxy derivative of resveratrol analogue selectively induced activation of the mitochondrial apoptotic pathway in transformed fibroblasts. Br. J. Cancer 92, 513-521.]. Here we extended the study to over 20 trans-stilbene derivatives and their cis-isomers to explore structure activity relationship. Among them, 3,4,5,4'-tetramethoxy-cis-stilbene (MC-4), the cis-isomer of MR-4, was most potent, with IC50 of 20 nM for growth inhibition. MC-4 induced a rapid perinuclear mitochondrial clustering, membrane permeability transition, cytochrome c release and DNA fragmentation. To determine whether trans- and cis-stilbene polyphenols may share a common mechanism, we compared the effects of MC-4 and MR-4 in three isogenic cell lines derived from the colorectal carcinoma HCT116 cells: p53+/+ (p53-wt), p53-/- (p53-null) and p21-/- (p21-null). Deletion of either p53 or p21 neither blocked the effects of MC-4 or MR-4 on mitochondrial clustering nor inhibited apoptosis, indicating that the actions of both stilbenes are independent of p53 and p21. Although microtubule disruption has been proposed to account for the action of some cis-stilbene polyphenols, we did not observe differences in microtubule dynamics between cells treated with MC-4 and MR-4. These findings suggest that MC-4 and MR-4 may share a common mechanism whereby the perinucear mitochondrial clustering, rather than p53, p21, or microtubule depolymerization, is critical for their pro-apoptotic action.

N-Acetylhexosaminidase inhibitory properties of C-1 homologated GlcNAc- and GalNAc-thiazolines.

Several C-1 homologated GlcNAc- and GalNAc-thiazolines, as well as a related GalNAc-thiazole, have been prepared. The compounds are analogues of GlcNAc-thiazoline, a potent transition-state-mimicking inhibitor of retaining beta-N-acetylglycosaminidases. Kinetic evaluation of these fused pyranose-heterocycles against the bacterial N-acetylhexosaminidase SpHex suggests active site steric restrictions around the substrate anomeric carbon.