Coupling-Condensation Strategy for the Convergent Synthesis of an Imidazole-Fused 2-Aminoquinoline NLRP3 Agonist.

The development of a convergent route to the NLRP3 (nucleotide-binding domain and leucine-rich repeat-containing protein 3) agonist BMS-986299 is reported. The synthesis relies on a key Miyaura borylation and a tandem Suzuki-Miyaura coupling between an iodoimidazole and an o-aminochloroarene, followed by acid-mediated cyclization to afford the aminoquinoline core. The subsequent Boc cleavage and regioselective acylation afford the target compound. Two routes to the iodoimidazole intermediate are presented, along with the synthesis of the o-aminochloroarene via Negishi coupling. The convergent six-step route leads to an 80% reduction in process mass intensity compared to the linear enabling synthesis.

Generation of quinolone antimalarials targeting the Plasmodium falciparum mitochondrial respiratory chain for the treatment and prophylaxis of malaria.

There is an urgent need for new antimalarial drugs with novel mechanisms of action to deliver effective control and eradication programs. Parasite resistance to all existing antimalarial classes, including the artemisinins, has been reported during their clinical use. A failure to generate new antimalarials with novel mechanisms of action that circumvent the current resistance challenges will contribute to a resurgence in the disease which would represent a global health emergency. Here we present a unique generation of quinolone lead antimalarials with a dual mechanism of action against two respiratory enzymes, NADH:ubiquinone oxidoreductase (Plasmodium falciparum NDH2) and cytochrome bc(1). Inhibitor specificity for the two enzymes can be controlled subtly by manipulation of the privileged quinolone core at the 2 or 3 position. Inhibitors display potent (nanomolar) activity against both parasite enzymes and against multidrug-resistant P. falciparum parasites as evidenced by rapid and selective depolarization of the parasite mitochondrial membrane potential, leading to a disruption of pyrimidine metabolism and parasite death. Several analogs also display activity against liver-stage parasites (Plasmodium cynomolgi) as well as transmission-blocking properties. Lead optimized molecules also display potent oral antimalarial activity in the Plasmodium berghei mouse malaria model associated with favorable pharmacokinetic features that are aligned with a single-dose treatment. The ease and low cost of synthesis of these inhibitors fulfill the target product profile for the generation of a potent, safe, and inexpensive drug with the potential for eventual clinical deployment in the control and eradication of falciparum malaria.

Synthesis and evaluation of the antimalarial, anticancer, and caspase 3 activities of tetraoxane dimers.

The synthesis of a range of mono spiro and dispiro 1,2,4,5-tetraoxane dimers is described. Selected molecules were examined in in vitro assays to determine their antimalarial and anticancer potential. Our studies reveal that several molecules possess potent nanomolar antimalarial and single digit micromolar antiproliferative IC(50)s versus colon (HT29-AK and leukemia (HL60) cell lines.

Broadly neutralizing aptamers to SARS-CoV-2: A diverse panel of modified DNA antiviral agents.

Since its discovery, COVID-19 has rapidly spread across the globe and has had a massive toll on human health, with infection mortality rates as high as 10%, and a crippling impact on the world economy. Despite numerous advances, there remains an urgent need for accurate and rapid point-of-care diagnostic tests and better therapeutic treatment options. To contribute chemically distinct, non-protein-based affinity reagents, we report here the identification of modified DNA-based aptamers that selectively bind to the S1, S2, or receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Several aptamers inhibit the binding of the spike protein to its cell-surface receptor angiotensin-converting enzyme 2 (ACE2) and neutralize authentic SARS-CoV-2 virus in vitro, including all variants of concern. With a high degree of nuclease resistance imparted by the base modifications, these reagents represent a new class of molecules with potential for further development as diagnostics or therapeutics.

Examination of the cytotoxic and embryotoxic potential and underlying mechanisms of next-generation synthetic trioxolane and tetraoxane antimalarials.

Semisynthetic artemisinin-based therapies are the first-line treatment for P. falciparum malaria, but next-generation synthetic drug candidates are urgently required to improve availability and respond to the emergence of artemisinin-resistant parasites. Artemisinins are embryotoxic in animal models and induce apoptosis in sensitive mammalian cells. Understanding the cytotoxic propensities of antimalarial drug candidates is crucial to their successful development and utilization. Here, we demonstrate that, similarly to the model artemisinin artesunate (ARS), a synthetic tetraoxane drug candidate (RKA182) and a trioxolane equivalent (FBEG100) induce embryotoxicity and depletion of primitive erythroblasts in a rodent model. We also show that RKA182, FBEG100 and ARS are cytotoxic toward a panel of established and primary human cell lines, with caspase-dependent apoptosis and caspase-independent necrosis underlying the induction of cell death. Although the toxic effects of RKA182 and FBEG100 proceed more rapidly and are relatively less cell-selective than that of ARS, all three compounds are shown to be dependent upon heme, iron and oxidative stress for their ability to induce cell death. However, in contrast to previously studied artemisinins, the toxicity of RKA182 and FBEG100 is shown to be independent of general chemical decomposition. Although tetraoxanes and trioxolanes have shown promise as next-generation antimalarials, the data described here indicate that adverse effects associated with artemisinins, including embryotoxicity, cannot be ruled out with these novel compounds, and a full understanding of their toxicological actions will be central to the continuing design and development of safe and effective drug candidates which could prove important in the fight against malaria.

Circumventing glass vial and diluent effects on solution stability of small molecule analytes during analytical method development and validation.

Solution stability of analytes plays an important part in qualitative analysis, especially in conducting accurate, quantitative analyses. Sample diluents and glass vials as sample containers for HPLC analyses can play a critical role and should be evaluated during chromatographic method development. We have encountered several instances during pharmaceutical development where the glass vial/diluent combination has negatively impacted method performance. One case encompasses adsorption of piperazine, a secondary amine, to non-silanized glass vials, resulting in recovery failures during analytical method transfer. Two further cases describe the propensity for peracetylated C-aryl glucosides being subject chemical transformations relating to sample diluent. The first reports transesterification with methanol-based diluents and the second describes hydrolysis with acetonitrile/water diluents mediated by the mild alkalinity of certain brands of Type I borosilicate vials. A final case explores development of a related substance method, it was found that an impurity was prone to hydrolysis and another impurity with a primary amine tended to be adsorbed on glass vials. Diluents of appropriate pH and buffer strength were strategically selected to neutralize the mild alkalinity of the glass vials as well as to mitigate adsorption of the amine analyte on glass vials. As a result, excellent sample stability and reproducibility were achieved, regardless the quality and brand of Type I glass vials used. Here we present four case studies that demonstrate how the negative impact of Type I glass vials on those susceptible analytes can be effectively eliminated by using appropriate sample diluents, which is essential to ensure accurate analytical data and provide for a smooth method validation and transfer.

Quantitative GC determination of sodium triacetoxyborohydride (STAB).

Sodium triacetoxyborohydride (STAB) is a mild, selective, and frequently used reducing agent for reductive amination transformations. STAB has been reported to undergo degradation when exposed to air, often requiring additional sub-stoichiometric reagent charges to drive reactions to completion. We report the development and qualification of a rapid, quantitative GC derivatization method using 3,4-dihydroisoquinoline to enable accurate hydride assay determination of STAB. Assay data generated from this method has been directly compared to a recently published HPLC method using bromosalicaldehyde as the derivatization reagent. The degradation kinetics of STAB have also been further investigated by Raman spectroscopy to demonstrate how exposure of STAB to air can negatively impact the reagent's performance.

A chemometric model for the quantitative determination of isotopic impurities in d3-methylamine hydrochloride by Fourier-transform infrared spectroscopy.

Deuterated drug molecules are of increasing interest to the pharmaceutical industry due to their capacity to slow metabolism and the potential for improved pharmacokinetics or improved pharmacodynamics they may offer over their non-deuterated counterparts. The desired level of deuteration or isotopic purity is a critical quality attribute for these compounds that can be essential for drug efficacy or patient safety. Deuterated reagents are often used to introduce a deuterated moiety into the drug substance; as such, isotopic impurities in these deuterated input materials need to be tightly controlled. A novel Fourier-transform infrared (FTIR) spectroscopic method was developed and evaluated as a fast and straightforward technique to quantify low-level isotopic impurities in the deuterated reagent d3-methylamine hydrochloride. Using data acquired through LC-MS analysis, the resulting chemometric model was validated according to ICH Q2(R1) guidelines achieving limits of quantitation of 0.31, 0.31, and 0.34 wt% for d0-, d1- and d2-methylamine hydrochloride impurities respectively.

Design, synthesis and antimalarial/anticancer evaluation of spermidine linked artemisinin conjugates designed to exploit polyamine transporters in Plasmodium falciparum and HL-60 cancer cell lines.

A series of artemisinin-spermidine conjugates designed to utilise the upregulated polyamine transporter found in cancer cells have been prepared. These conjugates were evaluated against human promyelocytic leukaemia HL-60 cells and chloroquine-sensitive 3D7 Plasmodium falciparum and several show promising anticancer and antimalarial activity. Although some limitations in this vector-based approach are apparent, a number of high potency Boc-protected analogues were identified with activity against malaria parasites as low as 0.21nM.

Discovery of novel non-peptide inhibitors of BACE-1 using virtual high-throughput screening.

A novel series of isatin-based inhibitors of beta-secretase (BACE-1) have been identified using a virtual high-throughput screening approach. Structure-activity relationship studies revealed structural features important for inhibition. Docking studies suggest these inhibitors may bind within the BACE-1 active site through H-bonding interactions involving the catalytic aspartate residues.

Synthesis and biological evaluation of extraordinarily potent C-10 carba artemisinin dimers against P. falciparum malaria parasites and HL-60 cancer cells.

A series of artemisinin dimers incorporating a metabolically stable C-10 carba-linkage have been prepared, several of which show remarkable in vitro antimalarial activity (as low as 30 pM) versus Plasmodium falciparum and in vitro anticancer activity in the micromolar to nanomolar range versus HL-60 cell lines.

Antimalarial and antitumor evaluation of novel C-10 non-acetal dimers of 10beta-(2-hydroxyethyl)deoxoartemisinin.

Four series of C-10 non-acetal dimers were prepared from key trioxane alcohol 10beta-(2-hydroxyethyl)deoxoartemisinin (9b). All of the dimers prepared displayed potent low nanomolar antimalarial activity versus the K1 and HB3 strains of Plasmodium falciparum. The most potent compound assayed was phosphate dimer 14a, which was greater than 50 times more potent than the parent drug artemisinin and about 15 times more potent than the clinically used acetal artemether. In contrast to their potent activity versus malaria parasites, virtually all of the dimers expressed poor anticancer activity apart from the trioxane phosphate ester dimers 14a and 14b, which expressed nanomolar growth inhibitory (GI50) values versus a range of cancer cell lines in the NCI 60 human cell line screen. Further detailed studies on these dimers in vitro in HL60 cells demonstrate that both phosphate ester dimers (14a and 14b) are more potent than the anticancer agent doxorubicin. Interestingly, phosphate ester monomers 9c and 9d, antimalarially active in the low nanomolar region versus P. falciparum, are inactive as anticancer agents even at concentrations in the millimolar region. This observation emphasizes the importance of two trioxane units for high antiproliferative activity, and we propose that the nature of the linker in dimers of this type plays a crucial role in imparting potent anticancer activity.

Discovery of biphenylacetamide-derived inhibitors of BACE1 using de novo structure-based molecular design.

ß-Secretase (BACE1), the enzyme responsible for the first and rate-limiting step in the production of amyloid-ß peptides, is an attractive target for the treatment of Alzheimer's disease. In this study, we report the application of the de novo fragment-based molecular design program SPROUT to the discovery of a series of nonpeptide BACE1 inhibitors based upon a biphenylacetamide scaffold. The binding affinity of molecules based upon this designed molecular scaffold was increased from an initial BACE1 IC50 of 323 µM to 27 µM following the synthesis of a library of optimized ligands whose structures were refined using the recently developed SPROUT-HitOpt software. Although a number of inhibitors were found to exhibit cellular toxicity, one compound in the series was found to have useful BACE1 inhibitory activity in a cellular assay with minimal cellular toxicity. This work demonstrates the power of an in silico fragment-based molecular design approach in the discovery of novel BACE1 inhibitors.

Identification of novel antimalarial chemotypes via chemoinformatic compound selection methods for a high-throughput screening program against the novel malarial target, PfNDH2: increasing hit rate via virtual screening methods.

Malaria is responsible for approximately 1 million deaths annually; thus, continued efforts to discover new antimalarials are required. A HTS screen was established to identify novel inhibitors of the parasite's mitochondrial enzyme NADH:quinone oxidoreductase (PfNDH2). On the basis of only one known inhibitor of this enzyme, the challenge was to discover novel inhibitors of PfNDH2 with diverse chemical scaffolds. To this end, using a range of ligand-based chemoinformatics methods, ~17000 compounds were selected from a commercial library of ~750000 compounds. Forty-eight compounds were identified with PfNDH2 enzyme inhibition IC(50) values ranging from 100 nM to 40 µM and also displayed exciting whole cell antimalarial activity. These novel inhibitors were identified through sampling 16% of the available chemical space, while only screening 2% of the library. This study confirms the added value of using multiple ligand-based chemoinformatic approaches and has successfully identified novel distinct chemotypes primed for development as new agents against malaria.

Comparison of the reactivity of antimalarial 1,2,4,5-tetraoxanes with 1,2,4-trioxolanes in the presence of ferrous iron salts, heme, and ferrous iron salts/phosphatidylcholine.

Dispiro-1,2,4,5-tetraoxanes and 1,2,4-trioxolanes represent attractive classes of synthetic antimalarial peroxides due to their structural simplicity, good stability, and impressive antimalarial activity. We investigated the reactivity of a series of potent amide functionalized tetraoxanes with Fe(II)gluconate, FeSO(4), FeSO(4)/TEMPO, FeSO(4)/phosphatidylcholine, and heme to gain knowledge of their potential mechanism of bioactivation and to compare the results with the corresponding 1,2,4-trioxolanes. Spin-trapping experiments demonstrate that Fe(II)-mediated peroxide activation of tetraoxanes produces primary and secondary C-radical intermediates. Reaction of tetraoxanes and trioxolanes with phosphatidylcholine, a predominant unsaturated lipid present in the parasite digestive vacuole membrane, under Fenton reaction conditions showed that both endoperoxides share a common reactivity in terms of phospholipid oxidation that differs with that of artemisinin. Significantly, when tetraoxanes undergo bioactivation in the presence of heme, only the secondary C-centered radical is observed, which smoothly produces regioisomeric drug derived-heme adducts. The ability of these tetraoxanes to alkylate the porphyrin ring was also confirmed with Fe(II)TPP and Mn(II)TPP, and docking studies were performed to rationalize the regioselectivity observed in the alkylation process. The efficient process of heme alkylation and extensive lipid peroxidation observed here may play a role in the mechanism of action of these two important classes of synthetic endoperoxide antimalarial.

Rationale design of biotinylated antimalarial endoperoxide carbon centered radical prodrugs for applications in proteomics.

The semisynthetic artemisinin derivatives such as artesunate and artemether, along with the fully synthetic endoperoxide antimalarials (e.g., OZ277, Nature 2004, 430, 900-904), are believed to mediate their antimalarial effects by iron-induced formation of carbon-centered radicals capable of alkylating heme and/or protein. Here, we describe the design and synthesis of a series of biotinylated endoperoxide probe molecules for use in proteomic studies. The target molecules include derivatives of the artemisinin and OZ families, and we demonstrate that these conjugates express nanomolar in vitro activity versus cultured strains of Plasmodium falciparum. We also describe the synthesis of chemically cleavable linked conjugates designed to enable mild elution of labeled proteins during target protein identification.

Endoperoxide carbonyl falcipain 2/3 inhibitor hybrids: toward combination chemotherapy of malaria through a single chemical entity.

We extend our approach of combination chemotherapy through a single prodrug entity (O'Neill et al. Angew. Chem., Int. Ed. 2004, 43, 4193) by using a 1,2,4-trioxolane as a protease inhibitor carbonyl-masking group. These molecules are designed to target the malaria parasite through two independent mechanisms of action: iron(II) decomposition releases the carbonyl protease inhibitor and potentially cytotoxic C-radical species in tandem. Using a proposed target "heme", we also demonstrate heme alkylation/carbonyl inhibitor release and quantitatively measure endoperoxide turnover in parasitized red blood cells.

Evidence for the involvement of carbon-centered radicals in the induction of apoptotic cell death by artemisinin compounds.

Artemisinin and its derivatives are currently recommended as first-line antimalarials in regions where Plasmodium falciparum is resistant to traditional drugs. The cytotoxic activity of these endoperoxides toward rapidly dividing human carcinoma cells and cell lines has been reported, and it is hypothesized that activation of the endoperoxide bridge by an iron(II) species, to form C-centered radicals, is essential for cytotoxicity. The studies described here have utilized artemisinin derivatives, dihydroartemisinin, 10beta-(p-bromophenoxy)dihydroartemisinin, and 10beta-(p-fluorophenoxy)dihydroartemisinin, to determine the chemistry of endoperoxide bridge activation to reactive intermediates responsible for initiating cell death and to elucidate the molecular mechanism of cell death. These studies have demonstrated the selective cytotoxic activity of the endoperoxides toward leukemia cell lines (HL-60 and Jurkat) over quiescent peripheral blood mononuclear cells. Deoxy-10beta-(p-fluorophenoxy)dihydroartemisinin, which lacks the endoperoxide bridge, was 50- and 130-fold less active in HL-60 and Jurkat cells, respectively, confirming the importance of this functional group for cytotoxicity. We have shown that chemical activation is responsible for cytotoxicity by using liquid chromatography-mass spectrometry analysis to monitor endoperoxide activation by measurement of a stable rearrangement product of endoperoxide-derived radicals, which was formed in sensitive HL-60 cells but not in insensitive peripheral blood mononuclear cells. In HL-60 cells the endoperoxides induce caspase-dependent apoptotic cell death characterized by concentration- and time-dependent mitochondrial membrane depolarization, activation of caspases-3 and -7, sub-G(0)/G(1) DNA formation, and attenuation by benzyloxycarbonyl-VAD-fluoromethyl ketone, a caspase inhibitor. Overall, these results indicate that endoperoxide-induced cell death is a consequence of activation of the endoperoxide bridge to radical species, which triggers caspase-dependent apoptosis.