A Tandem Ring Closure and Nitrobenzene Reduction with Sulfide Provides an Improved Route to an Important Intermediate for the Anti-Tuberculosis Drug Candidate Sutezolid.

Sutezolid is an in-development thiomorpholine derivative of the FDA-approved tuberculosis (TB) treatment linezolid. Current synthetic routes for preparing sutezolid start with thiomorpholine as a key structural building block; unfortunately, this material was identified as a major cost driver for the API, which will limit the potential uptake of this treatment in lower income regions. In this work, an alternative, lower-cost synthetic strategy to a known p-phenylenediamine intermediate to sutezolid has been demonstrated. The key step in this process is the construction of the thiomorpholine ring by a nucleophilic sulfide ring closure on an activated bis(2-hydroxyethyl)-functionalized aniline, which was in turn made by reaction of 3,4-difluoronitrobenzene and diethanolamine. This sulfide treatment has the added benefit of affecting a Zinin reduction of the nitro functional group, which alleviates the need for the transition metal reduction used in previous routes. After optimization, this key reaction was able to provide the desired aniline intermediate in yields between 65 and 80% and, after a standard charcoal treatment, purity of >94%. Initial demonstrations of the full 3-step strategy were successfully conducted on scales up to 100 g with overall yields of 53-68%. This preliminary work will serve as the foundation for a broader low-cost redesign of the sutezolid synthetic process.

Practical and Scalable Two-Step Process for 6-(2-Fluoro-4-nitrophenyl)-2-oxa-6-azaspiro[3.3]heptane: A Key Intermediate of the Potent Antibiotic Drug Candidate TBI-223.

A low-cost, protecting group-free route to 6-(2-fluoro-4-nitrophenyl)-2-oxa-6-azaspiro[3.3]heptane (1), the starting material for the in-development tuberculosis treatment TBI-223, is described. The key bond forming step in this route is the creation of the azetidine ring through a hydroxide-facilitated alkylation of 2-fluoro-4-nitroaniline (2) with 3,3-bis(bromomethyl)oxetane (BBMO, 3). After optimization, this ring formation reaction was demonstrated at 100 g scale with isolated yield of 87% and final product purity of >99%. The alkylating agent 3 was synthesized using an optimized procedure that starts from tribromoneopentyl alcohol (TBNPA, 4), a commercially available flame retardant. Treatment of 4 with sodium hydroxide under Schotten-Baumann conditions closed the oxetane ring, and after distillation, 3 was recovered in 72% yield and >95% purity. This new approach to compound 1 avoids the previous drawbacks associated with the synthesis of 2-oxa-6-azaspiro[3,3]heptane (5), the major cost driver used in previous routes to TBI-223. The optimization and multigram scale-up results for this new route are reported herein.

Development of a Practical Synthesis of the 8-FDC Fragment of OPC-167832.

A concise and practical synthesis has been developed to provide the 8-fluoro-5-hydroxy-3,4-diydrocarbostyril (8-FDC) fragment of OPC-167832 in 41% yield and in >99% purity over four steps from 3-amino-4-fluorophenol. The key feature of this process is the development of a telescoped one-pot synthesis of the quinolone via a chemoselective amidation/acid-induced cyclization that allows for simple product isolation without the need for column chromatography.

Synthesis of an Oxathiolane Drug Substance Intermediate Guided by Constraint-Driven Innovation.

A new route was developed for construction of the oxathiolane intermediate used in the synthesis of lamivudine (3TC) and emtricitabine (FTC). We developed the presented route by constraining ourselves to low-cost, widely available starting materials-we refer to this as supply-centered synthesis. Sulfenyl chloride chemistry was used to construct the framework for the oxathiolane from acyclic precursors. This bond construction choice enabled the use of chloroacetic acid, vinyl acetate, sodium thiosulfate, and water to produce the oxathiolane.

Expanding Access to Remdesivir via an Improved Pyrrolotriazine Synthesis: Supply Centered Synthesis.

Pyrrolotriazine 1 is an important precursor to remdesivir. Initial results toward an efficient synthesis are disclosed consisting of sequential cyanation, amination, and triazine formation beginning from pyrrole. This route makes use of highly abundant, commoditized raw material inputs. The yield of triazine was doubled from 31% to 59%, and the synthetic step count was reduced from 4 to 2. These efforts help to secure the remdesivir supply chain.

An Economical Route to Lamivudine Featuring a Novel Strategy for Stereospecific Assembly.

An economical synthesis of lamivudine was developed by employing a new method to establish the stereochemistry about the heterocyclic oxathiolane ring. Toward this end, an inexpensive and readily accessible lactic acid derivative served the dual purpose of activating the carbohydrate's anomeric center for N-glycosylation and transferring stereochemical information to the substrate simultaneously. Both enantiomers of the lactic acid derivative are available, and either ß-enantiomer in this challenging class of 2'-deoxynucleoside active pharmaceutical ingredients can be formed.

Surface charge controls the fate of Au nanorods in saline estuaries.

This work reports the distribution of negatively charged, gold core nanoparticles in a model marine estuary as a function of time. A single dose of purified polystyrene sulfonate (PSS)-coated gold nanorods was added to a series of three replicate estuarine mesocosms to emulate an abrupt nanoparticle release event to a tidal creek of a Spartina -dominated estuary. The mesocosms contained several phases that were monitored: seawater, natural sediments, mature cordgrass, juvenile northern quahog clam, mud snails, and grass shrimp. Aqueous nanorod concentrations rose rapidly upon initial dosing and then fell to stable levels over the course of approximately 50 h, after which they remained stable for the remainder of the experiment (41 days total). The concentration of nanorods rose in all other phases during the initial phase of the experiment; however, some organisms demonstrated depuration over extended periods of time (100+ h) before removal from the dosed tanks. Clams and biofilm samples were also removed from the contaminated tanks post-exposure to monitor their depuration in pristine seawater. The highest net uptake of gold (mass normalized) occurred in the biofilm phase during the first 24 h, after which it was stable (to the 95% level of confidence) throughout the remainder of the exposure experiment. The results are compared against a previous study of positively charged nanoparticles of the same size to parameterize the role of surface charge in determining nanoparticle fate in complex aquatic environments.

Combinatorial parameter space as an empirical tool for predicting water chemistry: Fe(II) oxidation across a watershed.

Fe(II) oxidation kinetics in surface waters are a complex function of the concentration of several dissolved species that vary geographically and temporally across watersheds. This work reports an empirical, combinatorial investigation of Fe(II) oxidation that simultaneously evaluated these variations across the pH, Fe(II), PO4³â», Cl⁻, Br(-), CO3²â», and natural organic matter (NOM) axes. The work assayed the effects of independent and dependent variables through application of a novel experimental design that varied Fe(II), PO4³â», Cl⁻, Br⁻, and CO3²â» along the pH axis. Each factor was varied across concentration ranges corresponding to the natural variation between typical fresh and salt water. The system was designed to describe the oxidation of Fe(II) that occurs when Fe(II)-rich groundwaters are mixed rapidly with oxic overlaying waters as a result of tidal movement, bioturbation, dredging, and other mixing/resuspension events. Factors and interfactor interactions were statistically evaluated to determine their importance to Fe(II) oxidation at the 95% level of confidence. Significant factors were retained and used to construct predictive numerical models of Fe(II) oxidation rates. Two models (M1 and M2) were constructed to represent the conditional endmembers of unrestricted Fe cycling (M1) and restricted Fe cycling (due to forced precipitation of Fe(III), M2). The models were challenged to predict net Fe(II) oxidation rates across a watershed (the Congaree/Santee rivers, sampled at ten different locations in South Carolina). The models were generally capable of predicting Fe(II) oxidation rates to within the 95% confidence interval, although M2 consistently overpredicted the rate relative to M1. The minimum initial Fe(II) concentration needed to observe Fe cycling is estimated based on the model output.

Short-term Fe cycling during Fe(II) oxidation: exploring joint oxidation and precipitation with a combinatorial system.

The net oxidation of Fe(II)aq by dioxygen initiates a suite of reactions including the oxidation of multiple Fe(II) complexes, generation of secondary oxidants, Fe(III) reduction, and precipitation of insoluble products. This work reports application of a multifactorial strategy to describe the oxidation of Fe(II) under conditions that correspond to those found where Fe(II)-rich groundwaters mix rapidly with overlying oxygenated waters. Response surfaces were constructed describing the relationship of the net oxidation process with mixtures of the common ligands chloride (Cl-), bromide (Br-), total carbonate (CO3(2-)), Fe(II), and Suwannee River natural organic matter (SRNOM) at pH 8.00. Response surfaces were generated in the presence and absence of added phosphate, representing conditional end members corresponding to geographical locations where Fe(III) precipitation is respectively forced and unconstrained. Comparison of net Fe(II) oxidation rates in the presence and absence of phosphate then enabled resolution of the relative contributions of Fe(II) oxidation and Fe(III) reduction to the overall process. The differences between the two surfaces demonstrated the importance of Fe(II) regeneration on the rapid (min) time scale during net oxidation. The minimum Fe(II) concentration necessary to initiate measurable cycling is reported. The presence of reactive oxygen species was evaluated through the use of probes added to the center point condition of the experimental matrix. Analysis of the statistical significance of the Fe(II)-factor relationships demonstrated that over the conditional scale of the experiments complexation of Fe(II) by the selected ligands did not correlate to the experimental outcome.

Multivariate examination of Fe(II)/Fe(III) cycling and consequent hydroxyl radical generation.

The introduction of Fe(II)(aq) into aerated solutions resulted in net Fe(II) oxidation with concomitant, rapid Fe(II)/Fe(IIII) cycling and concurrent generation of reactive oxygen species. The effect of mixtures of naturally occurring solutes on Fe(II)/Fe(III) cycling and the concurrent oxidation of dissolved organics is reported. The experimental strategy was based on a multivariate, microscale, high-throughput approach for evaluating the effect of covarying concentrations of bromide, iodide, Suwannee River natural organic matter (SRNOM), chloride, and total carbonate species. Superoxide and HO• were evaluated at the center point condition of the experimental design with selective scavengers (superoxide dismutase and benzoic acid). The rate of Fe(II) oxidation decreased in the presence of these scavengers, indicating it is a function of oxygen, superoxide, and HO•. HO• generated during Fe(II)/Fe(III) cycling was quantified with the selective probe 1,3-dicyanotetrachlorobenzene (DCTCB). Through the range of experimental conditions of this design, the ratio of the number of moles of HO• produced to the number of moles of Fe(II) consumed varied from 3 to 750, corresponding to approximately 10 to 2200 Fe(II)/Fe(III) cycles, respectively. The implications of these findings with respect to organic oxidation during the aeration of anoxic Fe(II) rich groundwaters are discussed.

The adsorption of saxitoxin to clays and sediments in fresh and saline waters.

The adsorption of saxitoxin to Na- and Ca-montmorillonite, kaolin (crystalline and amorphous), kaolinite, Bread and Butter Creek sediment (an estuarine tidal creek), Gulf of Mexico sediment, and Santa Barbara Basin sediment in deionized water and 32 per thousand salinity simulated seawater (Instant Ocean) is reported. Adsorption was partially reversible for all cases and best described using a Freundlich isotherm. The corresponding Freundlich constants (K(F)) ranged from 8.83 x 10(3)micromol/kg to 6.76 x 10(4)micromol/kg for freshwater and 4.73 x 10(3)micromol/kg-1.11 x 10(4)micromol/kg for seawater. There is a positive linear correlation seen between the K(F) values and the cation-exchange capacity of the adsorbents. The release of saxitoxin from previously equilibrated adsorbents was determined in freshwater (0-18%) and seawater (4-53%).

Adsorption of domoic acid to marine sediments and clays.

Conditional solid-water distribution coefficients (K(d)) for the adsorption of domoic acid (DA) to a series of complex sediments and clays were determined in artificial seawater. K(d) ranged from 5.11 L g(-1) to 0.97 L g(-1), with a corresponding ranking of: kaolinite > Gulf of Mexico sediment > Santa Barbara Basin sediment > Bread and Butter Creek sediment > poorly crystallized kaolin > Ca-montmorillonite > Na-montmorillonite > well crystallized kaolin > diatomaceous earth. Adsorption correlated with the anion exchange capacity of the clays tested (R(2) = 0.98), but not the more structurally complex sediments. The effect of added transition metals (Fe(iii), Cu(ii), Al(iii)) and terrestrially derived dissolved organic matter (Suwannee River DOM, SRDOM) on DA adsorption to Na-montmorillonite, well crystallized kaolin, and Gulf of Mexico sediment, was also tested. The addition of transition metals led to increased adsorption to all surfaces by a factor of 2-7, presumably by enabling the adsorption of DA-metal complexes. SRDOM enhanced DA adsorption by a factor of approximately 2.5. The release of adsorbed DA from sediments was also examined. Under our conditions, adsorbed DA equilibrated with the overlying aqueous phase within minutes with approximately 50% release.

Photostability of kainic acid in seawater.

The environmental degradation of a mixture of domoic acid (DA) and kainic acid (KA) in seawater with and without added transition metals is reported. The association constants for kainic acid with Fe (III) and Cu (II) were determined using (1)H nuclear magnetic resonance (NMR; K1,Fe(III) = 2.27 x 10(12), K2,Fe(III) = 8.99 x 10(8), K1,Cu(II) = 1.38 x 10(10), and K2,Cu(II) = 4.35 x 10(7)). The photochemical half-life of kainic acid has been determined to be significantly longer (40-100 h) than that of domoic acid in corresponding marine systems (12-34 h). The significance of this finding was highlighted by a comparison of the quantification of a mixture of kainic and domoic acids during photodegradation by liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques and the widely used competitive enzyme-linked immunosorbent assay (cELISA; Biosense Laboratories) method. The MS-based analysis showed that approximately 50% of the DA was photodegraded within 15 h. In contrast, the domoic acid cELISA assay reported that the concentration essentially remained unchanged over this period. The possibility of interference from naturally occurring kainic acid during cELISA measurements could lead to the overestimation of total domoic acid, especially if they occur in mixtures in sunlit waters.