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.

Assessing availability, prices, and market share of quality-assured malaria ACT and RDT in the private retail sector in Nigeria and Uganda.

BACKGROUND: An estimated 50% of suspected malaria cases in sub-Saharan Africa first seek care in the private sector, especially in private medicine retail outlets. Quality of care in these outlets is generally unknown but considered poor with many patients not receiving a confirmatory diagnosis or the recommended first-line artemisinin-based combination therapy (ACT). In 2010, a subsidy pilot scheme, the Affordable Medicines Facility malaria, was introduced to crowd out the use of monotherapies in favour of WHO-pre-qualified artemisinin-based combinations (WHO-PQ-ACTs) in the private health sector. The scheme improved the availability, market share, and cost of WHO-PQ-ACTs in countries like Nigeria and Uganda, but in 2018, the subsidies were halted in Nigeria and significantly reduced in Uganda. This paper presents findings from six retail audit surveys conducted from 2014 to 2021 in Nigeria and Uganda to assess whether the impact of subsidies on the price, availability, and market share of artemisinin-based combinations has been sustained after the subsidies were reduced or discontinued. METHODS: Six independent retail audits were conducted in private medicine retail outlets, including pharmacies, drug shops, and clinics in Nigeria (2016, 2018, 2021), and Uganda (2014, 2019, 2020) to assess the availability, price, and market share of anti-malarials, including WHO-PQ-ACTs and non-WHO-PQ-ACTs, and malaria rapid diagnostic tests (RDTs). RESULTS: Between 2016 and 2021, there was a 57% decrease in WHO-PQ-ACT availability in Nigeria and a 9% decrease in Uganda. During the same period, non-WHO-PQ-ACT availability increased in Nigeria by 41% and by 34% in Uganda. The price of WHO-PQ-ACTs increased by 42% in Nigeria to $0.68 and increased in Uganda by 24% to $0.95. The price of non-WHO-PQ-ACTs decreased in Nigeria by 26% to $1.08 and decreased in Uganda by 64% to $1.23. There was a 76% decrease in the market share of WHO-PQ-ACTs in Nigeria and a 17% decrease in Uganda. Malaria RDT availability remained low throughout. CONCLUSION: With the reduction or termination of subsidies for WHO-PQ-ACTs in Uganda and Nigeria, retail prices have increased, and retail prices of non-WHO-PQ-ACTs decreased, likely contributing to a shift of higher availability and increased use of non-WHO-PQ-ACTs.

Application of Chiral Transfer Reagents to Improve Stereoselectivity and Yields in the Synthesis of the Antituberculosis Drug Bedaquiline.

Bedaquiline (BDQ) is an important drug for treating multidrug-resistant tuberculosis (MDR-TB), a worldwide disease that causes more than 1.6 million deaths yearly. The current synthetic strategy adopted by the manufacturers to assemble this molecule relies on a nucleophilic addition reaction of a quinoline fragment to a ketone, but it suffers from low conversion and no stereoselectivity, which subsequently increases the cost of manufacturing BDQ. The Medicines for All Institute (M4ALL) has developed a new reaction methodology to this process that not only allows high conversion of starting materials but also results in good diastereo- and enantioselectivity toward the desired BDQ stereoisomer. A variety of chiral lithium amides derived from amino acids were studied, and it was found that lithium (R)-2-(methoxymethyl)pyrrolidide, obtained from d-proline, results in high assay yield of the desired syn-diastereomer pair (82%) and with considerable stereocontrol (d.r. = 13.6:1, e.r. = 3.6:1, 56% ee), providing BDQ in up to a 64% assay yield before purification steps toward the final API. This represents a considerable improvement in the BDQ yield compared to previously reported conditions and could be critical to further lowering the cost of this life-saving drug.

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.

Facile and Scalable Methodology for the Pyrrolo[2,1-f][1,2,4]triazine of Remdesivir.

Pyrrolo[2,1-f][1,2,4]triazine (1) is an important regulatory starting material in the production of the antiviral drug remdesivir. Compound 1 was produced through a newly developed synthetic methodology utilizing simple building blocks such as pyrrole, chloramine, and formamidine acetate by examining the mechanistic pathway for the process optimization exercise. Triazine 1 was obtained in 55% overall yield in a two-vessel-operated process. This work describes the safety of the process, impurity profiles and control, and efforts toward the scale-up of triazine for the preparation of kilogram quantity.

Toward a Practical, Nonenzymatic Process for Investigational COVID-19 Antiviral Molnupiravir from Cytidine: Supply-Centered Synthesis.

A scalable four-step synthesis of molnupiravir from cytidine is described herein. The attractiveness of this approach is its fully chemical nature involving inexpensive reagents and more environmentally friendly solvents such as water, isopropanol, acetonitrile, and acetone. Isolation and purification procedures are improved in comparison to our earlier study as all intermediates can be isolated via recrystallization. The key steps in the synthesis, namely, ester formation, hydroxyamination, and deprotection were carried out on a multigram scale to afford molnupiravir in 36-41% yield with an average purity of 98 wt % by qNMR and 99 area% by HPLC.

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.

Analysis and Isolation of Potential Artemisinin Precursors from Waste Streams of Artemisia Annua Extraction.

High-performance liquid chromatography, liquid chromatography-mass spectrometry, and gas chromatography-mass spectrometry methods were developed to analyze the process waste streams of Artemisia Annua extraction. Results from these methods suggested that the final waste from the extraction process could serve as a source of dihydroartemisinic acid (DHAA) that could be converted to additional artemisinin. Two additional impurities were isolated and identified in the waste material as well as in A. annua leaf samples. That these impurities also appear as side-products in chemical transformations of DHAA to artemisinin supports the conclusion that the in vivo transformation proceeds as nonspecific oxidations. These impurities do not appear in isolated artemisinin. A simple, high-yielding procedure for recovery of DHAA from the primary waste stream was developed.

Practical Synthesis of the Bicyclic Darunavir Side Chain: (3R,3aS,6aR)-Hexahydrofuro[2,3-b]furan-3-ol from Monopotassium Isocitrate.

A practical synthesis of (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-ol-a key intermediate in the synthesis of darunavir-from monopotassium isocitrate is described. The isocitric acid salt, obtained from a high-yielding fermentation fed by sunflower oil, was converted in several steps to a tertiary amide. This amide, along with the compound's ester functionalities, was reduced with lithium aluminum hydride to give, on acidic workup, a transient aminal-triol. This was converted in situ to the title compound, the bicyclic acetal furofuranol side chain of darunavir, a protease inhibitor used in treatment of HIV/AIDS. Key to the success of this process was identifying an optimal amide that allowed for complete reaction and successful product isolation. N-Methyl aniline amide was identified as the most suitable substrate for the reduction and the subsequent cyclization to the desired product. Thus, the side chain is produced in 55% overall yield from monopotassium isocitrate.

Identification of impurities in artemisinin, their behavior in high performance liquid chromatography and implications for the quality of derived anti-malarial drugs.

Previous work [1] on the HPLC analysis of artemisinin tentatively identified the two impurities present above trace levels. This identification was based on LC-MS results and NMR of impurities isolated from artemisinin. In this work the impurities have been synthesized allowing verification of their identity by LC-MS. It is found that the previously suggested elution order is incorrect. A determination of relative response factors strongly impacts suggested limits on impurity levels and explains the erroneous peak assignment. The fates of the identified impurities are explored in the transformation of artemisinin to its derivative active pharmaceutical ingredients. A survey of a wide variety of artemisinin samples isolated from different geographical regions, different growing seasons, different plant backgrounds and using different extraction and purification approaches showed that artemisinin has sufficient purity for its intended use as a raw material for anti-malarial drug products.

High performance liquid chromatographic evaluation of artemisinin, raw material in the synthesis of artesunate and artemether.

A barrier to the development of artemisinin derivative based combination treatment of malaria is the lack of defined specifications and purity test methods for the raw material artemisinin. An HPLC method previously published in the International Pharmacopoeia to evaluate purity of artemisinin as an active pharmaceutical ingredient is adapted for use. Excellent method precision and linearity are demonstrated along with observations of robustness. In support of the development of specifications major impurities are identified using high resolution HPLC-MS, isolation via preparative HPLC followed by NMR. The identified impurities differ from those previously claimed.

Use of elevated flow rates in preparative subcritical fluid chromatography.

The benefits of using high flow rates in preparative subcritical fluid chromatography are explored. It is demonstrated that chromatograms loaded to onset of peak coalescence do not deteriorate as flow increases. This allows separation of material in very short time periods leading to dramatically increased production rates. A key factor to accessing elevated flows is the use of shorter columns and the resulting decrease in pressure drop.

The effect of acidic and basic additives on the enantioseparation of basic drugs using polysaccharide-based chiral stationary phases.

The enantioseparation of nine commercially available basic drugs was achieved on polysaccharide-based chiral stationary phases with the acidic additive ethanesulfonic acid and the basic additive butylamine. Seven different commercially available CSPs were used for the study (AD, AS, OD, OJ, OG, OB, and OC). Mobile phase additives have been proven to be essential in obtaining satisfactory enantio-resolution in terms of both efficiency and selectivity. Significantly improved selectivities were obtained for the basic probe drugs with the acidic additive, ethanesulfonic acid, rather than the basic additive, butylamine. This is best seen with Chiralpak AS CSP. No enantioseparation for the nine drugs was observed when butylamine was used as an additive; however, satisfactory enantioseparation for the nine drugs was achieved using ethanesulfonic acid. Higher column efficiencies were observed with the acidic additive, especially when isopropanol was used as a modifier. Higher sensitivity was also achieved with ethanesulfonic acid because of the significantly lower background at the UV detection wavelength. The acidic additive was demonstrated to be superior to the basic additive for the enantioseparation of basic drugs using seven different polysaccharide-based CSPs. These results are counterintuitive to the common "rule of thumb" in enantioseparation that states acidic additives work best for acidic analytes and basic additives work best for basic analytes. The beneficial effects of acidic additive in enantioseparations observed in this study could significantly improve the applicability of polysaccharide-based CSPs for the enantioseparation of basic analytes.

Chiral separations on polysaccharide stationary phases using polar organic mobile phases.

About 30% of a chemically diverse set of compounds were found to separate on four polysaccharide chiral stationary phases using polar organic mobile phases. No structural features appeared to correlate to successful separations. Titrations between normal and polar organic mobile phases suggested that separation mechanisms do not differ between these mobile phases. Attempts made to control retention met with varying degrees of success. Addition of hexane to alcohols had minor effects on retention although this was occasionally beneficial. Addition of water to alcohols increased retention. Addition of water to acetonitrile decreased retention. Addition of alcohol to acetonitrile also proved beneficial to the separation of some compounds. Loading studies performed to mimic preparative separations indicated that the benefits of polar organic mobile phases are largely due to increased solubility.

Chiral separation of amines by high-performance liquid chromatography using polysaccharide stationary phases and acidic additives.

A dramatic and beneficial effect of ethanesulfonic acid (ESA) on the chiral HPLC separation of basic compounds was found. Using a single chiral column and a starting mobile phase, more than half of a diverse set of amines was baseline separated. Changing alcohol content and alcohol type increased the success rate. Methanesulfonic acid (MSA) proved even more successful. The mechanism of this unexpected finding appears to be a combination of ion-pair salt formation in the mobile phase and increased binding with the chiral stationary phase (CSP) arising from a localized decrease in pH.

Chiral separation of amines in subcritical fluid chromatography using polysaccharide stationary phases and acidic additives.

The chiral separation of basic compounds by subcritical fluid chromatography (SFC) is often unsuccessful, due possibly to multiple interactions of the analyte with the mobile and stationary phase. Incorporation of a strong acid, ethanesulfonic acid (ESA), into the sample diluent and mobile phase modifier gives a dramatic improvement in these separations. Screening with ethanol containing 0.1% ESA on CHIRALPAK AD-H gave separation of 36 of 45 basic compounds previously not separated in SFC. The mechanism appears to involve the separation of an intact salt pair formed between the basic compound and ESA. Other modifiers, other acids and one additional stationary phase were examined and found to yield additional separations.

Origin of enhanced chiral selectivity by acidic additives for a polysaccharide-based stationary phase.

The effects of ethanesulfonic acid (ESA) and n-butylamine as additives were studied for a wide variety of chiral compounds using the polysaccharide chiral stationary phase (CSP), Chiralpak AD. The mobile phase consisted of hexane-ethanol (90:10, v/v). The additives typically had small effects, with one exception: the acidic additive had an enormous effect on the chiral selectivity of amino acid esters. The improved chiral selectivity was largely due to the longer retention of the later eluting enantiomer. Retention behavior of amines indicated that the higher selectivity for amino acid esters owes to increased hydrogen-bonding donation by the amine group of the analyte. Computation establishes the feasibility of a planar complex between the analyte and the cliral stationary phase, involving a pair of complementary hydrogen-bonding groups on each species, enabled by protonation of the analyte. Retention behaviors for a range of structures point to steric hindrance as the third interaction to comprise the requisite three interactions in chiral recognition.