HPLC Purification Research Articles

Stereoselective synthesis of 2-deoxy-2-disubstituted ribonolactones through a TiCl4-mediated Evans-Aldol reaction

A convenient and novel route for the stereoselective synthesis of 3,5-Di-O-benzoyl-2-deoxy-2-chloro-2-C-methyl-ᴅ-ribono-γ-lactone 5a and its analogues is developed by an Evans-Aldol reaction in the presence of TiCl4. Starting from 2-chloropropionyl chloride, 5a is efficiently and regioselectively produced on a kilogram scale in an overall yield of 32% with an HPLC purity of 98% by area. In addition, this approach allowed an efficient synthesis of 3,5-Di-O-benzoyl-2-deoxy-2-fluoro-2-C-methyl-ᴅ-ribono-γ-lactone 5b in a total 20% yield.

Development and Process Intensification of an Efficient Flow–Cascade Reaction Sequence in the Synthesis of Afizagabar

Aromatic nitration and catalytic hydrogenation are among the most dangerous reactions in the chemical industry. The traditional, batchwise pilot plant manufacturing process of a key intermediate of our drug candidate afizagabar (S44819) involved these kinds of transformations (besides a Dakin–West-type reaction, a ring closure, and a keto reduction step). To mitigate some of the hazards associated with this sequence, a flow chemical approach was developed. First, a flow–cascade process was elaborated, which furnished the product with a throughput of 1.52 g/h with an HPLC purity of 95.6%. The bottleneck of the procedure in terms of output was the heterogeneous catalytic hydrogenation; therefore, our subsequent process intensification efforts primarily concentrated on this step. Finally, application of higher concentrations and an upscaled hydrogenation reactor combined with the corresponding adjustment of parameters of further reaction steps resulted in an efficient process with an effective product yield of 11.95 g/h and an increased HPLC purity (97.1%). The 4-step uninterrupted process described here is based on a newly developed heterogeneous flow reactor system and a custom-made liquid–liquid extractor, providing an instructive case study on handling hazardous processes in a safe and efficient way.

Environmentally Sensible Organocatalytic Fmoc/t-Bu Solid-Phase Peptide Synthesis.

Despite numerous reports on catalytic amide bond formation (ABF), these methods have thus far had a minimal impact on the universal fluorenylmethoxycarbonyl (Fmoc)/t-Bu solid-phase peptide synthesis (SPPS) methodology. We now report a proof-of-principle Fmoc/t-Bu SPPS in which both couplings and Fmoc deprotections were catalyzed by readily available reagents in an inexpensive green solvent. Couplings were carried out with >99% stereoselectivity, employing 1.1 equiv of Fmoc amino acids (AAs), using diisopropylcarbodiimide (DIC) as a coupling agent and 1-hydroxy-1,2,3-triazole-5-carboxylic acid ethyl ester (HOCt) (TON ∼ 30) as a catalyst, while Fmoc deprotections were carried out using 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) (TON ∼ 7), facilitating synthesis of a model pentapeptide in 95% HPLC purity while also enabling minimization of solvent washing.

Evaluation of an 131I-labeled HER2-specific single domain antibody fragment for the radiopharmaceutical therapy of HER2-expressing cancers

Radiopharmaceutical therapy (RPT) is an attractive strategy for treatment of disseminated cancers including those overexpressing the HER2 receptor including breast, ovarian and gastroesophageal carcinomas. Single-domain antibody fragments (sdAbs) exemplified by the HER2-targeted VHH_1028 evaluated herein are attractive for RPT because they rapidly accumulate in tumor and clear faster from normal tissues than intact antibodies. In this study, VHH_1028 was labeled using the residualizing prosthetic agent N-succinimidyl 3-guanidinomethyl 5-[131I]iodobenzoate (iso-[131I]SGMIB) and its tissue distribution evaluated in the HER2-expressing SKOV-3 ovarian and BT474 breast carcinoma xenograft models. In head-to-head comparisons to [131I]SGMIB-2Rs15d, a HER2-targeted radiopharmaceutical currently under clinical investigation, iso-[131I]SGMIB-VHH_1028 exhibited significantly higher tumor uptake and significantly lower kidney accumulation. The results demonstrated 2.9 and 6.3 times more favorable tumor-to-kidney radiation dose ratios in the SKOV-3 and BT474 xenograft models, respectively. Iso-[131I]SGMIB-VHH_1028 was prepared using a solid-phase extraction method for purification of the prosthetic agent intermediate Boc2-iso-[131I]SGMIB that reproducibly scaled to therapeutic-level doses and obviated the need for its HPLC purification. Single-dose (SKOV-3) and multiple-dose (BT474) treatment regimens demonstrated that iso-[131I]SGMIB-VHH_1028 was well tolerated and provided significant tumor growth delay and survival prolongation. This study suggests that iso-[131I]SGMIB-VHH_1028 is a promising candidate for RPT of HER2-expressing cancers and further development is warranted.

Macrolactin Metabolite Production by Bacillus sp. ZJ318 Isolated from Marine Sediment.

A total of 172 microbial strains were screened and isolated from Arctic Ocean marine sediments at a depth of 42 ~ 3,763m. A microorganism with strong antibacterial activity against Staphylococcus aureus was identified as Bacillus sp. ZJ318 according to the results of 16S rDNA sequencing and phylogenetic tree analyses. Bioactivity-guided isolation of the new/novel metabolite in the ethyl acetate (EA) extract obtained from the fermentation broth of this strain was followed by chromatographic fractionation and subsequent HPLC purification, leading to the isolation of one known macrolactin. The chemical structure of the macrolactin, which indicated macrolactin J isolation from marine microorganisms for the first time, was assigned based on a high-resolution electrospray ionization mass spectrometer system (HR-EMI-MS), nuclear magnetic resonance (NMR) spectral analyses, and a literature review. To improve macrolactin J production, the corresponding effects of nitrogen sources were investigated, and (NH4)2SO4 was determined to produce the best effect. In addition, the optimal culture conditions were determined by an orthogonal experiment. Under these conditions, the yield of macrolactin J was increased to 2.41mg/L, which was 2.2 times the original yield. This work lays a foundation for follow-up mechanistic and application research on macrolactin J.

PHENYX – A flexible pipetting-based platform for automated microvolume radiochemistry

Recent droplet-based radiochemistry platforms have enabled reactions in < 10 µL volumes, providing many advantages including minimal reagent consumption, fast synthesis times, high yields, high molar activity, and scalable production amount. However, in the open droplet format there can be at times unwanted evaporation, or loss of activity as volatile species. To address this limitation, we developed a pipette-based radiosynthesizer platform, in which microvolume reactions are performed in a small well that can be sealed using a removable heated lid. The overall system comprises a pipettor mounted on a XYZ motion gantry to access a pipette tip rack, specialized pipette attachments, and a disposable cassette. All synthesis operations are performed by the pipette, including trap and release of [18F]fluoride from the QMA cartridge, liquid transfers among reservoirs and the reactor, and installation or removal of the heated lid, enabling a wide range of radiosynthesis protocols to be implemented. As a proof-of-concept demonstration, [18F]Fallypide was synthesized on the platform. Up to 2 mL of aqueous [18F]fluoride (2 – 540 MBq) was concentrated to ∼ 34 μL by trapping on a preconditioned micro-QMA cartridge (3 mg resin) and eluting with TBAHCO3 (0.35 μmol). The concentrated radioisotope was evaporated in the Reactor, and then 8 μL of precursor solution (0.62 μmol in MeCN/thexyl alcohol) was added and heated at 110 °C for 7 min. After collection and analytical-scale HPLC purification, the radiochemical yield was 71 ± 6% (n = 3), and the product exhibited high radiochemical purity (>99%) and molar activity of 290–670 GBq/μmol.

Engineering a Transaminase for the Efficient Synthesis of a Key Intermediate for Rimegepant

Transaminases have been increasingly utilized as efficient biocatalysts in the synthesis of pharmaceutical intermediates, but a major drawback is their poor substrate acceptance, especially the limitation for the synthesis of sterically hindered chiral amines. Herein we report the engineering of a transaminase that can convert the ketone (6S,9R)-6-(2,3-difluorophenyl)-9-hydroxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-5-one to (5S,6S,9R)-5-amino-6-(2,3-difluorophenyl)-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-ol, a key intermediate for the synthesis of rimegepant, a CGRP antagonist for the treatment of migraine. Starting from an enzyme backbone with no detectable activity toward the desired ketone, a rational design approach enabled us to produce an enzyme variant with detectable trace activity. Then, by following various evolution strategies, including iterative saturation mutagenesis focused on a key loop and random mutagenesis of the whole sequence, further improvement of the activity was achieved. The resultant variant showed 99.0% conversion and >99.5% de for the desired reaction at the gram scale as well as at the kilogram scale to afford the product (5S,6S,9R)-5-amino-6-(2,3-difluorophenyl)-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-ol in 80.2% yield with 99.9% HPLC purity, thus showcasing promising potential for industrial application.

Chemical modification of uridine modulates mRNA-mediated proinflammatory and antiviral response in primary human macrophages

In vitro transcribed (IVT)-mRNA has been accepted as a promising therapeutic modality. Advances in facile and rapid production technologies make IVT-mRNA an appealing alternative to protein- or virus-based medicines. Robust expression levels, lack of genotoxicity, and their manageable immunogenicity benefit its clinical applicability. We postulated that innate immune responses of therapeutically relevant human cells can be tailored or abrogated by combinations of 5′-end and internal IVT-mRNA modifications. Using primary human macrophages as targets, our data show the particular importance of uridine modifications for IVT-mRNA performance. Among five nucleotide modification schemes tested, 5-methoxy-uridine outperformed other modifications up to 4-fold increased transgene expression, triggering moderate proinflammatory and non-detectable antiviral responses. Macrophage responses against IVT-mRNAs exhibiting high immunogenicity (e.g., pseudouridine) could be minimized upon HPLC purification. Conversely, 5′-end modifications had only modest effects on mRNA expression and immune responses. Our results revealed how the uptake of chemically modified IVT-mRNA impacts human macrophages, responding with distinct patterns of innate immune responses concomitant with increased transient transgene expression. We anticipate our findings are instrumental to predictively address specific cell responses required for a wide range of therapeutic applications from eliciting controlled immunogenicity in mRNA vaccines to, e.g., completely abrogating cell activation in protein replacement therapies.

Industrial Cunninghamia lanceolata carbon supported FeO(OH) nanoparticles-catalyzed hydrogenation of nitroarenes

The development of green and efficient methods for hydrogenation of nitroarenes is still highly demanding in organic synthesis. Herein, we report an industrial Cunninghamia lanceolata carbon supported FeO(OH) nanoparticles process for the synthesis of aryl amines with good yields via hydrogenation of nitroarenes. Nine key anti-cancer drug intermediates were successfully achieved with protocol. And Osimertinib intermediate 4m can be smoothly synthesized at a 2.67 kg-scale with >99.5% HPLC purity. This protocol features cheap carbon source, highly catalytic activity, simple operation, kilogram-scalable and recyclable catalysts (eight times without observable losing activity).

Neuroinflammation in Low-Level PM2.5-Exposed Rats Illustrated by PET via an Improved Automated Produced [18F]FEPPA: A Feasibility Study.

Background [18F]FEPPA is a potent TSPO imaging agent that has been found to be a potential tracer for imaging neuroinflammation. In order to fulfill the demand of this tracer for preclinical and clinical studies, we have developed a one-pot automated synthesis with simplified HPLC purification of this tracer, which was then used for PET imaging of neuroinflammation in fine particulate matter- (PM2.5-) exposed rats. Results Using this automated synthesis method, the RCY of the [18F]FEPPA was 38 ± 4% (n = 17, EOB) in a synthesis time of 83 ± 8 min from EOB. The radiochemical purity and molar activities were greater than 99% and 209 ± 138 GBq/μmol (EOS, n = 15), respectively. The quality of the [18F]FEPPA synthesized by this method met the U.S. Pharmacopoeia (USP) criteria. The stability test showed that the [18F]FEPPA was stable at 21 ± 2°C for up to 4 hr after the end of synthesis (EOS). Moreover, microPET imaging showed that increased tracer activity of [18F]FEPPA in the brain of PM2.5-exposed rats (n = 6) were higher than that of normal controls (n = 6) and regional-specific. Conclusions Using the improved semipreparative HPLC purification, [18F]FEPPA has been produced in high quantity, high quality, and high reproducibility and, for the first time, used for PET imaging the effects of PM2.5 in the rat brain. It is ready to be used for imaging inflammation in various clinical or preclinical studies, especially for nearby PET centers without cyclotrons.

ROUTE DEVELOPMENT, ANTIVIRAL STUDIES, FIELD EVALUATION AND TOXICITY OF AN ANTIVIRAL PLANT PROTECTANT NK0238

<List> <ListItem><ItemContent> • Developed a two-step synthetic route to anti-plant-virus candidate NK0238. </ItemContent></ListItem> <ListItem><ItemContent> • NK0238 exhibited a broad antivirus spectrum in greenhouse. </ItemContent></ListItem> <ListItem><ItemContent> • NK0238 showed comparable antivirus activities as controls in field trials. </ItemContent></ListItem> <ListItem><ItemContent> • NK0238 was safe to birds, fish, bees and silkworms. </ItemContent></ListItem> <ListItem><ItemContent> • NK0238 has a very good prospect in commercial development. </ItemContent></ListItem></List> It has previously been shown that tryptophan, the biosynthesis precursor of Peganum harmala alkaloids, and its derivatives have anti-TMV activity both in vitro and in vivo. Further exploration of this led to the identification of NK0238 as a highly effective agent for the prevention and control of diseases caused by plant viruses, but the existing routes are unsuitable for its large-scale synthesis. This study optimized a route for two-step synthesis of this virucide candidate via reaction of l-tryptophan with triphosgene to produce l-tryptophan-N-carboxylic anhydride, which then reacts with n-octylamine to give NK0238 at up to 94% yield and nearly 97% HPLC purity. In addition, the route was used for the preparation of NK0238 on a>40 g scale permitting further assessment of its antivirus activity in the greenhouse and field experiments, and toxicity tests. NK0238 exhibited useful antiviral activities against a variety of viruses both in greenhouse and field experiments. The toxicity tests showed that NK0238 was not acutely toxic to birds, fish, honey bees and silkworms. The optimized route provides a solid foundation for its large-scale synthesis and subsequent efficacy and toxicity studies, its excellent activity and safety make NK0238 a promising drug candidate for further development.

First-Generation Asymmetric Synthesis of the Selective Estrogen Receptor Degrader GDC-9545 (Giredestrant) Featuring a Highly Efficient Pictet–Spengler Reaction and a C–N Coupling Reaction

An asymmetric synthesis of the selective estrogen receptor degrader GDC-9545 (1) is described. The synthesis features a Friedel–Crafts indole functionalization and a strain-release aminoazetidine formation to construct the two key starting materials 2 and 4, respectively, a diastereoselective Pictet–Spengler reaction (98% yield, 95:5 dr) to assemble the tetrahydrocarboline core, and a highly efficient Pd-catalyzed C–N coupling (90% yield) using [t-BuBrettPhos Pd(allyl)]OTf as the catalyst and DBU as the base to furnish the final C–N bond. This expedient route produces GDC-9545·tartrate active pharmaceutical ingredient in a longest linear sequence of six steps in 37% overall yield with 99.0 area % HPLC purity without chromatographic purification.

Extraction, purification and anti-TMV effects of α (β)-2,7,11-cembratriene-4,6-diol from tobacco leaves

Acetone ethanol extracts from tobacco leaves have antiviral activity against tobacco mosaic virus (TMV), but the antiviral effects of their specialized metabolites have not been systematically studied yet, especially the underlying mechanism is still unexplored. The tobacco cembranoids α(β)-2,7,11-cembratriene-4,6-diol (α(β)-CBD) were extracted and purified with an effective protocol including innovatively added 5% phosphoric acid for elution, one time silica gel chromatographic column separation and impurity removal and further HPLC purification. The in vivo anti-TMV activities showed that α(β)-CBD have higher protective effects of 73.2% and 71.6%, at 75.0 μM, respectively, than control agent Ningnanmycin (53.1%). Notably, The results of ELISA and TMV-GFP fluorescent optical imaging assay indicated a obviously reduced viral protein and weaker GFP fluorescence signal and smaller infection area, which confirmed their antiviral effects at protein level. Furthermore, the enhanced production of SA and JA and the significantly increased transcription of JA (COI1 and PDF1.2) and SA (PR1, NPR1 and EDS1) signaling pathway genes in treated plants further conformed that exogenously applied α(β)-CBD can effectively elicit the tobacco plant immunity against TMV. The α(β)-CBD mainly stimulates disease resistance of tobacco plants to resist TMV and it can be used as bioagents to control TMV in the future.

Preparative Synthesis of an R P-Guanosine-3',5'-Cyclic Phosphorothioate Analogue, a Drug Candidate for the Treatment of Retinal Degenerations.

Cyclic guanosine monophosphorothioate analogue 1a is currently showing potential as a drug for the treatment of inherited retinal neurodegenerations. To support ongoing preclinical and clinical work, we have developed a diastereoselective synthesis via cyclization and sulfurization of the nucleoside 5′-H-phosphonate monoester, which affords the desired RP-3′,5′-cyclic phosphorothioate in 9:1 ratio to the undesired SP-diastereomer. This route was made viable as a result of the silyl protection sequence used, which achieved >80% selectivity for 2′,5′-hydroxyls over 3′,5′-hydroxyls. Finally, the chromatography-free process allowed for a scale-up, as intermediates and the final product were isolated by crystallization to give 125 g of 1a (13.8% total yield) with over 99.9% HPLC purity.

Stereoselective synthesis of homochiral paracyclophanylindenofuranylimidazo[3.3.3]propellanes

The reaction between N-paracyclophanyl-substituted hydrazine-carbothioamides and dicyanomethylene-1,3-indanedione (CNIND) furnished planar and central chiral substituted (epiminomethanoimino)-indenofuranyl-1,4-(1,4-dibenzenocyclohexaphane)-1,2-carboxamides as a type of paracyclophanylfuroimidazo[3.3.3]propellanes in 35–81% yields as a major product as single diastereomers, and substituted spiro[indene-2,4′-indenopyran]-3′-carbonitrile (9–12% yields) as a minor product. To prepare the homochiral paracyclophanylpropellane, oxidation of scalemic formylparacyclophane afforded the corresponding acid. Afterward, the acid chloride formation of [2.2]paracyclophane was obtained by chlorination of the corresponding acid. By repeating the previous procedure and utilizing a final HPLC purification, we succeeded in obtaining the target homochiral propellane. Various spectroscopic techniques such as mass spectrometry, 1H NMR, 13C NMR, and X-ray analysis have been used for structure determination and confirmation of the stereochemistry of the isolated products.Graphic abstract

Screening of Acetyl Donors and the Robust Enzymatic Synthesis of Acetyl-CoA by 10-Deacetylbaccatin III-10-β-O-acetyltransferase

Acetyl-CoA is the precursor of many bio-manufacturing products and is also the hub of the cellular metabolism of energy and substances. However, acetyl-CoA is not a bulk commodity and its application is hindered due to its high cost and low yield. In this study, we screened acetyl donor candidates and utilized 10-deacetylbaccatin III-10-β-O-acetyltransferase (DBAT) in the synthesis of acetyl-CoA with CoASH as the acetyl acceptor. Among the tested candidates, acetylsalicylic acid methyl ester was identified to be the best acetyl donor, followed by acetyl-trans-resveratrol, acetylsalicylic acid ethyl ester, acetylsalicylsalicylic acid, and 4-acetoxyacetanilide. The enzymatic reaction conditions were optimized and the maximum yield of acetyl-CoA reached 14.82 mg/mL, which is the highest yield among all reported approaches to date. Meanwhile, 4.22 mg/mL of the by-product salicylic acid methyl ester, which is another industrial material, was produced. Additionally, a preliminary purification process for acetyl-CoA was established, in which 40 mg acetyl-CoA (HPLC purity &gt; 98%) was acquired from the finished 20 mL reaction system (feeding 46 mg CoASH and 34 mg ASME) with a recovery rate of 86%. Our study lays the foundation for the large-scale production of acetyl-CoA by an enzymatic approach and will promote its application in different fields.

Ethylenediamine derivatives efficiently react with oxidized RNA 3' ends providing access to mono and dually labelled RNA probes for enzymatic assays and in vivo translation.

Development of RNA-based technologies relies on the ability to detect, manipulate, and modify RNA. Efficient, selective and scalable covalent modification of long RNA molecules remains a challenge. We report a chemical method for modification of RNA 3′-end based on previously unrecognized superior reactivity of N-substituted ethylenediamines in reductive amination of periodate-oxidized RNA. Using this method, we obtained fluorescently labelled or biotinylated RNAs varying in length (from 3 to 2000 nt) and carrying different 5′ ends (including m7G cap) in high yields (70–100% by HPLC). The method is scalable (up to sub-milligrams of mRNA) and combined with label-facilitated HPLC purification yields highly homogeneous products. The combination of 3′-end labelling with 5′-end labelling by strain-promoted azide-alkyne cycloaddition (SPAAC) afforded a one-pot protocol for site-specific RNA bifunctionalization, providing access to two-colour fluorescent RNA probes. These probes exhibited fluorescence resonance energy transfer (FRET), which enabled real-time monitoring of several RNA hydrolase activities (RNase A, RNase T1, RNase R, Dcp1/2, and RNase H). Dually labelled mRNAs were efficiently translated in cultured cells and in zebrafish embryos, which combined with their detectability by fluorescent methods and scalability of the synthesis, opens new avenues for the investigation of mRNA metabolism and the fate of mRNA-based therapeutics.

Synthesis, Radiolabeling, and Biological Evaluation of 68Ga-Mucin1 and Its Folate Hybrid Peptide Conjugates for the Diagnosis of Ovarian Cancer

Because of our interest in developing new hybrid peptide radioconjugates with suitable biochemical properties for multiple-receptors targeting properties that are overexpressed on many human cancers especially ovarian cancer, we have synthesized 68Ga-NODAGA-MUC1 and 68Ga-NODAGA-MUC1-FA hybrid peptide conjugates using a straightforward and one-step simple reaction. Radiochemical yields were found to be higher than 95% (decay corrected), with a total synthesis time of less than 20 min. Radiochemical purities were always higher than 95% without HPLC purification. In vitro studies on KB cancer cells showed that substantial amounts of the radioconjugates were associated with cell fractions and held great affinities and specificities toward the KB cell line. In vivo characterization in normal female Balb/c mice revealed rapid blood clearance of these radioconjugates with excretion predominantly by the urinary system. Biodistribution studies in nude mice bearing human KB cell line xenografts demonstrated significant tumor uptake and favorable biodistribution profile for 68Ga-NODAGA-MUC1-FA hybrid peptide conjugate compared to the 68Ga-NODAGA-MUC1 peptide monomeric counterpart. The uptake in the tumors was blocked by the excess injection of hybrid peptide, suggesting a receptor-mediated process. These results demonstrate that 68Ga-NODAGA-MUC1-FA hybrid peptide conjugate may be useful as a molecular probe for early detection and staging of folate and MUC1 receptor-positive cancers such as ovarian cancer and their metastasis as well as monitoring tumor response to treatment.

A Comprehensive Review of Vaccines against Covid-19

A Comprehensive Review of Vaccines against Covid-19

The Non-Anhydrous, Minimally Basic Synthesis of the Dopamine D2 Agonist [18F]MCL-524.

The dopamine D2 agonist MCL-524 is selective for the D2 receptor in the high-affinity state (D2high), and, therefore, the PET analogue, [18F]MCL-524, may facilitate the elucidation of the role of D2high in disorders such as schizophrenia. However, the previously reported synthesis of [18F]MCL-524 proved difficult to replicate and was lacking experimental details. We therefore developed a new synthesis of [18F]MCL-524 using a "non-anhydrous, minimally basic" (NAMB) approach. In this method, [18F]F- is eluted from a small (10-12 mg) trap-and-release column with tetraethylammonium tosylate (2.37 mg) in 7:3 MeCN:H2O (0.1 mL), rather than the basic carbonate or bicarbonate solution that is most often used for [18F]F- recovery. The tosylated precursor (1 mg) in 0.9 mL anhydrous acetonitrile was added directly to the eluate, without azeotropic drying, and the solution was heated (150 °C/15 min). The catechol was then deprotected with the Lewis acid In(OTf)3 (10 equiv.; 150 °C/20 min). In contrast to deprotection with protic acids, Lewis-acid-based deprotection facilitated the efficient removal of byproducts by HPLC and eliminated the need for SPE extraction prior to HPLC purification. Using the NAMB approach, [18F]MCL-524 was obtained in 5-9% RCY (decay-corrected, n = 3), confirming the utility of this improved method for the multistep synthesis of [18F]MCL-524 and suggesting that it may applicable to the synthesis of other 18F-labeled radiotracers.