Engineering Hydroxylase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of a Key Intermediate to Belzutifan.

Biocatalytic oxidations are an emerging technology for selective C-H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non-heme iron and α-ketoglutarate-dependent dioxygenase for the direct stereo- and regio-selective hydroxylation of a non-native fluoroindanone en route to the oncology treatment belzutifan, replacing a five-step chemical synthesis with a direct enantioselective hydroxylation. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high-yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan.

Highly Diastereoselective Synthesis of a HCV NS5B Nucleoside Polymerase Inhibitor.

An asymmetric synthesis of HCV NS5B nucleoside polymerase inhibitor (1) is described. This novel route features several remarkably diastereoselective and high-yielding transformations, including construction of the all-carbon quaternary stereogenic center at C-2 via a thermodynamic aldol reaction. A subsequent glycosylation reaction with activated uracil via C-1 phosphate and installation of the cyclic phosphate group using an achiral phosphorus(III) reagent followed by oxidation provides 1.

Synthesis of the γ-Secretase Modulator MK-8428.

The synthesis of the γ-secretase modulator MK-8428 (1) is described. The synthesis is highlighted by an enzyme-catalyzed reaction to access 3,4,5-trifluoro-(S)-phenylglycine, a 1-pot activation/displacement/deprotection sequence to introduce the aminooxy functionality and a dehydrative intramolecular cyclization under mild conditions to form the oxadiazine heterocycle of 1. In situ reaction monitoring was employed to understand the deleterious role of water during the formation of a methanesulfonate ester in the 1-pot activation/displacement/deprotection sequence.

Stereoselective addition of 2-phenyloxazol-4-yl trifluoromethanesulfonate to N-sulfinyl imines: application to the synthesis of the HCV protease inhibitor boceprevir.

The stereoselective addition of 2-phenyloxazol-4-yl trifluoromethanesulfonate to N-sulfinylimines is described. Vinyl anions derived from enol triflate 2 undergo 1,2-addition with a variety of aldimines to afford the corresponding secondary sulfonamides as single diastereomers. The absolute stereochemistry was confirmed by X-ray crystallography which provides support that the reaction proceeds through an open, nonchelate transition state. This methodology has been applied to the synthesis of the ketoamide fragment of the protease inhibitor boceprevir.

Engineered Ribosyl-1-Kinase Enables Concise Synthesis of Molnupiravir, an Antiviral for COVID-19.

Molnupiravir (MK-4482) is an investigational antiviral agent that is under development for the treatment of COVID-19. Given the potential high demand and urgency for this compound, it was critical to develop a short and sustainable synthesis from simple raw materials that would minimize the time needed to manufacture and supply molnupiravir. The route reported here is enabled through the invention of a novel biocatalytic cascade featuring an engineered ribosyl-1-kinase and uridine phosphorylase. These engineered enzymes were deployed with a pyruvate-oxidase-enabled phosphate recycling strategy. Compared to the initial route, this synthesis of molnupiravir is 70% shorter and approximately 7-fold higher yielding. Looking forward, the biocatalytic approach to molnupiravir outlined here is anticipated to have broad applications for streamlining the synthesis of nucleosides in general.

Development of a Robust Manufacturing Route for Molnupiravir, an Antiviral for the Treatment of COVID-19.

Herein is described the development of a large-scale manufacturing process for molnupiravir, an orally dosed antiviral that was recently demonstrated to be efficacious for the treatment of patients with COVID-19. The yield, robustness, and efficiency of each of the five steps were improved, ultimately culminating in a 1.6-fold improvement in overall yield and a dramatic increase in the overall throughput compared to the baseline process.

Synthesis of the N-(tert-butyloxycarbonyl)-O-triisopropylsilyl-D-pyrrolosamine Glycal of Lomaiviticin A & B via Epimerization of L-Threonine.

An efficient synthesis of the N-(tert-butyloxycarbonyl)-O-triisopropylsilyl-D-pyrrolosamine glycal of lomaiviticin A (1) and lomaiviticin B (2) is described. The synthesis is highlighted by the epimerization of the L-threonine-derived oxazolidine 10 to oxazolidine 11. This key epimerization reaction, which serves to establish the correct relative configuration of the carbohydrate unit, was made possible only after conformational analysis indicated that substituted oxazolidines may adopt conformations that preclude enolization.

Palladium-Catalyzed Enantioselective Synthesis of Cyclic Sulfamidates and Application to a Synthesis of Verubecestat.

An enantioselective arylation reaction catalyzed by palladium complexed with substituted phosphinooxazoline (PHOX) ligands is described. Aza-quaternary stereocenters are readily accessible through the arylation reaction between cyclic iminosulfates and a wide variety of arylboronic acids, including electron-poor and ortho-substituted arylboronic acids. This reaction was applied to the preparation of verubecestat, which is currently undergoing clinical evaluation for the treatment of Alzheimer's disease.

A Next Generation Synthesis of BACE1 Inhibitor Verubecestat (MK-8931).

The development of a commercial manufacturing route to verubecestat (MK-8931) is described, highlights of which include the application of a continuous processing step to outcompete fast proton transfer in a Mannich-type ketimine addition, a copper-catalyzed amidation reaction, and an optimized guanidinylation procedure to form the key iminothiadiazine dioxide core.

Macrocyclic glycopeptide chiral selectors bonded to core-shell particles enables enantiopurity analysis of the entire verubecestat synthetic route.

Verubecestat is an inhibitor of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) being evaluated in clinical trials for the treatment of Alzheimer's disease. Synthetic route development involves diastereoselective transformations with a need for enantiomeric excess (ee) determination of each intermediate and final active pharmaceutical ingredient (API). The analytical technical package of validated methods relies on enantioselective SFC and RPLC separations using multiple 3 and 5 µm coated polysaccharide-based chiral stationary phases (CSPs) and mobile phases combinations. Evaluation of recently developed chiral columns revealed a single chiral selector (Teicoplanin) bonded to 2.7 µm core-shell particles using H3PO4 in H2O/ACN and triethylammonium acetate: methanol based eluents at different isocratic compositions allowed good enatioseparation of all verubecestat intermediates. EE determination of verubecestat is easily performed on NicoShell, another macrocyclic glycopeptide chiral selector bonded to 2.7 µm superficially porous particles. This approach enables fast and reliable enantiopurity analysis of the entire verubecestat synthetic route using only two chiral columns and mobile phases on a conventional HPLC system, simplifying technical package preparation, method validation and transfer to manufacturing facilities.

Prostate brachytherapy postimplant dosimetry: a comparison of suture-embedded and loose seed implants.

PURPOSE: To compare postimplant dosimetry and seed embolization rates for prostate brachytherapy implants using suture-embedded and loose seeds. METHODS AND MATERIALS: Dosimetric analysis of the whole prostate, prostate quadrants, rectum, and surrogate urethra was performed on 54 loose seed and 81 RAPIDStrand (RS) patients. Seed embolization rates were determined from chest radiographs. RESULTS: Whole prostate V100 and D90 did not differ significantly for the loose seed (V100 = 90.5%, D90 =153.2 Gy) and RS groups (V100 = 91.5%, D90 = 151.6 Gy) (p = 0.43 and 0.65, respectively), but V150, V200, and contiguous V200 were higher (p < or = 0.003) for the RS group (59.9%, 28.3%, and 23.2%, respectively) than the loose seed group (52.5%, 22.8%, and 16.1%, respectively). Extraprostatic measures (conformity index and external index) were also different at the p < 0.05 level. The embolization rate was 40% in the loose seed group and 14% in the RS group. CONCLUSIONS: The most significant difference between the two study groups was a decrease in the embolization rate. Although some statistically significant changes in postimplant dosimetry were observed, they were nevertheless small.

Learning-Based Multi-Label Segmentation of Transrectal Ultrasound Images for Prostate Brachytherapy.

Low-dose-rate prostate brachytherapy treatment takes place by implantation of small radioactive seeds in and sometimes adjacent to the prostate gland. A patient specific target anatomy for seed placement is usually determined by contouring a set of collected transrectal ultrasound images prior to implantation. Standard-of-care in prostate brachytherapy is to delineate the clinical target anatomy, which closely follows the real prostate boundary. Subsequently, the boundary is dilated with respect to the clinical guidelines to determine a planning target volume. Manual contouring of these two anatomical targets is a tedious task with relatively high observer variability. In this work, we aim to reduce the segmentation variability and planning time by proposing an efficient learning-based multi-label segmentation algorithm. We incorporate a sparse representation approach in our methodology to learn a dictionary of sparse joint elements consisting of images, and clinical and planning target volume segmentation. The generated dictionary inherently captures the relationships among elements, which also incorporates the institutional clinical guidelines. The proposed multi-label segmentation method is evaluated on a dataset of 590 brachytherapy treatment records by 5-fold cross validation. We show clinically acceptable instantaneous segmentation results for both target volumes.

Stereoselective synthesis of tetrahydropyran-4-ones from dioxinones catalyzed by scandium(III) triflate.

[reaction: see text] A scandium triflate catalyzed, diastereoselective cyclization between aldehydes and beta-hydroxy dioxinones has been discovered. This process capitalizes on the untapped nucleophilicity of the embedded enol ether within the dioxinone core. The bicyclic compounds from the resulting cyclization can be isolated, or alternatively, alkoxide nucleophiles can be directly added. This in situ addition fragments the dioxinone rings and delivers the 3-carboxy-substituted tetrahydropyran-4-ones in good yields with high levels of diastereoselectivity.

Radical trimodality therapy for patients with locally advanced bladder cancer: The British Columbia Cancer Agency experience.

PURPOSE: To assess the outcomes of patients with locally advanced bladder cancer (clinically T3b-T4 or N+and M0) who were referred to the British Columbia Cancer Agency and treated with radical trimodality therapy (RTMT). RTMT consists of transurethral resection of the tumor, followed by both chemotherapy and radiation. METHODS: Between 1997 and 2007, 380 patients with cT3b-cT4 or N+ M0 bladder cancer were referred to the British Columbia Cancer Agency. Of these patients, 50 (13%) were treated using RTMT (all with platin-based chemotherapy and median radiation dose of 60Gy). Patient and disease characteristics as well as treatment data were retrospectively recorded through a chart review. Study end points included overall survival (OS), bladder cancer-specific survival (BCSS), and local relapse-free survival (LRFS). RESULTS: Median follow-up period for surviving patients was 8.53 years. At 5 and 10 years, OS was 30% and 17%, BCSS was 31% and 27%, and LRFS was 60% and 50%, respectively. Complete local response on first cystoscopy following treatment was the only significant predictor of OS, BCSS, and LRFS on univariate analysis, and it was also a significant predictor for LRFS on multivariable analysis. CONCLUSIONS: RTMT is a reasonable alternative to radical cystectomy in patients with locally advanced disease who are either unfit for or unwilling to undergo cystectomy.

A multi-atlas-based segmentation framework for prostate brachytherapy.

Low-dose-rate brachytherapy is a radiation treatment method for localized prostate cancer. The standard of care for this treatment procedure is to acquire transrectal ultrasound images of the prostate in order to devise a plan to deliver sufficient radiation dose to the cancerous tissue. Brachytherapy planning involves delineation of contours in these images, which closely follow the prostate boundary, i.e., clinical target volume. This process is currently performed either manually or semi-automatically, which requires user interaction for landmark initialization. In this paper, we propose a multi-atlas fusion framework to automatically delineate the clinical target volume in ultrasound images. A dataset of a priori segmented ultrasound images, i.e., atlases, is registered to a target image. We introduce a pairwise atlas agreement factor that combines an image-similarity metric and similarity between a priori segmented contours. This factor is used in an atlas selection algorithm to prune the dataset before combining the atlas contours to produce a consensus segmentation. We evaluate the proposed segmentation approach on a set of 280 transrectal prostate volume studies. The proposed method produces segmentation results that are within the range of observer variability when compared to a semi-automatic segmentation technique that is routinely used in our cancer clinic.

Statistical Biomechanical Surface Registration: Application to MR-TRUS Fusion for Prostate Interventions.

A common challenge when performing surface-based registration of images is ensuring that the surfaces accurately represent consistent anatomical boundaries. Image segmentation may be difficult in some regions due to either poor contrast, low slice resolution, or tissue ambiguities. To address this, we present a novel non-rigid surface registration method designed to register two partial surfaces, capable of ignoring regions where the anatomical boundary is unclear. Our probabilistic approach incorporates prior geometric information in the form of a statistical shape model (SSM), and physical knowledge in the form of a finite element model (FEM). We validate results in the context of prostate interventions by registering pre-operative magnetic resonance imaging (MRI) to 3D transrectal ultrasound (TRUS). We show that both the geometric and physical priors significantly decrease net target registration error (TRE), leading to TREs of 2.35 ± 0.81 mm and 2.81 ± 0.66 mm when applied to full and partial surfaces, respectively. We investigate robustness in response to errors in segmentation, varying levels of missing data, and adjusting the tunable parameters. Results demonstrate that the proposed surface registration method is an efficient, robust, and effective solution for fusing data from multiple modalities.

Prostate brachytherapy training with simulated ultrasound and fluoroscopy images.

In this paper, a novel computer-based virtual training system for prostate brachytherapy is presented. This system incorporates, in a novel way, prior methodologies of ultrasound image synthesis and haptic transrectal ultrasound (TRUS) transducer interaction in a complete simulator that allows a trainee to maneuver the needle and the TRUS, to see the resulting patient-specific images and feel the interaction forces. The simulated TRUS images reflect the volumetric tissue deformation and comprise validated appearance models for the needle and implanted seeds. Rendered haptic forces use validated models for needle shaft flexure and friction, tip cutting, and deflection due to bevel. This paper also presents additional new features that make the simulator complete, in the sense that all aspects of the brachytherapy procedure as practiced at many cancer centers are simulated, including simulations of seed unloading, fluoroscopy imaging, and transversal/sagittal TRUS plane switching. For real-time rendering, methods for fast TRUS-needle-seed image formation are presented. In addition, the simulator computes real-time dosimetry, allowing a trainee to immediately see the consequence of planning changes. The simulation is also patient specific, as it allows the user to import the treatment plan for a patient together with the imaging data in order for a physician to practice an upcoming procedure or for a medical resident to train using typical implant scenarios or rarely encountered cases.

An automatic multi-atlas segmentation of the prostate in transrectal ultrasound images using pairwise atlas shape similarity.

Delineation of the prostate from transrectal ultrasound images is a necessary step in several computer-assisted clinical interventions, such as low dose rate brachytherapy. Current approaches to user segmentation require user intervention and therefore it is subject to user errors. It is desirable to have a fully automatic segmentation for improved segmentation consistency and speed. In this paper, we propose a multi-atlas fusion framework to automatically segment prostate transrectal ultrasound images. The framework initially registers a dataset of a priori segmented ultrasound images to a target image. Subsequently, it uses the pairwise similarity of registered prostate shapes, which is independent of the image-similarity metric optimized during the registration process, to prune the dataset prior to the fusion and consensus segmentation step. A leave-one-out cross-validation of the proposed framework on a dataset of 50 transrectal ultrasound volumes obtained from patients undergoing brachytherapy treatment shows that the proposed is clinically robust, accurate and reproducible.

Fusion of ultrasound B-mode and vibro-elastography images for automatic 3D segmentation of the prostate.

Prostate segmentation in B-mode images is a challenging task even when done manually by experts. In this paper we propose a 3D automatic prostate segmentation algorithm which makes use of information from both ultrasound B-mode and vibro-elastography data.We exploit the high contrast to noise ratio of vibro-elastography images of the prostate, in addition to the commonly used B-mode images, to implement a 2D Active Shape Model (ASM)-based segmentation algorithm on the midgland image. The prostate model is deformed by a combination of two measures: the gray level similarity and the continuity of the prostate edge in both image types. The automatically obtained mid-gland contour is then used to initialize a 3D segmentation algorithm which models the prostate as a tapered and warped ellipsoid. Vibro-elastography images are used in addition to ultrasound images to improve boundary detection.We report a Dice similarity coefficient of 0.87±0.07 and 0.87±0.08 comparing the 2D automatic contours with manual contours of two observers on 61 images. For 11 cases, a whole gland volume error of 10.2±2.2% and 13.5±4.1% and whole gland volume difference of -7.2±9.1% and -13.3±12.6% between 3D automatic and manual surfaces of two observers is obtained. This is the first validated work showing the fusion of B-mode and vibro-elastography data for automatic 3D segmentation of the prostate.