Small molecule APOL1 inhibitors as a precision medicine approach for APOL1-mediated kidney disease
Cell line generation and growth
Human embryonic kidney (HEK) cell lines containing a tetracycline (tet)-inducible expression system (T-REx™; Invitrogen, Carlsbad, CA) and pAAVS1-Puro-APOL1 G0, pAAVS1-Puro-APOL1 G1 or pAAVS1-Puro-APOL1 G2 were generated. The physiological haplotype sequence of APOL1 was used for all 3 APOL1 sequences with E150/I228/K255. Individual clones of each cell line were generated and clones G0 DC2.13, G1 DC3.25, and G2 DC4.44 were used in this study. Cell growth medium was Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Gaithersburg, MD; Catalog # 11960-077) supplemented with 10% (v/v) tet-free fetal bovine serum (FBS; Takara Bio USA, Mountain View, CA; Catalog # 631368), 2 mM L-glutamine (Gibco; Catalog # 25030-081), 5 µg/mL Blasticidin S HCl (Gibco; Catalog # A11139-03), 1 µg/mL puromycin dihydrochloride (Gibco; Catalog # A11138-03), and 100 U/mL penicillin-streptomycin (Gibco; Catalog # 15140-122).
Electrophysiology
HEK293 APOL1-inducible cells were induced with 15 ng/mL of tet for 16–20 h. Automated electrophysiology was performed by Qpatch48 (Sophion Biosciences). Cells were perfused with extracellular solution containing: 137 mM NMDG (Sigma Aldrich; Catalog # M2004), 5.4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM Glucose, 10 mM HEPES. The pH was adjusted to 7.2 with NaOH and adjusted to 300–305 mOsm with sucrose. The intracellular solution contained: 124 mM CsF, 2 mM MgCl2, 1 mM CaCl2, 11 mM EGTA, 10 mM HEPES, 1 mM ATP pH 7.2, 285–290 mOsm. Ouabain (100 μM) was added in the extracellular solution for blockade of Na+/K+ ATP-ase. NMDG replaced Na+ in the extracellular solution to isolate Cs+ efflux. We used Cs+ in the internal solution because we identified APOL1 channels to be permeable to Cs+. Cs+ was selected also to block endogenous voltage-gated K+ channels. Specifically, the reversal potential of the APOL1 ionic current in the presence of NaCl extracellularly and CsF intracellularly was near to 0 mV, supporting nonspecific monovalent cationic flow (Supplementary Fig. 2). Resistance of the QPatch plate chambers (holes) was 1.5–2.5 MΩ. Steady state inhibition of APOL1 current was measured using a ramp protocol in response to six consecutive compound concentrations (100 pM, 1 nM, 3 nM, 10 nM, 100 nM, 1 μM) with an exposure duration of 5 min to each concentration, 5 min after initial current recording (control). Each solution exchange of 5 mL volume was performed twice. The voltage protocol applied to the cells was as follows: Resting step: −80 mV for 100 ms; test pulse: −100 mV to 80 mV for 200 ms; resting step: −80 mV for 100 ms repeated at 10 s intervals, from a holding potential of −90 mV. Peak outward current was measured at the end of the 200 ms test pulse at +80 mV. Data acquisition and analysis was performed using the Sophion QPatch Assay Software (Sophion Biosciences). We continuously monitored the series resistance (MΩ) and the capacitance (pF). Cells were removed from compound analysis if the series resistance exceeded 15 MΩ, if the peak current amplitude was smaller than 100 pA, and if the reversal potential was smaller than −20 mV. The average peak outward currents before and after test compound application were used to calculate the percentage of current inhibited at each concentration. Outward currents in response to each concentration of VX-147 were normalized to its initial control current without VX-147. Concentration-response data were fit by GraphPad Prism using non-linear regression analysis with variable slope (four parameters).
Flow cytometry
HEK293 APOL1-inducible cells were grown in DMEM with 2% (v/v) doxycycline (dox)-free FBS in 96 well plates (Corning, 3904) at a seeding density of 50,000 cells/well in 100 μL of media and treated +/− 15 ng/mL doxycycline to induce APOL1 expression for 16–24 h. Cells were washed with 100 μL DPBS and detached with 100 μl of 1x Versene (Gibco, 15040066) at 37 °C for 15 min. Cells were then resuspended in complete media and transferred to a 96-well plate round bottom plate (Corning, 3799) and pelleted in a tabletop centrifuge at 300 × g for 5 mins. All staining reactions were performed in FACS buffer (DPBS supplemented with 2% FBS and 2 mM EDTA). Cells were stained for surface APOL1 expression in 50 μL of a 1:100 dilution of anti-APOL1 rabbit monoclonal antibody (Abcam, ab252218) in FACS buffer on ice for 30 min. Cells were washed in 200 μL of FACS buffer twice. Secondary staining was performed in 50 μL of a 1:2000 dilution of secondary goat anti-rabbit Alexa Fluor 647 antibody (ThermoFisher, A32733) on ice for 20 min. Cells were washed in 200 μL of FACS buffer twice. Cells were fixed in 50 μl of 4% paraformaldehyde for 15 min at room temperature. 4% PFA was washed off and replaced with 200 μL of FACS buffer for FACS analysis. Cells were immediately analyzed on a Beckman Coulter CytoFLEX instrument. The Alexa Fluor 647 signal was excited using the 638 nm laser and 660/10 nm BP filter. The Beckman coulter CytExpert software was used for data collection and FlowJo software was used for analysis. Live cells gated using FSC-A and SSC-A, then Alexa Fluor 647 intensity was analyzed.
Thallium assay methods
HEK293 APOL1-inducible cells were plated in DMEM with 2% (v/v) tet-free FBS with 15 ng/mL tet to induce APOL1 expression for 17–19 h. Cells were seeded into 384 well BioCoat™ Poly-D-Lysine coated, black/clear, flat-bottom, tissue culture-treated microplates at 2.6 × 104 cell/well. Loading buffer (HBSS, 20 mM HEPES, 5 mM Probenecid, 0.2 mM Ouabain, and components A and C from the FLIPR potassium assay bulk kit (Molecular Devices; Catalog # R8223) was added to the wells of the cell-containing assay plate. The cells were incubated for 30 min at room temperature (23 °C) and then compounds were added to the assay plate wells. Compounds were tested with a top concentration of 10 μM followed by a quarter-log dilution series with 20 total points down to 0.312 nM. An active control was used in each assay and the DMSO concentration was kept fixed at 0.2%. The cells were incubated with compound at room temperature (23 °C) for 30 min then the assay plate was placed on a FLIPR Tetra or Penta high-throughput cellular screening system to measure sample fluorescence intensity. Fluorescence readings were recorded every second for 10 s before the addition of thallium sulfate solution and an additional 60 seconds after the addition of thallium sulfate solution. Data were imported into Genedata for analysis and IC50 generation.
Trypanosome assay methods
Trypanosoma brucei brucei were obtained from ATCC and grown in ATCC medium 2834 modified HMI-9 medium. Trypanosomes in phenol red/FBS free media containing Serum Plus medium supplement (Sigma, 14008 C) were plated at a concentration of 120,000 parasites/well in 384-well plates containing compound and recombinant APOL1. The final concentration of APOL1 used in this assay was sufficient to kill approximately 90% of the parasites. AlamarBlue™ was added to the plates following 16 h incubation at 37 °C with APOL1 and compound, and then returned to the incubator for an additional 2.5 h37. The plate was then read on a SpectraMax plate reader with excitation/emission wavelengths of 555/585 nm. Data was imported into Genedata for analysis and dose response curves generated using the Smart Fit algorithm.
Multitox assay methods
HEK293 APOL1-inducible cells were plated in DMEM with 2% (v/v) tet-free FBS with 50 ng/mL tet to induce APOL1 expression for 24 h. Cells were plated into a 384-well Poly-D-Lysine coated plate at a density of 23,400 cells/well. Compounds were added to the plates for the full 24 h with a 10 point 3-fold dilution curve (top concentration, 10 μM) run in duplicate. The next day, assay buffer containing 2x MultiTox reagent from the MultiTox Multiplex cytotoxicity assay was added to each well and incubated an additional 30 min at 37 °C. The plates were read on an EnVision plate reader for viability (excitation: 400 nm, emission: 486 nm) and cytotoxicity (excitation: 485 nm, emission: 535 nm). A ratio of dead (cytotoxicity) to live (viability) cells was used to normalize data. The data was exported and analyzed in Genedata. Data was normalized using two controls, no tet treatment (100% viability) and 50 ng/mL tet treatment (0% viability) and dose response curves generated using the Smart Fit algorithm. NMDG media (109.50 mM N-Methyl-D-Glucamine, 5.4 mM NaCl, 1.8 mM CaCl2, 0.81 mM MgSO4, 25 mM glucose, 4.5 nM Ferric Nitrate, 10 mM HEPES) or High K+ media (115 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 25 mM glucose, 4.5 nM Ferric Nitrate, 10 mM HEPES) were used to isolate the ionic flux to assess the critical ion for cell death. The MultiTox Multiplex cytotoxicity assay was performed as described above with standard media.
Mouse model methods
To evaluate the pharmacodynamic effects of APOL1 inhibitors in vivo, we licensed transgenic APOL1 G2 mice from Beth Israel Deaconess Medical Center26. The mice were re-derived to allow for housing of the lines in pathogen-free facilities. Strains were propagated and maintained at Charles River Laboratories. The transgenic APOL1 G2 mice have a lysine at amino acid 150 in the APOL1 sequence. The mice were bred to homozygosity as hemizygous mice do not have a phenotype. Experiments were conducted in transgenic male and female FVB APOL1 multi-copy G2-expressing homozygous mice (APOL1 G2mc) (8–15 weeks old, Charles River, Wilmington, MA, USA). Mice were group-housed in a temperature-controlled environment (22 ± 1.5 °C, 30–70% relative humidity, 12 h light/dark) and were acclimatized in the animal facility for at least 3 days prior to use. FVB APOL1 G2 single copy (APOL1 G2sc) homozygous transgenic mice (with glutamate at amino acid 150) were generated at the Beth Israel Deaconess Medical Center (BIDMC) transgenic core facility26. The mice were re-derived to allow for housing of the lines in pathogen-free facilities. APOL1 G2sc mice were group-housed in a temperature-controlled environment (22 ± 1.5 °C, 30 to 70% relative humidity, 12 h light/dark) and were acclimatized in the animal facility for at least 3 days prior to use. Studies were conducted under a protocol that has been approved by Vertex Pharmaceuticals, Inc. Animal Care and Use Committee. Experiments were performed at the Vertex Boston site, which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC).
Prophylactic assessment of Compound 3
In APOL1 G2hom copy mice, increased proteinuria (mean urinary albumin-to-creatinine ratio, UACR) was induced by a single intraperitoneal (IP) injection of 150 µg/kg of murine IFNγ (R&D Systems, Catalog# 485-MI/CF) in a volume of 10 mL/kg. Blood (20 µL) was collected 1 h post IFNγ administration to evaluate serum IFNγ levels. Blood was allowed to clot for 30 min at room temperature and then centrifuged at 1500 relative centrifugal force (RCF) for 15 min. Serum IFNγ levels were quantified using the ProQuantum Mouse IFNγ Immunoassay (Invitrogen, Catalog# A41150) to confirm successful administration of IFNγ. Urine was collected at various time points throughout the experiment, which lasted no more than 72 h. For proteinuria analysis, urine was diluted appropriately, and urine albumin and creatinine were measured using a mouse albumin ELISA and creatinine companion kit (Bethyl Laboratories, Catalog# E99-134 and Ethos Biosciences, Catalog# 1012). Compound 3 (30 mg/kg, dosed as a crystalline suspension) or vehicle, which is composed of 1% Hydroxypropyl methylcellulose acetate succinate, type H, 0.25% of Polyvinylpyrrolidone K 30 and 2% of d-α-Tocopheryl polyethylene glycol 1000 succinate (1%HPMCAS-H/0.25%PVP-K30/2%TPGS, Shin-Etsu, MP Biomedicals and Antares manufacturers, respectively) was administered orally twice daily (bid) at 12 h interval in a volume of 10 mL/kg. Compound 3 was administered 1.5 h before IFNγ injection. UACR was calculated by dividing the average of albumin replicate values (expressed in ng/mL) to the average of creatinine replicate values (expressed to mg/dL) and then converted to the unit µg/mg. Area under the curve (AUC) was calculated per animal for UACR values from 0 to 48 h or 72 h, using GraphPad Prism, with baseline set to the mean of all animals of the same strain at baseline (day 0). Percent reduction of UACR AUC was calculated using the following equation:
((Meanvehicle – Meancompound 3)/(MeanVehicle)) * 100. Statistical analysis was performed using GraphPad Prism. UACR AUC data was log-transformed and analyzed with two tailed t-test comparing Vehicle vs IFNγ within a mouse strain or to compound 3. Statistical significance was set at p ≤ 0.05.
Therapeutic assessment of Compound 3 in plasmid chronic G2 single copy mice
Murine IFNγ cDNA was cloned in a CpG-free plasmid (Catalog #pcpgf-mcs) at Vertex laboratories26. The day before the hydro dynamic injection (HDI) individual body weight was collected. The day of the injection, CpG-free IFNγ plasmid was thawed on wet ice and diluted in sterile saline to administer a total of 0.3 μg in a total volume corresponding to 10% body weight in grams. Mice were left in their cage under a heat lamp for 2 min, transferred to a restrainer where the area of injection was cleaned with ethanol wipes. Syringes (3 mL) with a 25-gauge needle were used for the tail vein injection. The full volume was injected in a continuous motion over approximately 5–10 s. Mice were placed in a cage on a heating pad and monitored for 30 min immediately following injection.
Serum IFNγ levels were quantified using the ProQuantum Mouse IFNγ Immunoassay (Invitrogen, Catalog# A41150), to confirm successful administration of IFNγ plasmid in the chronic therapeutic mouse model. Blood (20 µL) was collected on days 1, 5, 9, 14 to evaluate serum IFNγ levels. Blood was allowed to clot for 30 min at room temperature and then centrifuged at 1500 relative centrifugal force (RCF) for 15 min. The serum fraction was collected and used to monitor IFNγ levels. For the ProQuantum Mouse IFNγ Immunoassay, the manufacturer’s protocol to quantify IFNγ was followed except for the following modifications to sample dilution: for day 1 dilution was 1:1000, for day 5 dilution was 1:50, for day 9 and 14 dilution was at 1:20. The standard curve was fit using a 4-parameter fit and sample values were interpolated using GraphPad Prism.
UACR and UACR AUCDay6-14 data were graphed as mean ± standard error of the mean (SEM). UACR AUCDay6-14 data was log-transformed and analyzed with One-way ANOVA followed by Tukey’s comparison test. Statistical significance was set at p ≤ 0.05.
Histopathology and immunohistochemistry
At the end of the study, animals were euthanized as per the IACUC-approved protocol. Euthanized animals were necropsied immediately, the left kidney was resected and fixed in 10% neutral buffered formalin (48 h), followed by transfer to 70% histology grade ethanol (Cat # R3154-1GA, Millipore Sigma) and submitted to the pathology core laboratory. Formalin-fixed kidneys were longitudinally bisected through the mid-sagittal plane to include cortex, medulla, and pelvis. Bisected kidney samples were routinely processed (Tissue-Tek tissue processing system, Sakura-Fintek), and paraffin-embedded. Serial sections from paraffin-embedded blocks were stained for a) hematoxylin and eosin (H&E), and b) periodic acid Schiff’s (PAS) using standard staining protocols as per Ventana Discovery Ultra autostainer protocols (Ventana Medical Systems, Roche). Specific protocols for APOL1 and nephrin immunohistochemistry are described below and were performed using Ventana Discovery Ultra autostainer (Ventana Medical Systems, Roche).
APOL1 and nephrin immunohistochemistry
Formalin-fixed paraffin-embedded (FFPE) tissue blocks were cut at 3 µm thickness, dried overnight at 37 °C, and baked at 60 °C for 1 h before use. All subsequent steps were performed on the Ventana Discovery Ultra platform (Ventana Medical Systems, Roche). The protocol for nephrin and APOL1 immunohistochemistry were similar with minor differences. FFPE sections were deparaffinized at 69 °C, followed by epitope retrieval (APOL1 – 91 °C, CC2 buffer for 32 min; nephrin – 95 °C, CC1 buffer for 64 min), peroxide block for 8 min (inhibitor conditioning medium), and primary antibody incubation for 1 h (APOL1 – rabbit anti APOL1 monoclonal antibody, Cat # ab252218, 1:250, Abcam; nephrin – rabbit anti-nephrin monoclonal antibody, Cat# ab216341, 1:1000, Abcam). Signals were amplified using OmniMap anti-Rabbit HRP kit (Cat # 760-4311) for 16 min and detected using DAB chromogen (Chromomap DAB Kit; Cat #760-159) for 8 min. Slides were counterstained for 8 min (Hematoxylin II, Cat # 970-2208), followed by bluing for 4 min (Bluing reagent, Cat # 760-2037) and coverslipped. Rabbit monoclonal IgG (clone DA1E, Cat # 3900, Cell Signaling Technologies) was used as isotype control for both APOL1 and nephrin primary antibodies. All reagents unless specified otherwise were provided by the manufacturer (Ventana Medical Systems). Coverslipped and immunolabeled glass slides were scanned using Aperio AT2 whole slide scanner (Leica Biosystems) at 40x objective magnification and were visualized using image scope/web scope software (Leica Biosystems).
Immunolabeled glass slides were scanned using Aperio AT2 whole slide scanner (Leica Biosystems) at 40x objective magnification and were visualized using image scope/web scope software (Leica Biosystems). All antibodies were diluted using the antibody diluent (Cat# ADB250, Ventana Medical Systems). All reagents unless specified otherwise were provided by the manufacturer (Ventana Medical Systems).
Morphometric analysis nephrin expression in glomeruli
Immunolabelled Stained glass slides were scanned at 40x magnification (Aperio AT2, Leica Biosystems) and quality checked for any artifacts (i.e., damaged tissue, wrinkles, particles, scanning lines). Scanned whole slide images (WSI) were imported into HALO v3.4 (Indica Labs), where images were manually annotated to include the renal cortex and exclude artifacts. An Artificial Intelligence- Convolution Neural Network I – CNN (DenseNet from HALO, Indica Labs V3.2) based classifier (“Kidney_Glomerulus_APOL1 DenseNet”), was trained to automatically identify glomeruli within kidney sections. The images were manually curated to remove any abnormalities or non-glomerular structures from further analysis. Morphometric image analysis was performed using an Area Quantification (v1.0) module in HALO (“APOL1- Kidney_Glomerulus_APOL1 DenseNet”). Intensity thresholds were set to detect weak, moderate, and strong intensity of DAB chromogen (brown) staining for APOL1 and nephrin with pathologist’s guidance. All the available glomeruli detected by the algorithm in a one longitudinal section of the kidney per animal were analyzed from each sample and a cumulative output (H-score) was obtained. H-score was calculated as the following:
\(H\; score=1\,\left(\%Area_weak\right)+2\,\left(\%Area_moderate\right)+3\,(\%Area_strong)\)
Statistical analysis was performed in GraphPad Prism 9 using the Kruskall-Wallis test with multiple comparisons. Graphs were generated in Microsoft Excel and Prism.
Podocyte exact morphology measurement procedure
A subset of mice was selected for a morphological assessment of the glomerular slit diaphragm. Mice were injected with a cocktail of 40 mg/mL ketamine and 2 mg/mL Xylazine. Level of anesthesia was then assessed with firm toe pinch. Once the subject was fully anesthetized, it was transferred to the working station for the perfusion. Area of incision was cleaned with an alcohol pad and tweezers were used to cut through the skin and expose the chest cavity. The diaphragm and the ribcage were cut to expose the heart. The ribcage was lifted, and a 25-gauge needle connected was inserted in the apex of the left ventricle. The right atrium was then cut, and the subject was perfused with 1X PBS/15,000-unit heparin at the rate of 20 mL/min for 3 min to washout the blood from the organs using a standard programmable infusion pump (Harvard Apparatus, Cat # 70-4500). To restrict fixation to the right kidney, the left kidney renal artery was clamped. Right kidney was perfused with 4% PFA at the rate of 20 mL/min for 5 min using an infusion pump (New Era Pump Systems, Inc. Cat # NE-9000). Right kidneys were then cut in half and left rotating at room temperature for 1 h inside a 15 mL conical tube. Sample were transferred to 4 °C before shipment to NIPOKA GmbH.
After deparaffinization and rehydration, formalin-fixed and paraffin-embedded kidney sections (3 µm) were boiled in Tris-EDTA buffer (10 mmol/l Tris, 1 mmol/l EDTA, pH 9) in a pressure cooker for 5 min, followed by the blocking step (1% FBS, 1% BSA, 0.1% fish gelatin, 1% normal goat serum) for 1 h. The following primary antibodies were incubated overnight at 4 °C: rabbit anti‐podocin 1:150 (IBL International, Hamburg, Germany, JP-29040) and mouse anti‐integrin alpha3 1:500 (Santacruz, Dallas, Texas, USA, sc-374242). After three washing steps in PBS, secondary antibodies were incubated for 1 h at room temperature (anti-rabbit Alexa Fluor 488‐conjugated IgG 1:600 (ChromoTek, Planegg, Germany, srbAF488-1-100) and anti-mouse Cy3‐conjugated IgG antibody 1:600 (Jackson ImmunoResearch, West Grove, PA, 115-166-006) for 1 h at room temperature. DAPI (1:100) was added to the slides for 5 min, followed by a washing step in PBS. Finally, the slides were incubated in H2O and mounted in Mowiol (Carl Roth, Karlsruhe, Germany) using high precision coverslips (Paul Marienfeld, Lauda-Königshofen, Germany). The evaluation of the filtration slit density (FSD) was performed using a recently established super-resolution microscopy-based methodology termed podocyte exact morphology measurement procedure (PEMP)33. For three-dimensional structured illumination microscopy (3D-SIM), z-stacks of 19 planes of both channels (488 and 561 nm) were acquired from the stained kidney section using an N-SIM super-resolution microscope (Nikon, Tokyo, Japan) equipped with a 100x silicone objective. The images were reconstructed into 3D-SIM images using NIS-Elements AR 5.30 (Nikon, Tokyo, Japan). The z-stacks were converted into a maximum intensity projection followed by the automatized identification of podocyte foot process areas that were selected based on the foot process positive antibody. Inside these areas, the filtration slit diaphragm length was automatically determined. The FSD was expressed as the ratio of the total filtration slit diaphragm length per podocyte foot process area. The FSD of 20 randomly selected glomeruli was determined for every animal.
Synthesis and characterization of chemical compounds
The chemical compounds (2 – 3, VX-147) in this manuscript were prepared according to the synthetic procedures detailed below. Purity assessment for final compounds was based on analytical UPLC: 2.1 mm × 50 mm Waters CSH C18 column, 1.7 μM, 130 Å. Mobile phases were as follows: A, H2O with 0.1% trifluoroacetic acid; B, acetonitrile with 0.1% trifluoroacetic acid; gradient, 10–60% B in 0.6 min with a 1.4 min run time. The flow rate was 0.6 mL/min. All compounds were ≥ 95% pure. Mass samples were analyzed on a Waters 3100 single quad mass spectrometer operated in positive MS mode with electrospray ionization. The mobile phase for all mass analysis consisted of acetonitrile–water mixtures with either 0.1% trifluoroacetic acid or ammonium formate. 1H NMR, 13C NMR, and 19F NMR spectra were recorded using either a Bruker Avance 400 (400 MHz) or a Bruker Avance II-300 (300 MHz) instrument. The screening hit, 1, is commercially available and was purchased from an external vendor. Compound 1 has a Chemical Abstracts Service Registry Number of 687574-42-1.
Synthetic route to (3S,4 R)-3-amino-4-hydroxypyrrolidin-2-one (S5)
Synthesis of Methyl (2S,3 R)-2,4-dibromo-3-hydroxy-butanoate (S1)
Potassium (2 R,3 R)-2,3,4-trihydroxybutanoate (280 g) was stirred with a 33% solution of HBr in acetic acid (1 L) at ambient temperature for 24 h. The reaction mixture was then poured into MeOH (5 L). The mixture was stirred at room temperature for 8 h, then at 65 °C for 4 h. The mixture was concentrated, and the crude residue was dissolved in MeOH (1.2 L) and then concentrated sulfuric acid (30 mL) was slowly added. The mixture was heated under reflux for 6 h, then concentrated. The crude residue was taken up with EtOAc (400 mL). The resulting solution was washed with water (250 mL), dried over Na2SO4, filtered, and concentrated to provide the product methyl (2S,3 R)-2,4-dibromo-3-hydroxy-butanoate as an oil which solidified upon storage at 4 °C. (375 g, 74%). 1H NMR (400 MHz, CDCl3) δ 4.71 (d, J = 3.4 Hz, 1H), 4.17-4.14 (m, 1H), 3.82 (s, 3H), 3.53-3.44 (m, 2H).
Synthesis of Methyl (2 R,3S)-3-(bromomethyl)oxirane-2-carboxylate (S2)
To a solution of methyl (2S,3 R)-2,4-dibromo-3-hydroxy-butanoate (200 g, 0.73 mol) in acetone (2.0 L) was added anhydrous K2CO3 (151.1 g, 1.1 mol) while the reaction temperature was maintained at 0 °C. The reaction was stirred at 0 °C for 2 h, then gradually warmed to ambient temperature over 4 h. The reaction mixture was filtered, and the filtrate was concentrated. The residue was distilled under vacuum 75–80 °C/200-300 Pa to provide the product as a colorless liquid (105 g, 74%). 1H NMR (300 MHz, CDCl3) δ 3.83 (s, 3H), 3.71–3.61 (m, 2H), 3.61–3.53 (m, 1H), 3.46 (dd, J = 9.9, 6.6 Hz, 1H) ppm. 13C NMR (75 MHz, CDCl3) δ 167.58, 55.89, 53.52, 52.77, 26.83 ppm.
Synthesis of Methyl (2 R,3 R)-3-(azidomethyl)oxirane-2-carboxylate (S3)
Methyl (2 R,3S)-3-(bromomethyl)oxirane-2-carboxylate (52.6 g, 269.7 mmol) was dissolved in DMF (500 mL) in a 3 L round-bottomed flask equipped with a magnetic stir bar. NaN3 (25.3 g, 388.4 mmol) was added, and the mixture was stirred at room temperature for 1 h. The reaction mixture was poured into water and extracted with EtOAc. The extract was washed with water, dried over MgSO4, and concentrated to afford a dark red oil. The oil residue was dissolved in CH2Cl2 and filtered over a plug of silica gel, eluting with CH2Cl2. The filtrate was concentrated to afford the product as a clear, light red oil (40.8 g, 96%). 1H NMR (300 MHz, CDCl3) δ 3.87-3.74 (m, 3H), 3.67-3.55 (m, 2H), 3.47 (dd, J = 13.3, 5.1 Hz, 1H), 3.38 (ddd, J = 6.3, 5.0, 4.4 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 167.76, 54.81, 52.67, 51.32, 48.74.
Synthesis of (1 R,5 R)-6-oxa-3-azabicyclo[3.1.0]hexan-2-one (S4)
A 2 L 3-neck flask with overhead stirrer was charged with methyl (2 R,3 R)-3-(azidomethyl)oxirane-2-carboxylate (67 g, 402.5 mmol) in toluene (500 mL), stirred for 10 min, and then warmed to 80 °C. Bu3SnH (220 mL, 817.8 mmol) and AIBN (2 g, 12.2 mmol) were dissolved in toluene (500 mL) and then added to the reaction over 3 h using an addition funnel. The resulting reaction mixture was stirred at 80–87 °C for 1 h, then cooled to ambient temperature, and concentrated under reduced pressure. The residue was partitioned between acetonitrile (2 L) and pentane (1 L), stirred for 10 min, and then the acetonitrile phase (bottom) was separated. The acetonitrile phase was washed with pentane (2 x 500 mL) and concentrated in vacuo to afford a light-yellow solid. The solid residue was triturated with pentane (200 mL) to afford the product as a yellow solid which was used without further purification (52 g, 98%). 1H NMR (300 MHz, CDCl3) δ 5.89 (s, 1H), 4.00 (q, J = 2.5 Hz, 1H), 3.74–3.50 (m, 2H), 3.44 (dd, J = 12.4, 2.4 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 173.24, 53.28, 52.18, 44.00.
Synthesis of (3S,4 R)-3-amino-4-hydroxypyrrolidin-2-one (S5)
A Parr vessel containing (1 R,5 R)-6-oxa-3-azabicyclo[3.1.0]hexan-2-one (60 g, 605.5 mmol) and NH3 (1.5 L, 58.6 mol) was pressurized to 200 psi and allowed to stir at 18 °C for 2 days. NH3 was released from the vessel to provide a grey solid. Heptanes was added and the mixture stirred for 30 min. The solid was filtered, and then the filter cake was isolated, and EtOAc and heptanes were used to dissolve the solid. The mixture was concentrated in vacuo to afford the product (55 g, 78%). 1H NMR (300 MHz, D2O) δ 4.13 (q, J = 7.2 Hz, 1H), 3.53 (dd, J = 10.4, 7.4 Hz, 1H), 3.36 (d, J = 7.5 Hz, 1H), 3.05 (dd, J = 10.4, 6.8 Hz, 1H).
Synthetic route to Compound 2
Synthesis of 4-fluoro-2-[2-(4-fluorophenyl)ethynyl]-6-methyl-aniline (S6)
To a stirred solution of 2-bromo-4-fluoro-6-methyl-aniline hydrogen bromide salt (25 g, 0.09 mol) and 1-ethynyl-4-fluoro-benzene (26.1 g, 0.22 mol) in toluene (500 mL) was added triethylamine (500 mL) at room temperature. The reaction was degassed for 20 min with nitrogen. To the reaction mixture was added copper iodide (1.67 g, 0.008 mol) and tetrakis(triphenylphosphine)palladium(0) (4.97 g, 0.004 mol), and the reaction was degassed for another 5 min. The reaction mixture was then stirred at 100 °C for 16 h. The reaction mixture was filtered through a pad of celite and washed with EtOAc (50 mL), then the filtrate was concentrated. The crude product was purified by flash column chromatography column using silica gel eluted with 3% EtOAc in heptanes, and concentrated to afford 4-fluoro-2-[2-(4-fluorophenyl)ethynyl]-6-methyl-aniline (12 g, 46%) as dark brown semi-solid. 1H NMR (400 MHz, CDCl3) δ: 7.48-7.52 (m, 2H), 7.02-7.07 (m, 2H), 6.80-6.94 (m, 2H), 4.11 (s, 2H), 2.18 (s, 3H). ESI-MS m/z calc. 243.09, found 244.06 (M + 1)+.
Synthesis of 5-fluoro-2-(4-fluorophenyl)-7-methyl-1H-indole (S7)
A solution of 4-fluoro-2-[2-(4-fluorophenyl)ethynyl]-6-methyl-aniline (6.09 g, 24.3 mmol) and palladium(II) chloride (434.9 mg, 2.43 mmol) was prepared in acetonitrile (243 mL) under argon, and stirred at 80 °C for 3 h. The reaction mixture was concentrated, and the crude product was purified by flash column chromatography column using silica gel eluted with 5% ethyl acetate in heptanes, then concentrated to afford 5-fluoro-2-(4- fluorophenyl)-7-methyl-1H-indole (4 g, 66%) as an off-white solid. 1H NMR (300 MHz, DMSO-d6) δ 11.16 (s, 1H), 8.01 – 7.91 (m, 2H), 7.37 – 7.27 (m, 2H), 7.09 (dd, J = 9.7, 2.5 Hz, 1H), 6.84 (d, J = 2.1 Hz, 1H), 6.76 (dd, J = 10.4, 2.4 Hz, 1H), 2.53 (s, 3H). ESI-MS m/z calc. 243.1, found 244.2 (M + 1)+.
Synthesis of Methyl 3-[5-fluoro-2-(4-fluorophenyl)-7-methyl-1H-indol-3-yl]propanoate (S8)
To 5-fluoro-2-(4-fluorophenyl)-7-methyl-1H-indole (502 mg, 2.06 mmol) in 10 mL toluene was added methanesulfonic acid (208 μL, 3.21 mmol) and triethylsilane (1.1 mL, 6.89 mmol), followed by methyl 3,3-dimethoxypropanoate (370 μL, 2.61 mmol). The reaction mixture was heated to 70 °C for 2 h. The reaction was quenched with water, extracted with ethyl acetate, and concentrated. The crude residue was dissolved in methanol and isopropanol (1:1, 30 mL), and 50 mg of 5% Pd/C added. The reaction mixture was hydrogenated at 50 psi for 4 h. The reaction mixture was filtered through celite, concentrated, and the crude residue purified by flash column chromatography column using silica gel eluted with 0-25% EtOAc/heptanes gradient to afford methyl 3-[5-fluoro-2-(4-fluorophenyl)-7-methyl-1H- indol-3-yl]propanoate (478 mg, 70%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 7.86 (s, 1H), 7.63 – 7.45 (m, 2H), 7.25 – 7.15 (m, 2H), 7.12 (dd, J = 9.5, 2.4 Hz, 1H), 6.81 (ddd, J = 9.9, 2.4, 0.9 Hz, 1H), 3.65 (s, 3H), 3.22 – 3.07 (m, 2H), 2.74 – 2.57 (m, 2H), 2.51 (d, J = 0.8 Hz, 3H). ESI-MS m/z calc. 329.12, found 330.27 (M + 1)+.
Synthesis of 3-[5-fluoro-2-(4-fluorophenyl)-7-methyl-1H-indol-3-yl]propanoic acid (S9)
To a solution of methyl 3-[5-fluoro-2-(4-fluorophenyl)-7-methyl-1H-indol-3-yl]propanoate (475 mg, 1.43 mmol) and LiOH (344 mg, 14.36 mmol) in 4 mL MeOH, was added 4 mL THF and 2 mL water. The reaction was stirred at 60 °C for 16 h. The reaction mixture was concentrated, then CH2Cl2 (60 mL) and water (40 mL) added, followed by acidification with aqueous HCl to pH = 1. The organic layer was washed with saturated aqueous NaCl and concentrated to provide 3-[5-fluoro-2-(4-fluorophenyl)-7-methyl-1H-indol-3-yl]propanoic acid (451 mg, 90%) 1H NMR (300 MHz, CDCl3) δ 7.88 (s, 1H), 7.63 – 7.44 (m, 2H), 7.26 – 7.02 (m, 4H), 6.82 (ddd, J = 9.8, 2.4, 0.9 Hz, 1H), 3.22 – 3.10 (m, 2H), 2.75 – 2.63 (m, 2H), 2.51 (d, J = 0.7 Hz, 3H). ESI-MS m/z calc. 315.11, found 316.23 (M + 1)+.
Synthesis of 3-[5-fluoro-2-(4-fluorophenyl)-7-methyl-1H-indol-3-yl]-N-[(3S)-2-oxopyrrolidin-3-yl]propanamide (Compound 2)
To a solution of 3-[5-fluoro-2-(4-fluorophenyl)-7-methyl-1H-indol-3-yl]propanoic acid, (19 mg, 0.05 mmol), (3S)-3-aminopyrrolidin-2-one (8 mg, 0.08 mmol) and HATU (28 mg, 0.07 mmol) in 1 mL DMSO was added triethylamine (30 μL, 0.22 mmol). The reaction mixture was stirred for 12 h at ambient temperature. The reaction mixture was filtered, then directly purified by reverse phase chromatography (C18 ACN/Water with TFA modifier) to afford the gave product 3-[5-fluoro-2-(4-fluorophenyl)-7-methyl-1H-indol-3-yl]-N-[(3S)-2-oxopyrrolidin-3-yl]propanamide (16 mg, 74%) as a white solid. 1H NMR (300 MHz, MeOD) δ 7.71 – 7.58 (m, 2H), 7.21 (t, J = 8.8 Hz, 2H), 7.13 (dd, J = 9.8, 2.4 Hz, 1H), 6.70 (dd, J = 10.2, 2.4 Hz, 1H), 4.45 (dd, J = 10.3, 8.7 Hz, 1H), 3.31 (m, 2H *obscured by MeOD solvent peak), 3.11 (t, J = 8.0 Hz, 2H), 2.64 – 2.52 (m, 2H), 2.49 (s, 3H), 2.44 – 2.31 (m, 1H), 1.84 (dq, J = 12.4, 9.4 Hz, 1H). 19F NMR (282 MHz, MeOD) δ -116.62, -127.29. 13C NMR (75 MHz, MeOD) δ 177.68, 175.81, 165.40, 162.14, 160.63, 157.55, 137.01, 133.65, 131.63, 131.53, 130.87, 130.82, 129.77, 129.64, 123.42, 123.30, 116.59, 116.30, 112.72, 112.65, 111.57, 111.23, 102.00, 101.69, 51.63, 40.02, 38.26, 29.34, 22.06, 17.00, 16.98. ESI-MS m/z calc. 397.16, found 398.18 (M + 1)+. Optical rotation: [α] D 19.6 = −21.05 (c = 1.0, 15 mg in 1 mL of MeOH).
Synthetic route to Compound 3
Synthesis of 3-[5-fluoro-2-(4-fluorophenyl)-7-methyl-1H-indol-3-yl]-N-[(3S,4 R)-4-hydroxy-2-oxo-pyrrolidin-3-yl]propanamide (Compound 3)
To a solution of 3-[5-fluoro-2-(4-fluorophenyl)-7-methyl-1H-indol-3-yl]propanoic acid (6.06 g, 16.34 mmol) and (3S,4 R)-3-amino-4-hydroxy-pyrrolidin-2-one (3.25 g, 21.30 mmol) in DMF (41 mL) was added 2-Chloro-4,6-dimethoxy-1,3,5-triazine (3.7 g, 21.07 mmol) and N-methylmorpholine (7.2 mL, 65.49 mmol). The reaction mixture was stirred for 16 h at ambient temperature. The reaction was quenched with water, extracted with EtOAc, washed with brine, dried over anhydrous MgSO4, and concentrated in vacuo. The crude product was recrystallized from 10% MeOH in water to provide 3-[5-fluoro-2-(4-fluorophenyl)-7-methyl-1H-indol-3-yl]-N-[(3S,4 R)-4-hydroxy-2-oxo-pyrrolidin-3-yl]propanamide (4.32 g, 62%) as a white solid. 1H NMR (300 MHz, MeOD) δ 7.69 – 7.59 (m, 2H), 7.27 – 7.17 (m, 2H), 7.18 – 7.11 (m, 1H), 6.71 (ddd, J = 10.1, 2.5, 1.0 Hz, 1H), 4.35 (td, J = 7.6, 6.8 Hz, 1H), 4.21 (d, J = 7.7 Hz, 1H), 3.56 (dd, J = 9.9, 7.6 Hz, 1H), 3.18 – 3.05 (m, 3H), 2.65 – 2.54 (m, 2H), 2.50 (s, 3H). 19F NMR (282 MHz, MeOD) δ -116.51, -127.10. 13C NMR (75 MHz, MeOD) δ 176.17, 174.97, 165.35, 162.09, 160.62, 157.54, 136.94, 133.65, 131.59, 131.48, 130.81, 130.76, 129.69, 129.55, 123.46, 123.34, 116.58, 116.29, 112.63, 112.57, 111.60, 111.25, 101.85, 101.54, 73.47, 60.00, 47.71, 38.02, 21.93, 17.01, 16.99. ESI-MS m/z calc. 413.16, found 414.01 (M + 1)+. Optical rotation: [α] D 19.6 = −40.80 (c = 1.0, 100 mg in 10 mL of MeOH).
Synthetic route to VX-147
Synthesis of 2,4-difluoro-6-[2-(4-fluorophenyl)ethynyl]aniline (S10)
To a flask containing 2,4-difluoro-6-iodo-aniline (134 g, 525.5 mmol) was added Et3N (1.3 L), followed by DMF (250 mL), 1-ethynyl-4-fluoro-benzene (83.5 g, 695.1 mmol), CuI (20.5 g, 107.6 mmol), and PdCl2(PPh3)2 (25 g, 35.6 mmol). The mixture was allowed to stir at room temperature for 2 h. Solvent was removed under reduced pressure and water (500 mL) was added. The mixture was extracted with ethyl acetate, filtered and concentrated. The product mixture was filtered through a silica gel plug (Eluent: CH2Cl2), followed by a second silica plug filtration (Eluent: 30-40% EtOAc in Heptane). The crude product was purified by flash column chromatography column using silica gel eluted with a gradient of 0-20% EtOAc in heptane to afford the product 2,4-difluoro-6-[2-(4-fluorophenyl)ethynyl]aniline as a pale-yellow solid. (87 g, 60%). 1H NMR (300 MHz, CDCl3) δ 7.58 – 7.45 (m, 2H), 7.14 – 7.02 (m, 2H), 6.92 (ddd, J = 8.8, 2.8, 1.7 Hz, 1H), 6.87 – 6.71 (m, 1H), 4.15 (s, 2H). ESI-MS m/z calc. 247.1, found 248.0 (M + 1)+.
Synthesis of 5,7-difluoro-2-(4-fluorophenyl)-1H-indole (S11)
To a solution of 2,4-difluoro-6-[2-(4-fluorophenyl)ethynyl]aniline (46 g, 167.5 mmol) in DMF (600 mL) was added CuI (1.9 g, 10.0 mmol) and the reaction was heated at reflux. Water (800 mL) was added and the mixture extracted with MTBE. The mixture was then washed with sat. NaCl solution, dried over Na2SO4 and then concentrated in vacuo to afford the product, which was used in subsequent steps without further purification (41 g, 87%). 1H NMR (300 MHz, CDCl3) δ 8.43 (s, 1H), 7.72 – 7.58 (m, 2H), 7.27 – 7.15 (m, 2H), 7.09 (dd, J = 9.0, 2.1 Hz, 1H), 6.85 – 6.63 (m, 2H). ESI-MS m/z calc. 247.1, found 248.0 (M + 1)+.
Synthesis of methyl (E)-3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]prop-2-enoate (S12)
A 12 L flask with overhead stirrer was charged with 5,7-difluoro-2-(4-fluorophenyl)-1H-indole (300 g, 1.2 mol), CH2Cl2 (3 L), methyl 3,3-dimethoxypropanoate (195 mL, 1.4 mol) and TFA (300 mL, 3.9 mol). The reaction was heated to reflux for 4 h. Additional CH2Cl2 was added to facilitate stirring. Upon cooling to room temperature, the solid product was filtered, washed with minimal CH2CI2 and dried to afford the product methyl (E)-3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]prop-2-enoate (388 g, 96%). 1H NMR (400 MHz, DMSO-d6) δ 12.66 (s, 1H), 7.77 – 7.57 (m, 4H), 7.56 – 7.37 (m, 2H), 7.19 (ddd, J = 11.0, 9.7, 2.1 Hz, 1H), 6.47 (d, J = 16.1 Hz, 1H), 3.69 (s, 3H). ESI-MS m/z calc. 331.1, found 332.4 (M + 1)+.
Synthesis of methyl 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoate (S13)
To a suspension of methyl (E)-3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]prop-2-enoate (80 g, 236.5 mmol) in EtOH (1.5 L) under a nitrogen atmosphere was added Pd(OH)2 (6 g of 20 % w/w, 8.5 mmol) and ammonium formate (160 g, 2.5 mol). The mixture was heated at reflux for 3 h, then filtered to remove catalyst. The filtrate was concentrated in vacuo to afford the product methyl 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3- yl]propanoate as an off-white solid, which was used without further purification (82 g, 100%). 1H NMR (300 MHz, CDCl3) δ 8.18 (s, 1H), 7.65 – 7.47 (m, 2H), 7.27 – 7.14 (m, 2H), 7.14 – 7.00 (m, 1H), 6.76 (ddd, J = 10.8, 9.4, 2.2 Hz, 1H), 3.65 (s, 3H), 3.27 – 3.04 (m, 2H), 2.75 – 2.49 (m, 2H). ESI-MS m/z calc. 333.1, found 334.3 (M + 1)+.
Synthesis of 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoic acid (S14)
LiOH (67 g, 2.8 mol) was added to a solution of methyl 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoate (217 g, 651.1 mmol) in THF (1 L) and water (100 mL). The mixture was heated at reflux for 2 h, and then allowed to cool overnight. THF was removed by concentration under reduced pressure, and water was added (approx. 1 L). The mixture was cooled on an ice bath and HCl (250 mL of 11.7 M, 2.9 mol) was added to adjust to pH = 4. EtOAc (300 mL) was added, and the aqueous layer extracted with further EtOAc (100 mL). The combined organic extracts were dried over Na2SO4, and filtered through a plug of silica gel, rinsing with EtOAc. The filtrate was concentrated in vacuo to afford an orange oil (50-75 mL). Heptanes (50 mL) were added and the mixture chilled on dry ice. Upon agitation, a crystalline solid formed. The mixture was allowed to stir on an ice-bath until to allow completion of the crystallization process. The solid was filtered, washed with heptane and air dried to afford the product 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoic acid (208 g, 96%). 1H NMR (300 MHz, CDCl3) δ 8.15 (s, 1H), 7.60 – 7.46 (m, 2H), 7.27 – 7.15 (m, 2H), 7.09 (dd, J = 9.1, 2.2 Hz, 1H), 6.77 (ddd, J = 10.8, 9.4, 2.2 Hz, 1H), 3.26 – 3.05 (m, 2H), 2.78 – 2.57 (m, 2H). ESI-MS m/z calc. 319.1, found 320.0 (M + 1)+.
Synthesis of 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-[(3S,4 R)-4-hydroxy-2-oxo-pyrrolidin-3-yl] propanamide (VX-147)
A 2 L 3-neck RB flask with magnetic stirrer, temperature probe and nitrogen inlet was charged with 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoic acid (90.5 g, 283.5 mmol) and (3S,4 R)-3-amino-4-hydroxy-pyrrolidin-2-one (39.9 g, 343.6 mmol) in DMF (1.65 L), and stirred for 15 min. CDMT (61.1 g, 348 mmol) was added. The mixture was then cooled to 2 °C in an ice bath. N-methylmorpholine was added (131 mL, 1.2 mol) dropwise over 20 min and the mixture was heated at 30 °C for 16 h. The reaction mixture was added into 4.5 L of ice water, and extracted with EtOAc (1.2 L x 4). The combined organic layers were washed with 1.2 L of 1 M HCl (x 3) and then water (1.2 L) and brine (1.2 L). The combined organic layers were dried over Na2SO4, filtered and concentrated. The mixture was washed through a silica gel plug (1.8 L of silica gel), first eluting with 25% EtOAc in dichloromethane (8 L) to remove impurities, followed by hot EtOAc (8 L), to elute the product. The EtOAc filtrate was concentrated in vacuo. TBME was then added (400 mL), and the mixture allowed to stir for overnight. Filtration of the resulting solid afforded the product 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-[(3S,4 R)-4-hydroxy-2-oxo-pyrrolidin-3-yl] propenamide as a white solid. 62 g, 52%). 1H NMR (300 MHz, MeOD) δ 7.70 – 7.58 (m, 2H), 7.29 – 7.13 (m, 3H), 6.73 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 4.34 (q, J = 7.5 Hz, 1H), 4.22 (d, J = 7.8 Hz, 1H), 3.57 (dd, J = 9.9, 7.5 Hz, 1H), 3.20 – 3.04 (m, 3H), 2.65 – 2.53 (m, 2H). 19F NMR (282 MHz, MeOD) δ -115.93, -124.40, -133.12. 13C NMR (75 MHz, MeOD) δ 174.55, 173.56, 164.21, 160.94, 158.22, 158.09, 155.11, 154.98, 150.15, 147.08, 146.89, 136.72, 131.34, 131.25, 131.20, 131.10, 130.21, 130.10, 128.74, 128.69, 120.93, 120.76, 115.40, 115.11, 111.75, 99.18, 99.13, 98.87, 98.82, 96.37, 96.09, 95.97, 95.69, 72.07, 58.58, 46.32, 36.40, 20.41. ESI-MS m/z calc. 417.13, found 418.20 (M + 1)+. Optical rotation: [α] D 19.6 = −13.46 (c = 1.0, 100 mg in 10 mL of MeOH).
Small Molecule X-ray Structure of VX-147
The single crystal X-ray diffraction studies were carried out on a Bruker Kappa diffractometer with a CPAD PHOTON II detector, utilizing Cu Kα radiation (λ = 1.54178(1)). A 0.380 × 0.128 × 0.050 mm colorless blade was mounted on a MiTeGen kapton Micromount with Paratone oil. Crystals were grown via evaporation of VX-147 from 1:1 dixoane:water at 40 °C. Data were collected at low temperature (100 K), under a nitrogen cold stream generated by an Oxford Cryosystems Cryostream 800 Plus, using ϖ and φ scans. Crystal-to-detector distance was 50 mm using variable exposure times depending on θ with a scan width of 1.0°. Data collection was 100% complete to 67.679° in θ. A total of 60490 reflections were collected covering the indices, −5 < =h < =5, −22 < =k < =22, −16 < =l < =16. 4443 reflections were found to be symmetry independent, with a Rint of 0.0402. Indexing and unit cell refinement indicated a primitive, monoclinic lattice, containing one molecule of API and one molecule of dioxane in the asymmetric unit. The space group was found to be P21. The absolute structure was determined to be correctly assigned by anomalous dispersion with a refined flack parameter of -0.06(4). The Bijvoet analysis conducted using PLATON produced Bayesian Statistics consistent with the correct assignment of absolute structure, P2(true) = 1.000 P3(true) = 1.000 P3(rac-twin) = 0.1 × 10−43 P3(false) = 0.3 × 10−161 with a final Hooft parameter of −0.04(4). The data were integrated using the Bruker SAINT software program and scaled using the SADABS software program. Solution by intrinsic phasing (SHELXT) produced a complete phasing model consistent with the proposed structure.
All nonhydrogen atoms were refined anisotropically by full-matrix least-squares (SHELXL-2014). All carbon bonded hydrogen atoms were placed using a riding model. Their positions were constrained relative to their parent atom using the appropriate HFIX command in SHELXL-2014. Crystallographic data are summarized in Supplementary Table 1.
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