3-(3-Hydroxypyrrolidine-1-Carbonyl)Pyrazine-2-Carboxylic Acid

In commercial campaigns targeting second-generation HCV NS3/4A protease inhibitors, the (3S)-3-hydroxypyrrolidine fragment embedded in 3-(3-Hydroxypyrrolidine-1-Carbonyl)Pyrazine-2-Carboxylic Acid becomes the critical topology-determining element for the P2 cap region. Manufacturing batches executed under ICH Q7 Chapter 8.3 employ this carboxylic acid as the electrophilic coupling partner, activated in anhydrous N,N-dimethylformamide with 1.05 to 1.15 molar equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl) and 0.10 equivalents of 1-hydroxybenzotriazole (HOBt) hydrate, relative to the amine nucleoside isostere that provides the P1–P3 backbone. The reaction mass is maintained at 0 °C ± 2 °C in a glass-lined 1,600 L vessel fitted with a triple-pitched retreat-curve impeller operating at 85 rpm tip speed; exothermic excursion beyond 5 °C triggers pyrazine ring N-oxide formation, detectable by LC–MS as a +16 Da adduct exceeding 0.7 area%. After aqueous work-up with 10 wt% citric acid, the methyl tert-butyl ether extract is concentrated on a wiped-film evaporator at 40 °C jacket temperature and 25 mbar vacuum, and the crude oil is crystallized from isopropyl acetate/n-heptane (3:7 v/v) with a seeding protocol that restricts supersaturation to ΔT < 8 °C relative to the cloud point. The isolated crystalline solid is dried in a double-cone rotary vacuum dryer at 45 °C and 5 mbar until Karl Fischer moisture reads below 0.10 wt%; batches that surpass 0.15 wt% water exhibit 2–4% yield erosion during subsequent HATU-mediated fragment condensation due to competing hydrolysis of the active ester. The resulting intermediate carries a certificate of conformance listing purity ≥ 98.5% by HPLC (C18, gradient acetonitrile/0.1% phosphoric acid, UV 210 nm), residual solvents compliant with ICH Q3C limits, and enantiomeric excess ≥ 99.0% determined on a Chiralpak IA-3 column. This intermediate is directly charged into the next GMP step assembling the full macrocyclic protease inhibitor, which, after spray-dried dispersion formulation with hypromellose acetate succinate, becomes an oral film-coated tablet for once-daily administration against genotype 1, 2, and 4 hepatitis C infections.

What Stoichiometric Imbalance Drives Amide Bond Formation with Hindered Piperazine Nucleophiles to >95% Conversion?

When the target architecture shifts from a linear peptide mimetic to a sterically congested biaryl ether bearing a 2,6-dimethylpiperazine terminus—encountered in certain ATP-competitive FLT3 kinase inhibitor scaffolds—the intrinsic reactivity of 3-(3-Hydroxypyrrolidine-1-Carbonyl)Pyrazine-2-Carboxylic Acid requires deliberate over-stoichiometry. Laboratory calorimetry data (Omnical SuperCRC, isothermal mode at 25 °C) indicate that the electronically deactivated pyrazine carbonyl exhibits an activation enthalpy ΔH‡ of 57 kJ·mol⁻¹ when attacking the axial conformer of the disubstituted piperazine, and the reaction stalls at 78–82% conversion under 1.05:1 acid-to-amine ratios. Process development consequently fixes the input ratio at 1.30:1 (acid/amine) combined with 2.0 equivalents of HATU and 1.5 equivalents of N,N-diisopropylethylamine in N-methyl-2-pyrrolidone at a total concentration of 0.45 M. This stream is processed through a Corning® Advanced-Flow™ G1 SiC reactor module (hydraulic diameter 0.6 mm, thermal fluid at -5 °C) with a residence time of 14 min to dissipate the instantaneous adiabatic temperature rise, which otherwise reaches +38 °C in batch mode and precipitates the N-acylurea by-product. The downstream purification integrates a continuous multi-column simulated moving bed (SMB) system equipped with eight Chiralpak AD columns (20 µm, 10 cm ID) using acetonitrile/water 85:15 v/v as eluent, isolating the desired (S)-piperazinyl amide with an optical purity of 99.4% ee and a throughput of 0.52 kg feed per kilogram of stationary phase per day. Operation is governed by ISO 13408-1:2008 aseptic processing principles during the final crystallization even though the intermediate itself is not sterile, a precaution enforced because degradation products exhibit cytotoxicity in the Ames II assay at levels above 15 ppm. The dried intermediate (residual NMP ≤ 290 ppm per ICH Q3C) serves as the penultimate fragment in the convergent synthesis of an investigational monotherapy for relapsed/refractory FLT3-ITD-positive acute myeloid leukemia, whose hydrochloride salt is lyophilized in 50 mL Type I borosilicate vials for intravenous infusion.

When a New Drug Application holder requests a Type II Drug Master File for the reference substance of 3-(3-Hydroxypyrrolidine-1-Carbonyl)Pyrazine-2-Carboxylic Acid, the purification pathway diverges sharply from bulk intermediate manufacturing because the limit for any single unspecified impurity must fall below 0.10 area% per USP <11> and ICH Q2(R1) requirements for a qualified working standard. The starting material—typically a technical-grade lot assaying at 97.2%—is first recrystallized from a ternary solvent system of acetone, water, and dimethyl sulfoxide (85:10:5 v/v/v) under a nitrogen stream to eliminate light-absorbing oligomers, then subjected to preparative reversed-phase high-performance liquid chromatography on a 50 mm ID column packed with 10 µm C18 fully end-capped silica. The mobile phase uses an isocratic composition of methanol and 0.05 vol% trifluoroacetic acid in water (48:52 v/v) at a flow rate of 80 mL·min⁻¹; fraction collection is triggered by a UV threshold of 2 mAU at 272 nm, corresponding to the π→π* transition of the pyrazine ring. Pooled fractions exhibiting single-peak purity are flash-frozen in a stainless-steel lyophilizer shelf at -45 °C and sublimated at 0.020 mbar for 72 h, yielding a fluffy white powder with a water content of 0.08 wt% by Karl Fischer coulometry. The standard is dispensed into 100 mg amber Type I vials under argon, and each vial is assigned an expiry of 18 months when stored at -20 °C and protected from light; periodic re-qualification conducted according to Ph. Eur. 5.18 employs differential scanning calorimetry (onset of melting endotherm at 187.2 °C) and quantitative ¹H NMR using dimethyl sulfone as internal standard to certify no detectable hydrolysis. Laboratories that integrate this reference material into compendial LC methods for process-related impurity analysis achieve a signal-to-noise ratio of >100:1 for the principal peak, enabling a limit of quantitation of 0.03 µg·mL⁻¹. Published data for this specific configuration in a pharmacopeial monograph is limited; nonetheless, the above protocol aligns with the general guidelines of WHO Technical Report Series No. 943 on chemical reference substances.

If the Ligand-to-Zirconium Cluster Ratio Is Adjusted to 1:1, the Resulting MOF Exhibits a BET Surface Area Exceeding 800 m²/g

Within the field of reticular chemistry, 3-(3-Hydroxypyrrolidine-1-Carbonyl)Pyrazine-2-Carboxylic Acid functions as a ditopic N,O-chelating linker that bridges Zr₆(μ₃-O)₄(μ₃-OH)₄ nodes in a fcu topology framework. The synthesis protocol employed on a 500 mL autoclave scale charges zirconium tetrachloride (ZrCl₄, sublimed grade, 99.99% trace metals basis) alongside the ligand at a molar ratio of 1.00:1.00 (metal to ligand), with the ligand constituting 18.5 wt% of the total solid mass and 14.2 wt% when including the modulator. N,N-dimethylformamide (DMF, anhydrous, water <50 ppm) serves as the solvent at a volume-to-solid ratio of 22 mL·g⁻¹, and formic acid (≥98%) is added as a competitive modulator at a 50 molar equivalent excess relative to Zr. The mixture is sonicated in a sealed PFA-lined vessel until optically clear, then heated in a gravimetric convection oven at 120 °C for 48 h under autogenous pressure, after which the crystalline precipitate is isolated by centrifugation at 8,000 × g for 15 min. Because the free pyrrolidine hydroxyl group is hygroscopic, all weighing operations are conducted inside a Vacuum Atmospheres glovebox with <1 ppm H₂O and <1 ppm O₂; exposure to ambient relative humidity above 60% for more than 15 min prior to solvothermal treatment leads to irreversible ligand hydration that depresses the BET surface area by 18–22%. Post-synthetic activation follows a two-stage solvent exchange: the as-synthesized MOF is immersed in fresh DMF for 24 h (three exchanges), then in anhydrous methanol for 48 h (four exchanges), before being degassed at 150 °C under dynamic vacuum (10⁻³ mbar) for 12 h. The resulting microcrystalline powder exhibits a Type I nitrogen sorption isotherm at 77 K with a Brunauer–Emmett–Teller surface area of 842 m²·g⁻¹ measured according to ISO 9277:2010 (linear region P/P₀ = 0.05–0.30) and a micropore volume of 0.31 cm³·g⁻¹ by the t-plot method. Thermogravimetric analysis under flowing air (ASTM E1131, ramp 10 °C·min⁻¹) shows framework decomposition commencing at 385 °C. Industrial research groups have evaluated this Zr-MOF in mixed-matrix membranes for post-combustion CO₂ capture, where its incorporation at 12 wt% loading into a Matrimid® 5218 polyimide matrix raises the CO₂/N₂ selectivity from 28 to 47 at 35 °C and 4 bar feed pressure.

Pyrazine-2-Carboxylate Building Blocks in Fungicide Lead Optimization Programs

Agrochemical discovery pipelines targeting Oomycete pathogens resistant to phenylamide fungicides have drawn on 3-(3-Hydroxypyrrolidine-1-Carbonyl)Pyrazine-2-Carboxylic Acid as a bioisostere of the natural product pyrazinecarboxylate pharmacophore. During the synthesis of candidate molecules for FRAC code group P5, the acid is first converted to the corresponding carbonyl chloride by refluxing in thionyl chloride (SOCl₂, 2.5 equivalents) with catalytic 0.5 mol% DMF in anhydrous toluene (8 vol relative to substrate) under a nitrogen sweep for 3.5 h. After evaporation under reduced pressure at 45 °C and co-stripping with toluene (2 × 200 mL), the lemon-yellow solid acyl chloride is used without further characterization and is added dropwise at -10 °C to a dichloromethane solution of the requisite 2-chloro-5-(trifluoromethyl)aniline (1.00 equivalent) and triethylamine (1.20 equivalents), keeping the internal temperature below -5 °C to avoid the formation of the symmetrical anhydride dimer. The batch is then warmed to 22 °C over 90 min, quenched with 5 wt% sodium bicarbonate, and the organic layer is dried over anhydrous magnesium sulfate. Flash chromatography on silica gel 60 Å (gradient ethyl acetate in hexanes 30% to 70%) furnishes the target N-(2-chloro-5-(trifluoromethyl)phenyl)-3-(3-hydroxypyrrolidine-1-carbonyl)pyrazine-2-carboxamide as a white solid in 87% isolated yield and 96.3% purity (HPLC, 254 nm). According to internal specifications referenced against CIPAC Handbook L, the technical-grade intermediate must exhibit a melting point range within 2.0 °C of the reference and a water content below 0.3 wt% before it is enrolled in GLP greenhouse efficacy trials. The formulated end product—typically a 200 g·L⁻¹ suspension concentrate containing the active ingredient along with naphthalene sulfonate dispersant and silicone anti-foam—is applied as a foliar spray at 100 g a.i.·ha⁻¹, providing curative activity against Phytophthora infestans isolates bearing the F129L cytochrome b mutation. Long-term stability studies guide storage at -20 °C under argon in opaque polyethylene drums fitted with desiccant cartridges because exposure to visible light and ambient moisture accelerates hydrolysis of the exocyclic amide bond, generating 3-hydroxypyrrolidine and pyrazine-2,3-dicarboxylic acid degradation fragments detectable by ion chromatography after 90 days at 40 °C / 75% RH.