Tert-Butyl (3R,4R)-3-Fluoro-4-Hydroxypyrrolidine-1-Carboxylate

What Limits the Gram-Negative Activity of Oral Carbapenems and How Fluorinated Pyrrolidine Side Chains Overcome This?

In the manufacturing route to next-generation oral carbapenem candidates structurally related to tebipenem pivoxil, the C‑2 side chain has been identified as the principal determinant of outer membrane penetration in Pseudomonas aeruginosa and extended-spectrum β‑lactamase‑producing Enterobacterales. The building block Tert-Butyl (3R,4R)-3-Fluoro-4-Hydroxypyrrolidine-1-Carboxylate serves as the direct precursor to a (3R,4R)‑3‑fluoro‑4‑mercaptopyrrolidine side chain that replaces the prototypical (2S,4S)‑4‑mercaptoproline motif. The substitution of hydrogen by fluorine at the C‑3 position lowers the pK_a of the pyrrolidinium nitrogen by approximately 0.8–1.2 log units, shifting the net charge at physiological pH and altering the hydrogen‑bonding footprint with penicillin‑binding protein 2; this has been correlated with a 4‑ to 8‑fold reduction in the MIC₉₀ against meropenem‑non‑susceptible Klebsiella pneumoniae isolates when the thioether‑linked fluoropyrrolidine is installed. Processing on a pilot‑scale GMP line commences with Boc‑deprotection using 4 M HCl/dioxane at 0–5 °C in a glass‑lined reactor, immediately followed by solvent displacement into anhydrous THF and dropwise addition of 1.05–1.15 equivalents of methanesulfonyl chloride in the presence of triethylamine, generating the corresponding mesylate without isolation. The subsequent thioacetylation with potassium thioacetate (1.3 eq) in DMF at 45 °C proceeds with full retention of configuration, and the crude thioester is hydrolysed with 0.5 M LiOH/MeOH to liberate the thiol. The free thiolate is then reacted in situ with the para‑nitrobenzyl‑protected carbapenem enolphosphate at −20 to −15 °C in acetonitrile, employing 1.20 equivalents of diisopropylethylamine to suppress ring‑opening side reactions. The terminal drug substance is an orally bioavailable pivaloyloxymethyl prodrug that delivers the active fluoro‑carbapenem after intestinal esterase hydrolysis. Industry‑specific compliance mandates full characterisation under ICH Q7 (GMP for Active Pharmaceutical Ingredients), residual solvent limits according to USP <467>, and elemental impurity control per ICH Q3D with specific focus on palladium and nickel. The bacterial endotoxin threshold, when the side chain is destined for sterile injectable formulations, is maintained below 0.15 EU/mg as verified by the USP <85> kinetic chromogenic LAL assay.

When Asymmetric Aldol Reactions Demand a Rigid Chiral Pocket: The Role of (3R,4R)-3-Fluoro-4-Hydroxypyrrolidine-Derived Siloxy Catalysts

The enantioselective construction of quaternary carbon centres in the synthesis of prostaglandin D₂ antagonists and selective glucocorticoid receptor modulators frequently relies on the Jørgensen–Hayashi catalyst class. Introduction of the C‑3 fluorine substituent into the pyrrolidine scaffold profoundly reshapes the catalyst’s conformational preorganisation. The fluorine gauche effect stabilises the exo envelope conformation of the pyrrolidine ring, thereby tightening the dihedral angle between the bulky diarylprolinol silyl ether and the enamine‑forming nitrogen by an estimated 6–8° relative to the non‑fluorinated analogue. Preparation of the catalyst starts with quantitative removal of the Boc protecting group from the titled building block using trifluoroacetic acid in dichloromethane (1:1 v/v, 0 °C to RT, 2 h) to furnish (3R,4R)‑3‑fluoro‑4‑hydroxypyrrolidine. This intermediate is then N‑alkylated with 3,5‑bis(trifluoromethyl)benzaldehyde under sodium triacetoxyborohydride conditions in 1,2‑dichloroethane at 15 °C, followed by silylation of the secondary hydroxyl with trimethylsilyl chloride (1.05 eq) and imidazole in dry DMF. The final siloxy catalyst is employed at a loading of 10–20 mol% relative to the aldehyde substrate. Operationally, the aldol reaction is executed in a low‑volume jacketed reactor with HPLC pump‑controlled syringe addition of neat aldehyde to a precooled (4 °C) solution of the silyl enol ether generated from the siloxy catalyst and propionaldehyde; the adiabatic temperature rise is clamped to ≤ 2 °C by adjusting the dosing rate. Under these conditions, anti‑aldol products with enantiomeric excesses exceeding 94% have been reported for α‑substituted acrolein acceptors. The downstream application in an API manufacturing setting requires compliance with ICH Q3A for catalyst residues, with a target purge factor for fluorine‑containing pyrrolidine derivatives validated through spiking studies on a Biotage® Isolera™ flash chromatography system. The terminal product types span chiral secondary alcohol building blocks destined for anti‑inflammatory or respiratory drug candidates currently in Phase II clinical evaluation. Residual solvent specifications are aligned with USP <467> Class 2 limits, and the absence of mutagenic nitrosamine impurities derived from triethylamine is confirmed by LC‑MS/MS with a reporting threshold of 0.03 ppm.

Replacement of L‑proline residues in ghrelin receptor inverse agonist sequences with (3R,4R)‑3‑fluoro‑4‑hydroxyproline has been demonstrated to enhance serum stability by shielding the adjacent peptide bond from endopeptidase cleavage, while the fluorine atom imparts a +0.6 log unit improvement in lipophilicity that facilitates passive membrane diffusion in intestinal epithelial Caco‑2 monolayers. In a solid‑phase peptide synthesis (SPPS) manufacturing environment operating under FDA 21 CFR Part 211, the titled compound is first converted to its Fmoc‑protected amino acid form: deprotected pyrrolidine hydrochloride is reacted with Fmoc‑OSu (1.1 eq) in aqueous sodium carbonate‑THF (pH 8.5–9.0) at 20–25 °C, with reaction progress monitored by HPLC (254 nm). The resulting Fmoc‑(3R,4R)‑3‑fluoro‑4‑hydroxyproline is incorporated into the growing peptidyl resin on a CS Bio® CS336X automated synthesizer using HATU/DIPEA activation. For each coupling cycle, 4.0 equivalents of the Fmoc‑fluoropyrrolidine building block are pre‑activated for 3 minutes in DMF prior to delivery, and the coupling time is extended to 120 minutes at 45 °C to overcome the reduced reactivity caused by the electron‑withdrawing fluorine substituent, which lowers the amine nucleophilicity by a factor of approximately 2.5 relative to proline. After cleavage with reagent K (TFA/phenol/water/thioanisole/EDT) and purification by preparative reversed‑phase C18 HPLC, the crude peptide is lyophilised to yield the final drug substance. The terminal product type is a metabolically stabilised peptide mimetic targeting the ghrelin‑O‑acyltransferase (GOAT) enzyme or a somatostatin subtype 5 antagonist; such peptides are typically processed under ICH Q3C residual solvent guidance, with a specification of ≤ 0.5 µg/day for individual genotoxic impurities as outlined in ICH M7. Quality assurance protocols mandate amino acid analysis with 6 M HCl hydrolysis and high‑resolution mass spectrometry to confirm the intact mass within ± 2 ppm.

Fluorine-Induced Pyramidalization at the Proline Nitrogen in TYK2 Pseudokinase Binders

TYK2 JH2 pseudokinase domain inhibitors that incorporate a prolyl amide isostere exhibit a well‑documented susceptibility to amide bond hydrolysis by human liver microsomes, which limits their oral exposure. The (3R,4R)‑3‑fluoro‑4‑hydroxypyrrolidine scaffold addresses this metabolic liability by inducing a 5.3° pyramidal bend at the nitrogen atom (χ₁ deviation from the planar sp² geometry) as measured by single‑crystal X‑ray diffraction of the Boc‑protected precursor, which translates into a 3.7‑fold improvement in hydrolytic stability at pH 7.4 and 37 °C. The manufacturing process for late‑stage functionalisation of the pyridopyrimidine core begins with Boc removal from the titled compound using 5–6 N HCl in isopropanol at 40 °C, yielding the hydrochloride salt that is filtered and dried under vacuum overnight. The free amine is generated with a 10% aqueous K₂CO₃ wash and directly subjected to a Schotten–Baumann‑type coupling with the activated ester of 2‑(difluoromethoxy)pyrido[3,4‑d]pyrimidine‑7‑carboxylic acid in a 2‑MeTHF/water biphasic system. The reaction is run at 0‑5 °C using 1.0 equivalent of the fluoropyrrolidine amine and 1.05 equivalents of the NHS‑ester, and the pH is maintained between 7.5 and 8.0 by automated dosing of 2 M NaOH to prevent protonation of the weakly basic pyrrolidine (pK_a ≈ 8.3). Following the coupling, the crude amide is reduced with sodium borohydride to convert residual aldehyde impurities into more polar alcohol derivatives that are easily removed during silica gel plug filtration. The final drug candidate is a selective TYK2 inhibitor with picomolar affinity for the pseudokinase domain. All processing steps performed under current Good Manufacturing Practice (cGMP) for Phase III clinical supply require adherence to ICH Q11 for starting material designation and a dedicated control strategy for nitrosamine risk based on EMA/511218/2020, which includes quantification of N‑nitrosoproline‑related impurities via UPLC‑HRMS with a limit of detection of 0.01 ppm. The downstream product type is an oral tablet formulation containing 6 mg of the TYK2 inhibitor anhydrous free base in a spray‑dried dispersion with HPMC‑AS.

Stabilising the Pyrrolidine Ring Against Oxidative Metabolism in DPP‑4 Antagonists

The predominant metabolic soft spot in omarigliptin‑class long‑acting DPP‑4 inhibitors is the α‑carbon of the pyrrolidine ring, which undergoes cytochrome P450‑mediated hydroxylation followed by dehydrogenation to an iminium species that is trapped by glutathione. Installation of a fluorine atom at the C‑3 position of the pyrrolidine, via incorporation of the titled building block, raises the oxidation potential by 0.25 V and shifts the site of metabolism to the distal piperazine‑2‑one ring, thereby extending the human half‑life to exceed 100 hours. In the commercial‑scale preparation of the key fluoropyrrolidine‑piperazinone intermediate, (3R,4R)‑3‑fluoro‑4‑hydroxypyrrolidine is first liberated from the Boc‑protected precursor by treatment with 98% formic acid at 50 °C for 4 hours, a method selected to avoid the formation of alkyl chloride adducts that are prevalent when HCl/dioxane is used. The resulting formate salt is exchanged to the free base on a Dowex® 550A OH ion‑exchange column, and the amine is immediately subjected to reductive amination with the N‑Boc‑piperazine‑2‑one‑3‑carbaldehyde (1.0 mol equivalent) in the presence of sodium triacetoxyborohydride (1.4 eq) in THF at 20 °C. The C‑4 hydroxyl group remains unprotected throughout the sequence and is selectively oxidised to the ketone in the final step using Dess–Martin periodinane (1.1 eq) in wet dichloromethane, yielding the saturated ketone that is the direct precursor to the active pharmaceutical ingredient. The addition ratio of the fluoropyrrolidine component is tightly controlled at 1.00 ± 0.02 equivalents to prevent over‑alkylation that generates a bis‑piperazinone impurity that is exceptionally difficult to purge by crystallisation. The terminal drug product type is a once‑weekly oral DPP‑4 inhibitor tablet, manufactured in a dedicated facility compliant with ISO 14644‑1 Class 8 cleanroom standards. Quality release of the fluoropyrrolidine intermediate requires a purity of ≥ 99.0% by HPLC‑CAD, enantiomeric excess ≥ 99.5% by chiral SFC, and residual heavy metals within the limits of ICH Q3D Option 1. Genotoxic impurity control is documented in an ICH M7‑compliant impurity fate and purge report, with confirmatory testing for mesityl oxide and triacetoxyborohydride‑derived N‑nitrosamines.

For ¹⁸F‑PET Imaging of Neuroinflammation: Non‑Radioactive Fluoro‑Pyrrolidine Reference Standard Manufacturing Under EudraLex Annex 3

Positron emission tomography tracers that quantify translocator protein (TSPO) expression or monoamine oxidase B density in neuroinflammatory plaques increasingly employ [¹⁸F]‑labelled pyrrolidine derivatives as metabolic trapping modules. While the radioactive analogue is synthesised from a tosylate‑activated precursor using the [¹⁸F]KF‑Kryptofix 2.2.2 complex, the corresponding 19F non‑radioactive reference standard is prepared in multi‑milligram quantities from the titled compound to establish chemical identity, radiochemical purity, and specific activity. The reference standard synthesis starts with Boc‑deprotection as described in prior sections, followed by acylation with 4‑fluorobenzoic acid using HATU (1.1 eq) and DIPEA (3.0 eq) in DMF at 25 °C. The crude product is purified by semi‑preparative HPLC on a Waters XBridge® C18 OBD™ column (19×150 mm, 5 μm) with a 0.1% TFA/water/acetonitrile gradient; the desired fraction is lyophilised to a white powder with a chemical purity exceeding 99.5% as determined by HPLC‑UV (λ=254 nm) and confirmed by Q‑TOF mass spectrometry. This material serves as the external reference for batch release of the [¹⁸F]‑labelled neuroinflammation tracer under EudraLex Volume 4 Annex 3 for radiopharmaceutical production. The addition ratio is not expressed as a formulation parameter but as a batch synthesis quantity: to qualify a new reference standard batch, a minimum of 25 mg is required to permit full ICH Q2(R1) validation of the HPLC method, including linearity (0.05–2.0 mg/mL), accuracy (recovery 97.0–103.0%), and precision (RSD ≤ 1.0%). The terminal product is a calibrated secondary chemical reference standard supplied with a Certificate of Analysis that reports residual solvents per USP <467>, elemental impurities per ICH Q3D, and enantiomeric purity by chiral HPLC. This standard directly supports the 21 CFR 212 and Ph. Eur. general monograph 1825 requirements for PET drug substance batch certification. In the hot cell production process, the fluorine‑19 standard is co‑injected with the ¹⁸F product onto an analytical radio‑HPLC to confirm identity by retention time matching; the accepted tolerance window is ± 2% of the calibrated retention time. Batch records also document the specific activity of the final clinical dose, which must exceed 37 GBq/µmol for a typical single human administration of 370 MBq.