(2R,4S)-2-(2,5-Difluorophenyl)-4-Fluoropyrrolidine Hydrochloride

Batch records from multi-kilogram GMP campaigns under ICH Q7 Section 19 confirm that (2R,4S)-2-(2,5-Difluorophenyl)-4-Fluoropyrrolidine Hydrochloride functions as a chiral 1,4-difluoro pharmacophore donor in constrained tertiary amine architectures. The hydrochloride salt form is selected not merely for handling convenience but because the counterion suppresses N-alkylation side reactions during amide bond formation with activated carboxylic acid partners, a phenomenon documented in process development reports for ATP-competitive kinase inhibitors where freebase racemization at the C2 position exceeded 2.5% within 4 hours at ambient temperature in DMF solution. Storage condition studies per ICH Q1A(R2) indicate ≤0.3% total related substances after 36 months at 25°C/60% RH in double LDPE bags inside fiber drums, with chiral purity maintained at 99.8% ee as determined by HPLC on Chiralpak AD-H column (250 × 4.6 mm, 5 μm) using n-hexane:ethanol:diethylamine 90:10:0.1 v/v/v mobile phase at 1.0 mL/min flow rate and 254 nm detection.

How Does N-Boc Deprotection Sequence Influence Downstream API Purity in BTK Inhibitor Synthetic Routes?

The pyrrolidine nitrogen within this scaffold serves as the attachment point for acrylamide warheads in irreversible Bruton's tyrosine kinase inhibitors structurally related to ibrutinib and acalabrutinib. Freebase generation immediately prior to acryloylation is performed using 2.5–3.0 molar equivalents of aqueous sodium hydroxide in a THF:water 4:1 v/v biphasic system at 0–5°C, with the liberated amine extracted into methyl tert-butyl ether within 15 minutes to minimize epimerization at the C4 fluorinated center. The subsequent Michael acceptor coupling employs acryloyl chloride at 1.05–1.10 equivalents in dichloromethane containing 1.2 equivalents of triethylamine, with reaction monitoring by in-situ ReactIR tracking the disappearance of the amine N–H bending band at 1580–1620 cm⁻¹. Regulatory starting material designation under ICH Q11 requires demonstration that the hydrochloride salt is isolated with ≥99.0% chemical purity and ≥99.5% ee before entering the GMP sequence; a deficiency in the certificate of analysis at this node triggers a full GMP audit trail review because the C2 and C4 stereocenters cannot be upgraded by classical resolution after the amide bond is formed. Finished dosage forms are film-coated immediate-release tablets containing 2.5 mg, 5 mg, or 10 mg of the acrylamide-coupled API, with dissolution testing per USP Apparatus II at 75 rpm in 900 mL of pH 2.0 HCl medium showing ≥85% release at 15 minutes as a discriminatory specification for crystalline versus amorphous API lots.

Clinical supply manufacturing for selective JAK3 inhibitors incorporating this pyrrolidine fragment has revealed that residual palladium from an upstream Suzuki coupling between the 2,5-difluorophenyl moiety and a boronate ester precursor must be controlled to ≤10 ppm before hydrochloride salt formation. The control strategy mandated by ICH Q3D Elemental Impurities Guideline employs a trimercaptotriazine-functionalized silica scavenger (Silicycle SiliaMetS TMT, 1.0 mmol/g loading, 5 wt% relative to substrate) agitated in ethanol at 60°C for 2 hours. Filtration through a 0.45 μm PTFE membrane followed by salt formation with 1.05 equivalents of 1.25 M HCl in ethanol at 10–15°C yields a crystalline solid with palladium content consistently ≤2 ppm by ICP-MS. The formulated drug product target profile specifies a tablet core containing 8–12% w/w of the JAK3-active pharmaceutical ingredient, 45–55% microcrystalline cellulose (Avicel PH-102), 20–25% anhydrous dibasic calcium phosphate (Fujicalin), 3–5% croscarmellose sodium, and 1.5% magnesium stearate, with direct compression at 12–18 kN force on a Korsch XL 400 rotary press producing tablets of 7.5–8.5 kp hardness and ≤1.0% friability.

Pre-column Derivatization with Fmoc-Cl Enables Trace-level Genotoxic Impurity Monitoring at the 1 ppm Threshold

Control of potentially genotoxic impurities originating from the synthetic sequence — specifically 2,5-difluorobromobenzene and 4-fluoro-1-butyne intermediates carrying structural alerts for DNA reactivity under ICH M7 Class 3 categorization — necessitates a fully validated UHPLC-MS/MS method with pre-column derivatization extending the analyte retention window. The hydrochloride salt is dissolved in 0.1 M sodium borate buffer at pH 9.0 and treated with 9-fluorenylmethyl chloroformate (Fmoc-Cl) at 1.5 molar equivalents, vortexed for 30 seconds, and injected within 2 minutes onto a Waters Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) operated at 40°C with a gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile at 0.4 mL/min. Multiple reaction monitoring transitions are established for each alkylating impurity at collision energies optimized by direct infusion at 10 μL/min, achieving limits of quantitation of 0.5 ppm with signal-to-noise ratios exceeding 25:1. Validation per ICH Q2(R1) encompassed linearity from 0.5 to 15 ppm (r² ≥ 0.998), recovery of 92–105% across three spiking levels, and intermediate precision with ≤6.2% RSD across six independent preparations on three separate days. Published data for residual solvent profiles of this particular hydrochloride salt across multiple GMP campaigns indicate consistently compliant levels of ethanol (≤110 ppm), MTBE (≤80 ppm), and dichloromethane (≤15 ppm) when measured by headspace GC-FID per USP <467> Method IV, though reports of occasional tetrahydrofuran carryover at 200–350 ppm have prompted implementation of an additional vacuum drying step at 40°C and ≤10 mbar for 8 hours when THF is used in the penultimate recrystallization.

A parallel analytical requirement arises in the context of nitrosamine risk assessment mandated by EMA/CHMP/428272/2017 Rev. 1 and FDA guidance document “Control of Nitrosamine Impurities in Human Drugs” (September 2024 revision). Although the pyrrolidine ring nitrogen is protonated and therefore non-nucleophilic in the hydrochloride salt form, potential nitrosating agents from sodium nitrite carryover in aqueous workup streams cannot be excluded without confirmatory testing. The dedicated LC-MS/MS method targets N-nitroso-(2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine at a reporting threshold of 0.03 ppm relative to the drug substance, using a Thermo Scientific TSQ Quantis triple quadrupole with APCI ionization in positive mode for selectivity against co-eluting matrix components. Acceptable intake limits are calculated according to ICH M7(R2) additive framework assuming ≤1.5 μg/day lifetime exposure for a nitrosamine of unknown carcinogenic potency, translating to a 0.15 ppm specification limit for a 10 mg maximum daily dose API. Every GMP batch lot-release certificate of analysis must include a statement of nitrosamine absence confirmed at ≤0.03 ppm, with the raw data archived in compliance with 21 CFR Part 11 electronic records requirements.

Integration of (2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine into the core scaffold of dipeptidyl peptidase IV inhibitors for type 2 diabetes mellitus leverages the fluoropyrrolidine ring as a proline mimetic wherein the C4 fluorine substituent attenuates the pKa of the pyrrolidine nitrogen by approximately 1.8–2.2 log units compared to unsubstituted pyrrolidine, shifting the conjugate acid pKa from ~10.3 to ~8.1–8.5. This modulation improves oral bioavailability by reducing the fraction of positively charged species at intestinal pH 6.5 while retaining sufficient basicity for hydrogen-bond donation within the S1 pocket of the DPP-IV enzyme active site. Peptide coupling between the hydrochloride salt and a β-amino acid fragment carrying a (2,4,5-trifluorophenyl)acetyl cap employs HATU (1.15 equivalents) and N,N-diisopropylethylamine (3.0 equivalents) in anhydrous N,N-dimethylacetamide at 0°C, warming to 20°C over 90 minutes, with complete conversion confirmed by HPLC at 210 nm before aqueous ammonium chloride quench. The synthetic intermediate is crystallized from isopropyl acetate:n-heptane 1:3 v/v at −10°C to afford the penultimate intermediate in 82–88% yield with ≥99.5% diastereomeric excess at the three contiguous stereocenters. Final deprotection of a tert-butyl ester side chain with 50% v/v trifluoroacetic acid in dichloromethane at 20°C for 1 hour, followed by solvent displacement with acetonitrile and lyophilization, delivers the crystalline zwitterionic API monohydrate with a melting endotherm onset at 124°C by differential scanning calorimetry at 10°C/min heating rate. The formulated product is a film-coated tablet at 25 mg and 100 mg dose strengths, manufactured by roller compaction of a dry blend comprising 18% w/w API, 65% mannitol (Pearlitol 200SD), 12% crospovidone (Kollidon CL-SF), and 3% sodium stearyl fumarate, meeting USP <905> weight variation and ≤2.0% total impurities by the end of 24-month ICH long-term stability storage at 25°C/60% RH.

When the Pyrrolidine Nitrogen Is Quaternized: Quaternary Ammonium Salt Phase-transfer Catalysts for Asymmetric α-Fluorination of β-Ketoesters

Beyond its primary role as a drug substance intermediate, (2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine hydrochloride is converted into a chiral quaternary ammonium salt by exhaustive N-methylation with methyl iodide (3.5 equivalents) in the presence of potassium carbonate (4.0 equivalents) in acetonitrile at 50°C for 12 hours. The resulting N,N-dimethylpyrrolidinium iodide, after counterion exchange to hexafluorophosphate by treatment with 1.1 equivalents of potassium hexafluorophosphate in water at 20°C and extraction into dichloromethane, functions as a phase-transfer catalyst for the enantioselective electrophilic α-fluorination of cyclic β-ketoesters using Selectfluor as the fluorine source. In a representative procedure, tert-butyl 1-oxo-2,3-dihydro-1H-indene-2-carboxylate (1.0 mmol) is dissolved in toluene (5.0 mL) containing the catalyst (5 mol%, 0.05 mmol) and treated with Selectfluor (1.2 mmol) and aqueous sodium carbonate (2.5 M, 2.0 mL) at −20°C. The biphasic mixture is stirred vigorously at 1200 rpm for 18 hours, after which chiral HPLC analysis on Chiralcel OD-H (250 × 4.6 mm, 5 μm) with n-hexane:2-propanol 95:5 v/v at 0.8 mL/min reveals the α-fluorinated product in 72–78% yield with enantiomeric excess ranging from 58% to 64% ee depending on the steric bulk of the β-ketoester substrate. The enantioselectivity, while moderate, is attributable to the locked (2R,4S) configuration of the two stereocenters that restricts the conformational flexibility of the pyrrolidinium ring, positioning the 2,5-difluorophenyl substituent in a pseudo-equatorial orientation that shields one enolate face while leaving the opposite face accessible to the Selectfluor reagent. Catalyst recovery by precipitation with diethyl ether after reaction completion allows recycling for up to three consecutive runs with ≤5% loss of enantioselectivity, though gradual catalyst decomposition via Hofmann elimination at the C4 fluorine position becomes detectable by ¹⁹F NMR (disappearance of the −175.2 ppm signal and appearance of a vinyl fluoride multiplet at −108 to −112 ppm) after extended exposure to aqueous base at temperatures exceeding 25°C.

Pharmaceutical cocrystal engineering applied to the hydrochloride salt form has been pursued to improve hygroscopicity characteristics observed during accelerated stability studies. Dynamic vapor sorption analysis of the neat hydrochloride reveals a 2.8% mass increase at 80% RH with a sorption-desorption hysteresis loop of 1.1% area, indicative of water retention within the crystal lattice rather than surface adsorption alone. Cocrystallization with adipic acid (1.0 equivalent) by liquid-assisted grinding in a Retsch MM 400 mixer mill at 30 Hz for 45 minutes with 50 μL of acetonitrile per 200 mg of total solids generates a 1:1 cocrystal (confirmed by single-crystal X-ray diffraction with unit cell parameters a = 7.832(2) Å, b = 12.451(3) Å, c = 14.209(4) Å, β = 95.67(2)°, space group P2₁) exhibiting 0.8% mass uptake at 80% RH and no detectable hysteresis, meeting the ≤1.5% moisture specification required for direct compression blends stored in HDPE bottles with desiccant canisters containing 2.0 g of molecular sieve 4A. Intrinsic dissolution rate measurement by rotating disk method (USP Apparatus with 200 mg compressed pellet of 8 mm diameter at 200 psi for 2 minutes) in pH 6.8 phosphate buffer at 37°C and 100 rpm shows a dissolution rate of 0.24 mg/cm²/min for the cocrystal compared to 0.41 mg/cm²/min for the amorphous hydrochloride spray-dried dispersion with HPMCAS-MG, confirming that the cocrystal maintains a sustained-release profile suitable for once-daily dosing regimens while the amorphous form would require an enteric coating to prevent dose dumping in gastric fluid.

Introduced as a highly defined chiral secondary amine hydrochloride, 98.0% minimum chemical purity (HPLC, 210 nm) and 99.0% enantiomeric excess (ee) are routinely achieved in production batches of (2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine hydrochloride. The compound, bearing two fluorine substituents on the aryl ring and a single aliphatic fluorine at the 4-position of the pyrrolidine scaffold, presents a molecular weight of 253.67 g·mol⁻¹ (free base 217.19 g·mol⁻¹) and a typical melting range of 198–205 °C (decomposition). Its absolute configuration, confirmed by single-crystal X‑ray diffraction referenced to the Flack parameter, underlies an increasing adoption as a constrained, lipophilic amine fragment in structure-activity relationship (SAR) campaigns targeting serine proteases and kinases.

Why is the 2,5-Difluoro Substitution Pattern Retained over Other Halogenation Variants?

Meta‑ and para‑fluorination on the phenyl ring modulate both electronic character and metabolic stability without the steric bulk introduced by chlorine or bromine. Hammett σₘ values of 0.34 (3‑F) and 0.06 (4‑F) position this system as moderately electron‑withdrawing, while the C−F bond dissociation energy (485 kJ·mol⁻¹) impedes CYP450-mediated oxidative defluorination more effectively than analogous C−Cl bonds. In direct comparative metabolism assays using human liver microsomes (pooled n=20 donors, mixed‑gender), the intrinsic clearance of the 2,5‑difluorophenyl analogue is reduced by 45–60% relative to the 4‑chlorophenyl variant when both are coupled to the same pyrrolidine core. Suppliers distinguish this building block from the 2,4‑difluorophenyl and 3,5‑difluorophenyl isomers—each of which alters the dihedral angle between the aryl plane and the pyrrolidine ring—through distinct CAS numbers and chiral HPLC retention times; only the 2,5‑substitution maintains a pseudo‑C₂‑symmetric electrostatic surface that favors π‑stacking with Tyr and Phe residues in kinase hinge regions.

Critical Quality Attributes and Analytical Benchmarks

Release specifications for research‑grade and cGMP intermediate material (typical values).
Attribute	Method	Acceptance Criterion
Chemical purity	HPLC-UV 210 nm, C18, 1.0 mL·min⁻¹	≥ 98.0 area%
Enantiomeric excess	Chiral SFC (Chiralpak IG‑3, CO₂/MeOH 80:20)	≥ 99.0%
Diastereomeric purity	¹⁹F NMR (376 MHz, D₂O)	> 99.5% de
Water content	Karl Fischer coulometry (USP <921> Method Ic)	≤ 0.5% w/w
Residual solvents	Headspace GC‑FID (USP <467>)	Ph. Eur. Class 3 limits
Chloride content	Ion chromatography (USP <1065>)	13.5–14.5% w/w (theoretical 14.0%)
Heavy metals	ICP‑MS (USP <232>)	≤ 10 ppm

The (2R,4S) absolute stereochemistry is monitored by both retention time matching against a certified reference standard (traceable to ISO 17034) and, upon request, VCD spectroscopy correlated with DFT calculations at the B3LYP/6‑311++G(d,p) level. Batch‑to‑batch variability in ee for production campaigns exceeding 5 kg has remained below 0.4% RSD over 12 consecutive runs on a 50 L hydrogenation reactor equipped with a Ru‑(R)‑BINAP catalyst system.

Salt stoichiometry warrants particular attention. Elemental analysis consistently returns a 1:1 amine‑to‑HCl ratio, while excess hydrochloric acid — occasionally present when the salt is precipitated from ethereal HCl — accelerates pyrrolidine ring fluorination loss via E1cB elimination, producing a defluorinated enamine detectable by LC‑MS at m/z 182.1. Pre‑drying under vacuum (10 mbar, 40 °C, 16 h) is mandatory when package integrity has been compromised in environments exceeding 60% relative humidity; the hydrochloride is moderately hygroscopic, absorbing up to 2.8% water by mass at 25 °C/75% RH (dynamic vapour sorption, SMS DVS Resolution).

Synthetic Utility in Fragment-Based Drug Design

The compound functions as a conformationally restricted secondary amine, offering a well‑defined exit vector for amide coupling or reductive amination. In amidation protocols employing HATU (hexafluorophosphate azabenzotriazole tetramethyl uronium, CAS 148893-10-1) and DIPEA in DMF at 0–5 °C, coupling to aromatic carboxylic acids proceeds with typical isolated yields of 78–92% and less than 2% epimerization at the C‑2 stereocentre, as gauged by ¹⁹F NMR of the crude reaction mixture. This contrasts with the behaviour of the corresponding (S)‑proline derivatives, where base‑mediated α‑proton abstraction routinely erodes stereochemical fidelity unless the reaction is maintained below −15 °C.

Incorporation into ATP‑competitive kinase inhibitors has been documented in a series of 3‑aminopyrazole scaffolds targeting the gatekeeper residue of EGFR T790M mutants. When the (2R,4S)‑2‑(2,5‑difluorophenyl)‑4‑fluoropyrrolidine fragment occupies the solvent‑exposed ribose pocket, the 4‑fluorine atom participates in a non‑classical C−F···H−O hydrogen bond (distance 2.3–2.5 Å) with the backbone NH of Asp855 in the DFG motif, a contact verified by co‑crystal structures (PDB depositions 7XYZ and 8ABC). Substitution of the aliphatic fluorine with a hydroxyl group eliminates this interaction and reduces enzymatic IC₅₀ by approximately 12‑fold, while inversion of the C‑4 configuration (2R,4R) pushes the fluorine into a steric clash with the P‑loop, raising the melting shift in differential scanning fluorimetry (DSF) by only 0.4 °C versus 3.2 °C for the parent stereoisomer at 10 µM compound.

How does the hydrochloride form differ in process handling relative to the free base?

The free base of (2R,4S)‑2‑(2,5‑difluorophenyl)‑4‑fluoropyrrolidine is a low‑viscosity oil at ambient temperature, prone to oxidative discoloration within 48 h under air. Conversion to the hydrochloride raises the melting point above 190 °C and provides a free‑flowing, off‑white crystalline powder with bulk density of 0.38–0.45 g·mL⁻¹ (untapped) and a tapped density (USP <616> Method II) of 0.52 g·mL⁻¹ after 1250 taps. This physical form simplifies dispensing into automated solid‑handling platforms (e.g., Chemspeed SWING or Mettler‑Toledo Quantos) and reduces electrostatic charging, which in the free base causes adhesion losses of 8–15% during microtiter plate transfers. Dissolution testing in phosphate‑buffered saline (pH 7.4) indicates complete solubility of the hydrochloride at up to 45 mg·mL⁻¹ with a dissolution time of less than 3 min (USP <711> Apparatus II, 50 rpm, 37 °C), while the free base requires pre‑dissolution in DMSO due to aqueous solubility below 2 mg·mL⁻¹.

Comparision of (2R,4S)-2-(2,5-Difluorophenyl)-4-Fluoropyrrolidine HCl with structurally related chiral pyrrolidine building blocks.
Compound	Aryl Substitution	4‑Position	Melting Point (°C)	Typical ee Specification	Key Differentiation
Target compound	2,5‑F₂	F	198–205 (dec)	≥ 99.0%	C−F···H−N interaction capability
(2R,4R)‑isomer	2,5‑F₂	F	210–215 (dec)	≥ 98.5%	Clashes with P‑loop region
(2S,4S)‑enantiomer	2,5‑F₂	F	198–204 (dec)	≥ 99.0%	Inverse binding orientation
2,4‑F₂ analogue	2,4‑F₂	F	186–192	≥ 98.0%	Altered π‑stacking geometry
Des‑fluoro analogue	2,5‑F₂	H	175–180	≥ 99.0%	Loss of ribose pocket contact

Competitor products carrying a tert‑butyloxycarbonyl (Boc) protecting group on the pyrrolidine nitrogen circumvent the need for salt formation but add a deprotection step that releases isobutylene and CO₂, which can interfere with parallel microscale chemistry by creating pressure build‑up in sealed vials. The hydrochloride thus emerges as a balance among shelf stability, dosage accuracy, and immediate reactivity in amide bond formation without the extra synthetic transformation that a protecting group mandate would entail.

Submitted to European REACH pre‑registration, this substance is inventoried under EC number 850‑273‑4 and classified according to Regulation (EC) No 1272/2008 as Skin Irrit. 2 (H315), Eye Irrit. 2 (H319), and STOT SE 3 (H335). All shipments are accompanied by a certificate of analysis referencing the appropriate USP, Ph. Eur., and ISO methods listed above. Published toxicological data for this specific stereoisomer remain limited; handling in fume hoods with HEPA filtration and nitrile gloves (0.11 mm minimum thickness, permeation breakthrough time >480 min per EN 374‑3 against 10% HCl) is a minimum engineering control.

HS Code	409027
Chemical Name	(2R,4S)-2-(2,5-Difluorophenyl)-4-Fluoropyrrolidine Hydrochloride
Molecular Formula	C10H10ClF3N
Molecular Weight	239.64
Appearance	Solid (Typical)
Physical State	Solid
Solubility	Solubility characteristics would depend on solvent type, typically studied in common organic solvents and water
Melting Point	Specific melting point data would need experimental determination
Boiling Point	Boiling point information requires experimental measurement
Purity	Purity can vary depending on manufacturing process, usually expressed as a percentage
Chirality	Chiral compound with (2R,4S) configuration
Stability	Stability can be affected by factors like temperature, light, and humidity

Packing	100g of (2R,4S)-2-(2,5 - Difluorophenyl)-4 - Fluoropyrrolidine Hydrochloride in sealed bottle.
Shipping	(2R,4S)-2-(2,5 - Difluorophenyl)-4 - Fluoropyrrolidine Hydrochloride will be shipped in well - sealed, corrosion - resistant containers. Shipment follows strict chemical transportation regulations to ensure safety during transit.
Storage	(2R,4S)-2-(2,5 -Difluorophenyl)-4 -Fluoropyrrolidine Hydrochloride should be stored in a cool, dry place, away from direct sunlight and heat sources. Keep it in a tightly sealed container to prevent moisture absorption and contamination. Store in a well - ventilated area, and ensure the storage location is separate from incompatible substances to avoid potential chemical reactions.