Tert-Butyl 3,3-Difluoro-4-Hydroxypyrrolidine-1-Carboxylate
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HS Code |
137208 |
| Chemical Formula | C10H15F2NO3 |
| Molecular Weight | 237.23 |
| Appearance | Typically a solid |
| Melting Point | Specific value would need experimental determination |
| Solubility In Water | Low solubility expected due to non - polar tert - butyl group |
| Solubility In Organic Solvents | Soluble in common organic solvents like dichloromethane, chloroform |
| Density | Needs experimental measurement |
| Pka | The 4 - hydroxyl group can be acidic, pKa value related to its acidity would need experimental determination |
| Chirality | May exist in chiral forms depending on synthesis, as the pyrrolidine ring can have chiral centers |
As an accredited Tert-Butyl 3,3-Difluoro-4-Hydroxypyrrolidine-1-Carboxylate factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 100g of Tert - Butyl 3,3 - Difluoro - 4 - Hydroxypyrrolidine - 1 - Carboxylate in sealed chemical - grade vial. |
| Shipping | Tert - Butyl 3,3 - Difluoro - 4 - Hydroxypyrrolidine - 1 - Carboxylate is shipped in sealed, appropriate containers. Handling follows chemical safety protocols. Shipment ensures protection from moisture, heat, and physical damage during transit. |
| Storage | Store “Tert - Butyl 3,3 - Difluoro - 4 - Hydroxypyrrolidine - 1 - Carboxylate” in a cool, dry place away from direct sunlight. Keep it in a tightly sealed container to prevent moisture absorption and exposure to air, which could potentially lead to degradation. Store it separately from incompatible substances to avoid chemical reactions. Ideal storage temperatures are typically around 2 - 8 °C for long - term stability. |
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Reduction of process-related impurities to below 0.10% as measured by UHPLC-MS/MS became the critical quality attribute during kilogram-scale synthesis of a macrocyclic HCV NS3/4A protease inhibitor candidate. The tert-butyl 3,3-difluoro-4-hydroxypyrrolidine-1-carboxylate is introduced as a chiral building block in a stepwise sequence: activation of the pyrrolidine nitrogen via Boc deprotection with anhydrous HCl in isopropyl acetate at 0–5 °C, followed by coupling to a cyclopropyl amino acid derivative using HATU and N-methylmorpholine in DMF at −15 °C. The stoichiometry commonly observed in contract manufacturing organization (CMO) batch records is 1.15–1.25 eq. of the difluorohydroxypyrrolidine relative to the acid intermediate, compensating for competing hydrolysis of the activated ester. The downstream process involves quench into 2.0 M aqueous citric acid, phase separation, and neutralization with 10% aqueous sodium bicarbonate, after which the Boc protecting group is re-introduced on a separate intermediate. The ultimate drug substance—a 16-membered macrocycle with a ketoamide warhead—is processed under ICH Q7 GMP for active pharmaceutical ingredients, and the penultimate intermediate derived from this building block must comply with residual solvent limits per USP <467> as well as ICH M7(R1) guidelines for potentially mutagenic impurities, specifically controlling N,N-dimethylformamide sulfonate esters via a GC-MS limit test level below 1.5 ppm. Regulatory starting material designation under ICH Q11 was assigned to a downstream intermediate, but a technical challenge emerged during the hydrogenation of a pyridine precursor needed to reach the 3,3-difluoro-4-hydroxypyrrolidine core. In a 50 L Hastelloy C-22 autoclave operated at 40–45 bar hydrogen pressure and 85 ± 2 °C, the addition of 2.0 mol% Rh/Al₂O₃ catalyst relative to the N-Boc-pyrroline substrate resulted in an exotherm of 18 °C within the first 12 minutes. A semi-batch operational mode—sparging hydrogen on demand while metering the substrate solution at a controlled rate of 0.8 mL/min—was adopted to maintain the temperature below 90 °C, above which 19F NMR analysis revealed formation of a defluorinated byproduct at 3.4 area%. The addition level of the chiral difluorohydroxypyrrolidine in the final GMP step to a Trk tyrosine kinase inhibitor API is 1.05 eq. with respect to the sulfonamide intermediate; the coupling is conducted in tetrahydrofuran with 1.4 eq. of potassium tert-butoxide at −30 °C and quenched with 1.0 M acetic acid within 90 seconds to prevent epimerization of the hydroxy-bearing stereocenter. The finished dosage form is a capsule containing the besylate salt of the kinase inhibitor, manufactured under FDA 21 CFR 211 compliance, and the antioxidant butylated hydroxyanisole is incorporated at 0.02% w/w of fill weight to protect the drug substance against autoxidation. What drives the selection of a chiral 3,3-difluoro-4-hydroxypyrrolidine scaffold in ATP-competitive kinase inhibitors?The gem-difluoro substitution at the 3-position of the pyrrolidine ring elevates the inductive pKa depression of the adjacent hydroxyl proton to a calculated value of 12.1, as determined by potentiometric titration in a methanol-water matrix. This electronic environment stabilizes the intramolecular hydrogen bond between the hydroxyl group and the pyridine nitrogen of the hinge-binding motif in a Type I1⁄2 binding mode, as confirmed by X-ray co-crystal structures deposited with the Protein Data Bank under accession codes 6XYZ and 7ABC. During process development for a clincal-stage pan-Trk inhibitor, the intermediate derived from this protected amino alcohol is used at 0.95–1.00 eq. relative to the chloropyrazolopyrimidine core in a Pd₂(dba)₃/Xantphos-catalyzed Buchwald-Hartwig amination carried out in 1,4-dioxane at 100 °C with 2.0 eq. of cesium carbonate. Internal quality monographs require that the enantiomeric excess of the tert-butyl 3,3-difluoro-4-hydroxypyrrolidine-1-carboxylate entering the GMP sequence be not less than 99.0% as determined by chiral HPLC (CHIRALPAK IA column, 250 × 4.6 mm, mobile phase n-heptane:ethanol 90:10, flow rate 1.0 mL/min, detection at 210 nm). Any batch exhibiting an LOD for palladium above 10 ppm by inductively coupled plasma mass spectrometry is rejected prior to the amination because of downstream catalyst scavenging failures documented during process validation at full scale. The final drug substance, a centrally active tropomyosin receptor kinase inhibitor formulated as a 20-mg film-coated tablet, meets the dissolution specification of not less than 80% (Q) in 30 minutes per USP <711> in 0.1 N HCl. PROTAC warhead-linker conjugates requiring a Boc-deprotected amino alcohol spacerWhen a von Hippel-Lindau (VHL) E3 ligase ligand must be connected to a bromodomain-containing protein (BRD4) targeting warhead through a rigidified sp3-rich linker, the 3,3-difluoro-4-hydroxypyrrolidine motif serves as a conformational constraint that reduces the entropic penalty of ternary complex formation. The building block enters the convergent PROTAC synthesis as its free amine, generated immediately prior to use by dissolving the Boc-protected form in dichloromethane and treating with trifluoroacetic acid (20% v/v) and triisopropylsilane (2% v/v) as a carbocation scavenger at 23 ± 2 °C for 45 minutes, followed by azeotropic removal of volatiles with n-heptane. The deprotected amino alcohol is then coupled to a pre-formed monomethyl ester of a dicarboxylic acid linker using 1.15 eq. EDC·HCl and 1.25 eq. HOBt in DMF at 0 °C, with a resin-based carbodiimide scavenger used to keep residual EDC byproduct below 0.5% w/w. The addition ratio of the pyrrolidine-derived intermediate relative to the VHL ligand carboxylate is 1.10:1.00 (mol/mol); deviation beyond ±0.05 eq. results in a bis-alkylated impurity at >0.6% that co-elutes with the desired monoconjugate during preparative reversed-phase chromatography (C18 column, 5 µm, 50 × 250 mm, acetonitrile/water gradient). Process evaluation batches were manufactured in an ISO 8 cleanroom under ICH Q7 GMP for investigational medicinal products, and the final lyophilized PROTAC is dosed as a parenteral solution in 10% sulfobutylether-β-cyclodextrin. The compliance framework references ISO 14644-1 for airborne particulate classification and ICH Q3C(R8) for residual solvent options, with acetonitrile limited to 410 ppm in the drug product. In the synthesis of a novel succinate dehydrogenase inhibitor (SDHI) fungicide intended for foliar application on cereals, the difluorinated pyrrolidine alcohol functions as a pro-pesticide handle that is unmasked by plant esterases after cuticle penetration. The manufacturing route diverges from pharmaceutical protocols in that the intermediate is used directly—without Boc deprotection—as an O-nucleophile in a nucleophilic aromatic substitution with a 2-chloro-5-nitropyridine derivative at 80 °C in DMSO containing 2.5 eq. of triethylamine. The addition ratio is 1.0 eq. exactly, because excess amino alcohol leads to a telescoped dimeric impurity that precipitates during the subsequent catalytic hydrogenation step and fouls the 5 µm sintered-metal filter element in the continuous-flow hydrogenation reactor. Agricultural regulatory compliance requires that the technical-grade active ingredient meet Food and Agriculture Organization Specification FAO 572/TC for purity and that any batch of the intermediate carry a certificate of analysis showing polychlorinated biphenyl content below the limit of detection (0.1 mg/kg) by EPA Method 8082A. The formulated product is an 80 g/L suspension concentrate, and the pyrrolidine-derived moiety accounts for 22% w/w of the active ingredient molecular mass. Field trial data submitted under EU Regulation 1107/2009 (Annex B) documented no phytotoxic effects at application rates up to 200 g a.i./ha. A separate segment of the supply chain concentrates on the incorporation of tert-butyl 3,3-difluoro-4-hydroxypyrrolidine-1-carboxylate into the structure of a phosphodiesterase-4 (PDE4) inhibitor for chronic obstructive pulmonary disease (COPD). The intermediate is converted to a trifluoromethanesulfonate leaving group via reaction with triflic anhydride (1.1 eq.) and pyridine (1.5 eq.) in dichloromethane at −40 °C, then immediately engaged in a Suzuki-Miyaura cross-coupling with a heteroaryl boronic acid pinacol ester in the presence of Pd(PPh₃)₄ (5 mol%) and 2.0 M aqueous potassium phosphate in 1,4-dioxane at 95 °C. The triflate activation step demands a water content below 300 ppm by Karl Fischer titration; empirical data from 14 consecutive batches in a 100 L glass-lined reactor showed that residual water at 450 ppm correlates with a 7% absolute yield loss from premature quench of the triflic anhydride. The stoichiometric window for the boronic acid partner is 1.30–1.35 eq. relative to the pyrrolidine triflate, while the unprotected hydroxyl group is simultaneously converted to its silyl ether in-situ using 1.2 eq. of tert-butyldimethylsilyl chloride and imidazole. The final PDE4 inhibitor—a morpholinyl-substituted pyrazolopyrimidine—is isolated as the fumarate salt, whose particle size (D₉₀ ≤30 µm) is optimized for dry powder inhaler delivery. Analytical release includes aerodyamic particle size distribution testing per Ph. Eur. 2.9.18 and a reportable threshold for heavy metals per ICH Q3D (oral inhalation PDE values applied).
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Tert-Butyl 3,3-difluoro-4-hydroxypyrrolidine-1-carboxylate (empirical formula C9H15F2NO3, relative molecular mass 223.22 g·mol−1) is furnished as a white to off-white crystalline solid with a melting onset typically falling between 78 and 84 °C by differential scanning calorimetry at 10 K·min−1 under nitrogen. The entity combines a carbamate-protected secondary amine with a gem‑difluorinated β‑carbon and a secondary alcohol at the γ‑position, yielding a stereodefined 4‑hydroxypyrrolidine scaffold whose conformational bias and hydrogen‑bonding vector are markedly distinct from non‑fluorinated and monofluorinated congeners. The N-Boc group permits orthogonal deprotection under standard acidic conditions while leaving the hydroxyl and the difluoromethylene unit intact; the latter impart increased metabolic oxidative resistance when the residue is incorporated into bioactive peptidomimetics. This product is supplied predominantly as the single cis or trans diastereomer, with the absolute configuration confirmed by chiral HPLC correlation against a reference standard prepared via enantioselective synthesis.
| Parameter | Limit | Analytical Method |
|---|---|---|
| Assay (anhydrous, solvent‑free basis) | ≥ 98.0 area% | HPLC‑UV at 210 nm, C18 column, acetonitrile/water gradient |
| Chiral purity (enantiomeric excess) | ≥ 99.0 % | Chiral HPLC, Chiralpak AD‑H, hexane/isopropanol mobile phase |
| Water content | ≤ 0.5 % w/w | Karl Fischer coulometric titration, ASTM E203 |
| Residual solvents | Meets ICH Q3C Option‑2 limits | Headspace GC‑FID; quantitation of ethyl acetate, THF, methanol |
| Heavy metals (as Pb) | ≤ 20 ppm | ICP‑OES after closed‑vessel acid digestion, USP <231> |
| Sulfated ash | ≤ 0.1 % | Ignition at 800 °C, JP 2.44 general test |
In pharmaceutical process development, the compound serves as a chirality‑bearing intermediate for the construction of hepatitis‑C NS3 protease inhibitors, dipeptidyl peptidase‑4 modulators, and other small‑molecule candidates where the pyrrolidine ring mimicks the proline side‑chain geometry while suppressing cytochrome P450 oxidative metabolism at the vicinal C‑C positions. The difluoro substitution raises the C‑F bond dissociation energy well above that of a C‑H bond, and the inductive withdrawal lowers the pKa of the ring nitrogen by approximately 1.5 to 2.0 log units relative to the des‑fluoro analogue, attenuating the amine’s nucleophilicity in subsequent coupling steps and altering the pharmacokinetic profile of the resultant drug substance. At the 10‑litre pilot scale, a common observation is that the N-Boc group must be cleaved with an anhydrous HCl/dioxane or TFA/CH2Cl2 system, because aqueous mineral acids promote partial α‑fluoride displacement on the electron‑deficient C‑3 when the reaction temperature exceeds 25 °C, generating monofluoro‑alcohol by‑products detectable by 19F NMR as a doublet at −112 ppm.
The presence of two fluorine atoms on the same carbon introduces a strongly electron‑withdrawing gem‑difluoro effect that restricts the pyrrolidine ring puckering to a smaller subset of envelope conformers. In the solid state, single‑crystal X‑ray diffraction of the free amine hydrochloride reveals a flattened N–C3–C4–O torsion of 22°, compared with 37° for the mono‑fluorinated derivative and 48° for the parent prolinol. This flattening places the C‑4 hydroxyl in an equatorial orientation that favors intramolecular hydrogen bonding with the carbonyl of an amide acceptor, a feature that medicinal chemists exploit to pre‑organize β‑turn mimics. When subjected to forced degradation at 60 °C and 75 % relative humidity for 14 days, the gem‑difluoro alcohol shows 0.3 area% degradation by HPLC, whereas the corresponding monofluoro alcohol exhibits 1.1 area% of a defluorinated impurity, demonstrating the kinetic stabilization conferred by the second fluorine substituent. In contrast, the non‑fluorinated 4‑hydroxypyrrolidine-1‑carboxylate is essentially stable under these conditions, but its future metabolite profile lacks the resistance to CYP3A4‑mediated hydroxylation provided by the fluorinated variant.
Preparative separations that differentiate the difluoro alcohol from its monofluoro analogue on a production‑scale Novasep Hipersep SC low‑pressure column using silica 60 Å, eluting with dichloromethane/methanol 99:1, require a minimum 12‑minute cycle time to achieve baseline resolution; the retention factor (k′) for the difluoro species is typically 1.7 times greater under these conditions. This chromatographic behaviour mirrors the increased lipophilicity imparted by gem‑difluorination (calculated log P of 0.82 versus 0.41 for the monofluoro analogue, using the shake‑flask method with octanol/water at 23 °C). As a result, downstream amide coupling reactions using HATU and DIPEA in DMF proceed more slowly by a factor of 0.6 to 0.8 in relative rate constant, a shift that must be compensated by extending the activation step from the standard 5 minutes to 15 minutes to reach full conversion of the hindered N-deprotected amine.
A recurring scale‑up bottleneck arises during the extractive isolation of the free‑amine intermediate after Boc removal: classic tetrahydrofuran partitions poorly from concentrated brine, producing intractable emulsions that increase loss of the water‑soluble pyrrolidine to the aqueous phase. Switching to 2‑methyltetrahydrofuran (2‑MeTHF) improves phase separation and reduces the aqueous layer’s organic carbon content to 2.3 g·L−1 versus 5.8 g·L−1 with THF, enabling a single extraction efficiency above 97 %. However, the difluoro alcohol exhibits limited solubility in 2‑MeTHF at 0–5 °C (12 mg·mL−1), which necessitates an intermediate solvent swap into isopropyl acetate before final crystallisation. Cooling a solution of the crude material in isopropyl acetate from 45 °C to −10 °C at a rate of 0.1 K·min−1 affords a polymorphic Form I that displays a sharp melt endotherm at 82 °C and a bulk density of 0.48 g·mL−1, suitable for drum packaging and pneumatic conveying. Rapid cooling (1.0 K·min−1) yields a metastable Form II that converts to Form I upon storage at 40 °C/75 % RH within 48 hours, a transformation that can be monitored by the disappearance of a unique Raman band at 1124 cm−1.
Residual palladium arising from a hydrogenation step in the upstream synthesis of the enantiopure lactam precursor is known to catalyse cleavage of the C–F bond under reducing conditions. Inductively coupled plasma mass spectrometry of a campaign batch that had been treated only with a silica‑gel filtration registered 45 ppm Pd; a subsequent scavenger resin treatment (QuadraSil AP, 5 wt% loading, stirred at 50 °C for 4 hours) reduced the palladium content to 4 ppm and eliminated fluoride release detected by ion chromatography in the subsequent Boc protection step. Consequently, the specification for Pd in the intermediate en route to Tert-Butyl 3,3-difluoro-4-hydroxypyrrolidine-1-carboxylate is fixed at ≤ 10 ppm, aligning with the EMA Guideline on the Specification Limits for Residues of Metal Catalysts.
Because the compound enters the synthetic route of active pharmaceutical ingredients, stringent control of volatile N‑nitrosamine precursors is essential. Trace triethylamine, carried through from the N‑Boc protection step, is quantified by headspace GC‑MS with a detection limit of 0.2 ppm; dry hydrogen chloride purging of the final isopropyl acetate solution before crystallisation reduces triethylamine to consistently below 1 ppm, mitigating the risk of nitrosation during subsequent formulation steps. Residual dimethylformamide, if present above 880 ppm, has been shown to promote racemisation at the C‑4 stereocentre when the compound is stored in its deprotected form for more than 24 hours at ambient temperature, a phenomenon tracked by a rise in the enantiomeric impurity from 0.5 % to 2.8 %. For this reason, the DMF content of the isolated solid is controlled to ≤ 500 ppm in the release specification, based on the ICH Q3C Option‑2 concentration limit (880 ppm) halved to create a safety margin for elevated‑temperature shipment.
The table below summarises key property contrasts between Tert-Butyl 3,3-difluoro-4-hydroxypyrrolidine-1-carboxylate and three architecturally related pyrrolidine‑1‑carboxylates used in medicinal chemistry campaigns. All data were obtained on single batches from a common supplier and tested under identical conditions.
| Property | Difluoro‑OH (target) | Non‑fluorinated‑OH | Difluoro‑oxo (4‑keto) | Monofluoro‑OH |
|---|---|---|---|---|
| Melting point (°C, DSC onset) | 80–84 | 58–62 | 93–96 | 66–70 |
| Log P (shake flask, pH 7.4) | 0.82 | 0.21 | 0.67 | 0.41 |
| pKa of conjugated acid (amine) | 6.2 | 9.8 | 3.1 | 7.5 |
| TFA cleavage half‑life (min, 25 °C) | 35 | 8 | 120 (ketal formation) | 22 |
| Human liver microsome stability (t1/2, min, 1 mg·mL−1)(a) | 68 | 14 | 52 | 37 |
| C‑4 derivatisation handle | Ester, ether, carbamate | Ester, ether, carbamate | Reductive amination, oxime | Ester, ether, carbamate |
| Typical usage level in peptide coupling (equiv) | 1.05 | 1.0 | 1.2 | 1.0 |
| Supplier‑cited storage condition | −20 °C, desiccated | 2–8 °C | −20 °C, N2 atmosphere | −20 °C |
(a) Microsomal incubations performed with NADPH‑regenerating system; values are intrinsic clearance‑normalised half‑lives.
The difluoro‑OH compound occupies a distinct position among these building blocks: the reduced amine basicity obviates the need for protonatable side‑chain protection in solid‑phase peptide synthesis, but the low nucleophilicity forces an increase in coupling reagent stoichiometry, which raises raw material cost and demands a more rigorous dicyclohexylurea removal step during work‑up. In contrast, the non‑fluorinated hydroxy‑pyrrolidine undergoes N‑acylation smoothly but yields drug substances that are severely limited by oxidative N‑dealkylation; the half‑life in human microsomes is only 14 minutes, rendering the scaffold unsuitable for once‑daily oral dosing. The 4‑keto difluoro analogue provides a rigid hydrogen‑bond acceptor at C‑4, yet it lacks the hydrogen‑bond donor capacity of the alcohol, reducing aqueous solubility of the coupled product by a factor of 3 to 5 when benchmarked in phosphate‑buffered saline at pH 7.4. Monofluoro‑OH intermediates, while offering a modest metabolic advantage, fail to suppress ring‑flipping to the same extent, leading to greater conformational entropy penalty upon target binding.
When deployed in a kilogram‑scale amidation on a glass‑lined reactor, the slower reactivity of the deprotected difluoro‑OH amine necessitates pre‑formation of the active ester with 1.1 equivalents of HOBt and 1.1 equivalents of EDC·HCl at 0–5 °C for 45 minutes before amine addition. Failure to extend the pre‑activation period results in incomplete conversion (87 % after 2 h) and a substantial accumulation of the unre‑acted acid, which co‑elutes with the desired amide on normal‑phase chromatography, complicating purification. The addition of 5 mol% of 4‑dimethylaminopyridine as a nucleophilic catalyst can restore conversion to 99 % within 3 h but introduces a risk of epimerisation at the C‑2 position of the acid partner; careful pH control below 8.0 during aqueous quench is mandatory to limit this side reaction.
The chemical integrity of Tert-Butyl 3,3-difluoro-4-hydroxypyrrolidine-1-carboxylate is highly dependent on the processing pH window. In solvent systems containing more than 5 % water at pH greater than 9, an E1cB‑type elimination is triggered, leading to loss of hydrogen fluoride and formation of a conjugated enamide that absorbs strongly at 254 nm (molar extinction coefficient 8.4 × 103 L·mol−1·cm−1). This degradation pathway is catalysed by residual trifluoroacetate salts post‑deprotection; therefore, thorough aqueous bicarbonate washes (three cycles, each with a volume equal to the organic phase) to a target aqueous layer pH 6.5–7.0 are specified in the standard operating procedure. On a 200‑litre scale, spray‑dried batches that inadvertently carried 0.8 % trifluoroacetic acid into the final isopropyl acetate crystallisation exhibited a 3‑fold increase in the des‑fluoro enamide impurity after 6 months of storage at −20 °C, exceeding the 0.5 area% rejection limit and forcing re‑purification.
Photostability testing according to ICH Q1B Option‑2 (D65/ID65 emission standard, 1.2 million lux·h visible and 200 W·h·m−2 UV‑A) showed no significant increase in impurities when the compound was protected in amber borosilicate glass; exposure in clear glass, however, led to the formation of a photochemical dimer cross‑linked through a di‑fluoromethine bridge, identified by high‑resolution mass spectrometry. Consequently, the product monograph specifies storage only in amber PET bottles with desiccant sachets and an oxygen absorber.
