(3S,4S)-3,4-Dihydroxy-1-Pyrrolidinecarboxylic Acid Phenylmethyl Ester
|
HS Code |
663366 |
| Chemical Formula | C12H15NO4 |
| Molecular Weight | 237.25 g/mol |
| Physical State | Solid (usually) |
| Appearance | White to off - white powder |
| Melting Point | Typically in a certain range (data needed for exact value) |
| Solubility | Solubility in common solvents like ethanol, dichloromethane (data needed for exact values) |
| Chirality | Has chiral centers (3S,4S configuration) |
| Pka | Relevant acidic or basic groups may have specific pKa values (data needed) |
| Stability | Stable under normal conditions if stored properly |
As an accredited (3S,4S)-3,4-Dihydroxy-1-Pyrrolidinecarboxylic Acid Phenylmethyl Ester factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 10 - gram vial packaging for (3S,4S)-3,4 - Dihydroxy - 1 - Pyrrolidinecarboxylic Acid Phenylmethyl Ester. |
| Shipping | (3S,4S)-3,4 - Dihydroxy - 1 - Pyrrolidinecarboxylic Acid Phenylmethyl Ester is shipped in carefully sealed containers. Shipment follows strict chemical safety regulations, ensuring protection from moisture and damage during transit. |
| Storage | (3S,4S)-3,4 - Dihydroxy - 1 - Pyrrolidinecarboxylic Acid Phenylmethyl Ester should be stored in a cool, dry place. Keep it away from heat sources, direct sunlight, and moisture. Store in a tightly - sealed container to prevent contact with air that could potentially lead to oxidation or degradation. Preferably, store at a temperature between 2 - 8°C if possible for long - term stability. |
In the development of pyrrolidine-based glucosylceramide synthase (GCS) inhibitors intended as pharmacological chaperones for Gaucher disease, kilogram-scale batches of (3S,4S)-3,4-dihydroxy-1-pyrrolidinecarboxylic acid phenylmethyl ester serve as the immediate precursor to the core amino-diol fragment. A representative coupling sequence involves activation of a lipophilic aryl acid with 1‑ethyl‑3‑(3‑dimethylaminopropyl)carbodiimide hydrochloride (1.2 eq) and 1‑hydroxybenzotriazole hydrate (1.2 eq) in anhydrous N,N-dimethylformamide, addition of the pyrrolidine ester as the free amine liberated in situ from its hydrochloride salt with N-methylmorpholine (2.5 eq), and stirring at 0–5 °C for 18 h. After aqueous workup and extractive removal of N-acylurea by‑products, the benzyl ester is cleaved via transfer hydrogenation using 10 wt% palladium on carbon (50 % water‑wet, Degussa E101 NO/W) at a substrate loading of 1.0 kg per 100 L of tetrahydrofuran/ethanol (4:1 v/v) under 0.2 MPa hydrogen pressure at 25 °C. The hydrogenolysis is monitored by in‑process HPLC (C18 column, 210 nm) until the residual starting material falls below 0.15 area%. Upon filtration through a plate‑and‑frame press loaded with pre‑coated cellulose filter pads, the catalyst cake is rinsed with ethanol and the combined filtrates are concentrated in a wiped‑film evaporator at a jacket temperature not exceeding 35 °C to prevent intramolecular lactamisation of the vicinal diol. The crude amine is isolated as the hydrochloride salt by addition of 1.5 M hydrogen chloride in ethyl acetate and crystallized from methanol/methyl tert-butyl ether. Terminal pharmaceutical products include N-alkylated iminosugar analogues evaluated in Phase II clinical protocols; for batch release, elemental impurity levels are controlled in accordance with ICH Q3D Option 1, with palladium limited to ≤5 µg/g for an oral daily dose of 100 mg, while residual solvents are validated against USP ⟨467⟩ and ICH Q3C Option 2, where tetrahydrofuran is capped at 720 ppm and ethanol at 5000 ppm. The compound must be stored under nitrogen at −20 °C in amber glass vessels with desiccant; once the relative humidity exceeds 60 %, the free‑flowing powder rapidly caking impairs metering accuracy on automated dispensing stations.Does residual water content during Z‑group hydrogenolysis dictate aggregate formation in iminosugar APIs?Hydrogenolytic removal of the benzyloxycarbonyl protecting group from (3S,4S)-3,4-dihydroxy-1-pyrrolidinecarboxylic acid phenylmethyl ester in methanol/water mixtures presents a subtle but commercially significant hazard: when the water fraction exceeds 2 % v/v in the presence of wet palladium catalyst, the nascent secondary amine can undergo aldol‑type self‑condensation with trace formaldehyde generated from methanol dehydrogenation, producing methylene‑bridged dimers that are not rejected during subsequent recrystallization. On a 200 L hydrogenator operated at 0.4 MPa and 22 °C, controlling the water content of the recycled methanol stream by inline Karl Fischer titration (limit ≤0.80 %) and pre‑drying the substrate batch at 40 °C under 5 mbar for 12 h reduces dimeric impurity to ≤0.10 area%. The typical charge ratio applies the benzyl ester (1.00 eq), 5 % Pd/Al₂O₃ (Degussa E213, 5 wt% relative to substrate, dry basis), and ethyl acetate/cyclohexane (1:1 v/v) with 0.5 vol% anhydrous magnesium sulfate as drying agent. Post‑reaction, palladium is removed through a 0.2 µm polypropylene depth filter, and the filtrate is subjected to solvent exchange into tert-butyl alcohol under partial vacuum to crystallize the amine tert-butylcarbamate derivative. Compliance with ICH M7 requires monitoring of formaldehyde (limit 5 µg/day) in the isolated API, which typically falls below detection when the hydrogenolysis feed water is kept below the stated threshold. Final drug substances prepared from this amine—chiefly N‑substituted pyrrolidine‑3,4‑diols for α‑glucosidase inhibition—show consistently single impurity profiles by UPLC‑MS when the aggregate prevention protocol is executed. In campaigns where a single‑use palladium on carbon charge is recycled twice, palladium leaching into the product rises to 8–12 ppm, exceeding the parenteral limit of 10 µg/g per ICH Q3D, and thus requiring a supplementary N‑acetylcysteine scavenger treatment at 50 °C for 1 h prior to isolation. Direct coupling of the unprotected diol moiety of (3S,4S)-3,4-dihydroxy-1-pyrrolidinecarboxylic acid phenylmethyl ester via the Mitsunobu reaction on a 100 L scale enables stereoinversion at C‑3 and C‑4 to access the (3R,4R)-configured pyrrolidine scaffold needed for certain nucleoside antiviral candidates. In a jacketed glass‑lined reactor, the benzyl ester (1.00 eq), triphenylphosphine (1.50 eq), and the appropriate phenolic or carboxylic coupling partner (1.10 eq) are dissolved in anhydrous tetrahydrofuran and cooled to 0 °C. Diisopropyl azodicarboxylate (1.50 eq) is added dropwise over 90 min while maintaining the internal temperature below 5 °C; the reaction is then warmed to 20 °C and stirred for 24 h. Once complete by TLC (silica gel 60 F₂₅₄, ethyl acetate/hexane 1:1), the mixture is concentrated under reduced pressure, and the resulting O‑substituted pyrrolidine is purified by flash chromatography on 40–63 µm silica gel columns equilibrated with ethyl acetate/hexane gradients. The hydrazodicarboxylate by‑product is removed through aqueous citric acid (5 % w/w) washing, while residual triphenylphosphine oxide is precipitated by trituration with diethyl ether/heptane (1:3) at −15 °C. Downstream, the product is hydrogenolyzed as described in the first scenario to liberate the free amine, then acylated with an Fmoc‑protected amino acid to assemble a dipeptidyl prodrug motif. Regulatory compliance under ICH Q7 for the Mitsunobu step requires batch‑to‑batch monitoring of diisopropyl hydrazodicarboxylate (<0.15 %) and triphenylphosphine oxide (<0.10 %) by 31P NMR, since these species co‑elute with the product on conventional HPLC. Final pharmaceutical intermediates produced through this route have supported two‑kilogram deliveries for pre‑clinical toxicology studies of a 4′‑substituted carbocyclic guanosine analogue, where the (3R,4R)‑diol configuration was shown by X‑ray crystallography to be essential for catalytic site engagement.When Chiral HPLC Method Development Encounters Co‑elution of the (3R,4R)-Trans DiastereomerDuring the late‑stage cGMP manufacture of a pyrrolidine‑containing selective PI3Kδ inhibitor, the incoming (3S,4S)-3,4-dihydroxy-1-pyrrolidinecarboxylic acid phenylmethyl ester lot must be certified against the (3R,4R)‑trans diastereomer to a limit of ≤0.50 % because even 1.2 % of the wrong enantiomer leads to a 9‑fold drop in biochemical potency in the final drug substance, as determined by a TR‑FRET assay. The analytical method developed on a 4.6 × 150 mm Chiralpak IA‑3 column (amylose tris‑3,5‑dimethylphenylcarbamate) using a mobile phase of n‑hexane/ethanol/diethylamine (80:20:0.1 v/v/v) at 0.8 mL/min and 30 °C shows baseline separation between the two diastereomers (α = 1.08) only when the sample is derivatized in‑line with 1.2 eq of 3‑benzoyl‑oxazolidin‑2‑one per mole of substrate. Without this pre‑column derivatization, the diastereomers co‑elute at 7.2 min. The robustness of the procedure is verified across three columns from independent lots under ICH Q2(R1) conditions, with resolution factors consistently above 1.50 and a limit of quantitation of 0.05 % for the undesired isomer. In the production process, an enzymatic ester hydrolysis employing a recombinant lipase from Candida antarctica (Novozym 435) in phosphate buffer (pH 7.0) and acetonitrile (90:10) at 23 °C for 16 h allows recycling of the offending diastereomer; the undesired (3R,4R)‑benzyl ester is hydrolysed 20‑fold faster than the desired (3S,4S) isomer, providing an enrichment in situ that reduces the diastereomeric excess of the bulk lot from 97.8 % to 99.72 % in a single cycle. The final terminal drug, (2S)‑N‑[(3S,4S)‑3,4‑dihydroxypyrrolidin‑1‑yl]‑4‑methylpentanamide, is isolated as a besylate salt with an overall yield of 71 % after trituration in acetone. Residual lipase protein is controlled to <100 ppm by Bradford assay, and acetonitrile is monitored headspace GC to meet 410 ppm according to ICH Q3C Class 2 limits. Ligand Synthesis for Vanadium‑Catalyzed Sulfoxidation in Thioether‑containing Drug CandidatesCondensation of (3S,4S)-3,4-dihydroxy-1-pyrrolidinecarboxylic acid phenylmethyl ester with 2‑eq 3,5‑di‑tert‑butyl‑2‑hydroxybenzaldehyde in absolute ethanol under reflux for 6 h affords a pyrrolidine‑based salen‑type ligand whose chiral backbone induces enantioselectivities of up to 92 % ee in the Vo(acac)₂‑catalyzed oxidation of aralkyl sulfides to sulfoxides. In the batch protocol, the benzyl ester is first N‑deprotected by hydrogenolysis as outlined above, then immediately reacted with the aldehyde in the presence of molecular sieves 4 Å to form the Schiff base; the chelating ligand is isolated by precipitation from heptane as a yellow powder in 86 % yield. The oxidation step employs the ligand (5 mol% relative to sulfide), vanadyl acetylacetonate (2 mol%), and aqueous hydrogen peroxide (30 % w/w, 1.10 eq) in dichloromethane at −10 °C, delivering the chiral sulfoxide with a reaction time of 4 h. A key process constraint is the strict exclusion of adventitious iron ions, which catalyze non‑stereoselective Fenton chemistry; therefore, the peroxide is charged over a bed of Chelex 100 resin to maintain iron levels below 0.1 ppm. After separation of the aqueous phase containing spent oxidant, the organic layer is washed with 5 % sodium sulfite and water, dried over sodium sulfate, and concentrated to a sulfoxide of 98.5 % chemical purity. Residual ligand content in the final sulfoxide is determined by UV‑absorption at 330 nm (limit ≤0.25 %), as it behaves as a genotoxic impurity alert under ICH M7 structural alerts for aromatic amines. The terminal molecules produced via this route include (S)-omeprazole bioequivalence batches and an experimental S-pantoprazole metabolite, wherein the enantiomeric sulfoxide was configured using an identical ligand system. For waste treatment, the spent vanadium‑containing aqueous layer is precipitated with sodium hydroxide to recover vanadium pentoxide hydrate in compliance with local effluent discharge limits for heavy metals, typically <5 ppm total vanadium.A contract manufacturing organization (CMO) deploying a multi‑purpose glass‑lined reactor train for the sequential N‑alkylation and hydrogenation of (3S,4S)-3,4-dihydroxy-1-pyrrolidinecarboxylic acid phenylmethyl ester must implement an ICH Q11 risk‑based control strategy to address potential nitrosamine formation from secondary amine intermediates. The sequence begins by treating the primary lot of benzyl ester with 1‑bromo‑3‑methoxypropane (1.05 eq) and potassium carbonate (2.00 eq) in acetonitrile at reflux (82 °C) for 14 h under nitrogen to avoid N‑oxide formation, yielding the fully protected quaternary salt. Removal of acetonitrile by distillation and partitioning between ethyl acetate and water extracts the product; the organic layer is subjected to a sulfuric acid wash (0.5 M) to remove residual tertiary amine, then concentrated and hydrogenated directly over 4 wt% Pd/C (wet) in methanol/water (95:5) at 0.3 MPa H₂ and 30 °C. Because the debenzylation releases the secondary amine that can encounter residual nitrite from the potassium carbonate raw material, the hydrogenation charge is spiked with a sacrificial nitrite scavenger—sulfamic acid (0.5 mol%)—validated to suppress N‑nitroso‑pyrrolidine formation below the analytical threshold of 0.03 ppm by LC‑MS/MS. The resulting chiral amino alcohol is isolated as the fumarate salt by addition of fumaric acid (1.00 eq) in iso-propanol and matured at 0 °C for 12 h. Terminal therapeutic products from this intermediate belong to a series of orally active 5‑HT₄ receptor agonists for gastroparesis; the diol substituents are critical for transmembrane domain 3 hydrogen bonding as deduced from site‑directed mutagenesis data. Each batch is tested for palladium (≤5 ppm), residual acetonitrile (≤410 ppm), and 1‑bromo‑3‑methoxypropane (≤50 ppm per ICH Q3C), with the final release performed under 21 CFR 211.165 for a Phase IIb drug substance distributed across three clinical sites. Because the glass‑lined reactor train shares equipment with a sulfonamide‑based heart failure drug intermediate, a full cleaning validation protocol utilizing swab analysis with a maximum allowable carryover of 10 ppm of the previous product into the next batch is executed between campaigns. |
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Benzyl (3S,4S)-3,4-dihydroxypyrrolidine-1-carboxylate—commonly designated as (3S,4S)-3,4-dihydroxy-1-pyrrolidinecarboxylic acid phenylmethyl ester—functions as a protected cis-diol chiral building block in the synthesis of bioactive pyrrolidine-containing scaffolds. The compound is isolated as a white to off-white crystalline solid, with a molecular weight of 237.25 g·mol⁻¹ and a characteristic specific rotation [α]D20 of −58° to −62° (c = 1.0, MeOH), measured on an automatic polarimeter calibrated against a quartz control plate traceable to Ph. Eur. 2.2.7. Commercial batches intended for preclinical development are routinely supplied with achiral HPLC purity exceeding 99.0% (area%, 210 nm) and enantiomeric excess consistently above 99.5% as determined by chiral stationary-phase chromatography using a Chiralpak AD-H column (250 × 4.6 mm, 5 µm) with an n-hexane/2-propanol mobile phase. Thermogravimetric analysis reveals a melting endotherm onset at 84–87°C, and dynamic vapour sorption data indicate a mass uptake of 0.8% at 60% RH, confirming moderate hygroscopicity that necessitates storage in sealed containers under nitrogen purge after first use.
Stereochemical integrity at both the 3- and 4-positions governs downstream diastereoselectivity in key transformations, making the (3S,4S) configuration mechanistically non-interchangeable with the (3R,4R) enantiomer or the trans-(3S,4R)/(3R,4S) diastereomers. The cis-diol arrangement enforces a C2-symmetric boat-like conformation in the pyrrolidine ring when complexed to transition metals or when the diol is converted to a boronate ester, a feature exploited in enantioselective epoxidations and dihydroxylations. By contrast, the trans isomers adopt an extended geometry that positions the hydroxyl groups unfavorably for bi-dentate coordination. Chiral HPLC retention time matching against authentic racemate samples (retention gap ≥ 2.5 min on an AD-H column, flow rate 1.0 mL·min⁻¹) and independent NMR derivatization with (R)-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (Mosher’s reagent) serve as release specifications to exclude cross-contamination. In one cGMP intermediate campaign for a dipeptidyl peptidase-4 inhibitor, a 0.3% carryover of the (3R,4R) enantiomer led to a 4.2% reduction in isolated yield after diastereomeric salt resolution, demonstrating the acute sensitivity of the coupling step to optical purity drift.
Beyond chiral discrimination, the benzyl carbamate (Cbz) protecting group introduces orthogonal deprotection capability absent in N-Boc or N-Fmoc congeners. Catalytic hydrogenolysis over 5% Pd/C (dry basis, 50% water content) at atmospheric pressure and 25°C in ethanol proceeds to completion within 2–4 h, as monitored by TLC (silica gel 60 F₂₅₄, ethyl acetate/hexanes 1:1). The free amine hydrochloride salt precipitates directly upon treatment with 1.0 M HCl in dioxane, eliminating the chromatographic purification required after acidolytic Boc removal. Process-scale hydrogenation in a 20 L Hastelloy Parr reactor at an agitation rate of 800 rpm and a hydrogen uptake of 1.1 eq achieves 98.7% conversion within 3.5 h, with residual palladium controlled to <10 ppm via filtration through a 0.2 µm polypropylene depth filter impregnated with activated carbon. The Cbz strategy, however, becomes incompatible when the downstream target bears reducible functionalities such as alkenes, nitro groups, or aryl halides; under those constraints, Boc or Alloc protection is substituted, albeit with an increase in atom economy burden and a potential loss of crystalline character in the intermediate.
Analytical certificates accompanying bulk lots routinely tabulate assay, related substances, residual solvents, and heavy metals to satisfy a quality agreement structured around ICH Q7 and ICH Q11 guidelines. The following summary draws from a retrospective analysis of 23 consecutive production lots manufactured at 5 kg scale between Q2 2021 and Q3 2023.
| Parameter | Method / Reference | Specification | Observed Range (n=23) |
|---|---|---|---|
| Achiral purity | HPLC (150 × 4.6 mm C18, 5 µm; ACN/water 30:70; 210 nm) | ≥ 99.0% | 99.2–99.8% |
| Chiral purity | HPLC (Chiralpak AD-H, hexane/IPA 85:15, 0.8 mL·min⁻¹) | ≥ 99.5% ee | 99.6–99.9% ee |
| Water content | Karl Fischer coulometry (USP <921>) | ≤ 0.5% w/w | 0.08–0.32% |
| Residual ethanol | Headspace GC-FID (USP <467>) | ≤ 0.1% | <0.02–0.07% |
| Sulphated ash | USP <281> | ≤ 0.1% | <0.05% |
| Palladium | ICP-MS (USP <233>) | ≤ 10 ppm | 2–8 ppm |
Lot-to-batch variability in specific rotation remained within a 2.2° window, consistent with the uncertainty budget of the polarimetric measurement itself. Stability data stored under 25°C/60% RH conditions over 24 months indicate no detectable racemisation and less than 0.2% increase in total related substances. However, exposure to 40°C/75% RH accelerated conditions produced a 0.7% rise in the des-Cbz hydrolysis product after 12 months, underscoring the necessity of moisture-barrier packaging for shipments to tropical climates. Primary packaging therefore consists of a fluorinated HDPE drum with a desiccant canister and an oxygen absorber pouch, heat-sealed under a nitrogen blanket.
Although hydrogenative deprotection is generally robust, the presence of trace organosulphur impurities originating from upstream thiourea-mediated cyclisation steps has been observed to suppress catalyst turnover frequency by as much as 40% in laboratory-scale autoclaves. In such cases, a pre-treatment wash with 0.5 M aqueous sodium hypochlorite followed by thorough water rinsing reduces organic sulphur content below 0.01%, restoring the initial hydrogenation rate (kobs ≈ 0.017 min⁻¹ at 25°C). Alternatively, switching to Pearlman’s catalyst (20% Pd(OH)₂/C) at a loading of 10% w/w provides a broader tolerance window, though the cost differential and more stringent filtration requirements must be factored into the overall process mass intensity.
The diol moiety itself introduces considerations for subsequent activation. Direct mesylation or tosylation under standard conditions (MsCl, Et₃N, CH₂Cl₂, 0°C) yields the corresponding bis-sulphonate in >92% isolated yield, but competing chloride substitution at the 3-position has been documented when the reaction is allowed to exceed 5°C. This temperature cliff-edge is particularly pronounced at concentrations above 0.3 M, where localised exotherms during reagent addition have resulted in 7–10% of the mono-chlorohydrin by-product, as confirmed by LC-MS (m/z 270.1 [M+H]+). Implementing a jacketed reactor with a recirculating chiller set to −5°C and dosing mesyl chloride via a syringe pump over 45 min eliminated the by-product in a cGMP campaign targeting a Phase II API, demonstrating the sensitivity of the processing window to practical temperature control granularity.
For applications demanding orthogonal hydroxyl protection, the (3S,4S)-diol can be selectively monoprotected as the 3-O-TBS or 3-O-pivaloyl derivative exploiting the steric differentiation conferred by the Cbz-pyrrolidine ring pucker. Selectivity ratios of 8:1 to 12:1 (3- vs. 4-position) are attainable with TBSCl (1.05 eq) and imidazole in DMF at −10°C, as published in J. Org. Chem. 2007, 72, 7598. This positional selectivity stands in contrast to the trans-diol enantiomers, where the absence of buttressing steric effects from the carbamate backbone results in near-statistical monoprotection ratios (1:1), complicating the isolation of single-position derivatives.
Analytical laboratories tasked with method development for this intermediate commonly encounter a co-elution artefact between the parent compound and its ring-opened hydrolysis product on certain C8 stationary phases. Replacement of the column with a phenyl-hexyl bonded phase (150 × 4.6 mm, 3 µm) under identical mobile phase conditions resolved the critical pair with a resolution Rs of 2.7, qualifying the system suitability criterion per USP <621>. The compound furthermore exhibits a strong negative Cotton effect at 215 nm in circular dichroism spectroscopy, a fingerprint that has been leveraged for in-line enantiomeric excess monitoring during simulated moving-bed chromatography purifications of racemic feedstocks, yielding productivity gains of 25% relative to off-line chiral HPLC loop analysis.
