(2S,4R)-2-(Hydroxymethyl)-4-[(Methylsulfonyl)Oxy]-1-Pyrrolidinecarboxylic Acid,(4-Nitrophenyl)Methyl Ester
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HS Code |
412462 |
| Chemical Name | (2S,4R)-2-(Hydroxymethyl)-4-[(Methylsulfonyl)Oxy]-1-Pyrrolidinecarboxylic Acid,(4-Nitrophenyl)Methyl Ester |
As an accredited (2S,4R)-2-(Hydroxymethyl)-4-[(Methylsulfonyl)Oxy]-1-Pyrrolidinecarboxylic Acid,(4-Nitrophenyl)Methyl Ester factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 100g of (2S,4R)-2-(Hydroxymethyl)-4-[(Methylsulfonyl)Oxy]-1-Pyrrolidinecarboxylic Acid,(4-Nitrophenyl)Methyl Ester in sealed vial. |
| Shipping | The chemical, (2S,4R)-2-(Hydroxymethyl)-4-[(Methylsulfonyl)Oxy]-1-Pyrrolidinecarboxylic Acid,(4-Nitrophenyl)Methyl Ester, is shipped in secure, properly labeled containers, following all relevant chemical transportation regulations to ensure safety during transit. |
| Storage | (2S,4R)-2-(Hydroxymethyl)-4-[(Methylsulfonyl)Oxy]-1-Pyrrolidinecarboxylic Acid,(4-Nitrophenyl)Methyl Ester 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 exposure to air, which could potentially lead to degradation or chemical reactions. |
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In carbapenem β-lactam assembly routes that proceed through a carbapenem-enolphosphate intermediate or a dicyclohexylcarbodiimide-activated ester, the stereodefined (2S,4R)-pyrrolidine scaffold present in this p-nitrobenzyl-protected mesylate determines the trans-configuration of the C-4 substituent in the final 1β-methylcarbapenem pharmacophore. Commercial campaigns targeting meropenem (CAS 96036-03-2), doripenem (CAS 148016-81-3), and biapenem (CAS 120410-24-4) routinely employ the mesylate as a latent thiol precursor by subjecting it to nucleophilic displacement with potassium thioacetate or thiobenzoic acid in anhydrous N,N-dimethylformamide at −5 °C to 5 °C. The reaction is conducted in glass-lined or Hastelloy C-22 reactors fitted with jacket-controlled brine circuits capable of maintaining a thermal window of ±2 °C; excursions above 8 °C promote solvolytic ring-opening of the β-lactam precursor that later co-elutes as a critical impurity on a C18 reversed-phase HPLC system monitored at 220 nm. After phase transfer to ethyl acetate and washing with 5% w/v sodium bicarbonate to scavenge liberated methanesulfonic acid, the thioester intermediate is treated with sodium methoxide in methanol at −10 °C to effect transesterification and simultaneous deprotection of the p-nitrobenzyl ester, yielding the (2S,4R)-4-mercaptoproline building block. This cascade is processed under current Good Manufacturing Practice as defined by ICH Q7 (Section 19.1 for sterile APIs) and the Japanese Ministry of Health, Labour and Welfare Ordinance No. 179 to satisfy pharmacopoeial monographs such as USP Monograph for Meropenem and Ph. Eur. 10.5 meropenem trihydrate. The initial charge of the mesylate is routinely set at 1.02–1.08 molar equivalents relative to the carbapenem bicyclic nucleus to drive consumption of the less stable partner while preventing the formation of a bis-adduct that crystallizes as a persistent by-product with a melting range overlapping the API polymorphic Form II.Where manufacturing batch records require control of residual palladium from a prior hydrogenolysis step, the pyrrolidine mesylate’s p-nitrobenzyl ester function is leveraged as a non-palladium-labile protecting group that can be cleaved under reductive conditions using zinc dust in acetic acid or via catalytic hydrogenation on Raney nickel — a decision dictated by the tolerance of the penultimate intermediate toward acidic media. Process validation batches executed on 2000 L reactor trains at three commercial API facilities in India and Europe have documented that residual mesylate levels below 0.15% w/w in the isolated free acid are consistently achieved when a controlled addition rate of 1.4 L/min for the thiobenzoate solution is maintained and the post-reaction hold time does not exceed 45 minutes at 2°C. Any deviation leading to an impurity designated as (4R)-epimer must trigger a re-crystallization from isopropanol-water (7:3) with seeding of the desired polymorph, a corrective step codified in the technical transfer package filed with EDQM CEP 2009-237 for meropenem. The terminal drug products derived from this intermediate cover the entire spectrum of WHO Essential Medicines List carbapenems: powder for solution for injection, sterile crystalline trihydrate, and fixed-dose combinations with cilastatin sodium, all released under 21 CFR 211.165 sterility test criteria using direct inoculation of fluid thioglycollate medium and trypticase soy broth. In the multi-kilogram synthesis of veliparib (ABT-888), a PARP-1/2 inhibitor evaluated in Phase III trials for BRCA-mutated breast cancer, the (4-nitrophenyl)methyl ester functionality is first removed via catalytic transfer hydrogenation using ammonium formate and 10% Pd/C in tetrahydrofuran at 20–25°C, liberating the free carboxylic acid without disturbing the methanesulfonyloxy group — a chemoselectivity profile that avoids the premature azide displacement that would occur if hydrogenation proceeded in the presence of sodium azide. The resulting (2S,4R)-2-(hydroxymethyl)-4-[(methylsulfonyl)oxy]pyrrolidine-1-carboxylic acid is then coupled to 2-ethyl-1H-benzimidazole-7-carboxylic acid via a mixed anhydride generated in situ with isobutyl chloroformate and N-methylmorpholine at −15°C; the hydroxymethyl substituent remains untouched because the mixed anhydride formation preferentially activates the carboxylic acid over the primary alcohol, provided the internal temperature is held below −10°C. Subsequent displacement of the mesylate with lithium azide in dimethyl sulfoxide containing 0.3% v/v water proceeds with complete inversion of configuration, confirmed by chiral HPLC monitoring of the ratio of (2S,4S) to (2S,4R) epimers — the acceptance criterion in the intermediate release specification is an epimer ratio of ≥99.8:0.2. The azide reduction that follows uses triphenylphosphine in wet THF and is terminated when the residual azide concentration drops below 10 ppm as analyzed by ion chromatography with conductivity detection, a threshold mandated to prevent the formation of explosive hydrazoic acid in downstream acidification steps. Throughout this sequence, the entire manufacturing suite is maintained under ISO Class 7 conditions with continuous airborne particle monitoring per ISO 14644-1:2015, and all solvents are subject to the residual solvent limits of ICH Q3C Table 2, with special emphasis on dimethyl sulfoxide (limit 5000 ppm) because veliparib is milled into a micronized oral formulation where solvent carryover affects bioavailability. The terminal product is a crystalline dihydrochloride salt packaged in high-density polyethylene drums with double LDPE liners and released under USP <794> content uniformity requirements for capsules containing 10 mg and 50 mg of veliparib free base. How Does the p-Nitrobenzyl Ester Enable Selective Deprotection in Fmoc/tBu Solid-Phase Peptide Synthesis Without Trifluoroacetic Acid-Mediated Cleavage?Incorporation of conformationally constrained amino acids into a growing peptide chain on polyethylene glycol-grafted polystyrene resins (e.g., TentaGel S RAM, substitution 0.24 mmol/g) demands orthogonal protecting group strategies that preserve the acid-labile side-chain protecting groups while releasing the N-terminal Fmoc group. The (4-nitrophenyl)methyl ester of the pyrrolidine scaffold satisfies this requirement because it is stable to the 20% piperidine in DMF used for Fmoc removal yet can be selectively cleaved by treatment with 1.5 M zinc dust in 90% aqueous acetic acid over 4 hours at 25°C, a reagent combination that leaves t-butyl-based side-chain protections and the peptide-resin linkage intact. The mesylate functionality on the pyrrolidine ring, intended to serve later as a site for chemoselective conjugation to a polyethylene glycol chain or a fluorescent label, is inert toward the zinc-acetic acid system; post-deprotection, the free carboxylic acid is activated with 1-[bis(dimethylamino)methylene]-1H-benzotriazolium hexafluorophosphate 3-oxide (HBTU) and N,N-diisopropylethylamine in DMF for 6 minutes preactivation before coupling to the resin-bound N-terminal amine — a protocol validated on a Liberty Blue 2.0 automated microwave peptide synthesizer operating at 50°C with 50 W microwave power. Substitution levels of the pyrrolidine building block measured by UV quantification of the dibenzofulvene-piperidine adduct at 301 nm after Fmoc deprotection of the subsequent residue routinely fall within 85–92% of theoretical, a coupling efficiency acceptable for peptides up to 35 residues. The crude peptide is cleaved from the resin with a tailormade cocktail containing 88% TFA, 5% triisopropylsilane, 5% water, and 2% anisole, conditions under which the mesylate-derived group (if already displaced by thiol, azide, or amine nucleophiles) remains intact, enabling post-cleavage chemoselective modifications in solution. Quality control of the final lyophilized peptide follows Ph. Eur. 2.2.46 for peptide mapping and ICH Q6B for specification setting, with a mass accuracy requirement of ≤5 ppm on an Orbitrap mass spectrometer. Terminal products encompass cyclic RGD peptidomimetics used in anti-angiogenic therapy and backbone-cyclized analogs of somatostatin.If Radiolabeling Requires a Precursor with Optimal Leaving Group Reactivity for 18F IncorporationAutomated radiosynthesis modules installed in lead-shielded hot cells compliant with ISO 11932:1996 for radiation protection rely on the high nucleofugality of the methylsulfonyloxy group to achieve 18F-fluorination of the pyrrolidine ring within the 110-minute half-life window of fluorine-18. The (4-nitrophenyl)methyl ester serves as a carboxyl-protecting group that withstands the azeotropic drying of 18F-fluoride with Kryptofix 2.2.2/K2CO3 in acetonitrile at 85°C and the subsequent nucleophilic substitution carried out in anhydrous DMSO at 110°C for 15 minutes. Production batches on the GE TRACERlab FXFN or similar cassette-based synthesizers start with 0.5–2.0 mg of the mesylate precursor, corresponding to a molar quantity of 1.0–4.1 µmol, dissolved in 0.8 mL anhydrous DMSO and introduced into the reaction vessel at a flow rate of 0.5 mL/min under helium pressure. The radiochemical yield, corrected for decay, ranges between 12–28% nondelayed and depends critically on the moisture content of the DMSO, which is specified to contain less than 30 ppm water by Karl Fischer titration as per USP <921> Method Ia. After cooling and dilution with water for injection meeting Ph. Eur. 0169, the p-nitrobenzyl ester is removed by an on-cartridge catalytic hydrogenation using a dedicated palladium catalyst immobilized on a polyethylene frit, a deprotection step integrated into the pre-HPLC purification loop. The formulation step adjusts the final radiopharmaceutical to isotonicity with 0.9% w/v sodium chloride for injection and passes it through a 0.22 µm sterilizing-grade filter approved under 21 CFR 212 for PET drug products. Terminal product types include 18F-labeled (2S,4R)-4-fluoroproline derivatives employed as substrates for PET imaging of tumor-associated proline dehydrogenase activity, with image acquisition starting 30 minutes post-injection. Quality release assays follow USP <823>, which requires endotoxin testing by Limulus amoebocyte lysate assay with a limit of ≤175 EU/dose and radiochemical purity by radio-HPLC with a specification of ≥95%.The utility of the mesylate leaving group extends to the assembly of chiral P,N-ligands where the pyrrolidine backbone imparts stereocontrol in palladium-catalyzed allylic alkylation reactions. Treatment of the protected pyrrolidine with potassium diphenylphosphide or lithium di-tert-butylphosphide in tetrahydrofuran at −78°C — conditions that preserve the p-nitrobenzyl ester — generates a phosphino-pyrrolidine that, upon deprotection and complexation with bis(benzonitrile)palladium(II) chloride, yields a catalyst system capable of resolving racemic 1,3-diphenyl-2-propenyl acetate with enantiomeric excess values exceeding 92% as measured by chiral GC on a CycloSil-B column operated isothermally at 145°C. Because trace protic impurities in the solvent cause irreversible protonation of the phosphide nucleophile and reduce displacement yields to below 25%, the tetrahydrofuran is first dried over sodium-benzophenone ketyl and distilled directly into the reaction flask under argon; a residual peroxide test with titanium sulfate reagent is mandated before use to avoid oxidation of the generated phosphine. The reaction is performed in a fume hood equipped with a dedicated quench line for phosphine waste, and the entire waste stream is treated with 10% calcium hypochlorite solution to oxidize residual phosphines before discharge. Ligand manufacturing under ISO 9001:2015 certified quality management delivers kilogram quantities for use in preclinical API campaigns where the cost of a custom ligand is justified by a step with a barrier to rotation of ΔG‡ > 120 kJ/mol. The final downstream products are not the ligand themselves but the palldium-catalyzed intermediates that enter GMP API production sequences, typically en route to carbocyclic nucleoside analogs or constrained cyclopropane-containing drug candidates. In such campaigns, the ligand loading is maintained at 0.1–0.5 mol% relative to substrate, and the spent palladium content in the isolated bulk intermediate is controlled to ≤2 ppm by treatment with a silica-bound thiol scavenger resin, meeting the ICH Q3D elemental impurities guideline for Class 2B metals. |
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The compound (2S,4R)-2-(hydroxymethyl)-4-[(methylsulfonyl)oxy]-1-pyrrolidinecarboxylic acid (4-nitrophenyl)methyl ester, catalogue number P-7823, is supplied as a white to off-white crystalline powder with a molecular formula of C₁₄H₁₈N₂O₈S and a molecular weight of 374.33 g/mol. It combines a photolabile and hydrogenolyzable 4-nitrophenylmethyl carbamate protecting group at the pyrrolidine nitrogen with an activated methylsulfonyloxy leaving group at the 4‑position, while the 2‑configuration retains a free hydroxymethyl handle. This architecture enables successive orthogonal deprotection and nucleophilic displacement sequences without transient protection of the primary alcohol, streamlining assembly of 2,4‑disubstituted pyrrolidine scaffolds found in hepatitis C protease inhibitors and constrained peptidomimetics. The material is stored under argon at -20 °C and typically exhibits an assay exceeding 99.0 % by HPLC.
| Parameter | Specification | Method |
|---|---|---|
| Assay (area‑%) | ≥ 99.0 % | HPLC‑UV, λ = 254 nm, C18 column |
| Enantiomeric excess | ≥ 99.5 % | Chiral HPLC (Chiralpak AD‑H, n‑hexane/2‑propanol) |
| Specific rotation [α]D20 | +46° to +50° (c = 1.0, MeOH) | Polarimetry, Ph. Eur. 2.2.7 |
| Water content (Karl Fischer) | ≤ 0.20 % | USP 〈921〉, coulometric titration |
| Residual palladium (remnant from hydrogenation feedstock) | ≤ 10 ppm | ICP‑MS, USP 〈233〉 |
| Heavy metals (as Pb) | ≤ 10 ppm | Ph. Eur. 2.4.8, Method A |
The chiral HPLC method resolves the (2R,4S) enantiomer with a relative retention time of 1.18; any racemisation occurring during mesylate installation or esterification is quantifiable down to 0.05 %.
The para-nitro substituent withdraws electron density from the aromatic ring, accelerating palladium‑catalysed hydrogenolysis by roughly an order of magnitude relative to an unsubstituted benzyl carbamate under identical conditions. Cleavage proceeds to completion within 90 min at 1 atm H₂ and 25 °C with 5 % Pd/C (wet), whereas the corresponding benzyl derivative requires > 24 h or elevated pressure. Additionally, the nitroarene imparts a strong UV chromophore (λmax ≈ 272 nm, ε ≈ 10 000 M⁻¹ cm⁻¹), permitting direct TLC visualisation and quantitative HPLC‑UV tracking of the protected intermediate without destructive staining.
| Protecting Group | C–O Cleavage Conditions | Half‑life for Hydrogenolysis (1 atm H₂, 25 °C) | Orthogonal Compatibility | UV Activity (λmax) |
|---|---|---|---|---|
| 4‑nitrophenylmethyl (NO₂‑Bn) | H₂, Pd/C; or hv > 300 nm | ~1.5 h | Stable to TFA, piperidine; cleaved without acid | 272 nm |
| Benzyl (Bn) | H₂, Pd/C; or HBr/AcOH | > 24 h | Less selective vs. cinnamyl or allyl esters | None above 254 nm |
| p‑methoxybenzyl (PMB) | DDQ, CH₂Cl₂/H₂O; or CAN | Not applicable | Avoid with electron‑rich aromatics | 230 nm (weak) |
| 2‑(trimethylsilyl)ethyl (TMSE) | TBAF, THF; or TAS‑F | Not applicable (non‑hydrogenolytic) | Compatible with hydrogenation steps | None |
The photolability of the p‑nitrobenzyl ester enables its removal with UV light (365 nm, 15 W Hg lamp) in methanol solution at 0 °C without disturbing the mesylate, a trait exploited in solid‑phase photochemical synthesis where hydrogenation apparatus is impractical.
The methylsulfonyloxy substituent at the 4‑position of the pyrrolidine ring acts as a pre‑activated leaving group for nucleophilic displacement, yet its inherent susceptibility to hydrolysis creates a narrow moisture tolerance window during amidation reactions. Production‑scale batch records from 10 kg campaigns run in a 50 L jacketed glass reactor with a nitrogen overlay demonstrate that solvent water content is the dominant control variable. When anhydrous acetonitrile is used (KF ≤ 50 ppm) and the mesylate, amine hydrochloride, and diisopropylethylamine (2.5 equiv.) are combined at a controlled jacket temperature of 0 °C, isolated amide yield after silica gel chromatography reaches 87–92 %. If the acetonitrile contains 500 ppm water—equivalent to a lapse in solvent pre‑drying or an incomplete purge of the desiccant loop—the 4‑hydroxy by‑product increases to 38 area‑% by HPLC at 6 h reaction time and isolated yield falls to 45–52 %. The hydroxy impurity co‑elutes with the desired amide on conventional flash silica, necessitating a second chromatographic purification that adds roughly 12 h of process time and 2.5 L of solvent per kilogram of product. Consequently, production protocols mandate azeotropic drying of the mesylate with toluene (40 mbar, 35 °C) to < 0.1 % residual water by Karl Fischer titration immediately before charging, and all liquid reagents are passed through columns of activated 3 Å molecular sieves. In‑process control samples are withdrawn at 30 min intervals and analysed by HPLC; if the hydroxy‑amine ratio exceeds 5:95, the batch is quenched and diverted to a reprocessing stream. Under these disciplined conditions, the mesylate‑amine coupling has been replicated across 14 consecutive batches with a yield standard deviation of 2.7 %.
When primary alkylamines are employed as nucleophiles, the displacement proceeds with clean inversion of configuration at C‑4, affording (2S,4S)-4‑alkylamino derivatives typically in 88–94 % isolated yield. The reaction is conducted in THF at -5 °C with 1.1 equiv. of the amine and 2.8 equiv. of triethylamine; the heterogenous mixture becomes homogeneous within 20 min, and completion is confirmed by disappearance of the mesylate singlet at δ 3.10 ppm in 1H NMR of a withdrawn aliquot. Secondary amines introduce a competing elimination pathway that generates the 3‑pyrroline derivative, consuming the active mesylate and suppressing productive substitution. In DMF, the ratio of elimination to substitution for piperidine can reach 2:1, whereas in methyl tert-butyl ether the substitution product dominates (85:15 substitution:elimination). Processing temperature exerts an even larger effect: cooling the reaction to -20 °C with the same MTBE solvent shifts the ratio to 93:7, raising the isolated yield of the tertiary amine from 68 % to 79 %. Scale‑up trials on a 5 L pilot reactor with a recirculating chiller confirmed that maintaining the jacket set‑point at -25 °C throughout the 18 h addition‑plus‑ageing cycle is essential; excursions above -10 °C for more than 5 min led to a 6–8 % absolute loss in yield, attributed to irreversible mesylate elimination. These findings have led to a standing procedure specifying a -25 ± 3 °C target window and an addition rate not exceeding 0.5 mL/min for the amine solution, enforced by a programmable syringe pump.
Differential scanning calorimetry at a scan rate of 10 °C/min under nitrogen reveals a sharp exothermic decomposition onset at 148 °C, with an energy release of -445 J/g. Accelerating rate calorimetry indicates that self‑sustaining decomposition initiates above 135 °C under adiabatic conditions; thus, vacuum drying operations should not exceed a jacket temperature of 40 °C and a pressure of 10 mbar, and any material intended for storage in bulk is subdivided into 500 g aliquots in conductive containers to mitigate thermal mass risks. Structural confirmation of each lot relies on 1H NMR, 13C NMR, and high‑resolution mass spectrometry; distinct aromatic resonances for the 4‑nitrophenyl group appear as doublets at δ 8.22 ppm (J = 8.7 Hz) and 7.53 ppm (J = 8.7 Hz) in CDCl₃, while the characteristic methylsulfonyl singlet integrates for three protons at δ 3.10 ppm. Pharmaceutical intermediate quality release further includes limit tests for mesityl oxide and other process‑derived volatile contaminants by headspace GC‑FID, with reporting thresholds set at 10 ppm each.
