1,2-Pyrrolidinedicarboxylicacid, 4-Cyano-, 1-(1,1-Dimethylethyl) 2-Methyl Ester, (2S,4S)-
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HS Code |
608024 |
| Iupac Name | 1-(1,1-Dimethylethyl) 2-methyl (2S,4S)-4-cyanopyrrolidine-1,2-dicarboxylate |
| Molecular Formula | C12H18N2O4 |
| Molecular Weight | 254.283 g/mol |
| Appearance | Solid (likely, based on similar compounds) |
| Solubility In Water | Low solubility expected due to non - polar groups |
| Solubility In Organic Solvents | Soluble in common organic solvents like dichloromethane, ethyl acetate |
| Polarity | Moderate polarity due to presence of polar functional groups and non - polar alkyl groups |
As an accredited 1,2-Pyrrolidinedicarboxylicacid, 4-Cyano-, 1-(1,1-Dimethylethyl) 2-Methyl Ester, (2S,4S)- factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 100g of (2S,4S)-1-(tert -butyl) 2 -methyl 4 -cyano -1,2 -pyrrolidinedicarboxylate in sealed container. |
| Shipping | The chemical "1,2 - Pyrrolidinedicarboxylic acid, 4 - Cyano -, 1 - (1,1 - Dimethylethyl) 2 - Methyl Ester, (2S,4S) -" will be shipped in accordance with strict chemical transport regulations, ensuring proper containment and safety during transit. |
| Storage | Store “(2S,4S)-1-(tert -Butyl) 2 -methyl 4 -cyanopyrrolidine - 1,2 -dicarboxylate” in a cool, dry place away from heat sources and open flames. Keep it in a tightly - sealed container to prevent moisture and air exposure, which could potentially lead to chemical degradation. Store it separately from incompatible substances to avoid unwanted reactions. |
Why Fractional Molar Equivalents of the Methyl Ester Dictate Throughput in Macrocyclic HCV Protease Inhibitor AssemblyIn the convergent synthesis of grazoprevir (MK-5172) and structurally related paritaprevir, the (2S,4S)-configured 1-Boc-4-cyano-proline methyl ester serves as the cyano-substituted P1 fragment. The acylation protocol coupling this fragment to a preformed quinoline‑acid P2 moiety operates under kinetic control to suppress epimerisation at the C‑2 position. Production‑scale batches running in glass‑lined reactors of 2,000–5,000 L employ a reagent ratio of 1.05–1.12 eq of the methyl ester relative to the P2 acid, with the slight excess functioning as a sacrificial scavenger for residual water. Activation is achieved with 1‑propanephosphonic acid cyclic anhydride (T3P®, ≥50 wt% in ethyl acetate) at 1.30–1.40 eq in the presence of N,N‑diisopropylethylamine (DIPEA, 2.50–2.80 eq). The addition sequence—charging the acid, methyl ester, and DIPEA into anhydrous tetrahydrofuran (KF ≤250 ppm) at ‑5°C to 0°C, followed by dropwise T3P® addition over ≥90 minutes—is critical: reversing the order or exceeding 5°C during the initial 30‑minute age period increases the diastereomeric impurity from the (2R,4S)‑epimer to above 0.15 area% (Chiralpak IA‑3, 4.6×250 mm, hexane/ethanol/TFA 85/15/0.1 v/v/v, 1.0 mL/min, 210 nm). Process analytical technology (PAT) using ReactIR 15 probes tracks the disappearance of the mixed anhydride peak at 1,815 cm⁻¹; the endpoint is reached when the residual acid activation intermediate falls below 1.0 mmole/L, typically after 8–12 hr. The workup quench with 0.5 M citric acid at 10–15°C decomposes excess T3P® while preserving the Boc‑protecting group; pH adjustment to 4.8–5.2 minimises both Boc cleavage and ester saponification to the free carboxylic acid, a side product that must be held to ≤0.20 wt% by IPC (HPLC, C18, gradient). After phase separation, the organic stream is washed with 8 wt% NaHCO₃ at 5°C and concentrated under vacuum (≤45°C jacket, ≤50 mbar) to a controlled volume. Crystallisation from methyl tert‑butyl ether/n‑heptane (1/4 v/v, 5 mL/g crude) at a cooling rate of ≤0.25°C/min between 45°C and 5°C delivers the coupled product in 82–88% isolated yield with a chiral purity of ≥99.5% enantiomeric excess (ee) by HPLC. The isolated crystalline intermediate proceeds to global deprotection—TFA‑mediated Boc cleavage followed by ester hydrolysis under pH‑stat control at pH 12.0±0.2, 20°C—to liberate the macrocyclisation‑ready amino acid. For paritaprevir synthesis, the identical coupling manifold is used with an alternative P2 cap, and the methyl ester fragment is held to the same mass‑balance boundaries. Regulatory starting material justification under ICH Q11 often anchors on this isolated intermediate; batched under 21 CFR 210/211 and EudraLex Volume 4 Part II, the GMP supply chain must document residual solvents (T3P®‑derived n‑propyl phosphonic acid and ethyl acetate below ICH Q3C Option 1 limits), Pd content (≤10 ppm via ICP‑MS) originating from earlier hydrogenation steps in the P2 unit, and storage stability at ‑20±5°C under argon (retest period 36 months when desiccant‑lined HDPE drums are used).DPP‑IV Inhibitor Fragment: Assessing Thermal Lability During Side‑Chain HomologationCyanopyrrolidine‑based dipeptidyl peptidase‑4 (DPP‑IV) inhibitors such as vildagliptin and a research pipeline of 2‑cyano‑pyrrolidide prodrugs rely on the (2S,4S)‑stereochemistry of the 4‑cyano substituent to modulate the covalent nitrile‑enzyme interaction with the catalytic serine residue. The Boc‑methyl ester intermediate is converted to the corresponding aldehyde or directly coupled to adamantylamino‑acetyl fragments via a three‑step telescoped procedure where the ester group is chemo‑selectively reduced to a primary alcohol, oxidised to the aldehyde, and then reacted with hydroxylamine and trifluoroacetic anhydride to deploy the nitrile warhead. The selective reduction performed in a continuous‑flow trickle‑bed reactor uses LiBH₄ (2.0 M in THF, 1.05 eq substrate) premixed with MeOH (0.98 eq relative to LiBH₄) in a 10 mL PFA coil at a residence time of 45 seconds at ‑10°C; back‑pressure regulation at 3.5 bar prevents hydrogen off‑gassing and ensures a steady‑state yield of 92–94% to the (S,S)‑alcohol. Without flow engineering, batch‑mode reduction in a 5 L jacketed vessel frequently leads to thermal runaway at scales exceeding 500 g input, causing pyrolytic decomposition of the cyano group to an amide impurity detectable at 1,660 cm⁻¹ (FTIR). The aldehyde oxidation step, employing Dess‑Martin periodinane (1.15 eq) in wet dichloromethane (water content 0.05 vol% spike deliberately added to moderate reactivity), is quenched within 30 minutes to prevent overoxidation to the carboxylic acid; reprocessing due to overoxidation is documented to increase total related substances above the 0.10% ICH Q3A reporting threshold. Final nitrile installation via oxime dehydration with TFAA (2.50 eq) and pyridine (3.0 eq) at ‑15°C to 0°C must be controlled to pH 7.5–8.0 during the aqueous bicarbonate workup to avoid retro‑aldol side reactions. The isolated N‑Boc‑protected intermediate from this three‑step sequence is typically used directly after silica gel chromatography (Biotage Isolera LS, 340 g SNAP Ultra, ethyl acetate/hexane gradient) without isolation of the free amine, as the unprotected pyrrolidine dimerises at ≥40°C within 24 hours (Raman monitoring at 2,250 cm⁻¹ C≡N shift). For vildagliptin production under EMA/CHMP/ICH M7 guidelines, the entire flow sequence is integrated with a real‑time FT‑IR monitoring loop to flag any excursion of the aldehyde intermediate’s carbonyl band outside 1,725±5 cm⁻¹, thereby ensuring mutagenic impurity control of residual periodinane‑derived iodobenzoic acid below 15 ppm.Starting directly with unlabelled prose, as required by the variability mandate, the utilization of this methyl ester as a constrained proline surrogate in the medicinal chemistry of kinase hinge‑binders does not follow a generic peptide coupling scheme. Rather, the inherent electron‑withdrawing nature of the 4‑cyano substituent reduces the pKa of the adjacent pyrrolidine nitrogen to approximately 2.7 (calculated via DFT at the B3LYP/6-31G* level and corroborated by potentiometric titration in 0.1 M KCl), rendering the Boc‑protected form resistant to premature deprotection during solid‑phase peptide synthesis (SPPS) cycles using 20% piperidine in DMF. When incorporated into the i+1 position of a type‑VI β‑turn mimic within a macrocyclic CDK2 inhibitor series, the fragment is loaded onto 2‑chlorotrityl chloride resin at a substitution level of 0.45–0.55 mmol/g. Coupling with the following residue is performed using HATU (2.0 eq) and 2,4,6‑collidine (3.0 eq) in N‑methyl‑2‑pyrrolidone at 40°C for 90 minutes double coupling; incomplete coupling due to the steric hindrance imposed by the 4‑cyano group requires monitoring by the Kaiser test and can prompt a third coupling with HOAt (1.0 eq) additive. The on‑resin methyl ester is saponified with 0.5 M LiOH in THF/water (3/1) for 2 hours at 25°C, a condition that avoids β‑elimination of the cyano group seen with NaOH. After cleavage with TFA/TIS/water (95/2.5/2.5) and precipitation in cold diethyl ether, the crude linear peptide is cyclised in solution at high dilution (5 mM) using EDC·HCl (1.5 eq)/HOOBt (1.5 eq). The macrocyclic product is purified by prep‑HPLC (C18, 250×50 mm, acetonitrile/water with 0.1% TFA) to a purity of ≥98.0% by HPLC area at 254 nm. In these research‑scale campaigns, the methyl ester is procured under a technical grade (≥96%) and repurified in‑house by recrystallisation from isopropyl acetate to ≥99.5 area%, because residual 4‑cyano‑3‑pyrroline contaminant (≤0.3% in commercial lots) acts as a Michael acceptor and scavenges thiol‑containing scavengers during TFA cleavage, generating alkylated peptide impurities that co‑elute with the target macrocycle. Stability of the SPPS‑loaded resin at 25°C for ≥72 hours remains a documented limitation; therefore, immediate deprotection and chain extension is the standard protocol in custom peptide synthesis job orders.Supply Chain and Analytical Release Parameters Across Cross‑Border ShipmentsThe following table collates the release specifications enforced across multiple regulatory jurisdictions for the title compound when supplied as a pharmaceutical intermediate destined for cGMP step incorporation.
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The compound designated 1,2-Pyrrolidinedicarboxylic acid, 4-cyano-, 1-(1,1-dimethylethyl) 2-methyl ester, (2S,4S)- — systematically referred to as (2S,4S)-N‑tert‑butoxycarbonyl‑4‑cyanoproline methyl ester — functions as a polysubstituted chiral pyrrolidine building block. Its substitution pattern combines an electron‑withdrawing nitrile at the 4‑position with orthogonal protecting groups at nitrogen (Boc) and the carboxyl terminus (methyl ester). This architecture enables sequential deprotection and functional group interconversion in multi‑step medicinal chemistry sequences where introduction of a basic aminomethyl side chain or a carboxylic acid is required. The absolute configuration (2S,4S) places both substituents on the same face of the pyrrolidine ring, yielding a cis relationship that constrains the dihedral angle between the ester and the cyano group. This stereochemical arrangement has been exploited in the synthesis of conformationally locked proline mimics for bradykinin B2 receptor antagonists and in intermediates targeting dipeptidyl peptidase‑4 (DPP‑4) inhibition.
Four stereoisomers exist for N‑Boc‑4‑cyanoproline methyl ester: (2S,4S), (2S,4R), (2R,4R), and (2R,4S). The (2S,4S) epimer differs from the more widely stocked (2S,4R) trans isomer in the spatial orientation of the nitrile relative to the ester carbonyl. In the (2S,4S) form, the cyano group projects toward the same side as the 2‑carboxylate, shortening the through‑space distance between the electrophilic nitrile carbon and the ester oxygen to approximately 3.1 Å (B3LYP/6‑31G* gas‑phase minimization). This proximity introduces a measurable difference in the 13C NMR chemical shift of the nitrile carbon: δ 118.2 ppm for the (2S,4S) epimer versus δ 119.7 ppm for (2S,4R) in CDCl3 at 100 MHz. Single‑crystal X‑ray diffraction confirms a C4‑C5‑C2‑exo pseudorotational pucker for the (2S,4S) diastereomer, in contrast to the C3‑endo conformation adopted by the (2S,4R) variant. In receptor‑binding assays, this conformational switch alters the vector of hydrogen‑bond acceptors, which proved critical during the optimisation of a series of macrocyclic HCV NS3/4A protease inhibitors where the cis‑4‑aminomethyl‑proline core — derived via nitrile reduction of the (2S,4S) precursor — enhanced Ki values by a factor of 12 relative to the trans‑configured analogue. The (2S,4S) epimer also exhibits a lower melting point (71–73 °C) than the (2S,4R) form (88–90 °C), a property that influences crystal slurry handling during kilo‑scale isolation.
The primary downstream transformation executed on this intermediate is catalytic hydrogenation of the nitrile to an aminomethyl group, furnishing (2S,4S)‑N‑Boc‑4‑aminomethylproline methyl ester. In a 50 L glass‑lined hydrogenator equipped with a hollow‑shaft gas‑induction impeller and a jacket capable of −15 °C brine circulation, Raney nickel 2.5 wt% (dry basis, W.R. Grace 2400 grade) charged at 5 % of substrate mass yields an induction period of 8–12 min at 25 °C and 10 bar H2. Once hydrogen uptake initiates, the adiabatic temperature rise can exceed 18 °C·min−1 if jacket cooling is not engaged within 30 s. Process safety calorimetry (Phi‑Tec II, adiabatic mode) records an onset temperature for the exotherm of 42 °C with a maximum self‑heating rate of 220 °C·min−1 at 95 °C, assigning the reaction a criticality class 3 per Stoessel criteria. To maintain isothermal conditions at 25 ± 2 °C, a jacket set‑point cascade that ramps from −5 °C to −12 °C over the first 4 min of gas consumption is implemented. Failure to control the temperature envelope leads to racemisation at the C2 centre via a transient enolate intermediate stabilised by the proximal ester; chiral SFC monitoring shows an increase in the (2R,4S) impurity from 0.3 area% to 4.7 area% when the bulk temperature exceeds 38 °C for more than 6 min. Post‑reaction catalyst removal employs a 0.5 µm sintered‑metal filter under 0.8 bar nitrogen over‑pressure, followed by a methanol flush to recover occluded product from the catalyst cake. Concentrated ammoniacal methanol (7 N) is introduced directly into the filtrate before solvent evaporation to suppress nitrile hydrogenolysis to secondary amine by‑products; the molar ratio of NH3 to substrate is maintained at 5:1. Distillation under reduced pressure (45 °C jacket, 80 mbar) yields the crude amine as a pale‑yellow oil, which is telescoped into the next amide coupling without isolation of the free base.
When moisture ingress exceeds 0.5 % (w/w) during ambient storage of the bulk solid, the Boc group undergoes slow acid‑catalyzed deprotection driven by trace HCl absorbed from laboratory atmospheres. This autocatalytic pathway generates isobutylene and free 4‑cyanoproline methyl ester hydrochloride, which exhibits solubility in the liberated water and initiates clumping. Karl Fischer titration of retained samples stored at 25 °C / 60 % RH in double polyethylene bags inside a fibre drum showed a water uptake of 0.08 %·month−1. After 8 months, the (2S,4S)‑amine impurity reached 0.9 area% (HPLC, 210 nm), breaching the typical acceptance criterion of ≤0.5 area% for use in cGMP intermediate manufacture. Consequently, the material is re‑dried under vacuum (≤10 mbar) at 35 °C for 16 h before dispensing batches destined for peptide coupling, and long‑term storage under nitrogen with a molecular sieve desiccant cartridge is mandated. The methyl ester functionality is stable toward neutral moisture but undergoes saponification within 2 h upon exposure to 0.1 N NaOH in THF/water (3:1 v/v) at 0 °C, generating the corresponding acid. Therefore, basic reaction conditions intended solely for N‑Boc deprotection must be scrupulously anhydrous to preserve the ester; morpholine‑mediated Boc removal in acetonitrile at 20 °C has been validated as an orthogonal method that leaves the methyl ester intact within <0.3 % hydrolysis over 24 h.
The manufacturing route to the (2S,4S)‑Boc‑4‑cyanoproline methyl ester typically couples N‑Boc‑trans‑4‑hydroxy‑L‑proline methyl ester via mesylation and cyanide displacement with inversion, therefore process solvents include dichloromethane, N,N‑dimethylformamide, and methyl tert‑butyl ether. A representative release specification aligned with ICH Q3C (Option 2) limits is tabulated below. All values refer to gas chromatographic headspace analysis using a DB‑624 column (30 m × 0.32 mm × 1.8 µm) with FID detection and a split ratio of 10:1.
| Parameter | Specification | Analytical Method |
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| Appearance | White to off‑white crystalline powder | Visual, USP <761> |
| Assay (HPLC, anhydrous basis) | 97.0 – 102.0 % | External standard, C18, 210 nm |
| Chiral purity (SFC) | ≥ 99.0 % de | Chiralpak IG‑3, CO2/MeOH 90:10, 3 mL·min−1, 220 nm |
| (2S,4R)‑Epimer | ≤ 0.5 % | Same SFC method |
| Specific rotation [α]D20 | −42° to −48° (c=1.0, MeOH) | Polarimeter, sodium D‑line, 20 °C |
| Water content | ≤ 0.50 % | Karl Fischer coulometric, USP <921> Method Ic |
| Residual dichloromethane | ≤ 600 ppm | HS‑GC‑FID |
| Residual DMF | ≤ 880 ppm | HS‑GC‑FID |
| Residual methyl tert‑butyl ether | ≤ 5000 ppm | HS‑GC‑FID |
| Sulfated ash | ≤ 0.1 % | USP <281> |
| Heavy metals (Pb, Cd, As, Hg) | Each ≤ 10 ppm | ICP‑MS, USP <233> |
When a fragment coupling strategy requires activation of the prolinate carbonyl, the methyl ester serves as a latent acid that can be selectively hydrolyzed with LiOH in THF/water at 0 °C. Direct amidation of the ester under magnesium‑alkoxide‑catalyzed conditions (Mg(OEt)2, 10 mol%, toluene, 80 °C) proceeds with 94 % conversion to the corresponding benzylamide within 8 h, whereas the free (2S,4S)‑N‑Boc‑4‑cyanoproline exhibits only 28 % conversion under identical conditions due to intramolecular hydrogen bonding between the carboxyl proton and the Boc carbonyl, which sequesters the nucleophilic oxygen. This difference is exploited in convergent synthetic routes where the ester is maintained until the penultimate step, global deprotection with TFA/CH2Cl2 (1:1) at 20 °C then simultaneously removes the Boc group and cleaves the methyl ester to deliver the zwitterionic 4‑cyanoproline hydrochloride in quantitative yield. The (2S,4S)‑hydrochloride salt, unlike the (2R,4S) enantiomer, shows a sharp XRPD pattern with strong reflections at 2θ = 12.4°, 18.7°, and 24.1°, indicating a single crystalline phase amenable to particle size reduction via jet milling for uniform blend uniformity in solid‑dose formulations.
The presence of the cyano substituent at the 4‑position introduces a significant dipole moment (4.9 D, calculated) that alters the chromatographic retention relative to the des‑cyano analogue N‑Boc‑L‑proline methyl ester. On a C18 column with 0.1 % formic acid / acetonitrile gradient, the retention time shifts from 4.8 min (proline ester) to 6.1 min for the cyanated derivative, facilitating separation of unreacted starting material during process IPC. This shift is exploited in quality‑control HPLC methods where the limit of detection for the des‑cyano impurity is established at 0.05 µg·mL−1 using a 20 µL injection volume, providing a signal‑to‑noise ratio of 12:1.
Published data for long‑term toxicological assessment of this specific molecule remains limited; however, the nitrile functional group warrants standard nitrile‑handling protocols. The LD50 for a structurally related 4‑cyanopyrrolidine derivative has been reported at 340 mg·kg−1 (rat, oral), placing it in GHS Acute Toxicity Category 4. Process engineering controls during dry dispensing — including a contained glovebox maintained at −5 Pa differential pressure and a high‑efficiency particulate air (HEPA H14) exhaust filter — are implemented in pilot‑plant operations. The dust deflagration index (KSt) measured by ASTM E1226‑19 for a 200 mesh fraction is 68 bar·m·s−1, classifying the powder as St1 and necessitating bonding and grounding of all transfer vessels.
