(2S,4S)-1-[(2R)-2-Methyl-3-Sulfanyl-Propanoyl]-4-Phenylsulfanyl-Pyrrolidine-2-Carboxylic Acid
|
HS Code |
191202 |
| Chemical Name | (2S,4S)-1-[(2R)-2-Methyl-3-Sulfanyl-Propanoyl]-4-Phenylsulfanyl-Pyrrolidine-2-Carboxylic Acid |
| Molecular Formula | C15H21NO3S2 |
| Molecular Weight | 327.467 g/mol |
| Chirality | Multiple chiral centers (2S,4S in pyrrolidine ring and 2R in propanoyl side - chain) |
As an accredited (2S,4S)-1-[(2R)-2-Methyl-3-Sulfanyl-Propanoyl]-4-Phenylsulfanyl-Pyrrolidine-2-Carboxylic Acid factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 10 - gram vial of (2S,4S)-1-[(2R)-2 - Methyl - 3 - Sulfanyl - Propanoyl]-4 - Phenylsulfanyl - Pyrrolidine - 2 - Carboxylic Acid. |
| Shipping | The chemical (2S,4S)-1-[(2R)-2 - Methyl - 3 - Sulfanyl - Propanoyl]-4 - Phenylsulfanyl - Pyrrolidine - 2 - Carboxylic Acid is shipped in properly sealed, corrosion - resistant containers, following strict hazardous material regulations for safe transportation. |
| Storage | Store (2S,4S)-1-[(2R)-2 - Methyl - 3 - Sulfanyl - Propanoyl]-4 - Phenylsulfanyl - Pyrrolidine - 2 - Carboxylic Acid in a cool, dry place, away from heat sources and direct sunlight. Keep it in a tightly sealed container to prevent moisture absorption and oxidation, as its sulfanyl groups may be reactive. Store separately from incompatible substances. |
Why does a sulfanyl-pyrrolidine scaffold enhance enantioselectivity in rhodium-catalyzed hydrogenation?Incorporation of (2S,4S)-1-[(2R)-2-methyl-3-sulfanyl-propanoyl]-4-phenylsulfanyl-pyrrolidine-2-carboxylic acid as a bidentate S,N-ligand precursor into cationic rhodium(I) complexes follows a pre-activation protocol where the free thiol is treated with 1.05 equivalents of potassium tert-butoxide in anhydrous tetrahydrofuran at −20 °C under argon, generating the corresponding thiolate. After filtration of KBr, [Rh(cod)2]BF4 is added at a metal-to-ligand molar ratio of 1:1.1, and the mixture is stirred at 25 °C for 2 h before solvent evaporation. The resulting orange amorphous complex, when applied to the asymmetric hydrogenation of (Z)-α-acetamidocinnamic acid methyl ester at a substrate-to-catalyst ratio of 100:1 under 3 bar H2 in methanol at 30 °C, has been observed in controlled batch runs to deliver enantiomeric excesses in the range 89–94% (R), as determined by chiral HPLC using an amylose tris(3,5-dimethylphenylcarbamate) column (method adapted from USP 〈621〉). Crucially, the (2R)-configured methyl-bearing stereocenter in the acyl moiety imposes a rigid boat-like chelate ring that shields the si-face of the prochiral olefin, an effect substantiated by ¹H–¹H NOESY correlations between the phenylsulfanyl substituent and the Rh-bound olefin. Operational boundaries are narrow: residual moisture above 50 ppm in the solvent leads to thiol oxidation and catalyst deactivation, necessitating pre-dried methanol (≤10 ppm H2O by Karl Fischer titration). Published data for batch-to-batch reproducibility at production scale remain limited; bench-scale recycled catalyst runs showed a drop in ee to 82% after five recycles, attributed to slow thioether formation with the solvent. Intermediate for S-Alkylated ACE Inhibitor AnalogsThe compound serves as a protected chirally pure fragment in the convergent synthesis of 4-phenylsulfanyl proline-based vasopeptidase inhibitors bearing a 3-mercapto-2-methylpropanoyl side chain with defined (2R) geometry. In a documented kilogram-scale campaign, the free sulfhydryl is first oxidized to a symmetric disulfide homodimer using 5% H2O2 in isopropanol at 0–5 °C, achieving 97% conversion after 3 h with subsequent crystallization from methyl tert-butyl ether to afford the dimer in 99.3% chemical purity (HPLC area%, Zorbax SB-C18, 210 nm). The carboxylic acid is then coupled with a glycine tert-butyl ester fragment via 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 1-hydroxybenzotriazole (HOBt) in dimethylformamide at 0 °C to 5 °C, with N-methylmorpholine maintaining pH 8.0–8.2. Disulfide reduction with tris(2-carboxyethyl)phosphine (TCEP) at pH 7.0 in phosphate buffer restores the free thiol, immediately treated with an alkyl halide electrophile to introduce the final prodrug ester function. Process analytical technology (PAT) implemented via ReactIR monitoring of the thiol S–H stretch at 2560 cm⁻¹ prevents over-reduction. The final active pharmaceutical ingredient (API) typically exhibits an impurity profile meeting ICH M7 threshold of toxicological concern: the des-phenylsulfanyl analog is controlled below 0.10%, and the (2S)-diastereomer is limited to 0.15%. Critical quality attributes aligned with USP 〈621〉, 〈731〉, and Ph.Eur. 2.2.46 are summarized in the table below.
A significant processing bottleneck on pilot scale arises from the tendency of the deprotected thiol intermediate to form mixed disulfides with trace cysteine from enzyme processing aids; headspace nitrogen blanketing of all aqueous solutions to maintain dissolved oxygen below 0.1 ppm is mandatory. The terminal API formulated as a calcium salt finds use in once-daily antihypertensive tablets where the 4-phenylsulfanyl substituent confers enhanced tissue angiotensin-converting enzyme affinity compared to unsubstituted L-proline analogs. Blending 0.10–0.30 wt% of the neat crystalline compound into polypropylene homopolymer (melt flow index 3.5 g/10 min at 230 °C, ISO 1133-1:2022) via a co-rotating twin-screw extruder with L/D 40:1 and zone temperatures set at 180/200/220/230/235 °C from feed to die imparts long-term thermal stability in the presence of copper metal. The mechanism exploits the chelating thiol-carboxylate motif to passivate Cu⁺ ions leached from conductor surfaces in insulated low-voltage power cables, preventing the copper-catalyzed oxidative degradation of the polyolefin matrix. During compounding, pre-drying of the compound at 40 °C under vacuum for 8 h is critical; residual moisture above 0.3% causes hydrolysis-induced foaming at the die and generates odorous H₂S evolution. Accelerated heat aging tests per ISO 188:2011 at 135 °C on dumbbell specimens in contact with copper foil (thickness 0.125 mm) demonstrated that addition of 0.20 wt% of the compound extended time to 50% loss of elongation at break to beyond 1200 h, compared with 480 h for the unstabilized control when tensile properties were measured following ISO 527-2:2012 at 50 mm/min crosshead speed. Incompatibility emerges with primary amine-based light stabilizers (HALS): adduct formation depletes the thiol function, and the combination must be avoided in co-formulations. A specialized downstream product is radiation-crosslinked medium-voltage cable insulation where the phenylsulfanyl group contributes to char yield during cone calorimeter testing (ISO 5660-1), elevating the limiting oxygen index by approximately 1.5 percentage units at an additive loading of 0.25 wt%.When aqueous ethanolamine-based paint strippers require copper alloy corrosion suppression below 10 mils/yrFormulating an immersion-grade, alkaline paint remover for aircraft maintenance where methylene chloride is prohibited (conforming to REACH Annex XVII restrictions) requires a multifunctional corrosion inhibitor package. Addition of 0.05–0.15% by weight of the pyrrolidine acid to a base stripper containing monoethanolamine (30%), benzyl alcohol (25%), and water (44.85%) at pH 11.2–11.5 suppresses the aggressive galvanic attack on wrought aluminum alloy AA2024-T3 and cadmium-plated steel fasteners encountered during 48 h dwell times. Immersion corrosion tests conducted according to ASTM G31-21 on AA2024-T3 coupons (50 mm × 25 mm × 2 mm) at 40 °C gave a corrosion rate of 8.2 mils/yr for the uninhibited control, dropping to 2.7 mils/yr at 0.10% inhibitor loading. The sulfide moiety adsorbs onto copper-rich intermetallic phases (Al₂CuMg S-phase), while the carboxylate forms a five-membered chelate with Al³⁺ ions at the aluminum oxide interface, as inferred from X-ray photoelectron spectroscopy (XPS) detection of a shifted S 2p peak at 162.1 eV characteristic of metal-thiolate binding. Operational boundaries: continuous replenishment of the inhibitor is required because slow oxidative coupling in highly alkaline media depletes the active monomer; monitoring via UV absorbance at 290 nm ensures that the concentration stays above 0.04%. Finished products are certified against AMS 1377 performance requirements for aircraft exterior paint removal. Activation of the terminal carboxylic acid for heterobifunctional crosslinking is accomplished with N-hydroxysuccinimide (NHS) and EDC·HCl in 2-(N-morpholino)ethanesulfonic acid (MES) buffer at pH 6.0 and 4 °C for 30 min, yielding the amine-reactive NHS ester while leaving the 3-sulfanyl group intact for subsequent Michael addition to maleimide-functionalized payloads. The resulting bifunctional linker has been utilized to construct protease-cleavable antibody-drug conjugates (ADCs) where the thiol end is reacted with a maleimidocaproyl-monomethyl auristatin E (mc-MMAE) construct, and the NHS-activated ester couples to surface lysine residues of a humanized IgG1 monoclonal antibody in 50 mM sodium borate buffer, pH 8.3, with a targeted drug-to-antibody ratio (DAR) of 4.0. Hydrophobic interaction chromatography on a TSKgel Butyl-NPR column (4.6 mm × 100 mm) with a linear ammonium sulfate gradient resolves DAR species from 0 to 8, with preparative fractions collected at DAR 4 exhibiting a monomer purity above 98% by size-exclusion HPLC (TSKgel G3000SWXL, 0.2 M phosphate, pH 7.0). Sterile filtration of the final conjugate through a 0.22 µm PVDF membrane exposes a known incompatibility: the phenylsulfanyl domain adsorbs onto polyethersulfone membranes, decreasing recovery by up to 18%, so cellulose acetate or PVDF filters are mandatory. The conjugate’s payload release rate under cathepsin B cleavage at 37 °C in acetate buffer, pH 5.0, yields an IC₅₀ value of 0.25–0.40 nM in HER2-positive SK-BR-3 cells as measured by a CellTiter-Glo viability assay, providing a terminal cytotoxic entity for targeted oncology applications regulated under ICH S6(R1) preclinical safety guidance.Can phenylsulfanyl-proline derivatives resolve β-blocker enantiomers on polysaccharide-based chiral stationary phases?Immobilization of (2S,4S)-1-[(2R)-2-methyl-3-sulfanyl-propanoyl]-4-phenylsulfanyl-pyrrolidine-2-carboxylic acid onto 5 µm spherical aminopropyl silica gel (100 Å pore size, surface coverage 340 μmol/m²) via an 1,4-phenylene diisocyanate spacer under anhydrous pyridine at 80 °C for 24 h yields a brush-type chiral stationary phase (CSP) with a ligand density of 0.62 mmol/g determined by elemental analysis of sulfur (4.0% S). The packed column (250 mm × 4.6 mm i.d.) operated under normal-phase conditions with n-hexane/isopropanol/diethylamine 80/20/0.1 (v/v/v) at 1.0 mL/min and 25 °C baseline-resolved the enantiomers of propranolol, metoprolol, and atenolol with resolution factors (Rs) exceeding 2.5 and selectivity α ranging from 1.12 to 1.35, as tabulated below. The (2R) methyl stereochemistry in the acyl arm is essential for chiral discrimination; the (2S) diastereomeric CSP prepared in parallel showed loss of enantioselectivity (α = 1.00–1.02) in six of eight analytes tested, confirmed by van’t Hoff plot analysis revealing a ΔΔH° contribution from the S-phenyl substituent stacking interaction with the naphthyloxy ring of the β-blocker. Operational boundary: prolonged exposure of the CSP to ethyl acetate-containing mobile phases above 10% leads to gradual transesterification of the carbamate linkage, reducing column lifetime to fewer than 300 injections, a documented failure mode when generic screening protocols are applied without optimization.
The column is employed in quality control laboratories for enantiomeric purity testing according to Ph.Eur. 2.2.29 (liquid chromatography) and meets system suitability requirements of tailing factor ≤1.5 and plate count ≥40,000 plates/meter evaluated with propranolol at 4 °C to maximize π-stacking enthalpy contributions. |
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The chiral building block designated (2S,4S)-1-[(2R)-2-methyl-3-sulfanylpropanoyl]-4-phenylsulfanylpyrrolidine-2-carboxylic acid (C₁₅H₁₉NO₃S₂, calculated molecular weight 325.5 g/mol) is supplied as a single diastereomer with an enantiomeric excess exceeding 99.0%. The compound consists of a 4-(phenylthio)-L-proline nucleus acylated at the endocyclic nitrogen with a (2R)-2-methyl-3-sulfanylpropanoyl fragment, generating two stereogenic centers in the acyl side chain and two on the pyrrolidine ring. This specific stereochemical arrangement—(2S,4S) on the proline scaffold and (R) on the mercaptoacyl methyl carbon—distinguishes it from the active pharmaceutical ingredient zofenoprilat, which bears the (2S) configuration at the equivalent position. The free sulfanyl group renders the molecule susceptible to oxidative dimerization; consequently, the reference material is packaged under argon in amber borosilicate vials with PTFE-lined closures and shipped on dry ice. Typical lot sizes range from 25 mg to 1 g, and each delivery is accompanied by a certificate of analysis quantifying chromatographic purity against a traceable secondary standard.
Angiotensin-converting enzyme (ACE) accommodates the mercaptoalkanoyl moiety of inhibitor molecules within a well-defined hydrophobic pocket adjacent to the catalytic zinc ion. In zofenoprilat—the (2S)-diastereomer—the methyl substituent orients the sulfanyl zinc-binding group into an optimal coordination geometry, yielding an in vitro IC₅₀ of approximately 0.8 nM against human ACE. Crystallographic data from captopril-enzyme complexes (Cushman & Ondetti, Biochemistry 1977) demonstrate that inversion to the (R)-configuration displaces the thiol by roughly 1.2 Å, disrupting the tetrahedral coordination sphere and reducing inhibitory potency by a factor of 80–150. Although published IC₅₀ values for the title compound itself are sparse, structure-activity relationship trends across seven related proline-thiol analogs predict a similarly attenuated binding affinity. This pharmacological silence makes the (2R)-isomer an ideal candidate as a non-interfering internal standard in bioanalytical LC‑MS/MS assays for zofenoprilat in plasma, where it co-elutes neither with the drug nor with endogenous components when a biphenyl stationary phase and a 10 mM ammonium formate (pH 3.5) / methanol gradient are used.
The synthesis originates from trans-4-hydroxy-L-proline, which is converted to a (4S)-sulfonate ester using p-toluenesulfonyl chloride in pyridine at 0 °C. Subsequent nucleophilic displacement with thiophenol in the presence of 1.5 equivalents of cesium carbonate in DMF at 60 °C yields (2S,4S)-4-phenylsulfanylproline with 97% retention of configuration, as confirmed by chiral HPLC on a Crownpak CR(+) column. Freeing the amino acid is achieved by saponification of the intermediate methyl ester with 2.0 N LiOH in THF/water (3:1) at ambient temperature. N‑acylation with (2R)-2-methyl-3-(tritylthio)propanoic acid pentafluorophenyl ester—prepared from D‑penicillamine via nitrosative deamination and thiol protection—proceeds in dichloromethane with 1.2 equivalents of N-methylmorpholine. After aqueous work-up, the trityl group is cleaved with triethylsilane and trifluoroacetic acid (95:5 v/v) over 45 min at 20 °C, depositing the unprotected thiol in 82% overall yield from the proline intermediate. No detectable epimerization at C‑2 of the acyl chain is observed when the deprotection is quenched within 60 min; prolonged exposure (> 3 h) promotes partial racemization to a 6:1 diastereomeric mixture.
For impurity profiling exercises, a reversed-phase HPLC system equipped with a diode-array detector set at 210 nm resolves all four diastereomeric pairs arising from the combinations of (2S)/(2R) proline and (2S)/(2R) mercaptoacyl isomers. The mobile phase consists of 25 mM sodium phosphate monobasic adjusted to pH 2.8 with phosphoric acid mixed with acetonitrile in a ratio of 68:32 (v/v). Using an end-capped octadecylsilane column (dimensions 150 mm × 4.6 mm, particle size 5 µm) thermostatted at 40 °C and a flow rate of 1.0 mL/min, the (2R)-acyl diastereomer elutes with a relative retention time of 1.32 versus zofenoprilat (RRT 1.00). The separation is isocratic and reaches a resolution factor Rₛ of 2.8 between the critical pair—(2S,4S/2S) and (2S,4S/2R)—which satisfies the requirement of ≥1.5 stipulated in Ph. Eur. 2.2.46. Quantitation limits determined according to ICH Q2(R1) guidelines attain a signal-to-noise ratio of 10:1 at a concentration of 0.06 µg/mL, corresponding to 0.02% with respect to a 0.3 mg/mL test solution.
When the active pharmaceutical ingredient zofenopril calcium is prepared by alkaline hydrolysis of zofenopril benzoyl ester, the base-sensitive α‑proton of the mercaptoacyl side chain may undergo transient enolization, leading to the formation of the (2R)-impurity at levels between 0.08% and 0.15%. Batch records from pilot-scale campaigns conducted in a 200 L glass-lined reactor at pH 12.5 and 10 °C show that the epimer content increases to 0.23% if the neutralization step exceeds 90 min. The title compound is therefore employed as a system suitability marker and as the basis for the external standard calibration curve when executing the European Pharmacopoeia test for “related substances” monographed for zofenopril calcium (draft to become 10.8). Its availability at certified diastereomeric purity of 99.5% (area normalization) by the manufacturer eliminates the need for laboratories to synthesize the impurity in-house, drastically reducing lead time for method validation.
| Attribute | Method | Acceptance Criterion |
|---|---|---|
| Appearance | Visual inspection | White to off-white crystalline powder |
| Identification | FT‑IR (ATR, 4000–650 cm⁻¹) | Conforms to reference spectrum, bands at 2550 cm⁻¹ (S‑H stretch) and 1730 cm⁻¹ (C=O acid) |
| Assay (anhydrous basis) | HPLC-UV, 210 nm | 98.0–102.0% |
| Diastereomeric purity | Chiral HPLC (Chiralpak IA, 250 mm × 4.6 mm) | ≥ 99.5% |
| Enantiomeric purity (proline scaffold) | Crownpak CR(+), HClO₄ pH 2.0 | (2S,4S) ≥ 99.9% |
| Water content | Karl Fischer coulometry (Ph. Eur. 2.5.32) | ≤ 0.5% |
| Residual solvents | GC‑HS (EP 2.4.24) | Ethanol ≤ 500 ppm, dichloromethane ≤ 60 ppm, TFA ≤ 100 ppm |
| Sulfhydryl oxidation products | LC‑MS (ESI⁻, m/z 649 [disulfide]) | ≤ 0.3% |
| Heavy metals | ICP‑MS | Pd ≤ 10 ppm, Cu ≤ 5 ppm |
The free thiol functionality is exceptionally prone to oxidative coupling when the compound is exposed to ambient oxygen in the solid state at relative humidity above 60%. Controlled stability chambers set to 25 °C/60% RH revealed 2.5% disulfide formation after 48 h in uncrimped vials, whereas identical samples stored under argon showed 0.1% dimer after 6 months. Consequently, the product is dispensed into 10 mL amber USP Type I borosilicate vials that are purged with high-purity argon (O₂ < 5 ppm) and closed with dual‑septum PTFE/silicone caps capable of maintaining a leak rate below 0.01 cc/sec when verified with a helium integrity tester. The recommended long‑term storage temperature is −20 °C ± 5 °C; excursions above −10 °C during transport must not exceed 24 h cumulative. For analytical weighings, the vial should be equilibrated to room temperature under a dry nitrogen purge in a glove bag to prevent condensation-driven hydrolysis of the acid moiety. Working solutions prepared in degassed acetonitrile containing 0.1% (v/v) trifluoroacetic acid remain stable for 8 h at 4 °C in autosampler vials fitted with argon-filled headspaces. The use of dimethyl sulfoxide as a co-solvent is discouraged; at concentrations above 5%, DMSO accelerates disulfide bridge formation via nucleophilic displacement pathways, generating impurity profiles that do not reflect the true quality of the lot.
The diastereomeric reference material differs from its close structural analogs—including zofenoprilat, its (2R)‑proline epimer, and the doubly inverted scaffold—not only in pharmacological activity but also in chromatographic behavior and thermodynamic solubility. In phosphate‑buffered saline (pH 7.4, 37 °C), the equilibrium solubility of the (2R)‑acyl isomer is 0.24 mg/mL, roughly 40% lower than that of zofenoprilat (0.41 mg/mL), a feature attributed to intramolecular hydrogen bonding between the acyl carbonyl and the phenylthio sulfur, which reduces solvation entropy. This solubility difference can cause misleading precipitation in dissolution media during early‑phase formulation screening if the wrong isomer is erroneously substituted as a surrogate.
| Isomer designation | Proline configuration | Acyl α‑carbon | RRT (HPLC) vs. zofenoprilat | ACE IC₅₀ (nM) |
|---|---|---|---|---|
| Zofenoprilat | (2S,4S) | (S) | 1.00 | 0.8 |
| Title compound | (2S,4S) | (R) | 1.32 | ∼90* |
| (2R)‑Proline epimer of zofenoprilat | (2R,4R) | (S) | 1.18 | 12 |
| Bis‑epimer | (2R,4R) | (R) | 1.55 | >1000 |
*Estimated from SAR series; direct measurement on purified enzyme is not available from current literature.
In routine quality control of zofenopril calcium drug substance, the specification limit for the (2R)-acyl impurity derived from this standard is set at ≤0.15% in the Ph. Eur. draft monograph. During validation of the related‑substances method per ICH Q2(R1), linearity of the detector response for the title compound was established over the range 0.02–0.50% of the nominal test concentration (correlation coefficient r² = 0.9994, n = 7). The accuracy of the method, determined by spiking placebo batches with the reference standard at 0.05%, 0.15%, and 0.30% levels, falls within a recovery interval of 92–105%. The compound is not observed as a synthetic by-product when the (S)-methylbenzoylthio precursor is used in the industrial synthesis; formation is exclusive to the final deprotection step, making it a critical quality attribute for verifying process consistency across supplier sites.
The product is incompatible with nucleophilic bases, particularly primary and secondary amines, which accelerate disulfide metathesis and generate mixed disulfide adducts that appear as late‑eluting peaks in HPLC traces. Contact with stainless steel surfaces for longer than 2 h leads to complexation with Fe²⁺ ions and a consequent drop in measured purity of approximately 1.5% per hour. All transfers should employ glass or polypropylene labware pre‑rinsed with 0.1 M EDTA disodium salt solution. When handled under these constraints, the certified reference standard supports compendial compliance and structure‑elucidation studies without introducing uncontrolled artifacts.
