(Z)-3-(Aminomethyl)-4-(Methoxyimino)Pyrrolidine Dimethanesulfonate

How Does the Methoxyimino Group Modulate β-Lactamase Stability in C-3 Pyrrolidinium Cephalosporins?

Industrial batches of a parenteral fourth-generation cephalosporin incorporating the (Z)-3-(aminomethyl)-4-(methoxyimino)pyrrolidine fragment begin with the crystalline dimethanesulfonate salt as the storage-stable amine source. The free base is liberated in situ by dosing 1.05–1.10 molar equivalents of the salt into a chilled (‑5 °C to 0 °C) anhydrous tetrahydrofuran slurry containing 1.05 eq of a tertiary amine such as N-methylmorpholine, immediately followed by addition of a pre-activated cephalosporanic acid mixed anhydride prepared from pivaloyl chloride. Maintaining the temperature at ‑15 °C ± 2 °C during the acylation step is critical: above ‑10 °C, base-catalysed Z‑to‑E isomerisation of the methoxyimino group accelerates, dropping the Z‑isomer content below the pharmacopoeial threshold of 99.5% within 90 minutes and reducing affinity for penicillin-binding protein PBP3 by up to 40-fold. The reaction is quenched with 2 M methanesulfonic acid after 6–8 hours, simultaneously regenerating the dimethanesulfonate form for subsequent crystallisation. Downstream processing employs a gradient cooling crystallisation from isopropanol/water (85:15 v/v, seeded at 45 °C, cooled to ‑5 °C at 0.15 °C/min) in an agitated glass-lined reactor. The filter cake is washed with cold acetone and dried in a vacuum tray dryer operating at 40 °C and ≤10 mbar with nitrogen breaking cycles to avoid moisture uptake. The terminal product is a sterile cephalosporin active pharmaceutical ingredient (API) lyophilised powder intended for reconstitutable injection vials, typically formulated at 1 g or 2 g potency. Manufacturing operations must comply with ICH Q7 Good Manufacturing Practice for Active Pharmaceutical Ingredients, with residual solvents controlled per ICH Q3C (THF limit 720 ppm, dichloromethane 600 ppm by headspace GC‑FID) and elemental impurities evaluated against ICH Q3D Step 2 PDEs; palladium catalyst carryover is monitored by ICP‑MS per USP <232>/<233> and maintained below 10 ppm. Process-related impurities, especially the des‑methoxyimino analogue and the E‑oxime isomer, are controlled to ≤0.10% each by HPLC‑UV at 254 nm using a C18 column and phosphate buffer/acetonitrile gradient.

Veterinary Cephalosporin Intermediates: Steric Shielding and Meat Residue Compliance

Veterinary formulation chemists evaluate the dimethanesulfonate salt as a crystalline, non-hygroscopic precursor for introducing the sterically demanding (Z)-4-methoxyimino pyrrolidine motif into the C‑3 quaternary ammonium side chain of animal‑exclusive fourth‑generation cephalosporins such as cefquinome. The methoxyimino group provides conformational shielding that retards hydrolysis by chromosomally encoded AmpC β‑lactamases of respiratory pathogens like Mannheimia haemolytica and Actinobacillus pleuropneumoniae. In the pivotal coupling step, the free amine is generated by partitioning the dimethanesulfonate salt between dichloromethane and 10% aqueous potassium carbonate at 0–5 °C; the organic layer containing the liberated amine is then added dropwise at 0.95–1.05 molar equivalents to a solution of the 3‑chloromethyl cephalosporin nucleus in dimethylacetamide in the presence of 1.2 eq of triethylamine. Agitation is maintained at 150 rpm in a baffled glass-lined reactor for 5 hours while the internal temperature is kept below 5 °C to minimise β‑lactam ring opening. After phase separation, the aqueous phase is extracted with fresh dichloromethane, and the combined organic layers are concentrated under reduced pressure at 30 °C. The crude quaternary ammonium product is precipitated by adding ethyl acetate, filtered, and recrystallised from methanol‑acetone. The terminal dosage form is a 2.5% cefquinome sulphate suspension for intramammary infusion or an injectable solution for cattle and swine. Every batch supplied to the veterinary GMP chain must adhere to VICH GL18 for residue control because the active moiety partitions into milk and edible tissues; analytical release includes bacterial endotoxin testing per Ph. Eur. 2.6.14 with a specification of ≤0.20 EU mg^-1, residual dimethyl sulfoxide below 5000 ppm by GC‑FID (Ph. Eur. 5.4), and methanesulfonic acid content ≤0.50% w/w by ion chromatography. Maximum residue limits established in EU Regulation 37/2010 (Annex I) for cefquinome are 20 μg kg^-1 in bovine milk and 50 μg kg^-1 in bovine muscle, necessitating a mass balance demonstrating ≥99.8% clearance of the intermediate-derived impurities from the final drug substance synthesis pathway.

Integrating a conformationally constrained pyrrolidine scaffold into the linker region of heterobifunctional proteolysis-targeting chimeras (PROTACs) requires a monomer that simultaneously provides a hydrogen-bond-accepting methoxyimino group and a primary aminomethyl handle for further derivatisation. The dimethanesulfonate salt is first converted to the Fmoc‑protected free amine via a Schotten‑Baumann procedure (Fmoc‑Cl, 10% Na₂CO₃, THF‑water at 0 °C) and subsequently incorporated into a biotin‑PEG₄‑linker‑VHL ligand assembly using standard solid‑phase peptide synthesis protocols. The protected monomer is coupled at a loading of 0.85–0.95 mmol g^-1 on Rink amide resin with HATU (4.0 eq) and DIPEA (8.0 eq) in DMF for 45 minutes at 22 °C. Cleavage with TFA‑triisopropylsilane‑water (95:2.5:2.5) liberates the free amino‑functionalised linker motif, which after RP‑HPLC purification is coupled to a cereblon ligand to complete the degrader architecture. The terminal product is a PROTAC candidate molecule intended for in vivo pharmacology studies in tumour xenograft models, such as those degrading BRD4 or androgen receptor. Because the salt generates methanesulfonic acid during the final deprotection, process analytical control is crucial: free methanesulfonate in the crude product is monitored by capillary electrophoresis and kept below 0.15% w/w before lyophilisation, and any residual DMF is restricted to ≤880 ppm per ICH Q3C Class 2 guidelines. Early‑stage non‑GMP batches follow an in‑house specification of HPLC purity ≥95.0% at 220 nm and are screened for potential genotoxic methanesulfonate esters down to a threshold of 1.5 μg day^-1 in accordance with ICH M7(R1).

Agrochemical Building Blocks for Strobilurin-Mimicking Succinate Dehydrogenase Inhibitors

Modern fungicide discovery programs extensively exploit the methoxyimino pharmacophore found in commercial strobilurins, yet introducing a basic pyrrolidine anchor via this dimethanesulfonate salt creates a confluence of systemic mobility and target-site affinity. The salt is employed as a halogen‑free amine donor in the assembly of novel succinate dehydrogenase inhibitor (SDHI) candidates connecting a pyrrolidine‑oxime bridge to a pyrazole‑4‑carboxamide warhead. Pilot‑scale batches are produced by dissolving the dimethanesulfonate salt (1.00 eq) in N,N‑dimethylformamide, adding pyridine (1.50 eq) as an acid scavenger, and feeding a DMF solution of the imidoyl chloride derivative (1.20 eq) dropwise over 30 minutes while the jacket temperature is held at 10 °C ± 2 °C. This narrow thermal envelope is essential: at 15 °C the rate of Z‑to‑E oxime rearrangement doubles, forming the pharmacologically inferior E‑oxime congener whose in vitro IC₅₀ against Zymoseptoria tritici succinate‑ubiquinone reductase exceeds 10-fold the value of the Z‑isomer. After 4 hours the mixture is poured into ice‑water (2 °C) and the precipitate is collected, washed with cold water, and dried in a fluid‑bed dryer at inlet temperature 45 °C to a moisture endpoint of ≤0.50% w/w (Karl Fischer, CIPAC MT 30.6). The isolated technical‑grade intermediate has a purity specification of ≥97.0% by external standard GC‑MS analysis, with the E‑oxime limited to ≤1.50% and the des‑aminomethyl impurity to ≤0.30%. It is then forwarded to formulate a 20% suspension concentrate (SC) or a 50% water‑dispersible granule (WG) for foliar application on cereals at rates of 75–150 g a.i. ha^-1. Regulatory submission for an agrochemical active substance demands compliance with CIPAC Handbook K analytical methods, while environmental safety data are generated under OECD 502/503 aquatic toxicity protocols; the acute fish toxicity endpoint (LC₅₀ >100 mg L^-1 for Danio rerio) and the Daphnia magna immobilisation EC₅₀ >85 mg L^-1 are documented on the five‑batch analysis report. Residual acetonitrile carried from the final recrystallisation is controlled below 410 ppm by headspace GC‑FID in accordance with FAO/WHO JMPR recommendations.

Non‑peptidic HIV‑1 protease inhibitors derived from cyclic sulfonamide or hydroxyethylamine isosteres frequently incorporate a (3‑aminomethyl‑pyrrolidine) scaffold to engage the S2′ subsite via a hydrogen‑bonding network. The dimethanesulfonate salt supplies the amine nucleophile under rigorously anhydrous conditions for coupling to a tetrahydrofuranyl carbamate‑activated P1′ fragment. In a typical campaign on a 20‑L jacketed reactor, the salt (1.20 eq) is suspended in acetonitrile and cooled to ‑10 °C before adding Hünig’s base (3.00 eq). A pre‑chilled solution of the chloroformate‑activated intermediate in acetonitrile is fed at a rate that keeps the internal temperature below ‑5 °C, and the coupling is complete within 90 minutes as verified by TLC (silica gel 60 F₂₅₄, ethyl acetate‑hexane 1:1). The crude product is concentrated under reduced pressure and purified by silica gel column chromatography using a gradient from 30% to 70% ethyl acetate in hexane. Fractions with R_f=0.35 are pooled and evaporated to afford the protected drug intermediate as a single isomer. Final deprotection with HCl in dioxane yields the hydrochloride salt of the protease inhibitor precursor, which is subsequently coupled with a sulfonamide warhead to obtain compounds structurally related to darunavir. All intermediate stages are released against an in‑house monograph requiring HPLC purity ≥98.0% (area% at 210 nm), with individual unknown impurities ≤0.50% and residual dichloromethane ≤600 ppm according to USP <467> Procedure A. Because trace methanesulfonate ion can alkylate the pyrrolidine nitrogen to form mutagenic N‑methylated by‑products under thermal stress, drying is limited to 35 °C under vacuum for no longer than 12 hours, and each batch is screened by LC‑MS/MS for N‑methyl‑4‑methoxyimino pyrrolidinium species with a reporting threshold of 2.5 ppm.