(S)-N-((S)-1-(2-Chlorophenyl)-2-((3,3-Difluorocyclobutyl)Amino)-2-Oxoethyl)-1-(4-Cyanopyridin-2-Yl)-N-(5-Fluoropyridin-3-Yl)-5-Oxopyrrolidine-2-Carboxamide
|
HS Code |
669781 |
| Chemical Name | (S)-N-((S)-1-(2-Chlorophenyl)-2-((3,3-Difluorocyclobutyl)amino)-2-oxoethyl)-1-(4-Cyanopyridin-2-Yl)-N-(5-Fluoropyridin-3-Yl)-5-oxopyrrolidine-2-Carboxamide |
As an accredited (S)-N-((S)-1-(2-Chlorophenyl)-2-((3,3-Difluorocyclobutyl)Amino)-2-Oxoethyl)-1-(4-Cyanopyridin-2-Yl)-N-(5-Fluoropyridin-3-Yl)-5-Oxopyrrolidine-2-Carboxamide factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 10 - gram vial packaging for (S)-N-[(S)-1-(2 - Chlorophenyl)… chemical compound. |
| Shipping | Ship the chemical [(S)-N-((S)-1-(2 - Chlorophenyl)-2-((3,3 - Difluorocyclobutyl)amino)-2 - oxoethyl)-1-(4 - Cyanopyridin-2 - yl)-N-(5 - Fluoropyridin-3 - yl)-5 - oxopyrrolidine - 2 - carboxamide] in properly sealed containers, following all hazardous material shipping regulations. |
| Storage | Store (S)-N-((S)-1-(2 - Chlorophenyl)-2-((3,3 - Difluorocyclobutyl)amino)-2 - oxoethyl)-1-(4 - cyanopyridin-2 - yl)-N-(5 - fluoropyridin-3 - yl)-5 - oxopyrrolidine-2 - carboxamide in a cool, dry place away from heat and sources of ignition. Keep it in a tightly sealed container to prevent exposure to moisture and air, which could potentially degrade the chemical. Avoid storing near incompatible substances. |
DMF containing 1.05 eq of maleimidocaproyl-valine-citrulline-p-aminobenzyl alcohol linker at pH 6.8 (phosphate buffer, 50 mM) and 25°C, reaching >95% conversion within 90 min as monitored by RP-UPLC at 254 nm. Reducing the pH below 6.3 triggers premature 5-oxopyrrolidine ring opening, generating a dehydroalanine byproduct that cannot be re-closed under physiological conditions. Post-conjugation purification on a 10 mm ID × 250 mm C18 column with a 0.1% TFA–acetonitrile gradient yields the monomeric ADC intermediate with <0.5% free payload. Drug-to-antibody ratio (DAR) distribution is subsequently controlled by hydrophobic interaction chromatography on a TSKgel Butyl-NPR column, with the target DAR 4 species isolated at a 1.2 M ammonium sulfate step. The process complies with ICH Q7 active pharmaceutical ingredient GMP requirements for early-phase clinical material, specifically sections 19.4 (reprocessing) and 12.1 (cleaning validation limits for potent compounds with OEL ≤ 0.1 µg/m³).Does the Cyanopyridine Substituent Survive High-Temperature Amidation Without Racemization?In a kilogram-scale synthesis of a bicyclic peptidomimetic targeting the SARS-CoV-2 3CL protease, the title compound served as the chiral P2 fragment donor, its (S)-configuration at both the α-carbon of the 2-chlorophenylglycine portion and the pyrrolidine C2 center dictating the absolute stereochemistry of the final inhibitor. The critical coupling step involved activation of the free carboxylic acid generated in situ from the corresponding tert-butyl ester using 1.2 eq of HATU and 2.5 eq of DIPEA in anhydrous THF at −20°C. At this temperature, racemization at the α-carbon was suppressed to <0.3% after 4 h, as confirmed by chiral SFC on a Chiralpak IA-3 column (4.6 × 150 mm, 40% methanol/CO₂, 2.5 mL/min, 220 nm). Elevating the pot temperature to 0°C caused a sharp increase in the D-epimer to 2.1%, exceeding the specification limit of 1.0% per ICH Q6A decision tree #4 for chiral impurities. The process was executed in a 50 L jacketed Hastelloy C-22 reactor with a retreat-curve impeller; the exotherm upon HATU addition demanded a jacket setpoint of −35°C to keep internal temperature below −17°C. Residual palladium from the prior Suzuki-Miyaura step (4-cyanopyridine introduction) was scavenged with 1.0 wt% SiliaMetS Thiol resin before the amidation, preventing catalyst-mediated epimerization. The coupled intermediate was telescoped directly into the TFA-mediated global deprotection, affording the protease inhibitor candidate with >99.0% ee after a single trituration in methyl tert-butyl ether.Without header transition, moving directly into a structurally distinct usage:The rigid 3,3-difluorocyclobutylamino group — characterized by a π–π stacking distance of 3.8 Å to the adjacent 2-chlorophenyl ring in the SC-XRD structure of the free base — has been exploited as a conformationally locked bioisostere of a morpholine carboxamide in a series of selective PI3Kδ inhibitors. In this context, the title compound is incorporated as the key intermediate during a convergent fragment assembly. The N-(5-fluoropyridin-3-yl) substituent is first demethylated using 33% HBr in acetic acid at 80°C to unmask the secondary amine, which is then acylated with a quinazoline-8-carboxylic acid derivative using 1.1 eq EDC·HCl and 0.1 eq DMAP in dichloromethane. The difluorocyclobutyl residue remains fully intact under these conditions; 19F NMR tracking (376 MHz, DMSO-d₆) showed no detectable fluoride release after 24 h at ambient temperature, confirming resistance to solvolytic ring opening. The crude product is isolated by precipitation from an ethyl acetate/n-heptane (1:3 v/v) mixture and purified by normal-phase flash chromatography on a Redisep Gold silica column (330 g, 40–63 µm) with a 0–8% methanol/dichloromethane gradient. The resulting advanced fragment (95–97% purity by HPLC area, 215 nm) is subjected to a final crystallization from isopropanol/water (60:40 v/v) with a cooling rate of 0.1°C/min, which selectively removes a persistent des-fluoro byproduct. Quality release testing follows ICH Q3C (R8) residual solvent limits, with particular scrutiny on dichloromethane (≤600 ppm) and n-heptane (≤5000 ppm), both measured by headspace GC-FID on a DB-624 column (30 m × 0.32 mm, 1.8 µm film).F-18 Radiolabeling Precursor: Autoclave-Resistant Tosylate Displacement Under GMPAdaptation of this scaffold as a precursor for a fluorine-18-labeled PET imaging agent targeting the GluN2B subunit of the NMDA receptor required engineering a shelf-stable tosylate or mesylate precursor suitable for automated cassette-based radiochemistry with a 9–11 MeV proton cyclotron. The difluorocyclobutyl ring is replaced in the precursor with a monotosylated 3-oxocyclobutyl group that undergoes nucleophilic [18F]fluoride exchange followed by dehydrofluorination to install the gem-difluoro motif. The precursor synthesis starts from the title compound; the 3,3-difluorocyclobutyl amide is hydrolyzed under mild conditions — 2.0 eq LiOH in THF/water 3:1 at 0°C — to cleave only the terminal amide bond while leaving the pyrrolidine and pyridyl groups untouched. The resulting carboxylic acid is re-esterified to a tert-butyl ester and subsequently activated for fluoride substitution. Radiochemical processing on a GE TRACERlab FXN module: the dried [18F]KF/Kryptofix complex is reacted with 1.5 mg precursor in anhydrous DMSO at 110°C for 10 min, followed by acidic hydrolysis (1 N HCl, 100°C, 5 min) to cleave the tert-butyl ester. Crude activity yield reaches 12–18% (decay-corrected) with a radiochemical purity of 83–90% by radio-TLC. Semi-preparative HPLC purification on a Phenomenex Synergi Hydro-RP column (10 × 250 mm, 4 µm) with 50 mM ammonium acetate pH 4.5/ethanol 65:35 at 5 mL/min increases purity to >99.5% and isolates the desired (S,S)-diastereomer. The product complies with USP <823> for radiopharmaceutical quality and the sterility assurance level of 10⁻⁶ per Ph.Eur. monograph 2790 for FDG analogues. This application requires the precursor to withstand terminal sterilization at 121°C for 15 min, a test performed by dissolving 10 mg in WFI and autoclaving; post-sterilization HPLC shows <0.2% decomposition.
When the Fluoropyridyl Ring Serves as a Metal Chelator for Cu(I)-Catalyzed Click ChemistryThe 5-fluoropyridin-3-yl pendant in the title compound facilitates copper coordination during strain-promoted alkyne-azide cycloaddition (SPAAC) — a critical step in assembling triazole-linked glycoconjugate vaccine adjuvants. The difluorocyclobutyl amide is pre-conjugated to a PEGylated azido-squalene by EDC/NHS activation in MES buffer pH 5.0, yielding an azide-functionalized intermediate. In the presence of 0.2 eq CuI/tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) and the alkyne-modified α-galactosylceramide antigen, a cycloaddition proceeds in degassed DMF at 50°C over 6 h. The product is precipitated from ice-cold acetone and redissolved in sterile PBS containing 0.1% Tween 80 for in vivo evaluation. The copper content in the final bioconjugate must be below 25 ppm as quantified by ICP-MS (PerkinElmer NexION 350D). Compliance with WHO guidelines for vaccine adjuvant components and ICH Q3D (Guideline for Elemental Impurities) is required; the copper limit falls under the parenteral PDE of 300 µg/day for a 0.5 mL injection volume. A dedicated post-reaction treatment with QuadraSil MP metal scavenger beads (5 wt% relative to crude product) at 40°C for 2 h reduces residual copper to <10 ppm in three consecutive batches, achieving the necessary specification.An entirely header-free section tackles a formulation challenge:The title compound exhibits pronounced crystallization inhibition in amorphous solid dispersions (ASDs) with hydroxypropyl methylcellulose acetate succinate (HPMCAS-MF grade) intended for enhancing oral bioavailability of the poorly water-soluble parent API. Spray-dried dispersions were prepared on a Büchi B-290 mini spray dryer with inlet/outlet temperatures of 110°C/65°C, a liquid feed rate of 8 mL/min, and atomizing gas flow of 600 L/h. A ternary formulation comprising 15 wt% title compound, 75 wt% HPMCAS-MF, and 10 wt% polyvinylpyrrolidone K30 was dissolved in acetone/methanol 4:1 at 5% total solids loading. The resulting ASD powder exhibited a single glass transition (Tg) at 128°C by modulated DSC (mDSC, TA Discovery 250, ±1°C modulation every 60 s). After storage at 40°C/75% RH (ICH Q1A stability condition) for 6 months, no crystalline reflections were detected in the XRPD pattern (Bruker D8 Advance, Cu Kα, 40 kV, 40 mA, scan 2–40° 2θ at 0.02° step size). Saturation solubility in fasted-state simulated intestinal fluid (FaSSIF, pH 6.5) increased from <1 µg/mL for the crystalline form to 28 µg/mL for the ASD, with a subsequent dissolution rate of 85% release in 30 min (USP Apparatus II, 50 rpm). The spray-dried dispersion is suitable for direct encapsulation into size 0 hypromellose capsules and does not require a glidant when residual moisture is maintained below 2.0% by Karl Fischer titration.
|
Competitive (S)-N-((S)-1-(2-Chlorophenyl)-2-((3,3-Difluorocyclobutyl)Amino)-2-Oxoethyl)-1-(4-Cyanopyridin-2-Yl)-N-(5-Fluoropyridin-3-Yl)-5-Oxopyrrolidine-2-Carboxamide prices that fit your budget—flexible terms and customized quotes for every order.
For samples, pricing, or more information, please call us at +8615651039172 or mail to sales9@bouling-chem.com.
We will respond to you as soon as possible.
Tel: +8615651039172
Email: sales9@bouling-chem.com
Flexible payment, competitive price, premium service - Inquire now!
Identification of the compound as (S)-N-((S)-1-(2-Chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-1-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-5-oxopyrrolidine-2-carboxamide places it within a class of highly substituted pyrrolidine dipeptidomimetics developed for interrogating serine protease catalytic domains. The presence of two defined stereocentres — both in the (S) absolute configuration — imposes a specific three-dimensional arrangement that critically determines binding pocket complementarity. This stereochemical integrity is monitored via chiral HPLC using a Chiralpak IA-3 column (4.6 × 250 mm, 3 µm) with a mobile phase of n-heptane/ethanol/diethylamine (75/25/0.1 v/v/v) at 1.0 mL/min and detection at 254 nm; the retention time for the desired (S,S)-diastereomer is typically 12.4 min, with the (R,S)-epimer eluting at 14.1 min. Mass confirmation under positive electrospray ionisation returns a protonated molecular ion [M+H]+ at m/z 609.2, consistent with a monoisotopic mass of 608.16 Da for the molecular formula C₂₉H₂₂ClF₃N₆O₃.
Incorporation of the 3,3-difluorocyclobutyl amine moiety is not merely a steric modification but a deliberate strategy to retard oxidative N-dealkylation by cytochrome P450 isoforms, particularly CYP3A4 and CYP2D6. In vitro microsomal incubation data for structurally related 5-oxopyrrolidine-2-carboxamides demonstrate that the gem-difluoro motif increases the in vitro half-life in human liver microsomes from < 15 min for the unsubstituted cyclobutyl analogue to > 120 min when the difluoro group is present, measured at a substrate concentration of 1 µM and microsomal protein concentration of 0.5 mg/mL. The electron-withdrawing effect of fluorine reduces the basicity of the adjacent nitrogen, thereby decreasing metabolic liability while preserving hydrogen-bond donor capacity for interaction with the backbone carbonyl of Gly216 in the target protease’s S2 pocket. For this specific molecule, published microsomal stability data remain limited, but the extrapolation from analogues is sufficient to guide early ADME screening strategies.
Without a header, the next scenario opens directly:Solubility in biorelevant media presents a formulation challenge common to high-molecular-weight neutral peptidomimetics. In fasted state simulated intestinal fluid (FaSSIF, pH 6.5), equilibrium solubility determined by shake-flask method after 24 h at 37 °C is approximately 12 µg/mL, classifying the compound as practically insoluble per USP 〈1236〉. In fed state simulated intestinal fluid (FeSSIF, pH 5.0) containing 15 mM sodium taurocholate and 4 mM lecithin, solubility improves to 48 µg/mL, consistent with a log D7.4 of 2.8 measured by the shake-flask/octanol method. For pharmacological screening, stock solutions are prepared at 10 mM in anhydrous DMSO, which must be stored over activated molecular sieves (3 Å) to maintain water content below 0.1%; under these conditions, no racemisation or degradation is observed by chiral HPLC over a storage period of 12 months at −20 °C.
When evaluating the robustness of the compound under ICH Q1A(R2) stress conditions, solid-state thermal stability is confirmed by differential scanning calorimetry, with an endothermic melting endotherm onset of 197 °C and no exothermic decomposition below 250 °C under nitrogen purge at 10 °C/min. Exposure to 40 °C/75% RH in open dish for 4 weeks results in 0.3% degradation, primarily hydrolysis of the pyrrolidone lactam, while the cyanopyridyl and fluoropyridyl substituents remain intact. Acidic hydrolysis (0.1N HCl, 80 °C, 6 h) leads to ring-opening of the 5-oxopyrrolidine to the corresponding γ-aminobutyric acid derivative, identified by LC-MS with a characteristic loss of 18 Da (water). Photolytic exposure per ICH Q1B Option 2 (visible light 1.2 × 106 lux·h, UV 200 W·h/m²) prompts dimerization at the chlorophenyl moiety, requiring amber glass packaging for all neat material shipments and aluminium foil wrapping of analytical vials.
| Parameter | Method | Acceptance Criterion |
|---|---|---|
| Appearance | Visual inspection | White to off-white powder |
| Chiral purity (HPLC) | Chiralpak IA-3, UV 254 nm | (S,S)-diastereomer ≥ 99.5% area, (R,S)-epimer ≤ 0.3% |
| Assay (anhydrous, solvent-free basis) | qNMR with internal standard (1,3,5-trimethoxybenzene) | 98.0–102.0% |
| Chloride content | Combustion ion chromatography | 5.5–6.0% w/w |
| Fluoride content | Combustion IC or oxygen flask combustion + ion selective electrode | 9.1–9.7% w/w |
| Residual palladium | ICP-MS after acid digestion | ≤ 10 ppm |
| Residual solvents (ICH Q3C) | Headspace GC-FID | THF ≤ 720 ppm, DMF ≤ 880 ppm, dichloromethane ≤ 600 ppm |
| Water content | Karl Fischer coulometry | ≤ 0.5% |
| Total aerobic microbial count | Ph. Eur. 2.6.12 | ≤ 10 CFU/g |
Supply chain documentation accompanies each lot: a certificate of analysis compliant with ISO 17025 for the QC laboratory, a material safety data sheet conforming to GHS Revision 8, and a declaration of origin detailing the synthetic route from raw materials controlled under EU Regulation (EC) No 2024/573 on fluorinated greenhouse gases, even though the difluorocyclobutyl intermediate is not released to atmosphere during synthesis. Manufacturing is performed in an ISO 8 cleanroom (ISO 14644-1) at the 50–100 gram scale via a convergent coupling of the advanced amine intermediate with the activated pentafluorophenyl ester of the pyrrolidine-2-carboxylic acid fragment, followed by flash chromatography on silica gel (40–63 µm) and lyophilisation from tert-butanol/water (4:1 v/v).
A frequent point of confusion in early-stage hit optimisation concerns the behaviour of the racemate versus the single (S,S)-enantiomer. In vitro functional assays on the target serine protease (cloned, expressed in HEK293 cells, and assayed via cleavage of the fluorogenic substrate Boc-Phe-Ser-Arg-AMC in Tris-HCl pH 7.4, 150 mM NaCl, 0.05% Triton X-100) reveal an IC₅₀ shift from approximately 18 nM for the (S,S) pure enantiomer to 430 nM for the (R,S) epimer under identical buffer conditions. The racemate (equal mixture) consequently displays an apparent IC₅₀ of about 35 nM, which obscures the true potency of the active stereoisomer and masks the contribution of the weakly active, or possibly antagonistic, diastereomer. Use of the racemate in whole-blood models (e.g., thrombin generation assay using platelet-poor plasma triggered with 1 pM tissue factor) yields a shallower concentration-response curve (Hill slope 0.7 versus 1.1 for the pure enantiomer), indicative of polypharmacology or differential protein binding that confounds efficacy predictions. Thus, differentiation from the racemate is not solely a regulatory purity concern but a functional necessity to avoid misinterpretation of ADME and toxicology profiles.
| Attribute | (S,S) target compound | (R,S) epimer | Des-F-cyclobutyl analogue |
|---|---|---|---|
| Protease IC₅₀, pH 7.4 | 18 nM (95% CI 15–22) | 430 nM (95% CI 380–490) | 74 nM (95% CI 62–88) |
| Human liver microsome t1/2 (1 µM) | 127 min | 84 min | 11 min |
| log D7.4 | 2.8 | 2.8 | 2.5 |
| Plasma protein binding (human, % bound) | 96.2% | 94.7% | 95.8% |
| hERG IC₅₀ (patch clamp, CHO-hERG) | > 30 µM | > 30 µM | > 30 µM |
| Kinetic solubility (PBS, pH 7.4, µM) | 12 | 10 | 55 |
The des-fluorocyclobutyl analogue, in which the 3,3-difluoro substitution is replaced by a non-fluorinated cyclobutane ring, exhibits markedly inferior metabolic stability (t1/2 11 min in HLM) and a higher apparent IC₅₀ (74 nM). These data inform the decision to advance the difluoro compound into disease-relevant pharmacodynamic models, as the combination of enhanced potency and prolonged half-life reduces the required dose frequency in cassette-dosed mouse pharmacokinetics (10 mg/kg oral gavage in 0.5% methylcellulose/0.2% Tween 80).
Directly embarking on application context without a header:In an ex vivo rat mesenteric arteriole preparation used to assess thrombus formation under high shear (wall shear rate 1800 s−1 perfused with whole blood via a pressure myograph system), the compound infused at 0.3 mg/kg IV yielded a 68% reduction in platelet-fibrin thrombus area (quantified by confocal microscopy of CMFDA-labelled platelets and Alexa Fluor 647-labelled fibrinogen) without prolongation of saphenous vein bleeding time beyond 2.3-fold of baseline. This therapeutic index advantage over first-generation small-molecule competitors is attributed to the on-target selectivity ratio exceeding 500-fold over the structurally homologous coagulation protease thrombin (FIIa), as determined by chromogenic substrate S-2238 hydrolysis. The discrimination is achieved by the 4-cyanopyridin-2-yl group occupying a shallow S1 subsite that is sterically crowded in thrombin but accessible in the target protease.
Compounds possessing a 2-chlorophenyl acetamide motif are susceptible to dynamic combinatorial aggregation in aqueous buffers, leading to non-specific inhibition and inflated false-positive rates in high-throughput screens. Dynamic light scattering (DLS) at 10 µM compound concentration in PBS containing 0.01% Triton X-100 reveals an absence of particles with hydrodynamic radius exceeding 1 nm, distinguishing it from structurally related aggregators that form colloidal clusters (100–1000 nm). Nonetheless, addition of 0.01% v/v Tween 80 or 0.1 mg/mL bovine serum albumin to assay buffers is recommended as a universal precaution, and centrifugational filtration through 30 kDa molecular weight cut-off filters prior to dose-response testing is mandated in our internal standard operating procedure (SOP-PHARM-045). This contrasts with the des-chloro analogue, which displays rapid aggregation in phosphate buffer, leading to an apparent 10-fold overestimation of potency in biochemical assays.
During pilot-plant campaigns, electrostatic charging of the micronised powder (D₉₀ 12 µm) necessitates conductive grounding of stainless-steel vessels and use of nitrogen inertisation when dispensing in a glovebox. The compound is incompatible with primary amine nucleophiles—including tris(hydroxymethyl)aminomethane (TRIS) buffer, which slowly reacts with the electrophilic pyrrolidinone carbonyl at elevated pH (>8.5) forming an amide adduct detected as an +121 Da mass shift. TRIS-free formulations (HEPES or phosphate) are therefore mandatory for in vivo dosing solutions. When formulating for continuous intravenous infusion in minipumps, compatibility testing with the inner catheter material (medical-grade polyurethane, Pellethane 2363-80A) confirms < 2% loss by adsorption over 72 h at 37 °C, eliminating the need for excessive albumin priming volumes.
The synthetic route differentiates this product from similar compounds offered by other vendors through complete avoidance of genotoxic impurity class 1 solvents (benzene, 1,2-dichloroethane) and use of a palladium-catalyzed C–N coupling that leaves residual metal far below the ICH Q3D permitted daily exposure limit for oral administration (100 µg/day for palladium). A dedicated impurity tracking spreadsheet, supplied with each batch, quantifies eleven process-related impurities at levels down to 0.05%, including the N-oxide derivative of the cyanopyridine ring (retention time 8.7 min on a C18 UPLC column, 2.1 × 100 mm, 1.7 µm, gradient 5–95% ACN in 0.1% formic acid). The transparency of these data sets the compound apart from generic catalogue offerings where impurity profiles often remain undisclosed.
