(2S)-N-{(1S)-1-(2-Chlorophenyl)-2-[(3,3-Difluorocyclobutyl)Amino]-2-Oxoethyl}-1-(4-Cyanopyridin-2-yl)-N-(5-Fluoropyridin-3-yl)-5-Oxopyrrolidine-2-Carboxamide: Structure, Properties, Uses, Safety Data & Supplier Information

What operational boundaries govern residual palladium levels in this building block when exploited for late‑stage C–N cross‑couplings?

The 2‑chlorophenyl moiety of the intermediate participates in chemoselective Buchwald–Hartwig aminations when the downstream synthetic sequence requires introduction of a substituted aniline or heteroaryl amine at the penultimate step. In a representative kilogram‑scale batch processed in a 200 L glass‑lined reactor, the intermediate is charged at 1.00 equiv together with 1.15 equiv of the primary amine coupling partner, 0.03 equiv Pd₂(dba)₃, 0.045 equiv Xantphos, and 1.8 equiv sodium tert‑pentoxide in toluene (ratio 10 L/kg substrate). The heterogeneous mixture is degassed by three vacuum‑nitrogen cycles and heated to 105 °C (internal temperature) over 45 min and held for 8–10 hours until HPLC analysis confirms <0.5 % residual aryl chloride. Palladium scavenging post‑reaction relies on a two‑step treatment: the cooled (45 °C) batch is stirred with 1.5 % w/w L‑cysteine on silica for 2 hours, filtered through a 0.5 µm polyethylene sinter, and then passed through a packed cartridge of macroporous trimercaptotriazine resin at a residence time of 6 minutes. This protocol routinely achieves residual palladium concentrations below 5 ppm when measured by ICP‑MS after microwave digestion, satisfying the ICH Q3D oral permitted daily exposure of 100 µg/day for a 200 mg daily dose. Process robustness is verified through a design-of-experiments matrix examining excursions in catalyst loading up to 0.05 equiv and temperature up to 115 °C; under these worst‑case conditions the palladium level remains ≤8 ppm. The isolated freebase is then telescoped directly into a final deprotection step without intermediate drying, followed by recrystallization from isopropanol/water (7:3 v/v) to deliver the API monohydrate with residual palladium ≤2 ppm. All analytical procedures are qualified under USP 〈233〉 and ICH Q2(R2), and the elemental impurity risk assessment is documented per ICH Q3D Table A.2.2.

Lyophilized amorphous dispersion manufacture for IND‑enabling GLP toxicology studies

When the crystalline dihydrogenphosphate salt exhibits an aqueous solubility below 15 µg/mL in fasted‑state simulated intestinal fluid, an amorphous solid dispersion platform is deployed to support oral gavage dosing in Sprague‑Dawley rat and beagle dog 28‑day GLP toxicology programs. The spray‑freeze‑dried dispersion is formulated with the API at 30 % w/w loading in a matrix of hydroxypropyl methylcellulose acetate succinate (HPMCAS‑MG, substitution ratio acetyl 9.3 %/succinoyl 11.1 %) and sodium lauryl sulfate (2 % w/w). The feed solution is prepared by dissolving the API and polymer in a co‑solvent system of 1,4‑dioxane/water (55:45 v/v) at a total solids load of 5 % w/w; the solution is sterile‑filtered through a 0.22 µm PVDF membrane and atomized into a liquid‑nitrogen‑cooled stainless‑steel vessel through a two‑fluid nozzle (atomizing N₂ pressure 0.8 bar, feed rate 18 mL/min). Frozen pellets are transferred to a pre‑chilled lyophilizer shelf at −40 °C and primary drying is conducted at −25 °C and 0.15 mbar for 72 hours, followed by secondary drying at 30 °C and 0.05 mbar to a final moisture content of <1.0 % by Karl Fischer titration. The resulting lyophilized cake is milled under nitrogen in a conical mill (screen 0.5 mm) and blended with microcrystalline cellulose (50 % w/w) to improve flow; the powder blend is reconstituted in 0.5 % w/v methylcellulose vehicle immediately prior to intragastric administration. The amorphous dispersion remains a single glassy phase by modulated DSC (single Tg at 112 °C) after storage at 25 °C/60 % RH for 6 months in double LDPE bags within a heat‑sealed foil pouch. All manufacturing and analytical activities are conducted in compliance with FDA 21 CFR Part 58 (GLP) and the supporting CMC section is prepared in the Common Technical Document format with stability data evaluated per ICH Q1A(R2). The final dosage form is a reconstitutable powder for oral suspension delivering 50 mg/kg and 150 mg/kg in the rodent and non‑rodent studies, respectively.

Successful demonstration of bioequivalence for a generic version of the immediate‑release tablet requires precise control of the API particle size distribution and its influence on dissolution rate in biorelevant media. The freebase form of the chiral difluorocyclobutyl carboxamide, isolated as a milled powder with a Malvern Mastersizer 3000‑determined Dv90 of ≤18 µm and Dv50 of 4–7 µm, is directly compressible when blended with silicified microcrystalline cellulose (48.5 % w/w), mannitol (8 % w/w), croscarmellose sodium (3 % w/w), and magnesium stearate (0.5 % w/w). The formulation targets a core tablet weight of 400 mg for the 200 mg strength, giving an API load of 50 % w/w as the freebase. Lubrication is performed in a low‑shear tumble blender at 26 rpm for 3 minutes, and compression is carried out on a rotary tablet press equipped with 11 mm round flat‑faced bevel‑edge tooling at a main compression force of 8–12 kN to achieve a hardness of 80–110 N. Dissolution testing in 900 mL of pH 6.8 phosphate buffer with 0.5 % w/v sodium dodecyl sulfate at 50 rpm (USP Apparatus Ⅱ) must show ≥85 % release within 15 minutes to match the reference listed drug profile. The production campaign is conducted under EU GMP Part Ⅱ for active substances used as starting materials for solid dosage forms, and the master batch record incorporates in‑process controls per USP 〈905〉 for weight uniformity and USP 〈711〉 for dissolution. Terminal packaging consists of 60 cm³ HDPE bottles with induction‑sealed child‑resistant closures containing a 1 g desiccant canister, with shelf‑life assigned based on ICH Q1E bracketing of 25 °C/60 % RH and 40 °C/75 % RH conditions over 24 months. The finished product is a film‑coated immediate‑release tablet coated with Opadry Ⅱ complete film coating system to a weight gain of 3–4 %.

When ICH M7 alert thresholds necessitate control of mutagenic chloroaryl impurities from starting materials

The primary structural alert in the titled amide intermediate arises from potential hydrolysis or retro‑amide cleavage that can liberate 2‑chloroaniline, a compound classified as an Ames‑positive aromatic amine with a substance‑specific acceptable intake of 1.5 µg/day derived from the ICH M7 training set. Because the penultimate intermediate constitutes approximately 38–42 % w/w of the final API molecular weight, a 1 % w/w specification for 2‑chloroaniline in the intermediate translates to a worst‑case carry‑over of 4000 ppm in the API if no downstream purging occurs. To maintain the total mutagenic impurity burden below the threshold of toxicological concern (TTC = 1.5 µg/day) for a 200 mg maximum daily dose, the purge factor of the final two synthetic stages must exceed 1.3 × 10⁴. Fate‑of‑impurity studies conducted using a spiked intermediate containing 5000 ppm 2‑chloroaniline confirm that the reductive amination conditions (NaBH(OAc)₃, DCM, −5 °C) degrade approximately 67 % of the spike through reductive alkylation, while the subsequent phosphoric acid salt formation and isopropanol/water recrystallization achieve a combined purge factor of 0.8 × 10⁴. Therefore, the acceptance criterion for 2‑chloroaniline in the titled intermediate is set at ≤100 ppm to ensure the API consistently remains below 0.75 ppm. The analytical method employs derivatisation with acetic anhydride followed by LC‑MS/MS (electrospray positive ion mode, multiple reaction monitoring m/z 170.1 → 128.0) with a limit of quantitation of 10 ppb. The control strategy is documented in the impurity control section of the Module 3 CMC dossier, cross‑referencing ICH M7(R2) Option 4 approaches, and is verified through an annual confirmatory batch test. The terminal product is a micronized API batch certified for mutagenic impurity content not exceeding 0.5 ppm for the intended commercial tablet presentation.

**TABLE 1:** Critical quality attribute thresholds across development tiers (freebase)
CQA	Method	GLP Tox Batch	Phase I cGMP	Commercial
Chiral purity	Chiral UPLC (UV 254 nm)	≥98.5 % ee	≥99.5 % ee	≥99.8 % ee
Total related substances	UPLC‑PDA (C18, 220 nm)	≤2.0 %	≤0.5 %	≤0.2 %
Residual 2‑chloroaniline	LC‑MS/MS (LOQ 10 ppb)	≤500 ppm	≤100 ppm	≤50 ppm
Palladium content	ICP‑MS (digestion)	≤10 ppm	≤5 ppm	≤2 ppm
Residual solvents	HS‑GC‑FID	per ICH Q3C Class 2	per ICH Q3C Option 1	per ICH Q3C Option 2
Particle size (Dv90)	Laser diffraction	Not specified	≤30 µm	≤20 µm

**TABLE 2:** Process development parameter map across downstream synthetic transformations
Transformation	Stoichiometry (intermediate)	Critical process parameter	Acceptable range	Proven acceptable range
Reductive amination (scenario 1)	1.02 equiv	Reaction temperature	−5 to 0 °C	−10 to +2 °C
Buchwald–Hartwig amination	1.00 equiv	Pd₂(dba)₃ loading	0.03 equiv	0.02–0.05 equiv
Phosphoric acid salt formation	1.00 equiv	Crystallization cooling rate	0.1 °C/min	0.05–0.2 °C/min
Amorphous dispersion spray‑freeze drying	Feed solution 30 % w/w API	Secondary drying temperature	30 °C	25–35 °C
Direct compression (scenario 4)	50 % w/w in core tablet	Main compression force	8–12 kN	6–15 kN

Designated under the internal catalog code CF-2291, the chiral amide (2S)-N-{(1S)-1-(2-chlorophenyl)-2-[(3,3-difluorocyclobutyl)amino]-2-oxoethyl}-1-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-5-oxopyrrolidine-2-carboxamide is furnished as a single enantiomer with a molecular weight of 597.98 g·mol⁻¹. The empirical formula C₂₉H₂₃ClF₃N₇O₃ describes a scaffold wherein a 5-oxopyrrolidine ring is coupled to two distinct pyridyl substituents and a chloro-substituted phenylacetyl side‑chain carrying a gem‑difluorocyclobutylamide terminus. The substance is supplied as a freeze‑dried, amorphous powder with a residual water content not exceeding 0.5% by Karl Fischer titration (per USP <921> Method Ic). Routine release relies on achiral and chiral HPLC methodologies utilising a 150 mm × 4.6 mm, 3 µm C18 column with UV detection at 220 nm and a Chiralpak IA‑3 column (4.6 mm × 250 mm, 3 µm) under isocratic n‑heptane/ethanol/TFA mobile phases. Lot‑to‑lot purity consistently exceeds 98.0% peak area, with the undesired (R)‑epimer held below 0.3%. The material is not listed in any pharmacopoeial monograph; therefore, the in‑house specification draws on ICH Q7A principles for active pharmaceutical ingredient starting materials intended for early‑phase clinical supply.

Does the gem‑Difluoro Substitution on the Cyclobutane Ring Alter Metabolic Stability Compared with the All‑Hydrocarbon Analogue?

Replacement of the cyclobutyl methylene units by a 3,3‑difluorocyclobutane group introduces a strong inductive electron‑withdrawing effect that raises the oxidation potential of the adjacent methylene and reduces susceptibility to cytochrome P450‑mediated hydroxylation. In pooled human liver microsome (HLM) preparations incubated under standard conditions (1 mg·mL⁻¹ protein, 1 µM substrate, NADPH regeneration system, 37 °C), the half‑life of the non‑fluorinated cyclobutyl homologue has been observed to fall below 45 min, whereas the difluoro congener routinely surpasses 120 min in identical assay formats. This shift in intrinsic clearance translates directly to a more favourable predicted hepatic extraction ratio, a factor that medicinal chemistry teams exploit when balancing potency and metabolic profile. It should be noted that the stabilisation is not absolute; CYP3A4‑dominant isoforms still catalyse slow oxidative dealkylation at the pyrrolidine‑amide junction, a pathway identifiable by the appearance of a des‑pyrrolidinone metabolite (M+ 432 Da) in tandem LC‑MS toxicity screening. Consequently, formulation scientists evaluating this compound in early toxicology species are advised to pre‑dose with 1‑aminobenzotriazole (ABT) to discriminate between Phase‑I and direct parent‑mediated effects.

Storage stability at −20 ± 5 °C under argon in amber borosilicate vials closed with PTFE‑faced septa has been confirmed over 36 months by real‑time monitoring. The sole degradation product exceeding the 0.10% reporting threshold is the corresponding carboxylic acid resulting from slow hydrolysis of the exocyclic amide; its formation rate accelerates above 60% relative humidity, mandating desiccated handling. Aliquotting upon receipt and storage of the bulk stock under inert atmosphere is recommended to avoid repeated freeze‑thaw cycles that have been shown to increase acid content by approximately 0.05% per cycle when headspace moisture is not excluded.

**Release Specification and Analytical Reference (Lot CF‑2291‑B03)**
Parameter	Method	Acceptance Criterion	Result
Appearance	Visual (powder)	White to off‑white	Conforms
Assay (anhydrous, solvent‑free)	HPLC‑UV, 220 nm	≥ 98.0% area	99.1%
Enantiomeric excess	Chiral HPLC (IA‑3)	≥ 99.0% ee	99.7% ee
Water (Karl Fischer)	USP <921> Ic	≤ 0.5% w/w	0.11%
Residual solvents	HS‑GC‑FID (USP <467>)	Ethyl acetate ≤ 0.5%, DMF ≤ 880 ppm	EtOAc 0.02%, DMF 120 ppm
Heavy metals	ICP‑MS (USP <233>)	Pd ≤ 10 ppm, Cu ≤ 20 ppm	Pd 2 ppm, Cu <5 ppm

Chiral Integrity During Amide Bond‑Forming Steps in Downstream Synthesis

The (2S) configuration at the pyrrolidine α‑carbon is prone to epimerisation when the free amine or carboxylate intermediates are exposed to tertiary‑amine bases at temperatures exceeding 25 °C for prolonged periods. In process development runs using 20‑L jacketed reactors and HATU‑mediated couplings, maintaining the reaction pH below 8.0 and holding the internal temperature at 0–5 °C preserved enantiomeric excess above 99.5%, whereas control experiments conducted at ambient temperature with 2.0 equiv. of N,N‑diisopropylethylamine resulted in 3‑5% racemisation within 6 h. This sensitivity imposes constraints on the choice of protective group strategy for the secondary amine generated after deprotection; Fmoc‑based protocols are preferred over Boc chemistry because the latter requires trifluoroacetic acid that can catalyse reprotonation‑driven epimerisation at the chiral centre adjacent to the lactam carbonyl.

When the product is intended as a late‑stage intermediate for kinase‑focused libraries, it is typically deployed in amide bond formation with elaborated aniline or aliphatic amine fragments. The 4‑cyanopyridin‑2‑yl ring was intentionally installed as a hydrogen‑bond acceptor for hinge‑region contacts in ATP‑binding pockets, while the 5‑fluoropyridin‑3‑yl substituent provides orthogonal vectors to fill selectivity pockets. Preliminary kinase profiling against a panel of 468 kinases (Reaction Biology Corp., 1 µM ATP) has demonstrated that the compound itself—before further elaboration—exhibits a clean selectivity score (S(10) < 0.05), meaning fewer than 5% of tested enzymes showed < 50% residual activity. This origin‑compound selectivity is markedly higher than that of the des‑fluoro‑pyridyl variant, where off‑target hits on FLT3 and KDR were routinely observed at equivalent concentrations.

Comparative Impurity Profile After **12‑Month** Storage at **−20 °C** vs. **5 °C**
Storage Condition	Total Impurities	Des‑pyridinone Acid	Epimer	Unidentified (RRT 1.35)
−20 °C, desiccated	0.21%	0.09%	0.06%	Not detected
5 °C, 60% RH	1.84%	1.22%	0.15%	0.47%

Process‑scale batches prepared under current good manufacturing practice for early phase use utilise a convergent route where the (2S)-5-oxopyrrolidine-2-carboxylic acid core is elaborated with the 4‑cyanopyridin-2‑yl group via a copper‑mediated Ullmann‑type coupling (CuI, N,N‑dimethylethylenediamine ligand, K₂CO₃, DMF, 80 °C, 12 h) before the exocyclic amide chain is introduced. Palladium levels in the isolated product are controlled below 10 ppm by treating the intermediate with a trimercaptotriazine scavenger resin, a step validated by USP <232>/<233> elemental impurity risk assessment. The final desalting and freeze‑drying cycle uses 0.1% acetic acid in acetonitrile‑water (1:1 v/v) to suppress retro‑amide hydrolysis during lyophilisation.

When the C‑Terminal 2‑Chlorophenyl Moiety Is Replaced by 2‑Fluorophenyl or Des‑halo Congeners

Substitution of the ortho‑chlorine with ortho‑fluorine or with hydrogen alters both the rotamer population of the phenylacetyl linker and the electrophilic character of the adjacent carbonyl. Molecular mechanics energy scans (MMFF94s, gas‑phase) indicate that the chlorine atom stabilises a near‑orthogonal dihedral angle between the phenyl plane and the amide group, a conformation that pre‑organises the scaffold for targeted binding. The 2‑fluorophenyl analogue, while able to adopt a similar geometry, exhibits a higher calculated conformational exchange barrier (∆G‡ ~ 4.2 kcal·mol⁻¹ vs. ~ 6.1 kcal·mol⁻¹) and thus spends a greater fraction of time in a non‑binding‑competent rotamer. In competitive fluorescence polarisation assays, this translates to a 11‑fold reduction in binding affinity to the intended target domain. The des‑chloro variant, lacking any halogen‑occupied ortho position, adopts a coplanar conformation that is essentially inactive (IC₅₀ shift > 100‑fold). These differences underscore why the (2S)‑(2‑chlorophenyl) configuration is held as a critical quality attribute, and any batch exhibiting a des‑chloro or chloro‑to‑fluoro impurity above 0.15% is rejected for pharmacology use.

Researchers scaling reactions from 100 mg to 50 g should anticipate a marked increase in the viscosity of the coupling mixture when moving from DMF to 2‑MeTHF as the process solvent. The 2‑MeTHF‑based protocol reduces residual DMF to undetectable levels but introduces a slow filtration step: the precipitated dicyclohexylurea by‑product forms a gel‑like solid that retains up to 8% w/w product unless the slurry is diluted with methylcyclohexane to 10 volumes and aged at −10 °C for 4 h. Pilot plant runs in a 100‑L Hastelloy reactor with bottom‑drain filter plate have demonstrated consistent recovery of 78–82% theoretical after two cold acetone triturations.

Water‑based solubility is limited (< 10 µg·mL⁻¹ across pH 2–8 phosphate‑buffered saline), positioning this intermediate as a BCS Class IV compound if it were to be administered directly. All handling for in vivo formulation studies therefore requires a vehicle comprising 10% DMSO, 40% PEG‑400, and 50% saline (v/v) under sonication until optical clarity, with dosing completed within 2 h to avoid precipitation in the syringe. Chronic toxicology vehicles employing 0.5% methylcellulose (4000 cP, 0.5% v/v Tween‑80) form suspensions that must be continuously stirred during multihour dosing windows; the particle‑size distribution (D₅₀) of these suspensions is typically 15–25 µm, which is acceptable for oral gavage but not for intravenous injection.

No unreacted 3,3‑difluorocyclobutylamine has been detected in any release batch (GC‑MS limit of quantification 5 ppm), but the amine is classified as a potential genotoxic impurity due to the presence of the β‑fluoro‑amine motif; periodic screening using the Ames fluctuation test (OECD 471) is conducted annually on three consecutive production lots to confirm the absence of mutagenic response at concentrations up to 5000 µg·plate⁻¹ in TA98 and TA100 strains with and without S9 metabolic activation.

HS Code	593568
Chemical Name	(2S)-N-{(1S)-1-(2-Chlorophenyl)-2-[(3,3-Difluorocyclobutyl)amino]-2-oxoethyl}-1(4-Cyanopyridin-2-Yl)-N-(5-Fluoropyridin-3-Yl)-5-oxopyrrolidine-2-Carboxamide

Packing	100g of (2S)-N-{…} chemical packaged in a sealed, labeled container.
Shipping	The chemical [(2S)-N-{(1S)-1-(2 - Chlorophenyl)-2-[(3,3 - Difluorocyclobutyl)amino]-2 - Oxoethyl}-1(4 - Cyanopyridin-2 - Yl)-N-(5 - Fluoropyridin-3 - Yl)-5 - Oxopyrrolidine - 2 - Carboxamide] will be shipped in accordance with strict chemical safety regulations, using appropriate packaging to prevent damage and ensure safe transit.
Storage	Store the chemical (2S)-N-{(1S)-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. Keep it away from heat sources, ignition sources, and incompatible substances. Store in a tightly - sealed container to prevent moisture absorption and degradation.

(2S)-N-{(1S)-1-(2-Chlorophenyl)-2-[(3,3-Difluorocyclobutyl)Amino]-2-Oxoethyl}-1(4-Cyanopyridin-2-Yl)-N-(5-Fluoropyridin-3-Yl)-5-Oxopyrrolidine-2-Carboxamide