(3S,4R)-3-Ethyl-4-(3H-Imidazo[1,2-A]Pyrrolo[2,3-E]Pyrazin-8-Yl)-N-(2,2,2-Trifluoroethyl)Pyrrolidine-1-Carboxamide

The compound (3S,4R)-3-ethyl-4-(3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazin-8-yl)-N-(2,2,2-trifluoroethyl)pyrrolidine-1-carboxamide (C₂₀H₂₂F₃N₇O, 433.44 g·mol⁻¹) is supplied as an off-white to pale yellow amorphous powder with a nominal purity of ≥ 95 % by reversed-phase HPLC area normalization (detection at 254 nm, C18 silica, acetonitrile/water + 0.1 % TFA gradient). No CAS registry number has been assigned to this custom-synthesised batch; the material is intended exclusively for laboratory-scale biochemical profiling and structure-activity relationship exploration. The tricyclic imidazo[1,2-a]pyrrolo[2,3-e]pyrazine core orients its heteroatoms in an arrangement that deviates from the pyrrolo[2,3-d]pyrimidine hinge-binder geometries prevalent in numerous ATP-competitive kinase inhibitors, altering the distance and angle of hydrogen‑bond donors presented to the backbone carbonyl and NH of the kinase hinge region.

What Differentiates the Imidazo[1,2-a]pyrrolo[2,3-e]pyrazine Core from Classical Pyrrolopyrimidine Hinge-Binders?

The angular disposition of the pyrazine nitrogen lone pairs, combined with the imidazole ring fusion, modifies the lone‑pair directionality and π-electron density distribution relative to a simple pyrrolo[2,3-d]pyrimidine. In co‑crystal structures of related imidazopyrazine‑based ligands with JAK family kinases (PDB entries 6BBV, 6BBW), the tricycle engages the hinge backbone via a bidentate donor‑acceptor network through the pyrazine N1 and the imidazole NH, while the pyroolo edge extends a water‑mediated contact with the catalytic lysine. This geometry diminishes sensitivity to gatekeeper residue polymorphisms that confer resistance to linear heterocycles. The (3S,4R) absolute configuration of the pyrrolidine carrier enforces a fixed spatial relationship between the ethyl side chain and the trifluoroethyl carboxamide vector; in silico conformational sampling (Merck Molecular Force Field, 10 000-step stochastic search) indicates that the 3S-ethyl group occupies a pseudo‑equatorial orientation, which projects the carboxamide substituent into the solvent‑exposed ribose‑binding pocket while minimising steric clash with the glycine‑rich loop.

When Solubility Limits in Cell‑Based Assays Require DMSO Stock Preparation at ≤ 10 mM

Thermodynamic aqueous solubility, measured by a shake‑flask method after 24 h equilibration at 25.0 ± 0.2 °C in phosphate‑buffered saline (pH 7.4), remains below 5 µM for the free base. Solubility rises to approximately 120 µM in fasted‑state simulated intestinal fluid (FaSSIF, biorelevant medium prepared per Ph. Eur. 2.9.3) but the presence of mixed micelles accelerates chemical degradation (see forced‑degradation data). Consequently, stock solutions for in‑vitro pharmacology are typically prepared in anhydrous dimethyl sulfoxide at a target concentration of 10 mM and dispensed into single‑use aliquots to limit freeze‑thaw cycles. Dynamic light scattering (Malvern Zetasizer Nano ZS, backscatter angle 173°) confirms that the compound does not form detectable colloidal aggregates at 10 µM in assay buffer containing 0.1 % (v/v) DMSO, passing a 1 mm path‑length turbidity threshold of 0.01 AU. Upon dissolution in dimethyl sulfoxide, the carboxamide rotamer equilibrium equilibrates within 15 min at 25 °C, as monitored by ¹H‑NMR (400 MHz, DMSO‑d₆). The chemical shift of the trifluoroethyl methylene protons shifts downfield by 0.12 ppm relative to the crystalline solid, indicative of complete solvation and disruption of intermolecular hydrogen bonding. DMSO stock stability at −20 °C exceeds 12 months when protected from moisture; water uptake above 0.5 % (v/v) promotes a slow ring‑opening side reaction of the carboxamide, generating the corresponding pyrrolidine precursor and N‑(2,2,2‑trifluoroethyl)isocyanate as a reactive intermediate. This degradation can be detected as an additional peak at retention time 3.2 min under the standard HPLC conditions and must be controlled by use of freshly activated 4 Å molecular sieves.

Stability Under Accelerated Conditions: Forced Degradation in Aqueous Buffers

Forced‑degradation studies performed in accordance with ICH Q1A(R2) stress‑testing guidelines reveal two primary degradation pathways. In 0.1 M hydrochloric acid at 40 °C, the imidazo[1,2-a] ring undergoes acid‑catalysed hydrolysis with a pseudo‑first‑order rate constant k = 6.7 × 10⁻³ h⁻¹, yielding a ring‑opened amino‑aldehyde that subsequently cyclises to a imidazoline by‑product. In 0.1 M sodium hydroxide, cleavage of the carboxamide bond dominates, with k = 1.2 × 10⁻² h⁻¹ and an activation energy estimated at 72 kJ·mol⁻¹ over the range 30‑50 °C. Oxidative stress (3 % H₂O₂, 25 °C) oxidises the pyrrolidine tertiary amine to the corresponding N‑oxide, identified by LC‑MS (m/z 450.2 [M+H]⁺) and retrospective synthesis of the authentic standard. Photostability testing per ICH Q1B option 2 (xenon‑arc lamp, 1.2 × 10⁶ lux·h visible, 200 W·h·m⁻² UV‑A) indicates no significant degradation, supporting storage in amber vials as adequate.

Stereochemical Purity and Chiral Chromatography Specifications

Manufacture via an asymmetric synthesis route starting from (R)‑N‑Boc‑3‑ethyl‑4‑oxopyrrolidine enforces the (3S,4R) configuration with a diastereomeric excess exceeding 99.5 %. Chiral purity is quantified by supercritical fluid chromatography on a Chiralpak IA‑3 column (4.6 × 150 mm, 3 µm) at 40 °C, maintaining a back‑pressure of 120 bar with a mobile phase of CO₂/MeOH (80:20, v/v) containing 0.2 % isopropylamine. Under these conditions the (3S,4R) enantiomer elutes at 4.8 min (capacity factor k′ = 2.7), while the (3R,4S) antipode, synthesised as a control, appears at 6.1 min with a resolution R_s = 3.2. The enantiomeric ratio is determined by integration of the 214 nm signal; the limit of quantification for the undesired enantiomer is 0.05 % area. Batch‑release specifications require ≤ 0.5 % single unspecified diastereomer and ≤ 0.1 % enantiomer.

Release specification panel

Parameter	Method	Acceptance criterion
Appearance	Visual, CIE L*a*b* colour space	L* > 85, b* < 15
Purity (HPLC)	RP‑HPLC‑UV 254 nm, C18, ACN/H₂O + 0.1% TFA	≥ 95.0% area
Chiral purity	SFC‑UV 214 nm, Chiralpak IA‑3	≤ 0.5% diastereomer, ≤ 0.1% enantiomer
Water content	Karl Fischer coulometry (Ph. Eur. 2.5.12)	≤ 0.3% w/w
Residual solvents	Headspace GC‑FID, ICH Q3C class 2/3	DMSO ≤ 500 ppm, dichloromethane ≤ 600 ppm
Elemental analysis	Combustion, CHN (theoretical: C 55.42%, H 5.12%, N 21.93%)	± 0.4% of theoretical

The trifluoroethyl carboxamide moiety, compared with a simple ethyl or isopropyl carboxamide, lowers the calculated partition coefficient (clogP 2.3, ChemAxon) while increasing the topological polar surface area to 94 Ų. This shift is advantageous for maintaining ligand efficiency in the polar ribose‑binding subpocket. A direct comparator, the N-ethyl analogue, exhibits 0.7 log units higher clogP and a 15‑fold reduction in microsomal half-life in human liver microsomes (HLM, 1 mg·mL⁻¹ protein, NADPH‑dependent depletion), demonstrating the role of the trifluoroethyl group in attenuating CYP‑mediated oxidation. Published data for this specific stereochemical configuration is limited to patent disclosures; however, biochemical profiling using recombinant JAK1 kinase domain (Invitrogen PV4774) in a TR‑FRET‑based LanthaScreen™ assay (1 mM ATP, 50 mM HEPES pH 7.5, 10 mM MgCl₂) returned an IC₅₀ of 12 nM (95 % CI 8‑18 nM), with > 100‑fold selectivity over JAK2 and > 500‑fold over JAK3 under identical conditions. The compound is not a substrate for P‑glycoprotein in Caco‑2 bidirectional transport (efflux ratio 0.9, apical pH 6.5/basolateral pH 7.4), indicating no pronounced transporter‑mediated resistance liability. In practice, equilibration of the carboxamide rotamer population after reconstitution from solid can produce small apparent potency shifts in cell‑based assays if solutions are used immediately; a minimum 30‑min incubation of the DMSO stock at room temperature before dilution is recommended. For consistent inter‑experiment results, the stock should be dispensed into low‑binding polypropylene vials and never stored in borosilicate glass, which can catalyse trans‑amidation side reactions at the amide bond in the presence of trace primary amines. When working at concentrations below 100 nM in serum‑free medium, adsorption losses to polystyrene labware can exceed 40 %; pre‑coating with 0.1 % (w/v) bovine serum albumin solution for 1 h at 37 °C prior to compound addition reduces loss to  < 5 %, as verified by spiked recovery experiments with LC‑MS/MS quantification (lower limit of quantification 0.1 nM). The high fluorine content renders the substance X‑ray amorphous under ambient drying, a phenomenon confirmed by powder X‑ray diffractometry (Cu Kα, 40 kV/40 mA, scan range 3‑40° 2θ), where only a broad halo centred at 12° 2θ is observed; amorphous dispersions with hydroxypropyl methylcellulose acetate succinate (LG grade) prepared by spray‑drying improve dissolution‑limited bioavailability in rodent pharmacokinetic studies.

Key property comparison with N-ethyl carboxamide congener

Property	Trifluoroethyl carboxamide	N-ethyl analogue
HLM t½ (min)	89 ± 7	6.1 ± 0.9
Kinetic solubility (µM, pH 7.4)	4 ± 1	22 ± 3
clogP	2.3	3.0
JAK1 IC₅₀ (nM)	12	98
Caco‑2 Papp (10⁻⁶ cm·s⁻¹)	18 ± 2	27 ± 3

Production‑scale synthesis (≥ 10 g batches) using an organocatalytic asymmetric Michael addition to construct the pyrrolidine ring has demonstrated consistent impurity profiles across three validation runs; the highest residual single impurity, the (3S,4S) diastereomer arising from epimerisation of the 4‑position during the borane reduction step, is routinely controlled below 0.3 % by crystallisation of the intermediate hydrochloride salt from ethyl acetate‑heptane (1:5 v/v). Experience on a 50 L jacketed reactor system at agitation rates below 150 rpm showed that sedimentation of the product during anti‑solvent addition created localised hot spots that raised the diastereomeric impurity to 0.7 %; this was mitigated by redesigning the impeller to a retreat‑curve blade turbine and maintaining a minimum Reynolds number of 4 × 10³ throughout the crystallisation. The final product is dried under vacuum ( < 1 mbar) at 40 °C for 18 h and milled to a volume mean diameter D [4,3] = 15 ± 5 µm by jet‑milling, a particle size that balances flowability and dissolution performance in solid dispersion screening. The compound is incompatible with strong oxidizing agents, and exposure to atmospheric moisture above 60 % RH for periods exceeding 24 h causes progressive crystallinity loss and concomitant formation of the free pyrrolidine hydrolysis product; a dry‑nitrogen‑purged glove‑box environment is recommended for aliquoting when ambient humidity exceeds this threshold.