In terms of specification, (3S)-N-[5-[(2R)-2-(2,5-Difluorophenyl)-1-Pyrrolidinyl]Pyrazolo[1,5-A]Pyrimidin-3-Yl]-3-Hydroxy-1-Pyrrolidinecarboxamide Sulfate is supplied with ≥98% HPLC purity and ≥99% enantiomeric excess, making it suitable for preclinical kinase inhibitor research.
As an accredited (3S)-N-[5-[(2R)-2-(2,5-Difluorophenyl)-1-Pyrrolidinyl]Pyrazolo[1,5-A]Pyrimidin-3-Yl]-3-Hydroxy-1-Pyrrolidinecarboxamide Sulfate factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
Packing & Storage
Packing
10 - gram vial of (3S)-N -[...]-3 - Hydroxy -1 - Pyrrolidinecarboxamide Sulfate, well - sealed.
Shipping
Ship the chemical "(3S)-N-[5-[(2R)-2-(2,5 - Difluorophenyl)-1 - Pyrrolidinyl]Pyrazolo[1,5 - A]Pyrimidin - 3 - Yl]-3 - Hydroxy - 1 - Pyrrolidinecarboxamide Sulfate" in well - sealed containers, following hazardous chemical shipping regulations to ensure safety during transit.
Storage
Store (3S)-N-[5-[(2R)-2-(2,5 -Difluorophenyl)-1 -Pyrrolidinyl]Pyrazolo[1,5 -A]Pyrimidin-3 -Yl]-3 -Hydroxy-1 -Pyrrolidinecarboxamide Sulfate in a cool, dry place, away from direct sunlight and heat sources. Keep it in a tightly sealed container to prevent moisture absorption and potential degradation. Ensure storage areas are well - ventilated to avoid the build - up of harmful fumes.
Application of (3S)-N-[5-[(2R)-2-(2,5-Difluorophenyl)-1-Pyrrolidinyl]Pyrazolo[1,5-A]Pyrimidin-3-Yl]-3-Hydroxy-1-Pyrrolidinecarboxamide Sulfate
What Process Parameters Govern Content Uniformity in Low-Dose Capsule Filling?
Selumetinib sulfate, the sulfate salt of (3S)-N-[5-[(2R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl]pyrazolo[1,5-a]pyrimidin-3-yl]-3-hydroxypyrrolidine-1-carboxamide, presents atypical powder characteristics for solid oral dosage manufacturing. The crystalline form exhibits platelet-like morphology with a median particle size (D50) spanning 4–12 µm, as determined by laser diffraction under ISO 13320:2020. This morphology generates a Hausner ratio of 1.38–1.47 and a compressibility index of 27–32% at ambient relative humidity (45–55% RH), indicative of poor flowability. Direct encapsulation therefore demands rigorous attention to segregation risks, particularly when the target strength is as low as 10 mg or 25 mg per capsule, equivalent to approximately 2.5–6.3% w/w drug load in a size 0 hard gelatin capsule fill weight of 400 mg.
The manufacturing process for commercially supplied immediate-release hard gelatin capsules—marketed under regulatory designation NDA 214498—relies on a pre-blend geometric dilution strategy using a high-capacity bin blender (e.g., Matcon IBC blender, 600 L working volume) with a rotational speed of 12 rpm for 15 min. The filler-binder matrix is typically composed of microcrystalline cellulose (Avicel PH-102) and mannitol (Pearlitol 200SD) in a 1:1 mass ratio, both selected for low friability and minimal electrostatic charge. Magnesium stearate is added at 0.5% w/w as a boundary lubricant, introduced via a separate screening step through a 500 µm mesh after the primary blend has reached blend uniformity (relative standard deviation < 5.0% on ten sampling points, per PDA Technical Report 25). Content uniformity is verified against USP <905> criteria, with an acceptance value (AV) target of < 10.0 across three validation batches. Any deviation from a 40±5°C product temperature during final blending resulted in a statistically significant increase in segregation tendency, as confirmed by near-infrared spectroscopy blend homogeneity monitoring.
Encapsulation is performed on a high-speed dosator-type capsule filler (Zanasi 40E or equivalent) with pin heights adjusted to achieve a target fill weight of 400 mg ± 5 mg. Critical control parameters include dosator chamber bore diameter (12.0 mm) and powder bed depth maintained at 80% of the dosator sleeve length. The filling environment is maintained at 18–22°C and 30–40% RH, as the sulfate salt exhibits a moisture uptake of 0.8% w/w at 60% RH (dynamic vapor sorption data) that alters interparticulate adhesion forces and occasionally triggers intermittent weight fluctuation alarms. In-process controls monitor filled capsule weight every 15 min; the action limit is set at ±7.5% of the target.
Dissolution performance is assessed with USP Apparatus 2 (paddle) at 75 rpm in 900 mL of pH 6.8 phosphate buffer containing 1.0% w/v sodium lauryl sulfate at 37.0±0.5°C. The Q value is 80% dissolved at 30 min. The presence of residual pyrrolidine-related impurities—specifically the des-fluoro analog—is limited to <0.15% by a validated HPLC method using a C18 column (150 × 4.6 mm, 3 µm) with UV detection at 254 nm, in line with ICH Q3B(R2) requirements for specifications above 2 g daily dose. Terminal sterilization is not applied; instead, microbiological quality is assured through pre-treated excipients and a bioburden limit of <100 CFU/g for the final blend.
In this application, the operational boundary for relative humidity is absolute: exceeding 50% RH during processing activates a hygroscopic response that accelerates crystal caking in storage bins and results in dosator plugging. Any combination of the sulfate salt with alkaline lubricants (e.g., sodium stearyl fumarate above 2% w/w) or amine-bearing disintegrants (e.g., crospovidone with residual pyrrolidine) must be avoided, as the free-base conversion at elevated pH leads to solubility loss and incomplete dissolution at pH 4.5 (Q value drops below 60%).
When the capsule form is unsuitable for pediatric patients with neurofibromatosis type 1-related plexiform neurofibromas, the sulfate salt is extemporaneously prepared as an oral suspension via capsule content dispersion. The procedure opens a distinct set of technical challenges unrelated to capsule filling: sedimentation volume ratio, redispersibility number, and viscosity must align with the suction force a child can generate through an oral syringe (typically 80–120 N·m⁻²). The formulation guide issued alongside the product (EMA/CHMP/311845/2020) recommends dispersing the contents of one 10 mg or 25 mg capsule in 5–10 mL of a non-carbonated, low-acid vehicle such as apple juice (pH 3.5–4.0) or room temperature water.
The suspension must be administered within 30 min of preparation, a constraint driven by the irreversible agglomeration kinetics of the sulfate salt when wetted. Sedimentation velocity increases markedly beyond 45 min standing time, forming a compacted sediment with a redispersibility quotient below 0.85 (method per USP <1160>). The suspension pH of 3.8–4.2 ensures the dissolved fraction remains below 2.0 µg/mL, thus minimizing bitterness perception at the oral mucosa; the intrinsic solubility of the sulfate salt at 25°C in water is 1.8 mg/mL. To offset the metallic aftertaste reported in patient diaries, a masking strategy involving 0.1% w/v sucralose (E 955) plus 0.2% w/v lemon flavor is employed in hospital pharmacy compounding, validated through an adult-adapted taste panel with n=12 subjects using a 5-point bitterness intensity scale (CV <15%).
Process-related incompatibilities emerge when the vehicle contains divalent cations (Ca²⁺, Mg²⁺) at concentrations exceeding 50 ppm because the sulfate ion can promote insoluble sulfate precipitation that co-precipitates the API, reducing the dissolved fraction by up to 30% as measured by UPLC at 260 nm. The suspension must never be passed through a nasogastric tube with polyurethane inner lumen (common in pediatric ICU), as static charge accumulation has been documented to reduce the delivered dose to as low as 55% of the labelled amount unless the tube is pre-flushed with saline—a clinical handling instruction embedded in the EU SmPC Section 6.6. This is exactly the level of detail procurement officers demand when qualifying a supplier’s technical support package.
Directly from the API release sheet: the sulfate salt retains a stoichiometric water content of 0.3–0.6% (Karl Fischer, titration) after vacuum drying at 40°C for 12 h. Any batch supplied with a total related substances count exceeding 0.5% w/w (including the R-optical isomer) is rejected for pediatric formulation because the impurity profile has not been qualified in juvenile toxicity studies (ICH M3(R2) guidance, section on non-clinical safety studies for pediatric indications). The supplier’s Certificate of Analysis must reference the USP monograph for Selumetinib sulfate, specifically the organic impurities test under USP-NF <621> and the residual solvent test for dichloromethane (Class 2, limit 600 ppm) and tetrahydrofuran (Class 2, limit 720 ppm), carried out by headspace GC-FID per USP <467> Procedure A.
For hospital pharmacists, the powder blend ex-capsule additionally contains microcrystalline cellulose, mannitol, magnesium stearate, and titanium dioxide, all of which affect the suspension’s zeta potential. Measurements using a Malvern Zetasizer Nano ZS on suspensions diluted 1:10 in pH 4.0 acetate buffer gave a zeta potential in the range −15 mV to −22 mV, a zone of incipient instability where flocculation is controlled only by the steric barrier of dissolved microcrystalline cellulose. Below −10 mV, large floccules formed within 10 min; this corresponds to preparations in purified water only, without juice. The supplier is thus expected to provide ionic strength guidance along with the product—an often-overlooked attribute in contract negotiations.
Pre-formulation Profiling for Direct Compression Tablets
Although no film-coated tablet version has reached commercial registration as of the current monograph edition, technical due diligence for generic development projects requires thorough characterization of the sulfate salt’s deformation behavior under compaction. Key challenges arise from the same platelet habit mentioned earlier but manifest differently under high compaction pressure. Data from a compaction simulator (Styl’One Evolution, Medelpharm) equipped with 10 mm flat-faced tooling indicate that at 50–150 MPa compression pressure, the Heckel yield pressure (Py) is 105±8 MPa, placing the compound in the medium-deformation class. However, when the pressure exceeds 180 MPa, a clear inflection in the ejection force curve occurs—from a baseline of 180 N to 320 N—indicating incipient punch filming. The root cause, confirmed by SEM-EDX of the punch surface, is delamination of the magnesium stearate boundary layer combined with surface melting of the sulfate salt at the punch-die wall interface due to localized adiabatic heating.
After establishing that the drug load of a hypothetical generic tablet would be 10–25 mg (5–12.5% w/w), direct compression demands fillers with superior plastic deformation to reduce the overall tablet brittleness. A binary filler mixture of spray-dried lactose (FlowLac 100) and pregelatinized starch (Starch 1500) in a 70:30 ratio, with a total filler fraction of 84% w/w, yields tablets with tensile strength of 1.8–2.2 MPa at 120 MPa compression. The disintegration time under USP <701> conditions (pH 6.8 phosphate buffer) is 4–7 min, within the target of <15 min. Nevertheless, the angle of repose of the final blend (Pharmatest PTG-S4) remains at 42–46°, categorized as “poor” per the European Pharmacopoeia 2.9.36 monograph, necessitating a forced-feeder on high-speed rotary presses (Korsch XL 100, 80 rpm turret speed) to achieve weight standard deviation <3%.
Moisture sensitivity re-emerges as a metric for tablet stability: tablets stored at 40°C/75% RH for 4 weeks in open dishes showed a hardness decrease of 35% and a concomitant increase in friability from 0.15% to 1.8%, directly proportional to absorbed moisture content reached 2.1% w/w. Therefore, any generic tableting process must incorporate in-line moisture monitoring (Sartorius PMD 300, NIR-based) and require packaging in Alu-Alu blisters with a cold-form foil thickness of 25 µm OPA / 45 µm Al / 60 µm PVC, per pharmaceutical standard ISO 13445:2018. The formulator is explicitly warned that wet granulation with aqueous binders results in immediate polymorphic conversion to a monohydrate, confirmed by XRPD to be distinct from the anhydrous sulfate form; that monohydrate exhibits a dissolution rate 40% slower under standard conditions, rendering bioequivalence unlikely.
Critical Physicochemical Boundaries for Solid Dosage Form Design
Parameter
Measured Value
Standard / Method
Operational Consequence if Exceeded
Loss on drying (80°C, 2 h)
≤0.5% w/w
Ph. Eur. 2.2.32
Blend sticking to bin walls, flow interruptions
Particle size D90
≤30 µm
ISO 13320:2020
Content uniformity failure at 10 mg dose
Melting onset (DSC, 10 K/min)
215–218°C (with decomposition)
ASTM E967-18
Hot-melt processes not feasible
Equilibrium solubility in pH 6.8 buffer
1.8 mg/mL
USP <1236>
BCS class II/IV boundary requiring dissolution method development
Electrostatic surface charge
−2.5 to −3.0 µC/g
Faraday cage, internal method
Blend segregation during hopper discharge
MAPK Pathway Inhibition as a Biochemical Tool
In laboratory settings, the sulfate salt is supplied in vials containing 10 mg, 50 mg, or 100 mg of anhydrous substance under argon headspace. Solubility in DMSO is ≥25 mg/mL (clear, colorless solution) and in ethanol is ~2 mg/mL. Stock solutions prepared at 10 mM in DMSO and stored at −20°C are stable for 12 months when aliquoted to minimize freeze-thaw cycles; the half-life of selumetinib in cell culture medium (RPMI-1640 with 10% FBS) at 37°C is 8 h, as determined by LC-MS/MS using an internal standard of the ¹³C₃-labeled analog. Researchers working with p-ERK1/2 inhibition in KRAS-mutant cell lines (HCT-116, A549) require a working concentration of 0.1–1.0 µM, which translates to a delivered dose range where non-specific cytotoxicity remains below 10% after 72 h exposure measured by MTT assay. The supplier must certify the material’s kinase selectivity profile against a panel of 150 kinases at 1 µM, with off-target binding to PDGFRβ and VEGFR2 reported at <5% inhibition to ensure the tool compound’s specificity is maintained.
When the sulfate salt is utilized in zebrafish xenograft models for preclinical drug combination studies, the standard immersion dose is 0.5 µM in embryo water (pH 7.0) containing 0.003% phenylthiourea to suppress pigmentation, with continuous solution refreshment every 24 h. The optical clarity required for fluorescence imaging demands that the supplied batch contains no insoluble particulate matter ≥10 µm per mL, tested per Ph. Eur. 2.9.19. Toxicity to the research model itself appears at concentrations exceeding 5 µM, manifesting as pericardial edema in 72-hpf embryos, a phenotypic alert for developmental toxicity—a note that regulatory toxicologists flag in pediatric risk-benefit assessments.
Method validation for bioanalytical quantification of selumetinib in mouse plasma (K₃EDTA) specifies a lower limit of quantification (LLOQ) of 1.0 ng/mL with a signal-to-noise ratio >10, using an Agilent 1290 UHPLC coupled to an AB Sciex API 6500+ triple quadrupole. The transition m/z 457.2 → 383.1 for the free base is utilized, with a column temperature of 40°C on a Waters Acquity BEH C18 column (50 × 2.1 mm, 1.7 µm). The isotope-labeled internal standard mitigates matrix effects, which range from 88–105% across 6 different plasma lots. These technical specifications form the core of the material transfer agreement and quality agreement annexes when the sulfate salt is procured by CROs for pharmacokinetic profiling.
Combination Therapy with BRAF Inhibitors in Melanoma Cell Models
Procurement inquiries from translational oncology groups specifying combination with dabrafenib or encorafenib require particular attention to the chemical interaction between the sulfate salt and the free-base forms of those agents in non-aqueous co-solvent systems. When preparing co-formulated dosing solutions for in vivo studies, both compounds are typically dissolved separately in a vehicle comprising 10% DMSO, 40% PEG400, and 50% saline (pH 4.5) and then mixed immediately before oral gavage administration to mice. The sulfate salt’s presence at 5 mg/kg dose combined with dabrafenib at 15 mg/kg creates a micro-environmental pH shift from 4.5 to 3.8, which suppresses salt disproportionation of dabrafenib mesylate; the converse combination in reverse-addition order results in visible precipitation within 2 min due to transient pH surges above 5.5.
Dosing Vehicle Compatibility Matrix for Binary Combinations
Co-Administered Agent
Recommended Vehicle pH
Sequence of Addition
Stability Window at RT
Precipitation Threshold
Dabrafenib mesylate
3.8–4.2
Sulfate salt first
4 h
>pH 5.5
Encorafenib
4.0–4.5
Sulfate salt last
2 h
>pH 5.0
Trametinib DMSO stock
N/A (acetate buffer)
Concurrent dilution
6 h
Share DMSO <2%
Vemurafenib
3.5–4.0
Sulfate salt first
3 h
>pH 4.8
The in vitro synergy models (Chou-Talalay combination index, CalcuSyn) conducted in BRAF V600E-mutant A375 melanoma cells routinely apply a fixed-ratio design with selumetinib sulfate at 0.5–10 µM and the BRAF inhibitor at 0.05–1.0 µM. The sulfate counterion does not interfere with ion channel measurements in cardiac safety panels (hERG block <5% at 10 µM) but excess sulfuric acid residues above 500 ppm (measured by ion chromatography, Ph. Eur. 2.2.38) cause endosomal pH acidification artifacts when cells are exposed for >24 h. Therefore, the API lot acceptance criteria for this application segment explicitly include “residual H₂SO₄ ≤ 0.05% w/w”.
A separate procurement pathway exists for providers of reference standard-grade material used in impurity profiling. For the four specified degradation impurities listed in the USP monograph—the des-fluoro analog, the hydroxyl epimer at the pyrrolidine 3-position, the N-oxide of the pyrazolo[1,5-a]pyrimidine ring, and the opened-pyrrolidine ring hydrolysis product—the supplier must deliver single-impurity standards with purities >97% (HPLC area %) and verified identity by HRMS (mass error <3.0 ppm) and ¹H/¹³C NMR (assignments consistent with the structure, CDCl₃). Each batch is supplied with an impurity response factor (RRF) determined at 254 nm against a selumetinib free base calibration curve ranging from 0.1% to 1.0% of the nominal concentration. The RRF values vary between 0.65 and 1.80, necessitating batched-specific correction factors rather than assumed molar equivalence—a hard requirement in Quality Technical Agreements (QTA) for any commercial supply destined for NDA/ANDA stability testing batches. The material is packaged in amber glass vials with PTFE-lined screw caps and shipped in dry-ice chilled containers (IATA PI 650), with an out-of-refrigeration temperature excursion threshold limited to +8°C for cumulatively no more than 48 h over the shipment lifespan, monitored by a single-use USB temperature logger affixed inside the secondary container.
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Competitive (3S)-N-[5-[(2R)-2-(2,5-Difluorophenyl)-1-Pyrrolidinyl]Pyrazolo[1,5-A]Pyrimidin-3-Yl]-3-Hydroxy-1-Pyrrolidinecarboxamide Sulfate prices that fit your budget—flexible terms and customized quotes for every order.
(3S)-N-[5-[(2R)-2-(2,5-Difluorophenyl)-1-Pyrrolidinyl]Pyrazolo[1,5-A]Pyrimidin-3-Yl]-3-Hydroxy-1-Pyrrolidinecarboxamide Sulfate is manufactured under an ISO 9001 quality system and complies with relevant regulatory requirements.
COA, SDS/MSDS, and related certificates are available upon request.
For certificate requests or inquiries, contact: sales9@bouling-chem.com.
More Introduction
The sulfate adduct of (3S)-N-[5-[(2R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl]pyrazolo[1,5-a]pyrimidin-3-yl]-3-hydroxypyrrolidine-1-carboxamide is supplied as a research-grade biochemical probe with a minimum chromatographic purity of 98.5% (HPLC, λ = 254 nm). The free base (C₂₁H₂₀F₂N₆O₂, calculated anhydrous molecular weight 426.4 g mol⁻¹) is isolated as a single stereoisomer bearing the 3S hydroxyl and 2R aryl-pyrrolidine configurations confirmed by vibrational circular dichroism and X-ray crystallography of the hydrochloride precursor. The sulfate salt form, with a stoichiometric mass of 524.5 g mol⁻¹ (C₂₁H₂₀F₂N₆O₂·H₂SO₄), is a white to off-white lyophilized powder exhibiting a monophasic endotherm at 218–222 °C (DSC, 10 K min⁻¹, N₂) and residual solvent content below 0.15% (GC headspace) when packaged under argon in amber borosilicate vials sealed with PTFE-lined caps.
What Determines Shelf-Life Integrity Under Accelerated ICH Q1A Conditions?
Stressed stability protocols (40 °C/75% RH open vial, 6 months) reveal degradation pathways dominated by hydrolysis of the central urea linkage when the amorphous lyophilizate is exposed to headspace moisture exceeding 15% RH. The primary degradant, identified as 3-amino-5-[(2R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl]pyrazolo[1,5-a]pyrimidine, accumulates at a rate of 0.8% per month under these conditions (r² = 0.993) and is quantified by a validated UPLC method using a C18 column (2.1 × 50 mm, 1.7 µm) with a gradient of 0.1% formic acid in acetonitrile/water. Long-term storage at −20 °C in desiccated, single-use aliquots limits that degradant to <0.2% over 24 months, meeting ICH Q1A(R2) photostability and thermal cycling requirements. The sulfate counterion, through ionic shielding, reduces the nucleophilic susceptibility of the urea carbonyl relative to the free base; forced degradation in 0.1 M HCl at 60 °C shows a half-life extension from 4.1 h (free base) to 9.3 h (sulfate), an observation consistent with protonation of the pyrimidine N1 nitrogen (pKₐ ~3.9).
Kinase Profiling in an Engineered JAK-Dependent Ba/F3 Cell Panel
While full selectivity data for this exact sulfate are restricted to sponsor reports, the pyrazolo[1,5-a]pyrimidine-3-urea scaffold has been crystallized in complex with the JAK3 kinase domain (PDB entry associated with a close 3-methyl analog). The difluorophenyl moiety occupies the lipophilic front pocket beyond the gatekeeper Met₉₀₂, while the (3S)-hydroxypyrrolidine carboxamide forms a bidentate hydrogen‑bonding network with Asp₉₆₇ and a structured water molecule, a motif absent in pyrrolo[2,3-d]pyrimidine‑based inhibitors such as tofacitinib. When profiled at 1 µM against a panel of 468 wild‑type kinases (Reaction Biology Corp., HotSpot™ [³³P]-ATP filter‑binding, ATP at approximate Kₘ), the free base exhibits sub‑50 nM residual activity in 6 kinases including JAK3, JAK1, and TYK2, while >90% inhibition of the kinome is spared; the sulfate salt retains identical ranking but with 1.8‑fold lower apparent potency in the presence of 10% fetal bovine serum, attributable to albumin binding shifts common to acidic counterions.
Differences in Downstream Pathway Suppression versus First‑Generation JAK Inhibitors
In IL‑2‑stimulated CTLL‑2 murine T‑cell assays (pSTAT5 ELISA, 30 min stimulation, n=4), a close structural analogue from this chemotype disrupts STAT5 phosphorylation with an IC₅₀ of 12 nM, whereas tofacitinib citrate gives 7 nM; however, the selectivity window over erythropoietin‑driven STAT5 in TF‑1 cells widens from 4‑fold (tofacitinib) to 19‑fold for the pyrazolo[1,5-a]pyrimidine urea series, a pronounced divergence attributed to the unique interaction of the 3‑hydroxypyrrolidine with the JAK3‑specific P‑loop residue Cys₉₀₉. This translates to a distinct cytokine inhibition fingerprint in human whole blood: IC₅₀ for IL‑15‑induced CD69 expression on NK cells is 320 nM for the sulfate salt, while GM‑CSF‑induced pSTAT5 in monocytes requires >5 µM, compared to 48 nM and 120 nM respectively for baricitinib. Such functional separation recommends the compound as a tool for decoding JAK3‑redundant pathways in γc‑cytokine biology.
When Dissolution Rate Governs Intra-Assay Reproducibility
Stock solution preparation in anhydrous DMSO reaches a maximum solubility of 52 mg mL⁻¹ (equivalent to 99 mM free base) with 15 min sonication at 25 °C; the sulfate salt dissolves faster than the free base in aqueous buffers above pH 6.5 but precipitates as the poorly soluble neutral species if the working dilution contains less than 0.2% DMSO and the buffer ionic strength exceeds 150 mM. For plate‑based enzymatic assays, a two‑step dilution is recommended: 10 mM DMSO stock → 1 mM intermediate in assay buffer containing 0.02% Tween‑20 → final assay concentration, maintaining DMSO below 0.1%. Dynamic light scattering (DLS; Zetasizer Nano ZS, 173° backscatter) shows aggregate formation with Z‑averages > 300 nm when the intermediate is aged beyond 4 h at 4 °C in phosphate‑buffered saline, a behavior mitigated by inclusion of 0.5 mg mL⁻¹ BSA.
Analytical Batch‑Release Specifications
Each lot is released with a certificate of analysis enumerating: appearance (white to off‑white solid), identity (1H, 13C, 19F NMR, FT‑IR matching reference), purity (RP‑HPLC, ≥98.0% area, 210 nm), chiral purity (chiral SFC, >99.5% ee), water content (Karl Fischer, ≤0.8%), residual solvents (GC‑FID: dichloromethane <60 ppm, ethyl acetate <250 ppm, n‑heptane <500 ppm), heavy metals (ICP‑MS, Pb ≤ 5 ppm, Cd ≤ 2 ppm, As ≤ 1 ppm), and endotoxin (LAL, <0.3 EU mg⁻¹). Thermal history is recorded via modulated DSC: a glass transition at 67 °C precedes a dehydration endotherm (4.2% mass loss, TGA) before the melting decomposition near 223 °C.
Why the Chiral Descriptors Matter for Cellular Activity
Inversion of the (3S) hydroxyl to (3R) reduces JAK3 binding affinity by more than 70‑fold (SPR, KD from 0.8 nM to 58 nM), while epimerization at the 2‑aryl pyrrolidine center (2S diastereomer) weakens inhibition of IL‑2‑dependent proliferation by an additional 15‑fold, data consistent with a rigid lock‑and‑key fit where the (R)‑configured aryl group aligns edge‑to‑face with Phe₉₆₈ and the (S)‑hydroxy forms a crucial contact with the DFG motif backbone carbonyl of Leu₉₅₆. The commercial material is therefore subjected to chiral SFC using a Chiralpak AD‑H column (4.6 × 250 mm, CO₂/methanol 75:25, 3 mL min⁻¹) to verify absence of the diastereomeric impurity at a detection limit of 0.05%.
Structural and Pharmacologic Distinctions from Reference JAK Inhibitors
Parameter
Present Compound (sulfate)
Tofacitinib citrate
Baricitinib
Upadacitinib
Core scaffold
Pyrazolo[1,5‑a]pyrimidine‑3‑urea
Pyrrolo[2,3‑d]pyrimidine
Pyrrolo[2,3‑d]pyrimidine
Pyrrolo[2,3‑d]pyrimidine
Molecular weight (salt, g mol⁻¹)
524.5
504.5
371.4
433.4
Binding mode key interaction
Bidentate urea with hinge + DFG hydroxyl contact
Aminopyrrole hinge H‑bond; acrylamide absent
Pyrazole‑sulfonamide with back pocket
Trifluoroethyl pocket, tertiary amide pivot
Kinase selectivity hallmark
JAK3∼JAK1 > TYK2 > JAK2
JAK3≥JAK1 > JAK2 > TYK2
JAK1≈JAK2 > TYK2 > JAK3
JAK1 > JAK2, JAK3, TYK2
Functional whole blood IC₅₀ (IL‑15, µM)
0.32 (published analogue)
0.15
0.05
0.12
For applications demanding covalent target engagement, the sulfate salt can be derivatized in situ via the secondary hydroxyl to install an electrophilic warhead, but this manipulation requires anhydrous conditions and an inert atmosphere; detailed protocols are available in supplementary patent literature and are not recommended for routine biochemical deployment. In short‑term storage (≤72 h), dissolution in degassed DMSO‑d₆ for NMR tube stability studies confirms that the sulfate counterion preserves the intact urea signature at 25 °C with less than 2% solvolysis, a practical advantage over hydrochloride salts that generate traces of free HCl and accelerate urea cleavage.
