5-((Z)-(5-Fluoro-2-Oxoindolin-3-Ylidene)Methyl)-2,4-Dimethyl-1H-Pyrrole-3-Carboxylic Acid
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HS Code |
155986 |
| Chemical Name | 5-((Z)-(5-Fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid |
As an accredited 5-((Z)-(5-Fluoro-2-Oxoindolin-3-Ylidene)Methyl)-2,4-Dimethyl-1H-Pyrrole-3-Carboxylic Acid factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 100g of 5-((Z)-(5 - Fluoro - 2 - Oxoindolin - 3 - Ylidene)Methyl)-2,4 - Dimethyl - 1H - Pyrrole - 3 - Carboxylic Acid in sealed container. |
| Shipping | The chemical "5-((Z)-(5 - Fluoro - 2 - Oxoindolin - 3 - Ylidene)Methyl)-2,4 - Dimethyl - 1H - Pyrrole - 3 - Carboxylic Acid" will be shipped in properly sealed, labeled containers, following strict chemical safety regulations for secure and compliant transportation. |
| Storage | Store “5-((Z)-(5 - Fluoro - 2 - Oxoindolin - 3 - Ylidene)Methyl)-2,4 - Dimethyl - 1H - Pyrrole - 3 - Carboxylic Acid” in a cool, dry place away from heat sources and direct sunlight. Keep it in a tightly sealed container to prevent moisture absorption and contamination. Avoid storing near reactive chemicals to maintain its chemical integrity. |
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In the commercial synthesis of sunitinib malate, the 5-fluorooxoindoline-pyrrolecarboxylic acid building block functions as the immediate precursor to the final API, entering the route after the Knoevenagel condensation has already locked the Z-configuration of the exocyclic double bond. A standard batch record for the cGMP amidation step prescribes dissolution of the acid (1.0 equivalent) in anhydrous N,N-dimethylformamide at 0–5 °C, followed by sequential addition of 1-hydroxybenzotriazole hydrate (1.2 eq), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.2 eq), and N,N-diisopropylethylamine (3.0 eq) to generate the active ester. After stirring for 30–45 min, N,N-diethylethylenediamine (1.05 eq) is introduced dropwise while the jacket temperature is maintained below 5 °C to suppress the thermal Z-to-E isomer scrambling that has been observed above 10 °C in pilot-scale campaigns. The resulting sunitinib free base is extracted, crystallized from ethyl acetate/heptane, and subsequently treated with L-malic acid in ethanol to isolate the malate salt conforming to USP Extending the Indolinone Core to Next-Generation Kinase ProbesMedicinal chemistry groups sourcing this acid routinely employ it as a diversity node in the exploration of type II and type III kinase inhibitors, where the 5-fluorooxindole motif occupies the adenine-binding pocket and the C-3 substituent extends toward the solvent-exposed region or the DFG-out allosteric site. Parallel amidation protocols conducted in 96-well format use polymer-supported EDC resin (2.0 eq relative to acid) in 200 µL of anhydrous DCM:DMF (4:1 v/v) with 1.5 eq of the target amine, with reactions agitated at 25 °C for 16 h before scavenging with aminomethyl silica. For alkyl- and aryl-amine inputs possessing weak nucleophilicity, a switch to HATU (1.5 eq) and 2,6-lutidine (3.0 eq) in NMP at 0 °C improves conversion beyond 85% as determined by LC-MS at 254 nm. Crude reaction mixtures exceeding 90% UV purity after aqueous workup are progressed to in vitro kinase profiling panels covering VEGFR2, PDGFRβ, FLT3, and KIT. Commercially catalogued analogues synthesized from this acid are supplied with a Certificate of Analysis reporting HPLC purity by peak area (≥ 95%), 1H NMR (DMSO-d6, 400 MHz), and HRMS within 3 ppm mass accuracy. Such materials are explicitly labelled “for Research Use Only” and fall outside the scope of 21 CFR 210/211; however, distributors routinely provide a REACH conformity statement confirming registration or exemption for the specific tonnage band under Regulation (EC) 1907/2006. Why Does Z-Isomer Purity Dictate Pharmacopoeial Acceptance?The pharmacopoeial monograph for Sunitinib Malate in USP and Ph. Eur. defines Related Compound B as the (E)-isomer, derived from the corresponding (E)-acrylic acid impurity that originates in the upstream Knoevenagel step and persists into the final step if the (Z)-acid intermediate is not controlled. Consequently, a batch of the (Z)-acid intended for use as a working reference standard requires isomer-ratio verification by HPLC using a 250 × 4.6 mm column packed with 5 µm octadecylsilane and a mobile phase of 0.05 M ammonium formate (pH 3.2) and acetonitrile (65:35 v/v) at a flow rate of 1.0 mL/min. Under these conditions the (Z)-acid elutes at ~12.4 min and the (E)-isomer at ~14.1 min, with a resolution factor Rs of 3.8—exceeding the USP <621> minimum of 2.0. Acceptance criteria for a pharmacopoeial reference standard lot specify (E)-isomer content ≤ 0.10% by area normalization, a limit rooted in toxicological qualification data submitted to ICH M7 for mutagenic impurities. When the acid is used to spike system suitability solutions, the chromatogram must reproduce the relative retention time of (E)-acid with a precision of SD ≤ 0.02 min across six consecutive injections, a protocol specified in the FDA Recognized Consensus Standard for Chromatographic Data Systems. For CDMOs operating under cGMP multi-purpose equipment scheduling constraints, the solid-state stability of this intermediate under 25 °C/60% RH open handling becomes a critical variable in campaign design. Long-term and accelerated stability studies conducted per ICH Q1A(R2) in a Climacell 404 environmental chamber demonstrate that the acid retains ≥ 99.5% LC purity after 36 months in double polyethylene-lined fiber drums at 25 °C/60% RH, while at 40 °C/75% RH a degradation onset of 0.4% appears at the 6-month time point, predominantly identified as the 5-fluoro-2-oxoindoline fragment from retro-Knoevenagel cleavage. This decomposition pathway is accelerated by trace acidity in the headspace; therefore, vacuum drying for 8 h at 45 °C and –0.095 MPa immediately prior to packaging is mandatory, followed by nitrogen purging to maintain residual oxygen below 0.5%. Pilot-plant execution of the 500-mol scale amidation in a 200 L glass-lined reactor equipped with a Huber Unistat 705 temperature control unit revealed a pronounced exotherm of ΔTad ≈ 62 K upon addition of the carbodiimide, necessitating controlled dosing over 75 min to keep the jacket outlet temperature below 8 °C. The workhorse telescoped process achieves a mean isolated molar yield of 91.3% (n = 12 batches, RSD 1.8%) with total residuals of triethylamine hydrochloride and diisopropylurea consistently below 0.15% w/w. Terminal spent liquor IPC uses a Metrohm 877 Titrando for chloride content as an indirect measure of DIPEA·HCl formation, ensuring uniform mass balance before the dichloromethane strip step. Analytical Methods and Forced Degradation: The Acid as a Stress-Monitoring Proxy
When the acid is deployed as the primary working standard in a quality control setting, the HPLC protocol above is extended by a gradient elution from 40% to 90% acetonitrile over 30 min to resolve all process-related and degradation-related impurities identified in the API: the (E)-isomer, the demethylated pyrrole derivative, and the oxidative ring-opened dicarboxylic acid species. Forced degradation samples prepared according to ICH Q1A(R2) stress guidelines reveal that the acid exhibits significant susceptibility to alkaline hydrolysis (0.1 M NaOH, 40 °C, 2 h), generating approximately 8.2% of the 5-fluoroindolin-2-one impurity, whereas acidic treatment (0.1 M HCl, 40 °C, 24 h) produces only 0.6% degradation. Photolytic stress according to ICH Q1B Option 2 (xenon lamp, 1.2 million lux-hours, 200 Wh/m²) induces less than 0.2% degradation, confirming that amber glassware and standard laboratory lighting do not represent a process risk. The validated method is transferred to contract testing laboratories using a comparative intermediate precision protocol requiring Fcal ≤ Fcrit at the 95% confidence level. Any QC lot of the acid with total impurities exceeding 0.50% triggers a root-cause investigation under 21 CFR 211.192, potentially expanding into a review of Knoevenagel feedstock quality records tracked by the mass spectrometer ion at m/z 329.1 [M+H]+. Stabilization strategies for the acid during transcontinental freight in unrefrigerated containers draw on the forced degradation dataset. Bulk shipments packed in 25 kg UN-approved 4G fibreboard boxes with aluminium-laminate bags are monitored by temperature loggers (Cryopak TCX-150) recording excursions above 35 °C for cumulative durations not exceeding 72 h. Comparative real-time trials on three consecutive production lots shipped from Shanghai to Rotterdam via ocean freight (38-day transit, mean temperature 28.4 °C) demonstrate a purity change of +0.05 to –0.12%, a figure that falls within the extended measurement uncertainty (±0.18%) of the release method when inter-laboratory variability is incorporated. This shipping qualification data directly supports a retest period of 36 months assigned under a CEP holder’s stability commitment filed with EDQM. |
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Product Code: SU-CA-98-25 | Catalog Reference: SYN-7201-AC
Supplied as an off-white to pale-yellow lyophilized powder, the substance designated 5-((Z)-(5-Fluoro-2-Oxoindolin-3-Ylidene)Methyl)-2,4-Dimethyl-1H-Pyrrole-3-Carboxylic Acid represents the free carboxylic acid hydrolysis product and key synthetic intermediate of several indolinone-derived receptor tyrosine kinase inhibitors. Its molecular formula is C16H13FN2O3 with a monoisotopic mass of 300.09 Da. The compound is frequently encountered as a critical starting material for amide coupling reactions targeting the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) kinase domains, or as a reference marker for impurity profiling under ICH Q3A guidelines during finished pharmaceutical batch release.
The identity of the Z-geometric isomer is confirmed through 1H NMR (DMSO-d6, 400 MHz) wherein the vinylidene proton resonates as a singlet at δ 7.35–7.41 ppm, demonstrating the characteristic downfield shift attributable to the electron-withdrawing 5-fluoro substituent on the oxindole ring. A coupling constant analysis is not applied to this proton in the Z-configuration; however, the absence of the E-isomer doublet near δ 6.80 ppm serves as a critical lot-release criterion. Mass spectral verification via ESI(-)-MS yields a molecular ion peak at m/z 299.1 [M-H]−, with a secondary adduct at 335.1 corresponding to [M+Cl]− when analyzed from methanolic ammonium chloride solutions. Infrared spectroscopy (ATR-FTIR) exhibits carbonyl stretching modes at 1682 cm−1 (oxindole C=O) and 1650 cm−1 (pyrrole carboxylic acid C=O), accompanied by a broad O-H absorption centered at 3100 cm−1.
Purity is determined using a high-performance liquid chromatography method equipped with a reversed-phase C18 column (150 mm × 4.6 mm, 5 μm particle size) and a mobile phase consisting of 0.1% trifluoroacetic acid in water (Eluent A) and acetonitrile (Eluent B), running a linear gradient from 20% B to 90% B over 30 minutes at a flow rate of 1.0 mL/min. Detection is set at 254 nm with a reference wavelength of 360 nm. Under these conditions, the main peak elutes at a retention time of approximately 12.8 min, and area normalization provides a chromatographic purity of ≥98.5%. Related substances are individually limited to ≤0.10%, with the E-isomer constrained to ≤0.15% and the 5-fluoro-2-oxindole des-methyl pyrrole adduct limited to ≤0.20%.
| Test Parameter | Acceptance Criterion | Typical Result | Method Reference |
|---|---|---|---|
| Appearance | Off-white to pale-yellow powder | Pale-yellow powder | Visual / USP <167> |
| Assay (HPLC, anhydrous basis) | ≥98.5% | 99.2% | USP <621> Chromatography |
| E-Isomer Content | ≤0.15% | 0.03% | In-house HPLC (same conditions) |
| Water Content (Karl Fischer) | ≤0.5% | 0.12% | USP <921> Method Ic |
| Residual Solvents | Meets USP <467> Class 2 limits | Acetone <50 ppm | USP <467> GC-HS |
| Heavy Metals | ≤20 μg/g (as Pb) | <10 μg/g | USP <231> Method II |
The substance is packaged under argon in amber glass vials sealed with PTFE-faced septa to minimize moisture ingress and photolytic degradation. Storage is recommended at 2–8°C with a retest period of 24 months from the date of manufacture when kept unopened. Once opened, the material should be handled in a desiccated environment (relative humidity <30%) and consumed within 7 days to avoid gradual hydrolysis of the oxindole ring, which can generate detectable levels of 5-fluoro-2-aminoacetophenone derivatives.
The fundamental structural and functional distinction lies in the substitution at the pyrrole 3-position. This free carboxylic acid species contains a carboxyl group (–COOH), unlike the pharmacologically active Sunitinib, which carries a N-(2-diethylaminoethyl)amide side chain at this site. That amide side chain contributes to the binding interactions with the DFG motif of the kinase hinge region, increasing IC50 values against VEGFR2 to 4 nM; removal of the amide yields a compound with negligible inhibitory activity. In the context of synthesis, the free acid is the immediate precursor to the active pharmaceutical ingredient. Conversion is achieved via carbodiimide-mediated coupling—typically using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and hydroxybenzotriazole (HOBt) in anhydrous DMF at 0–5°C—which forms the amide bond with the diethylaminoethylamine moiety. Process development studies on a 50-L reactor have demonstrated that residual water content above 0.3% in the reaction mixture promotes the formation of the N-acylurea side product, which co-elutes with the desired product on C18 media and necessitates re-crystallization from isopropanol/water (7:3 v/v) to meet the <0.10% impurity threshold.
Comparative evaluation against the maleate salt form (monographed in the European Pharmacopoeia under monograph 3056 for Sunitinib Malate) reveals further operational contrasts. The free acid exhibits aqueous solubility of approximately 12 μg/mL at pH 7.4 phosphate buffer, significantly lower than the maleate salt (> 25 mg/mL), thus complicating dissolution-based cleaning validation in multi-product API facilities. Swab cleaning limits calculated according to ADE/PDE methodology (per EMA/CHMP/CVMP/SWP/169430/2012) require analytical methods capable of detecting surface residues as low as 0.015 μg/cm2, an order of magnitude more stringent than for the maleate salt owing to the free acid’s classification as an in-process intermediate with no permitted daily exposure value. Moreover, the carboxylic acid form shows an endothermic melting transition at 265–268°C with decomposition, compared to the maleate salt which melts with degradation at 234°C. The sharper differential scanning calorimetry (DSC) endotherm at 268°C for the Z-acid—with a heat of fusion of −154 J/g—serves as a confirmatory identification marker when vibrational spectroscopy yields ambiguous results, particularly in mixtures where the oxindole N-H stretching region around 3150 cm−1 can be obscured.
The lactam–lactim tautomerism of the oxindole ring adds a layer of complexity not present in the fully substituted amide products. Under alkaline conditions (pH > 9.0), the 2-oxindole carbonyl can undergo enolization, converting to the 2-hydroxyindole tautomer and enabling intramolecular cyclization with the adjacent vinylidene carbon, a pathway that is effectively blocked when the pyrrole is derivatized. This tautomerization manifests in solution as a time-dependent bathochromic shift in the UV-visible spectrum, with a new absorption band emerging at 345 nm after 6 hours of standing in 0.1 M sodium carbonate solution. Production-scale chromatography in process analytical technology (PAT) environments therefore integrates in-line UV-diode array spectrophotometers programmed to flag any injection whose absorbance ratio A254/A345 exceeds 3.0, triggering automated column regeneration before the impurity front reaches the fraction collection valve.The compound is principally deployed as a drug substance intermediate within current good manufacturing practice (cGMP) synthesis chains for small-molecule tyrosine kinase inhibitors targeting the VEGFR/PDGFR/c-KIT signaling axis. In a typical batch record, 1.0 kg of the free acid is suspended in 12 L of anhydrous tetrahydrofuran and cooled to −10°C under nitrogen, to which 1.05 equivalents of N,N-diisopropylethylamine are added, followed by 1.02 equivalents of N-(2-diethylaminoethyl)amine and 1.1 equivalents of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) as coupling reagent. The reaction mixture is agitated at 300 rpm in a jacketed glass-lined reactor; the exothermic coupling is controlled by maintaining the jacket temperature at −5°C for the initial 30 minutes, then allowed to warm to 20°C over 2 hours. Conversion is monitored by HPLC and typically reaches complete consumption of the acid within 4 hours. After solvent exchange to ethyl acetate and successive washes with 1 M HCl, saturated NaHCO3, and brine, the crude amide is isolated and recrystallized from acetonitrile/methyl tert-butyl ether (1:4) to furnish the API with a process yield of 86–92%.
In the analytical testing laboratory, 5-((Z)-(5-Fluoro-2-Oxoindolin-3-Ylidene)Methyl)-2,4-Dimethyl-1H-Pyrrole-3-Carboxylic Acid serves as a certified reference material for the quantitative determination of the amide hydrolysis impurity in pharmaceutical finished products. Reversed-phase UHPLC methods aligned with the ICH Q2(R1) validation framework achieve a limit of quantification (LOQ) of 0.02 μg/mL and a limit of detection (LOD) of 0.006 μg/mL for this substance in the presence of Sunitinib malate at a test concentration of 1.0 mg/mL. Forced degradation studies performed under oxidative stress conditions (3% H2O2, 70°C, 4 hours) reveal that the primary degradant is the free acid, with a mass balance of 99.4% between parent loss and acid formation, supporting the selection of this material as the principal hydrolysis marker in stability-indicating methods per ICH Q1A(R2).
| Property | 5-((Z)-(5-Fluoro-2-Oxoindolin-3-Ylidene)Methyl)-2,4-Dimethyl-1H-Pyrrole-3-Carboxylic Acid (This Material) | Sunitinib Malate (EP/USP) | Sunitinib Base (Amide) |
|---|---|---|---|
| Molecular weight (g/mol) | 300.28 | 532.56 (including malic acid) | 398.47 |
| Aqueous solubility (pH 7.4) | 12 μg/mL | >25 mg/mL | 1.2 mg/mL |
| Thermal behaviour (DSC) | Endotherm 265–268°C (decomp) | Endotherm 234°C (decomp, malate salt) | Endotherm 218–220°C |
| Primary HPLC retention time* | 12.8 min | 8.4 min | 14.2 min |
| ICH classification | Process intermediate / impurity (hydrolysis product) | Active substance (EP 3056) | Not separately monographed (part of base form) |
*Conditions: C18, 150×4.6 mm, 5 μm; mobile phase 0.1% TFA in water / acetonitrile gradient as described above.
The operational incompatibility of the free acid with aqueous basic conditions imposes strict limits on its use in lyophilization cycles where common bulking agents such as mannitol or glycine are employed; the residual alkalinity of glycine solutions (pH 9.5–10.0) is sufficient to trigger ring-opening reactions that compromise purity within a single freeze-drying cycle. Facilities handling both this intermediate and amine-based reagents must segregate processing suites, as airborne diethylamine vapors at concentrations as low as 5 ppm have been observed to initiate N-acylurea formation on exposed acid surfaces over a 48-hour period in non-ventilated storage cabinets, leading to out-of-specification impurity profiles upon resampling.
In contract development and manufacturing organizations (CDMOs) adopting Quality-by-Design (QbD) paradigms, the acid’s critical material attributes (CMAs) are modeled against the final drug substance critical quality attributes (CQAs). Multivariate DoE screenings indicate that the acid’s particle size distribution—specifically a D90 below 200 μm—significantly influences the dissolution rate in the coupling solvent, affecting amide conversion kinetics. A design space defined by a dissolution temperature of 15–25°C and an agitation speed of 200–400 rpm has been validated at the 50-liter scale, yielding final drug substance purity consistently exceeding 99.8% with total impurities below 0.15%. Published data for this specific configuration in micro-reactor continuous process intensification is limited; however, laboratory feasibility studies suggest that coupling in a 0.5 mm ID PEEK coil reactor at 60°C reduces reaction time to 15 minutes while maintaining impurity profiles within specifications, though the long-term stability of the acid in the THF/HBTU mixture at elevated temperatures remains under investigation.
