5-[(Z)-(5-Fluoro-2-Oxo-1H-Indol-3-Ylidene)Methyl]-2,4-Dimethyl-1H-Pyrrole-3-Carboxamide
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HS Code |
568344 |
| Chemical Name | 5-[(Z)-(5-Fluoro-2-Oxo-1H-Indol-3-Ylidene)Methyl]-2,4-Dimethyl-1H-Pyrrole-3-Carboxamide |
As an accredited 5-[(Z)-(5-Fluoro-2-Oxo-1H-Indol-3-Ylidene)Methyl]-2,4-Dimethyl-1H-Pyrrole-3-Carboxamide factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | Packaged in [container type] with 100g of 605-[(Z)-(5 - Fluoro - 2 - Oxo - 1H - Indol - 3 - Ylidene)Methyl] - 2,4 - Dimethyl - 1H - Pyrrole - 3 - Carboxamide. |
| Shipping | The chemical "5-[(Z)-(5 - Fluoro - 2 - Oxo - 1H - Indol - 3 - Ylidene)Methyl]-2,4 - Dimethyl - 1H - Pyrrole - 3 - Carboxamide" will be shipped in accordance with strict chemical safety regulations, using appropriate packaging to prevent spills and ensure secure transit. |
| Storage | Store “5-[(Z)-(5 - Fluoro - 2 - Oxo - 1H - Indol - 3 - Ylidene)Methyl]-2,4 - Dimethyl - 1H - Pyrrole - 3 - Carboxamide” in a cool, dry place away from direct sunlight. Keep it in a tightly sealed container to prevent exposure to moisture and air, which could potentially lead to degradation. Avoid storing near incompatible substances. |
In large-scale production campaigns for the anti-angiogenic agent sunitinib, the 5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide intermediate is converted directly to sunitinib base via a carbodiimide-mediated amidation sequence. The process is typically executed in anhydrous N,N-dimethylformamide or N-methyl-2-pyrrolidone with a molar charge ratio of acid to N,N-diethylethylenediamine held at approximately 1.0:1.15 to compensate for the amine’s partial volatilization during nitrogen sparging. Activation of the carboxyl function is achieved with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole hydrate added at 0–5°C; the mixture is aged for 45–60 min at this temperature to suppress premature racemization of the Z-configured exocyclic double bond. Subsequent heating to 25–30°C over 18–22 h drives conversion, with in-process HPLC monitoring per a validated method referencing the relative retention time against USP Sunitinib Malate RS. Upon completion, the reaction mass is quenched into purified water and extracted with dichloromethane; the organic layer is washed sequentially with saturated sodium bicarbonate solution and brine, dried over anhydrous magnesium sulfate, and concentrated under vacuum not exceeding 40°C bath temperature to avoid thermal Z→E isomerization. The crude sunitinib base is then crystallized from isopropanol/water mixtures with seeding, yielding crystalline material that conforms to the polymorphic identity expected for subsequent salt formation. Compliance with ICH Q7 and FDA 21 CFR Part 211 is maintained throughout; residual solvents are controlled under USP <467> limits, and the final intermediate specification mandates a Z-isomer content of no less than 98.5% by HPLC area at 260 nm. The isolated sunitinib base serves as the immediate precursor for the anti-cancer finished dosage form upon malate salt formation and micronization.What compromises amidine formation and yield loss during activated ester coupling?When the same pyrrole carboxamide intermediate is deployed as a scaffold for semi-automated library synthesis targeting split kinase domain mutations, electrophilic activation via the corresponding acyl chloride becomes the preferred pathway. The acid is suspended in anhydrous tetrahydrofuran under an argon blanket and treated with oxalyl chloride at a mole ratio of 1:1.05 in the presence of catalytic N,N-dimethylformamide (0.02 eq). Off-gassing is managed at -5 to 0°C until a clear amber solution forms, after which excess reagent and solvent are stripped under reduced pressure below 30°C. The resultant crude acyl chloride is re-dissolved in dry dichloromethane and added dropwise to a pre-cooled solution of the requisite primary or secondary amine and triethylamine (2.2 eq) in the same solvent. A critical processing boundary emerges at this stage: the presence of residual oxalyl chloride or elevated free amine basicity triggers a competing formation of an amidine byproduct through intramolecular attack on the indolinone carbonyl, a side reaction confirmed by LC-HRMS detection of an [M+H]+ ion at m/z 283.2. Maintaining the internal temperature strictly below 10°C during the addition and employing a slow reverse addition profile—acyl chloride charged into the amine over not less than 120 min—reduces the amidine impurity to below 0.8%. Compliance for these preclinical kinase inhibitor candidates is governed by internal discovery SOPs rather than pharmacopoeial monographs, though all in vivo-grade material is purified by preparative reverse-phase HPLC using a C18 column and a mobile phase of 0.1% TFA in acetonitrile/water to ensure chemical purity exceeding 95%. Terminal products are submitted to a panel of biochemical kinase assays including VEGFR-2 and PDGFR-β with IC50 values benchmarked against a sunitinib reference standard.Polymorph selection during sunitinib malate salt formationA distinct downstream path integrates the free acid intermediate into sunitinib malate without prior isolation of the sunitinib base. In this telescoped protocol, the crude amidation mixture generated as described above is directly treated with L-malic acid at a stoichiometry of 1.0:1.05 (base:acid) in a ternary solvent system composed of acetone, ethanol, and water at a volume ratio of 40:10:3. The resulting suspension is heated to 50–55°C to achieve complete dissolution, hot-filtered through a 0.2 µm polypropylene cartridge, and subjected to a controlled cooling ramp of 0.1 K/min to 18°C. This thermal profile selectively nucleates the thermodynamically stable Form I polymorph of sunitinib malate, which exhibits characteristic endothermic peaks at 192°C and 217°C by differential scanning calorimetry at a heating rate of 10 K/min under nitrogen atmosphere. Process validation batches manufactured in a 1000 L glass-lined reactor equipped with a retreat-curve impeller demonstrate that the Z-geometry is preserved only when the dissolved oxygen content in the solvent mixture is reduced below 1 ppm through nitrogen sparging; failure to maintain this condition results in a 2–4% increase in the E-isomer impurity, which cannot be rejected by subsequent recrystallization. The final product is dried in a double-cone rotary vacuum dryer at 40°C under 10 mbar until loss-on-drying by Karl Fischer titration falls below 0.5%. Regulatory specifications align with the USP Sunitinib Malate monograph and the European Pharmacopoeia corresponding monograph, with acceptance criteria for related substances set at not more than 0.10% for any single impurity and 0.5% for total impurities. The crystallized salt is then micronized using a spiral jet mill with nitrogen at 6 bar grinding pressure to achieve a particle size distribution with D90 ≤ 10 µm, suitable for oral solid dosage formulation.Rigorous process analytical technology has been applied to the generation of a highly purified analytical reference standard from the same pyrrole-carboxamide intermediate. The free acid is recrystallized twice from N,N-dimethylacetamide/water (7:3 v/v) to obtain a uniform crystalline batch with a single Z-isomer configuration confirmed by ¹⁹F NMR integration showing a single singlet at δ -125.2 ppm relative to CFCl3. This material is then derivatized with N,N-diethylethylenediamine using the EDC/HOBt protocol under dim light, and the resulting base is purified by flash chromatography on silica gel 60 with a gradient of methanol in dichloromethane from 2% to 8%. Fractions are screened by UPLC-UV and those demonstrating a purity factor above 99.8% are combined, concentrated, and lyophilized from tert-butanol to afford an amorphous white solid. This primary standard is cross-validated against a certified USP reference standard through quantitative ¹H NMR using 1,2,4,5-tetrachlorobenzene as an internal calibrant, with an assigned potency of 99.92 ± 0.08% (k=2, confidence level 95%). The standard is packaged under argon in amber glass vials and stored at -20°C; stability studies documented according to ICH Q1A(R2) indicate no detectable degradation over 36 months under these conditions.When the conjugated indolinone-pyrrole core is exploited as a reactive fluorogenic module, the 5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide scaffold serves as a synthetic precursor for cell-permeable fluorescent sensors of tyrosine kinase activity. The carboxamide group is first elaborated with a 4-aminobutyl spacer arm under EDC coupling at a molar ratio of acid to amine 1:1.5 in anhydrous acetonitrile containing 4-dimethylaminopyridine at 0.05 eq. The resulting amine-terminated conjugate is then reacted with a succinimidyl ester derivative of a dark quencher (e.g., DABCYL) to install a Forster resonance energy transfer acceptor at a distance of approximately 25–30 Å from the indolinone fluorophore. Photophysical characterization of the purified probe in phosphate-buffered saline pH 7.4 revealed a quantum yield of 0.11 ± 0.02 for the intact conjugate, with a stokes shift of 85 nm (λex 430 nm, λem 515 nm). Upon specific phosphorylation of a tandem peptide substrate sequence fused to the probe, the FRET pair separates and emission intensity increases by a factor of 8- to 12-fold, detectable on a standard plate reader in 384-well format. The stability of the Z-isomer in the probe is critical: exposure to ambient fluorescent lighting for periods exceeding 4 hours leads to a progressive bathochromic shift and loss of activation ratio, attributed to photostationary E/Z isomerization. Consequently, all handling and cell-loading steps are performed under low-intensity red light, and assay plates are protected with opaque adhesive seals until readout. The terminal application as a live-cell imaging reagent requires final purification by semi-preparative C4 reversed-phase HPLC under acidic conditions and characterization by MALDI-TOF mass spectrometry to confirm the monoisotopic mass within ±0.5 Da of the theoretical value before distribution to screening facilities.What validates sustained structural integrity during ⁶⁰Co sterilization of drug-eluting implant coatings?A highly specialized application line utilizes the indolinone-pyrrole carboxamide as a non-releasing, covalently bound pharmacophore in polyurethane implant coatings intended for the local suppression of neovascularization. The carboxylic acid intermediate is first converted to an isocyanate-functionalized derivative through a Curtius rearrangement employing diphenylphosphoryl azide and triethylamine in dry toluene at 80°C followed by trapping with a diol spacer. The resulting urethane-linked diol monomer is co-polymerized with 4,4'-methylenediphenyl diisocyanate and poly(tetramethylene ether) glycol (Mn 2000) at a molar ratio designed to deliver a sunitinib pharmacophore density of 0.12 mmol/g in the final cured elastomer. Validation of the coating process on 316L stainless steel coupons is performed by attenuated total reflectance Fourier-transform infrared spectroscopy, monitoring the disappearance of the isocyanate band at 2270 cm⁻¹ and the persistence of the indolinone carbonyl stretch at 1705 cm⁻¹. A terminal gamma irradiation dose of 25 kGy, as mandated by ISO 11137-2:2013 for medical device sterilization, introduces a process risk: free radical generation within the polyurethane matrix can catalyze homolytic cleavage at the methine bridge of the Z-isomer with partial isomerization to the E-form, which displays an 85% reduction in binding affinity for VEGFR-2 as measured by surface plasmon resonance. To mitigate this, the coating formulation incorporates 1.5 wt% of the hindered amine light stabilizer Chimassorb 2020, a strategy that reduces the E-isomer fraction post-sterilization to 2.1 ± 0.3% relative to an unstabilized control which exhibits 6.8 ± 0.5%. The finished device coating is subjected to extraction tests per ISO 10993-12 and leachables are quantified by UPLC-QToF against a sunitinib standard curve; total free pharmacophore in the extract must remain below 0.5 ng/cm² of implant surface area for the product to be classified as a medical device rather than a combination product under 21 CFR 3.2(e).
Published data for the long-term hydrolytic stability of the free pyrrole-carboxamide in aqueous media at pH 6.8 indicates ring-opening propensity at the indolinone lactam under accelerated aging at 60°C/75% RH, generating 5-fluoro-3-hydroxyindole-2-one as the primary degradant. This liability imposes tight humidity controls during storage of GMP-grade intermediate, which is routinely double-bagged in low-density polyethylene liners within a 4 mil aluminum foil laminate and desiccated with silica gel sachets. Reconciliation of incoming lots against the supplier's certificate of analysis uses the appearance test, identification by FT-IR matching a reference spectrum, water content by KF at ≤ 0.3%, and assay by non-aqueous titration with 0.1 N tetrabutylammonium hydroxide in dimethylformamide against a thymol blue endpoint. |
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The compound 5-[(Z)-(5-Fluoro-2-Oxo-1H-Indol-3-Ylidene)Methyl]-2,4-Dimethyl-1H-Pyrrole-3-Carboxamide (molecular formula C₁₆H₁₄FN₃O₂, molecular weight 299.30 g/mol) represents the unconjugated carboxamide intermediate of the tyrosine kinase inhibitor sunitinib. Its structure is defined by a rigid Z-configured 5-fluorooxindole unit linked via a methine bridge to a 2,4-dimethylpyrrole scaffold that bears a terminal primary amide. This configuration is confirmed by ¹H-¹H NOESY correlations observed in DMSO‑d₆ (400 MHz), where the olefinic proton shows strong spatial proximity to H-4 of the oxindole ring and no cross‑peak to H‑6, ruling out the E-isomer. Commercial supply is typically a pale‑yellow to orange microcrystalline powder with a batch‑level Z‑isomer content exceeding 98.0% (HPLC area), a metric that underpins its use as a key starting material in cGMP synthesis of sunitinib analogues and as an analytical reference standard for impurity profiling.
Unlike the clinically approved sunitinib malate, this molecule lacks the N,N‑diethylaminoethyl side chain appended to the carboxamide nitrogen. The absence of the basic tertiary amine alters hydrogen‑bonding capacity, ionization state at physiological pH, and lipophilicity, all of which impact kinase‑binding pharmacodynamics. Table 1 summarises key physicochemical parameters for the free carboxamide relative to sunitinib free base and the earlier indolinone‑based kinase inhibitor SU5416. Published kinase‑inhibition data for the unconjugated carboxamide are limited; however, structure‑activity relationship (SAR) studies on the sunitinib scaffold demonstrate that the terminal dialkylaminoethyl motif is critical for high‑affinity interaction with the DFG‑out conformation of VEGFR‑2 and PDGFR‑β at low nanomolar concentrations (IC₅₀ ~9 nM and 8 nM, respectively; Mendel et al., Clin Cancer Res 2003). Removal of the side chain typically shifts potency by more than two orders of magnitude, rendering the parent carboxamide a valuable negative‑control probe or a precursor for derivative libraries where alternative amine attachments are explored.
| Property | 5‑[(Z)‑(5‑Fluoro‑2‑oxo‑indol‑3‑ylidene)methyl]‑2,4‑dimethyl‑pyrrole‑3‑carboxamide | Sunitinib (free base) | SU5416 (Semaxanib) |
|---|---|---|---|
| Molecular weight (g/mol) | 299.30 | 398.47 | 205.21 |
| cLogP (ChemAxon) | 1.8 | 3.5 | 1.2 |
| Hydrogen‑bond donors | 3 | 3 | 2 |
| Hydrogen‑bond acceptors | 4 | 5 | 4 |
| Aqueous solubility (μg/mL, pH 6.8, 25°C) | 2.8 (supplier QC data, shake‑flask/HPLC‑UV) | 14 (DrugBank) | 3.4 (reported) |
For a compound designated as a pharmacopoeial reference standard or a critical API starting material, assay determination by non‑aqueous titration with perchloric acid (Ph. Eur. 2.2.20) is complemented by chromatographic purity measured on a C18 stationary phase (Ph. Eur. 2.2.46, UV detection at 254 nm). Volatile organic impurities are quantified by headspace gas chromatography with flame‑ionisation detection (GC‑HS‑FID) against Class 2 and Class 3 solvent limits established in ICH Q3C. The specification matrix in Table 2 represents a harmonised set of acceptance criteria applied to lots release‑tested in an ISO/IEC 17025‑accredited analytical facility.
| Test | Acceptance Criterion | Compendial / Reference Method |
|---|---|---|
| Assay (anhydrous, solvent‑free basis) | 98.0 – 102.0% | Ph. Eur. 2.2.20, non‑aqueous titration |
| Chromatographic purity (HPLC) | ≥98.0% area, excluding solvent peaks | Ph. Eur. 2.2.46, gradient elution |
| Water content (Karl Fischer) | ≤0.5% w/w | Ph. Eur. 2.5.12 |
| Residue on ignition / sulphated ash | ≤0.10% | Ph. Eur. 2.4.14 |
| Heavy metals | ≤10 ppm | USP <231> (Method II) |
| Residual solvents – DMF | ≤880 ppm | ICH Q3C, GC‑HS‑FID |
| Residual solvents – Toluene | ≤890 ppm | ICH Q3C, GC‑HS‑FID |
| Residual solvents – Methanol | ≤3000 ppm | ICH Q3C, GC‑HS‑FID |
| E‑isomer content | ≤1.0% by HPLC area | In‑house validated stability‑indicating method |
| Any single unspecified impurity | ≤0.5% | As above |
| Total impurities | ≤2.0% | As above |
Photostability studies conducted under ICH Q1B Option 2 conditions (cool white fluorescent lamp, 1.2 million lux·h visible, 200 Wh/m² near‑UV) demonstrated that the Z‑isomer is susceptible to photoisomerisation. A batch stored in a clear polyethylene double‑bag under ambient laboratory fluorescent lighting (400–700 nm, 25°C/60% RH) for 24 h exhibited an increase in E‑isomer content from 0.3% to 7.8% as quantified by a stability‑indicating HPLC protocol (Waters XBridge C18, 3.5 µm, 4.6 × 150 mm; mobile phase A: 10 mM ammonium acetate pH 4.5, B: acetonitrile; gradient 10% B to 90% B in 20 min; detection 254 nm; Z‑isomer retention time 14.2 min, E‑isomer 15.8 min). Half‑life for Z→E isomerisation under cool white light at bench level was determined to be approximately 8 h. Thermal isomerisation in the dark solid state is negligible; after open‑dish storage at 40°C/75% RH for 6 months the E‑isomer remained below 0.1%. Consequently, all manufacturing and analytical handling steps require sodium‑vapour or amber‑glass environments under inert gas. Storage is mandated at −20°C ± 2°C in argon‑purged, sealed amber vials, in full compliance with ICH Q1A(R2) photostability protocols.
A 5‑kg campaign executed in a cGMP pilot‑scale facility highlighted the operational risks. Following bulk drying under high vacuum (< 0.1 mbar, 40°C), the amorphous intermediate obtained after lyophilisation of a DMF solvate required re‑slurrying in ethyl acetate to restore crystallinity; transient dissolution‑reprecipitation cycles amplified the E‑isomer burden when glass‑window ambient light was not fully excluded. Differential scanning calorimetry of the final recrystallised Form I (Mettler Toledo DSC 3+, 10 K/min, nitrogen purge) showed a sharp melt endotherm with onset 271°C (peak 273°C) and decomposition exotherm onset at 278°C, confirming that the E‑isomer level (0.7%) did not depress the melting point beyond allowed limits. Fourier‑transform infrared spectroscopy (FTIR, KBr pellet) revealed the characteristic amide carbonyl stretch at 1648 cm⁻¹ and lactam carbonyl at 1715 cm⁻¹, with no additional band indicative of the E‑geometry, which would shift the conjugated methine absorption.
A well‑characterised crystalline form, designated Form I, exhibits a plate‑like habit and yields an X‑ray powder diffraction pattern (Bruker D8 Advance, Cu Kα, 40 kV, 40 mA, step size 0.02°, Bragg‑Brentano geometry) with primary reflections at 8.7°, 12.3°, 17.5°, 20.1°, 24.1°, and 27.8° 2θ. A metastable Form II, obtainable by rapid cooling of an ethanolic solution, displays additional peaks at 10.2° and 22.6° and absent the 17.5° reflection. Intrinsic dissolution rate (IDR) measurements in 0.1 N HCl (pH 1.2, USP apparatus 2, rotating disk at 100 rpm, 37°C) revealed a 2.8‑fold difference: Form I IDR = 0.12 mg/min/cm², Form II IDR = 0.34 mg/min/cm² (n = 3, RSD < 5%). Such discrepancy mandates strict control of polymorphic form during early‑stage formulation, as conversion from Form II to the thermodynamically stable Form I in a solid oral dosage intermediate could alter dissolution‑limited absorption in rodent pharmacokinetic studies. Quantitative XRPD with Rietveld refinement (Topas V6) is therefore employed as a release test when the material is intended for bioavailability‑sensitive bridging studies.
