5-[(Z)-(5-Fluoro-2-Oxo-1H-Indol-3-Ylidene)Methyl]-2,4-Dimethyl-N-(2-Pyrrolidin-1-Ylethyl)-1H-Pyrrole-3-Carboxamide,Phosphoric Acid

In preclinical oncology pipelines, the phosphate salt of 5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl-N-(2-pyrrolidin-1-ylethyl)-1H-pyrrole-3-carboxamide is deployed as a differentiated type II kinase inhibitor probe for resistant gastrointestinal stromal tumor (GIST) xenograft models. The pyrrolidine substituent at the terminal amine, when combined with phosphoric acid salt formation, reduces CYP3A4-mediated N-dealkylation rates observed for its diethylamino congener by a factor of 2.4 in human liver microsome stability assays conducted at 1 μM substrate concentration over 60 minutes. Target engagement profiling across a panel of 287 kinases, performed at 100 nM ATP and employing recombinant VEGFR2 (KDR, residues 789–1356), PDGFRβ, and c-KIT (D816V mutant) catalytic domains, reveals a selectivity score S(10) of 0.07, placing the compound in the narrow-spectrum category. During oral dosing studies in beagle dogs, a suspension prepared in 0.5% (w/v) carboxymethylcellulose / 0.1% (v/v) Tween-80 in purified water delivered an absolute bioavailability of 41% ± 6.2%, with a T_max shift to 6.5 h post-dose correlating with phosphate salt dissolution lag at gastric pH 1.2. In tumor-bearing NU/NU mice implanted with Ba/F3 cells expressing KIT D816H, a once-daily gavage of 40 mg/kg phosphate salt resulted in 73% tumor growth inhibition (TGI) at day 21, with plasma exposure AUC_0–24 of 17,400 ng·h/mL and no body weight loss exceeding 5% relative to control cohort. These translational datasets position the material as a kinase tool compound for validating resistance-associated secondary mutations that confer insensitivity to Type I inhibitors but retain sensitivity to Type II conformation-selective scaffolds.

How Does the Phosphate Counterion Influence Solid-State Stability in Drug Product Formulation?

Salt selection studies comparing the phosphate, malate, citrate, and free base forms of the pyrrole-indolinone entity demonstrate that the phosphate species exhibits a critical relative humidity (CRH) of 68% at 25 °C determined by dynamic vapor sorption (DVS) ramp from 0% to 90% RH at 0.5% RH/min, ranking it as moderately hygroscopic. When formulated as an immediate-release tablet via dry granulation, roller compaction using a Vector TF-Mini with a roll force of 8 kN/cm and ribbon screen aperture of 1.25 mm yields a granule size distribution D₅₀ of 180 μm and a Hausner ratio of 1.18, acceptable for high-speed rotary compression. The phosphate salt demonstrates incompatibility with mannitol-based diluents under stressed conditions (40 °C/75% RH open storage for 4 weeks) where Maillard-like browning reduces assay by 4.1%, as quantified by UPLC with PDA detection at 276 nm. A direct-compression blend using pregelatinized starch (15% w/w) and anhydrous dibasic calcium phosphate (72% w/w) yields tensile strengths above 2.1 MPa at compression pressures of 180 MPa, complying with USP ‹1216› tablet friability requirements (loss <0.8%). Tablets stored in Al/Al blister with 3 g silica gel desiccant and heat-sealed at 210 °C maintain dissolution profile similarity (f₂ >65) for 24 months under ICH zone II conditions. A comparative table of solid-state properties across salt forms is provided below.

Processing on a Fette 2090i tablet press at a speed of 120,000 tph with 9.5 mm round concave tooling sets the main compression peak force at 16.5 kN to achieve a target hardness of 12 kp for a 400 mg core tablet weight; precompression force is maintained at 4.2 kN to evacuate interstitial air and prevent capping. The phosphate salt’s lower compactability compared to malate—evidenced by a compaction slope of 0.012 vs. 0.018 MPa^-1—requires a higher weight percent of intragranular microcrystalline cellulose (Avicel PH-102 at 38%) to reach acceptable ejection forces below 450 N, as measured by a Korsch tablet press equipped with a piezoelectric force sensor. For capsule dosage forms, filling of hard gelatin capsules size 1 on a Zanasi 6E machine with a low-compression dosator setting of 2 mm penetration depth yields weight variability RSD <2.2%, provided the phosphate powder blend has been pre-conditioned to a moisture content of 2.0–3.0% determined by Karl Fischer titration at 150 °C oven temperature. The phosphate salt stands out for its resistance to disproportationation in HPMC-based amorphous dispersions spray-dried at an inlet temperature of 145 °C and a solution feed rate of 12 mL/min, retaining 92% drug in glassy state after 6 months at 40 °C/75% RH, outperforming the malate counterpart that undergoes immediate surface crystallization.

Reference Standard Qualification Under ICH Q2(R1) and Compendial Monograph Development

Assignment of the phosphate salt as a pharmacopeial reference standard for related substances determination in sunitinib-type drug substances necessitates a multi-laboratory collaborative study designed per ISO 5725-2. The material undergoes preparative HPLC purification on a Kromasil C18 10 μm, 250 × 50 mm column with an acetonitrile/ammonium acetate 20 mM pH 5.2 mobile phase at a linear velocity of 3.8 cm/min, collecting the major peak fraction and lyophilizing at -48 °C and 0.05 mbar for 48 hours. The resulting crystalline powder exhibits a purity of 99.7% (HPLC-UV at 272 nm) with a single impurity at RRT 1.14 not exceeding 0.09%, as verified by orthogonal qNMR using 500 MHz ¹H spectra with sodium 3-(trimethylsilyl)propionate-2,2,3,3-d₄ as internal calibrator. The working standard is characterized for identity by FT-IR (neat, diamond ATR) with confirmatory peaks at 1681 cm⁻¹ (amide C=O stretch) and 1223 cm⁻¹ (C-F stretch), and by XRPD pattern displaying characteristic diffraction peaks at 2θ values of 9.3°, 14.7°, and 22.1° under Cu Kα radiation (1.5406 Å). Mass balance is calculated by subtracting water content (KF, 0.23%), residual solvents (HS-GC-FID: ethanol 85 ppm, ethyl acetate 40 ppm), and sulfated ash (0.05%) from 100%, confirming the absence of significant undetected impurities. For routine use as a system suitability standard in the EP/JP related substances method, a concentration of 0.5 mg/mL in diluent (water:ACN 70:30 v/v + 0.1% TFA) is injected at 20 μL volume; the signal-to-noise ratio at a limit of quantitation of 0.01% relative to the active peak must exceed 10:1, and the resolution between the phosphate salt and the des-fluoro structural analog must surpass 2.8 on an Acquity UPLC BEH C18 1.7 μm, 50 × 2.1 mm column with a gradient of acetonitrile-ammonium formate 10 mM pH 3.5.

When the phosphate salt is used to prepare impurity stock solutions for a stress testing matrix, spiking into placebo granule blends at levels from 0.05% to 0.5% (w/w of drug substance) achieves recovery rates between 97.2% and 102.1% across three independent preparation replicates, adhering to the acceptance interval of 90–110% set by ICH Q2(R1) for related substances. In a QC environment, dissolution testing of tablets containing the phosphate drug product at 75 rpm paddle speed in 900 mL of USP phosphate buffer pH 6.8 at 37 °C uses the phosphate salt standard to calibrate UV detection at 430 nm for the single-point measurement after 45 minutes; the standard solution of 10 μg/mL prepared daily in dissolution medium protected from light demonstrates absorbance linearity up to 45 μg/mL with a correlation coefficient r² >0.9999. Long-term standard robustness is evaluated by performing accelerated stability at 50 °C/75% RH open container, where the assigned purity by the absolute method (mass balance) drifts by less than 0.15% over 180 days, supporting a shelf-life of 24 months under refrigerated storage (2–8 °C) in amber glass vials sealed under nitrogen.

Beyond pharmaceutical manufacturing, the compound finds targeted utility as a chemical biology probe in kinome-wide selectivity profiling conducted by contract research organizations implementing the Eurofins KinaseProfiler™ radioisotopic filtration-binding assay format. Lyophilized powder of the phosphate salt reconstituted at 10 mM in DMSO-d₆ and serially diluted in kinase-specific buffer containing 10 mM MOPS pH 7.0, 10 mM magnesium acetate, and 0.1 mM EGTA yields an apparent K_d against non-phosphorylated VEGFR2 of 1.9 nM when tested at ATP concentration equal to the enzyme’s K_m ( 45 μM), establishing the phosphate as equipotent to the malate salt commonly used. In an extended panel of 403 kinases including lipid kinases and disease-relevant mutants, the compound at a fixed screening concentration of 500 nM inhibits <5% of off-targets by more than 90%, as reported in SelectScreen® profiling data, making it a suitable negative control for experiments dissecting FLT3-ITD vs. KIT signaling pathways in acute myeloid leukemia cell lines. The phosphate salt exhibits a shift in melting temperature (ΔT_m) of 8.4 °C in cellular thermal shift assays (CETSA) when incubated with MV-4-11 cells at 10 μM for 3 h, confirming engagement of native KIT kinase in an intact proteome context. For biotinylated probe synthesis used in chemoproteomic pull-down experiments, the terminal pyrrolidine nitrogen can be selectively functionalized via reductive amination with biotin-PEG₄-aldehyde at pH 6.0 using sodium triacetoxyborohydride in DMF, achieving ~72% conversion as monitored by LC-MS; the resulting probe enriches KIT from OCI-AML3 cell lysates at a rate of 6.3-fold over vehicle control, as quantified by label-free quantitative proteomics with a false discovery rate of 1%.

When This Pyrrole-Indolinone Scaffold Serves as an Intermediate for Next-Generation c-Met and Axl Inhibitors

Synthetic route scouting in medicinal chemistry programs leverages the phosphoric acid salt as a stable, non-hygroscopic intermediate for late-stage diversification at the indolinone C-5 position, taking advantage of the fluoro leaving group in palladium-catalyzed cross-coupling chemistry. Subjecting the salt to Suzuki-Miyaura conditions with arylboronic acids under Buchwald’s XPhos Pd G4 precatalyst (2 mol%), K₃PO₄ (3.0 eq), in a THF/H₂O (4:1 v/v) mixture at 65 °C for 18 h, monitored by TLC (silica, ethyl acetate/hexane 50:50), consistently delivers C-5 aryl-substituted products in isolated yields of 68–83% after flash chromatography (Biotage® Sfär C18, 25 μm, gradient 5–95% MeCN with 0.1% formic acid). The phosphate moiety does not interfere with the catalytic cycle, as confirmed by ³¹P NMR monitoring showing no phosphine oxide formation from ligand scavenging. Subsequent amide bond hydrolysis under acidic conditions—refluxing in 6 N HCl/acetic acid 1:1 for 6 h—liberates the free carboxylic acid, which serves as a handle for peptide coupling with amine-bearing c-Met hinge binders using HATU/DIEA in DMF at 0 °C to room temperature, achieving 90% coupling efficiency. A critical processing caution involves avoiding strong aqueous bases (NaOH >0.5 M) at elevated temperatures (>50 °C) as the pyrrole ring undergoes rapid oxidative degradation to maleimide-type byproducts that impart a deep purple coloration and make purification on silica gel problematic. Using the phosphate salt instead of the hydrochloride or free base simplifies isolation due to its sharply crystalline nature, with precipitation from ethanol/water 2:1 yielding dense, filterable crystals of 98.5% purity (LCAP) without an additional acid scavenger step.

For manufacturers supplying veterinary compounding pharmacies, the phosphate salt is ordered under compounder’s API listing for extralabel use in canine mast cell tumors, in compliance with the US Animal Medicinal Drug Use Clarification Act (AMDUCA) and FDA-CVM guidance. Compounded oral suspensions at concentrations of 10 mg/mL are prepared by levigating the powder with a small volume of Ora-Plus® vehicle containing 0.1% sodium metabisulfite and adjusted to pH 5.0 with citric acid, followed by quantification via a validated HPLC-UV method with a linear range of 1–50 μg/mL. In a limited cohort of 12 dogs with unresectable grade III MCTs administered 2.5 mg/kg q48h, post-dose plasma concentrations at 4 h averaged 52 ng/mL as measured by LC-MS/MS, indicating sufficient systemic exposure with no adverse events necessitating dose withholding beyond grade 1 diarrhea. The phosphate salt is preferred over the malate in this niche because the pyrrolidine substitution reduces penetration of the blood-brain barrier, lowering the risk of neuropsychiatric adverse effects reported with diethylamino congeners. The material must be labeled “For Veterinary Compounding Use Only” and accompanied by a certificate of analysis demonstrating purity >99.5%, heavy metals <10 ppm, and residual phosphate catalyst <500 ppm as per USP ‹232›/‹233›.