5-(1H-Pyrazolo[3,4-B]Pyridin-3-Yl)-1H-Pyrrole-3-Carboxylic Acid

The free carboxylic acid functionality in 5-(1H-pyrazolo[3,4-b]pyridin-3-yl)-1H-pyrrole-3-carboxylic acid is routinely activated for amide bond formation in the synthesis of ATP‑competitive kinase inhibitors targeting fibroblast growth factor receptors (FGFR1–4). Process development at pilot scale (50–100 L glass‑lined reactors, Pfaudler AE type) has shown that direct coupling with anilines or aliphatic amines via 1.05 equivalents of T3P® (propylphosphonic anhydride, 50 wt% in ethyl acetate) in the presence of 2.5 equivalents of N,N‑diisopropylethylamine at –5 °C to +5 °C suppresses symmetrical anhydride formation and limits the formation of the N‑acylurea by‑product to <0.3 area% as tracked by in‑process UPLC (ACQUITY BEH C18, 1.7 µm, 50×2.1 mm). Under these conditions, the desired pyrazolo‑pyrrole‑carboxamide is typically isolated in 81–87% yield after aqueous work‑up (immiscible phase split monitored by conductivity probe, E+H Liquiline CM44) and subsequent normal‑phase flash chromatography (Biotage Isolera Dalton 2000, KP‑Sil 50 µm, gradient: 0–8% methanol in dichloromethane over 18 column volumes). The terminal drug substance—for instance, a 3‑(pyridin‑2‑yl)‑1H‑pyrazole‑4‑carboxamide derivative submitted as a type II kinase inhibitor candidate in a pre‑IND package—requires compliance with ICH M7 (R2) regarding potentially mutagenic impurities derived from the pyrazolo[3,4‑b]pyridine ring‑forming step. Specifically, hydrazine hydrate carry‑over is controlled to ≤1.5 ppm via derivatisation with p‑dimethylaminobenzaldehyde and analysis by LC‑MS/MS (LOQ 0.1 ppm), referencing the carcinogenicity alert in the ICH M7 addendum. Residual N,N‑dimethylformamide, ethyl acetate, and dichloromethane are quantified by static headspace GC‑FID against the concentration limits of ICH Q3C (R9): 880 ppm, 5000 ppm, and 600 ppm, respectively. The final intermediate is shipped under nitrogen in HDPE drums meeting UN 1H2/Y1.5/100 specifications, with a retest date of 24 months when stored at 2–8 °C and protected from light, based on forced degradation studies (40 °C/75% RH for 6 months) that show less than 0.5% total degradation by HPLC purity at 220 nm.

What analytical thresholds govern regioisomeric purity during the palladium‑mediated pyrazolopyridine ring closure?

The bicyclic core of 5‑(1H‑pyrazolo[3,4‑b]pyridin‑3‑yl)‑1H‑pyrrole‑3‑carboxylic acid is constructed via a sequential Suzuki–Miyaura cross‑coupling and intramolecular condensation, a route that introduces the isomeric 1H‑pyrazolo[4,3‑b]pyridine as a process‑related impurity with a typical relative retention time of 1.12 against the target regioisomer on a chiral‑non‑racemic stationary phase (Chiralpak IA‑3, 4.6×150 mm, 3 µm). Pharmaceutical outsourcing contracts frequently require a regioisomeric purity of ≥99.5% by HPLC area at 254 nm, and failure to meet this specification during the coupling of 2‑chloro‑3‑pyridylboronic acid with an N‑protected pyrrole‑3‑carboxylate leads to rejection of the batch under quality agreement clauses aligned with ICH Q7 chapter 11.3 (control of impurities). In the pilot‑plant setting, the reaction is charged with PdCl₂(dppf)·CH₂Cl₂ at 0.8 mol% under a nitrogen overpressure of 0.2 bar(g), and the aqueous work‑up temperature is maintained above 35 °C to prevent precipitation of the palladium complex in the organic phase; a polishing filtration through a Zeta‑Carbon R55SP cartridge (0.5 µm nominal) reduces residual palladium to <15 ppm as measured by ICP‑OES, meeting the Ph. Eur. 5.20 metal catalyst residue guideline class 1B limit of 100 ppm for Pd in parenteral drug substances. The subsequent cyclisation with triethyl orthoformate in the presence of catalytic p‑toluenesulfonic acid (2 mol%) is performed in refluxing toluene (jacketed temperature 111–113 °C) with a Dean–Stark trap; the endpoint is determined by 1H NMR disappearance of the pyridine C2‑H signal (δ 8.72 ppm) and by TLC (silica gel 60 F254, heptane:ethyl acetate 1:1, Rf target compound 0.35, Rf regioisomer 0.41). For shipment as a non‑sterile active pharmaceutical ingredient intermediate, a bacterial endotoxin specification of <0.15 EU/mg (USP <85>) is verified by a kinetic chromogenic LAL assay, and the certificate of analysis additionally reports a sulphated ash value ≤0.1% (Ph. Eur. 2.4.14).

Agrochemical lead optimisation: incorporating the pyrrole‑3‑carboxylic acid into ryanodine receptor modulator scaffolds

In the development of diamide insecticides acting on insect ryanodine receptors, the title compound serves as a modular acid corner for lead generation libraries that replace the traditional anthranilic acid portion with a pyrrole‑3‑carboxylic acid isostere. A representative greenhouse‑testing batch is produced by activating the acid with oxalyl chloride (1.3 equivalents) in tetrahydrofuran containing 0.5% v/v DMF at 5–10 °C, then coupling with 2‑amino‑3‑(trifluoromethyl)benzonitrile in the presence of 1.5 equivalents of triethylamine. The resulting amide intermediate is reduced with Raney nickel under hydrogen (3 bar) to the corresponding benzylamine, which is further condensed with a substituted phthalic acid mono‑ethyl ester to yield the final diamide. For active ingredient registration under EU Regulation 1107/2009, the five‑batch analysis data package must demonstrate a purity of ≥97.0% (qNMR, internal standard maleic acid, 99.9% traceable to NIST SRM), accompanied by the characterization of relevant impurities at or above the 0.1% threshold: these typically include the de‑chloro analog and the N‑methyl regioisomer originating from imperfect chemoselectivity during the pyrazole nitrogen alkylation step. The toxicological profile requires that the extractable nitrosamine N‑nitroso‑5‑(1H‑pyrazolo[3,4‑b]pyridin‑3‑yl)‑1H‑pyrrole‑3‑carboxylic acid is controlled to ≤0.03 ppm based on the TD₅₀ of 0.16 mg/kg/day extrapolated from the closely related N‑nitrosopyrrolidine congener, a limit that is achieved by sparging the reaction mixtures with nitrogen containing <50 ppb NOₓ and by using quenchers such as 0.01% w/w ascorbic acid in the work‑up quench tank. Published data for the field‑level residual limits of this specific diamide on leafy vegetables remain limited; however, the FAO Plant Production and Protection Paper 221 (Submission and Evaluation of Pesticide Residues Data) requires submission of supervised residue trials in triplicate localities across three climatic zones, the results of which will dictate the eventual Codex MRL for the unrefined agricultural commodity.

For applications in DNA‑encoded chemical library technology (DECL), 5‑(1H‑pyrazolo[3,4‑b]pyridin‑3‑yl)‑1H‑pyrrole‑3‑carboxylic acid is loaded onto a bifunctional oligonucleotide conjugate via amidation of the terminal hexaethylene glycol amino linker attached to a 5′‑phosphate‑modified 21‑mer DNA tag. The aqueous solubility of the sodium salt—approximately 8.2 mg/mL in 100 mM sodium borate buffer, pH 9.5—enables coupling without co‑solvent, minimising DNA duplex denaturation. A pilot library of 1,024 members was constructed using split‑and‑pool synthesis on a Tecan Freedom EVO liquid‑handling platform, where the acid was pre‑activated with EDC·HCl (200 equivalents relative to DNA) and sulfo‑NHS (100 equivalents) in 50 mM MES buffer, pH 6.0, at 22 °C for 45 minutes. Subsequent ligation‑based encoding (T4 DNA ligase, 0.5 U/µL, 16 °C, 16 h) and size‑exclusion purification (NAP‑10 columns, Sephadex G‑25) yielded the DECL assembly that was panned against recombinant FGFR3 kinase domain. Enrichment factors of 15–30-fold for pyrazolopyridine‑containing barcodes relative to negative controls validated the scaffold’s engagement, and the hit compounds were re‑synthesised off‑DNA on a 0.2 mmol scale using the T3P® protocol described above, achieving IC₅₀ values in the 12–85 nM range in a Caliper EZ Reader II mobility shift assay (ATP concentration Km). The work is performed under ISO 15189:2022‑accredited laboratory conditions for the biomolecular interaction data, and the oligonucleotide waste is decontaminated via 0.5 M sodium hydroxide treatment per NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (Appendix K).

The data in Table 1 were obtained by headspace GC (Agilent 7697A/7890B, DB‑624 30 m×0.25 mm×1.4 µm) operated in the split ratio 5:1 with an oven program from 40 °C (10 min) to 240 °C at 20 °C/min. Each lot was homogenised by mortar grinding and sampled in triplicate, and the reported values represent the mean of three injections per vial. The water content by Karl Fischer coulometry (Metrohm 851 Titrando) was 0.09–0.15% across all lots, within the ≤0.5% release criterion.

When solid‑phase peptide synthesis (SPPS) employs the pyrrole‑3‑carboxylic acid for backbone‑cyclised depsipeptide mimics

Attachment of the pyrrole‑3‑carboxylic acid as a β‑turn‑inducing residue in solid‑phase peptide synthesis requires anchoring through the carboxylic acid function onto a trityl chloride resin (TCP, loading 1.6 mmol/g) with 2.0 equivalents of the acid and 3.0 equivalents of DIEA in dry dichloromethane for 2 hours at ambient temperature. Unreacted trityl sites are capped by methanol (0.8 mL/g resin) to prevent deletion sequences, and the loading is quantified spectroscopically by cleaving an aliquot with 20% hexafluoroisopropanol in dichloromethane and measuring the cleaved acid against a calibrated HPLC standard. Chain elongation then proceeds with Fmoc‑protected amino acids activated by HBTU/0.5 M Oxyma‑Pure in DMF, while the pyrazolo[3,4‑b]pyridine nitrogen is temporarily protected as its N‑oxide during repetitive piperidine deblocking by oxidising with m‑CPBA (1.2 equivalents) prior to resin loading; the N‑oxide is reduced back to the parent heterocycle with zinc dust in acetic acid after full‑length chain assembly. The final cyclisation between the N‑terminus and the immobilized pyrrole‑3‑carboxyl anchor is carried out on‑resin using DPPA (1.5 equivalents) and sodium bicarbonate (3.0 equivalents) in DMF (resin‑bound linear peptide concentration 5 mM) for 24 hours, giving 55–70% cyclic monomer after global deprotection and reverse‑phase C18 purification (Phenomenex Luna, 10 µm, 250×21.2 mm, gradient 20–60% acetonitrile in 0.1% aqueous TFA). The final cyclic depsipeptide mimics are characterized by high‑resolution mass spectrometry (Q‑TOF, mass error <3 ppm) and circular dichroism spectroscopy to confirm the predicted type II′ β‑turn conformation. The entire SPPS workflow is performed under an inert atmosphere within fume hoods meeting ANSI/AIHA Z9.5‑2012 standards, and the acetonitrile‑containing eluates are distilled for recovery using a dedicated Büchi Rotavapor R‑300 system to reduce laboratory solvent inventory.