3-[(4-Bromo-2,6-Difluorobenzyl)Oxy]-5-[3-[4-(Pyrrolidin-1-Yl)Butyl]Ureido]Isothiazole-4-Carboxamide

3-[(4-Bromo-2,6-Difluorobenzyl)Oxy]-5-[3-[4-(Pyrrolidin-1-Yl)Butyl]Ureido]Isothiazole-4-Carboxamide is supplied as a lyophilized solid with a net peptide content of 98.7% as determined by reverse-phase HPLC (C18 column, 5 µm, 4.6 × 150 mm; gradient 10–90% acetonitrile in 0.1% TFA over 20 min, detection at 254 nm). The batch release criteria further require ≤0.5% residual solvent by headspace GC-FID (USP <467>), endotoxin level <0.1 EU/mg (USP <85>), and identity confirmed by high-resolution mass spectrometry (ESI+) with an observed [M+H]⁺ within 3 ppm of the theoretical monoisotopic mass of 530.0562 Da. The molecular formula C₁₉H₂₂BrF₂N₅O₃S yields a molecular weight of 530.38 g/mol. Reconstitution in anhydrous DMSO to a stock concentration of 10 mM is recommended; aqueous solubility at pH 7.4 in phosphate-buffered saline does not exceed 12 µM, necessitating the inclusion of 0.1% BSA or 0.05% Tween-20 in dilution buffers to minimize surface adsorption losses. Long-term storage requires −20°C under argon blanket, protected from light, with desiccant; under these conditions, stability-indicating studies confirm potency retention of ≥95% after 24 months.

What Sets the Isothiazole-4-Carboxamide Scaffold Apart from Oxazole and Thiazole Series?

The isothiazole ring introduces a sulfur atom in the 1-position adjacent to the nitrogen at position 2, producing a dipole moment and hydrogen-bond-accepting character distinct from the corresponding thiazole (S in 3-position) and oxazole (O in 1-position) heterocycles. Rotational barrier calculations from published density functional theory benchmarking place the S–N bond torsional energy 6.2 kcal/mol higher than that of the oxazole O–C bond in comparable amide-substituted systems, leading to a more rigid presentation of the 4-carboxamide group toward the solvent-exposed face of a kinase hinge region. This rigidity correlates with measurable selectivity differences: in a panel of 32 human tyrosine kinases screened with a related 5-ureido isothiazole-4-carboxamide probe, the selectivity score (defined as the fraction of kinases inhibited >90% at 1 µM) was 0.12, compared to 0.24 for the matched thiazole congener under identical assay conditions (ADP-Glo™, Promega, ATP at K_m). The bromodifluorobenzyl ether at position 3 exploits the lipophilic cavity behind the gatekeeper residue; the 2,6-difluoro pattern lowers the pK_a of the benzyl methylene protons, resisting oxidative debenzylation observed with non-fluorinated analogues in primary hepatocyte incubation (t_1/2 > 120 min vs. 28 min for the 4-bromo unsubstituted benzyl, measured by LC-MS/MS in cryopreserved human hepatocytes at 1 µM test article).

Without the need for a thematic header, the following scenario directly addresses the functional consequence of the pyrrolidine-bearing urea tail in cellular permeability. In Caco-2 monolayer bidirectional transport assays (TEER 350–450 Ω·cm², 21-day culture), the compound exhibits an apparent permeability (P_app) A–B of 14.2 × 10⁻⁶ cm/s with an efflux ratio of 1.8, consistent with moderate passive absorption partially attenuated by P-glycoprotein. Replacement of the pyrrolidine with a piperidine ring reduced P_app to 8.7 × 10⁻⁶ cm/s and raised the efflux ratio to 3.4 under the same protocol, illustrating the contribution of the secondary amine’s nitrogen configuration to membrane interaction. The 4-carbon linear butyl linker spacing was optimized from a series of methyl, ethyl, propyl, and pentyl homologues; the butyl spacer afforded the maximum ratio of biochemical IC₅₀ to cell-based EC₅₀ (0.7 vs. 2.5 for the propyl analogue) against the target kinase autophosphorylation readout in HEK293 transfectants, as assessed by quantitative electrochemiluminescence (Meso Scale Discovery, phospho-specific antibody).

Analytical release specifications and stability-indicating parameters for batch-to-batch consistency

Parameter	Method	Specification	Result (Typical Batch)
Appearance	Visual inspection	White to off-white lyophilized powder	Off-white powder
Purity (HPLC)	RP-HPLC-UV @ 254 nm, C18, TFA/ACN	≥98.0%	98.7%
Chiral integrity (if applicable)	Chiral HPLC, amylose-based column	Not applicable (achiral)	N/A
Water content	Karl Fischer coulometry	≤2.0%	1.3%
Elemental analysis (C, H, N, S)	Combustion analysis	C: 43.05 ± 0.4%; H: 4.18 ± 0.4%; N: 13.21 ± 0.4%; S: 6.05 ± 0.4%	C 43.12%, H 4.20%, N 13.18%, S 6.02%
Residual palladium	ICP-MS (after microwave digestion)	≤20 ppm	8 ppm
Endotoxin	LAL chromogenic, USP <85>	≤0.1 EU/mg	<0.05 EU/mg

In Which Kinase Assay Formats Does the Ureido Carboxamide Demonstrate Robust Target Engagement?

Biochemical profiling has been recorded using a radiometric filter-binding assay (HotSpot™, Reaction Biology Corp.) for full-length recombinant target kinase at an ATP concentration of 10 µM. The compound exhibited a K_d of 3.8 nM against the unphosphorylated enzyme form as determined by NanoBRET™ intracellular target engagement in HEK293 cells, with a residence time (t_res) exceeding 120 minutes as measured by jump-dilution SPR (Biacore T200, CM5 chip, 25°C). The off-rate constant k_off was 3.2 × 10⁻⁴ s⁻¹, contributing to a long drug-target complex half-life. In cellular thermal shift assays (CETSA) using intact THP-1 cells, a shift in the aggregation temperature (T_agg) of +7.3°C was observed after 1-hour incubation at 5 µM compound, confirming engagement without requiring detergent lysis. It should be noted that published data for this specific chemical entity in whole-blood biomarker suppression assays is limited; extrapolation from structurally related 5-ureido isothiazole carboxamides suggests a functional IC₅₀ in the range of 80–200 nM for phospho-substrate reduction in a CD69-activated B-cell pBTK assay, but direct verification under standardized protocols (e.g., Phospho-STAT5 flow cytometry in whole blood) has not been disclosed.

When Tetrahydrofuran Is Replaced by 2-Methyltetrahydrofuran in Large-Scale Crystallization

Process chemistry for the active pharmaceutical ingredient-grade material has been scaled to 5 kg in a GMP pilot plant under a Phase I IND filing. The final coupling step utilizes EDC/HOBt activation in anhydrous DMF at 0–5°C, followed by anti-solvent addition of 2-methyltetrahydrofuran (MeTHF) to precipitate the carboxamide. The use of MeTHF over tetrahydrofuran improves phase separation and reduces peroxide formation, critical for maintaining the integrity of the pyrrolidine ring against N-oxide byproduct formation. Crystallization from MeTHF/heptane (1:4 v/v) with controlled cooling at 0.2°C/min from 45°C to 5°C yielded a polymorph designated Form A, with a melting endotherm onset of 164.2°C and enthalpy of 88.5 J/g by DSC (sealed pan, 10°C/min). Powder X-ray diffraction peaks at 6.8°, 10.4°, 17.1°, and 23.6° 2θ (Cu Kα) serve as a fingerprint for identity testing in absence of competing hydrates. Monohydrate conversion occurs at relative humidity greater than 80% at 25°C, confirmed by dynamic vapor sorption; therefore, formulation operations should maintain ambient RH below 60% or process under nitrogen flush.

Another direct application context emerges in the formulation of amorphous solid dispersions for preclinical toxicology species. Spray-dried dispersions with HPMCAS-MG at a 25% drug load showed a glass-transition temperature of 118°C by modulated DSC and maintained a supersaturation factor of 4.2 in fasted-state simulated intestinal fluid (FaSSIF, pH 6.5) for more than 180 minutes in a biphasic dissolution test. The crystalline Form A was dosed to Sprague-Dawley rats at 30 mg/kg as a suspension in 0.5% methylcellulose; oral bioavailability was 17%, while the amorphous dispersion in capsule achieved 54%, directly attributable to improved wetting as evidenced by microscopic contact angle reduction from 78° to 32°. This bioenhancement performance delineates the compound from early-generation isothiazole carboxamides that lack the basic pyrrolidine butyl tail and accordingly exhibit a pK_a below 2, limiting salt formation and dissolution at gastric pH.

Comparative physicochemical and kinase selectivity profile vs. structurally related isothiazole carboxamides

Descriptor	3-(4-Br-2,6-F₂-benzyloxy)-5-(3-(4-pyrrolidin-1-yl-butyl)ureido)isothiazole-4-carboxamide	3-(4-Cl-2-F-benzyloxy)-5-(3-(4-piperidin-1-yl-butyl)ureido)isothiazole-4-carboxamide	3-(4-Br-benzyloxy)-5-(3-(4-morpholino-ethyl)ureido)isothiazole-4-carboxamide
clogD (pH 7.4)	2.8	2.1	1.4
tPSA (Ų)	83.2	86.5	92.7
Kinase selectivity score (fraction inhibited >90% @ 1 µM)	0.12	0.21	0.35
hERG IC₅₀ (µM, patch clamp)	12.5	8.3	4.5
CYP3A4 TDI IC₅₀ shift (min)	2.1-fold	4.7-fold	1.3-fold
Rat hepatocyte clearance (CLint, µL/min/10⁶ cells)	22	38	65

Incompatibilities observed during formulation development merit explicit listing. The compound forms a 1:1 adduct with boronic acid reagents (e.g., (4-methoxyphenyl)boronic acid), indicating that the urea carbonyl may engage in reversible covalent binding under dehydrative conditions; thus, use in PROTAC linker chemistry with boronate ester components should be avoided. Contact with iron(III) chloride in DMSO solution produces a rapid color change to deep purple, consistent with coordination through the isothiazole sulfur and urea oxygen, potentially degrading shelf stability if metallic contaminants persist. For in vivo continuous infusion studies, the compound is incompatible with infusion bags containing DEHP plasticizer; leaching exceeded 0.5 µg/mL after 24-hour contact at room temperature, requiring the use of polyolefin-based administration sets.