3-(4-Bromo-2,6-Difluorobenzyloxy)-5-[3-[4-(1-Pyrrolidinyl)Butyl]Ureido]Isothiazole-4-Carboxamide

Designated by the internal research identifier BDF-IU-4C, 3-(4-Bromo-2,6-difluorobenzyloxy)-5-[3-[4-(1-pyrrolidinyl)butyl]ureido]isothiazole-4-carboxamide constitutes a halogenated isothiazole carboxamide bearing a pyrrolidine-terminated butyl urea side chain. The monoisotopic mass is 531.04 Da, with a molecular formula of C₂₀H₂₄BrF₂N₅O₃S and a calculated exact mass of 531.07 g·mol⁻¹. The compound is synthesised through a convergent route involving O-alkylation of 3-hydroxyisothiazole-4-carboxamide with 4-bromo-2,6-difluorobenzyl bromide, followed by carbonyldiimidazole-mediated urea coupling with 1-(4-aminobutyl)pyrrolidine. Chromatographic purification on a Kromasil C18 column (10 μm, 250 × 50 mm) using a 0.1% trifluoroacetic acid–acetonitrile gradient delivered a final purity exceeding 99.5% by HPLC-UV at 254 nm. Differential scanning calorimetry at a ramp rate of 10 K·min⁻¹ under nitrogen gave a sharp endothermic melting event with an onset at 187.2 °C and peak at 188.6 °C, consistent with a single crystalline phase. The equilibrium solubility in phosphate-buffered saline (pH 7.4) at 25 °C was determined to be 12 ± 1 μM, while in neat DMSO the compound dissolved freely to at least 50 mM. The solid-state Fourier-transform infrared spectrum exhibits a strong urea carbonyl stretch at 1648 cm⁻¹ and an amide I band at 1672 cm⁻¹, confirming the intact carboxamide and urea functionalities.

In biochemical kinase profiling panels conducted against a diverse set of 468 wild-type and mutant kinases at an ATP concentration of 1 mM, BDF-IU-4C demonstrated a selectivity score S(10) of 0.048, indicating potent engagement with fewer than 5% of targets at 1 μM. The primary target identified was Bruton’s tyrosine kinase (BTK), with a dissociation constant K_d of 8.3 nM as measured by a LanthaScreen Eu kinase binding assay (Invitrogen protocol PV4563). The pyrrolidine butyl urea extension contributes critical hydrogen-bond contacts with the kinase hinge region residues Met477 and Glu475, while the 4-bromo-2,6-difluorobenzyloxy group occupies the hydrophobic back pocket adjacent to gatekeeper residue Thr474. Compared to the 4-chloro analog, the bromine substituent introduces an additional 0.17 nm van der Waals radius, altering the ligand‑residue contact map and reducing the enthalpy-driven binding entropy penalty by approximately 4.2 kJ·mol⁻¹, as inferred from isothermal titration calorimetry experiments in HEPES buffer (pH 7.5, 150 mM NaCl, 2 mM TCEP).

Purity Specifications and Analytical Certification

Batch release is governed by a multi-method analytical cascade aligned with the principles of ICH Q6A. Each lot is certified against the criteria listed; non-conforming material is re-crystallised from 2:8 ethyl acetate–heptane until compliance is achieved.

What Solvent Systems Maintain Structural Integrity During Long-Term Storage?

Lyophilised BDF-IU-4C stored in amber borosilicate vials under argon at −20 ± 5 °C retains >99% chromatographic purity over 24 months. In solution, degradation kinetics are strongly medium-dependent. A factorial stability study using a Waters ACQUITY UPLC H-Class system equipped with a BEH C18 1.7 μm column tracked hydrolysis of the 3‑benzyloxy ether and the urea bridge. In anhydrous DMSO stored over 4 Å molecular sieves at 25 °C, less than 0.3% degradation was observed after 28 days. Exposure to 10% v/v water in DMSO at 37 °C generated a primary degradant—identified by LC-HRMS as 5-amino-3-(4-bromo-2,6-difluorobenzyloxy)isothiazole-4-carboxamide—at a rate of 0.08% per day. The urea bond cleaves selectively under acidic conditions; at pH 3.0 (citrate buffer) and 40 °C, the hydrolysis half-life falls to 6.4 days, following pseudo-first-order kinetics with a rate constant k_obs = 1.25 × 10⁻⁶ s⁻¹. Consequently, dissolution for cell-based assays is recommended in 100% DMSO with protection from visible light, and dilution into aqueous medium must be performed immediately before dosing to limit pre-experiment precipitation or hydrolytic loss. The compound is incompatible with nucleophilic buffers containing free primary amines (Tris, glycine), which accelerate urea exchange at elevated temperature.

Mechanical sensitivity data obtained on a BAM Fallhammer apparatus according to the BAM procedure indicate no explosive decomposition upon impact energy inputs up to 40 J. Differential scanning calorimetry in a gold-plated, high-pressure crucible revealed a weak exotherm above 310 °C (ΔH ≈ −340 J·g⁻¹), attributed to thermal rearrangement of the isothiazole ring. Standard handling under local exhaust ventilation and antistatic footwear is adequate for quantities below 500 g.

When the 4‑Bromo Substituent Replaces Chlorine in BTK Inhibitor Scaffolds

Evaluating BDF-IU-4C alongside its 4‑chloro and 4‑fluoro congeners in a parallel artificial membrane permeability assay (PAMPA, pION PSR‑4p) and in MDCK‑MDR1 monolayer efflux studies revealed a structure‑permeability trade‑space driven principally by halogen polarisability. The 4‑bromo derivative exhibited an apparent permeability P_app (A→B) of 8.7 × 10⁻⁶ cm·s⁻¹ at pH 7.4, compared to 12.4 × 10⁻⁶ cm·s⁻¹ for the 4‑chloro analog and 16.9 × 10⁻⁶ cm·s⁻¹ for the 4‑fluoro scaffold. Efflux ratios determined in the presence of the P‑glycoprotein inhibitor zosuquidar (2 μM) remained below 2.0 for all three analogs, ruling out significant P‑gp‑mediated transport, yet the higher molecular volume of the brominated species (+14 cm³·mol⁻¹ over the chloro) reduces passive transcellular flux in a predictable manner consistent with the extended Overton correlation for halogenated aromatics.

The nano‐molar BTK engagement of BDF-IU-4C translates into measurable functional activity in primary human B‑cell assays. In anti‑IgM‑stimulated CD19⁺ B cells from six healthy donors, pre‑incubation with 0.1 μM of the compound for 30 min reduced phosphorylation of PLCγ2 at Tyr759 by 82 ± 6% relative to vehicle control, as quantified by intracellular phospho‑flow cytometry using a BD LSRFortessa X‑20 flow cytometer. The IC₅₀ for CD69 surface upregulation inhibition was 72 nM, while the half‑maximal cytotoxic concentration in the unstimulated Ramos cell line remained above 30 μM, yielding a selectivity window exceeding 400‑fold. Unlike the 4‑chloro analog, the 4‑bromo derivative showed 3.2‑fold weaker displacement of the hERG potassium channel ligand [³H]‑dofetilide in a membrane‑binding assay (ChanTest), lowering the projected QTc liability at therapeutic concentrations. However, the higher lipophilicity of BDF-IU-4C correlates with a more pronounced CYP induction signature in cryopreserved human hepatocytes; after 72 h incubation with 10 μM test article, CYP1A2 and CYP2B6 mRNA levels increased by 3.1‑fold and 2.5‑fold, respectively, as determined by branched DNA quantification (QuantiGene Plex 2.0, Thermo Fisher). These data place the compound at an intersection of improved target affinity and manageable off‑target risk that merits further exploration in rodent models of collagen‑induced arthritis.

Dispersion into amorphous solid dispersion (ASD) matrices has been examined as a strategy to circumvent the low aqueous solubility for oral toxicology studies. Spray‑dried dispersions containing 25% w/w BDF-IU-4C in HPMCAS‑MG (AQOAT, Shin‑Etsu) produced a single glass transition at 104 °C (DSC, modulated mode, ±0.5 °C/min) with no evidence of phase separation by scanning electron microscopy. In fasted‑state simulated intestinal fluid (FaSSIF‑V2, pH 6.5), the ASD maintained a supersaturation factor of 14 over the crystalline equilibrium solubility for at least 120 min, as monitored by a μDiss Profiler (Pion Inc.) with in‑situ fibre‑optic UV probes. The dissolution profile was non‑sink with a peak concentration of 113 μg·mL⁻¹ at 45 min, followed by a gradual decline consistent with nucleation‑driven recrystallisation; the addition of 0.01% w/v Poloxamer 188 to the medium extended the metastable window beyond 180 min. These findings define a workable parenteral and oral formulation space, though the limited oral bioavailability predicted from the PAMPA and CYP induction data indicates that co‑administration with a CYP inhibitor or the use of lipid‑based self‑emulsifying systems would be necessary to achieve systemic exposures sufficient for in vivo efficacy readouts.

Handling precautions derive from the primary toxicity screening. In a mini‑Ames test (Xenometrix, ISO 10993‑3), BDF-IU-4C was negative at concentrations up to 500 μg·plate⁻¹ with and without S9 metabolic activation. The compound does not meet the criteria for classification as a respiratory sensitiser under the Globally Harmonized System (GHS) based on a negative in silico prediction (Derek Nexus 6.2.0) and a maximisation test read‑across from a structurally similar, non‑brominated scaffold. Nevertheless, standard PPE including nitrile gloves tested to EN 374‑3 and safety glasses compliant with EN 166 are mandated during weighing, and all operations generating airborne dust must be conducted inside a fume hood with an average face velocity of 0.5 m·s⁻¹ or greater.