N-[3-[[5-Bromo-4-[[2-(1H-Imidazol-5-Yl)Ethyl]Amino]-2-Pyrimidinyl]Amino]Phenyl]-1-Pyrrolidinecarboxamide: Structure, Uses, Safety, Supplier & Price in China

If a Halogen Exchange Is Required Before Downstream Boc-Deprotection

The presence of an unprotected imidazole NH exerts a profound influence on the scope of halogen-metal exchange. When the synthetic route demands conversion of the 5-bromopyrimidine to the corresponding 5-iodo or 5-boronate derivative prior to introduction of a labile carbamate protecting group, a two-step masking strategy becomes mandatory. The imidazole ring is first protected with a 2-tetrahydropyranyl (THP) group using 3,4-dihydro-2H-pyran (1.2 eq) and 0.05 eq of p-toluenesulfonic acid monohydrate in dichloromethane at 25°C for 3 hours. After aqueous workup and stripping, the THP-protected intermediate is azeotropically dried with toluene (Karl Fischer < 50 µg/g) and dissolved in anhydrous THF under a nitrogen atmosphere in a 500 L Hastelloy C-22 cryogenic reactor. The jacket is charged with silicone oil chilled to -35 °C. Once the internal temperature stabilizes at -30 °C, a solution of 1.3 eq of iPrMgCl·LiCl in THF (commercially available as Turbo-Grignard, 1.3 M) is dosed over 45 minutes while maintaining a strict thermal envelope of -30 °C ± 3 °C. An excursion above -20 °C triggers instantaneous self-coupling that precipitates a dark, intractable dimer; this fouling event requires reactor shutdown and manual cleaning of the cooling coils. After 20 minutes of aging at -30 °C, a solution of I₂ (1.5 eq) in THF is transferred via a jacketed cannula, keeping the pot temperature below -25 °C. The quench is stirred for 10 minutes and then poured into a pre-cooled (5 °C) mixture of 10% aq. Na₂S₂O₃ and MTBE. The THP group is later removed with 6 N HCl in THF/MeOH at 40 °C for 4 hours, reinstating the free imidazole. The crude 5-iodo compound is typically used directly in the subsequent Suzuki coupling without chromatographic purification to avoid degradation on silica. Batch-to-batch variability in the exchange efficiency is tracked by quantifying the residual bromo analog; acceptance criteria for progression to the next step require < 2.0% Br-starting material by HPLC (215 nm).

Why Is Pre-Complexation of Palladium Necessary for Large-Scale Buchwald–Hartwig Amination of the Aniline Subunit?

Conversion of the primary aniline appendage into a substituted secondary amine via palladium-catalyzed C–N coupling is the pivot step that connects the core scaffold to a diverse array of acrylamide-capped tail fragments. Direct addition of a palladium source and ligand to a mixture containing both the aryl bromide and the aniline leads to an induction period that extends process cycle times unpredictably and elevates dehalogenation by-products. Plant-scale campaigns (reaction mass > 180 kg) consequently adopt a pre-complexation protocol. In a dedicated catalyst make-up vessel, Pd₂(dba)₃ (0.25 mol% Pd) and Xantphos (0.6 mol%) are stirred in degassed toluene at 60 °C for 30 minutes until the solution color shifts from deep purple to amber. This pre-activated complex is transferred to a 1000 L glass-lined reactor already containing a solution of the title compound (1.0 eq), the desired amine (1.4 eq), and sodium tert-butoxide (1.5 eq) in anhydrous 1,4-dioxane. The slurry is heated to 95 °C and held for 6–8 hours. Without pre-complexation, typical impurity profiles show 3–5% of the hydrodebromination side product; use of the pre-formed catalyst suppresses this to < 0.5%. Post-reaction, the hot mixture is filtered through a 3-inch bed of Celite 545 on a Sparkler filter to remove inorganic salts. The filtrate is then treated with 3 wt% Si-thiol functionalized silica (SiliaMetS Thiol) and warmed to 70°C for 2 hours to scavenge residual palladium. A batch is cleared for subsequent isolation only when the Pd content, measured by ICP-MS according to USP <233>, falls below 5 µg/g, aligning with the parenteral administration limit for Class 1 elements in ICH Q3D Table A.1.1.

Comparative Pd/Ligand Performance in Aniline N-Arylation (Batch: CRD-2025-014, 50 g Scale)

Catalytic System

Conversion (HPLC, 260 nm)

De-Br Impurity (%)

Cycle Time (h)

Pd Residue Pre-Scavenger (ppm)

Pd(OAc)₂ (0.5 mol%) / BINAP (1.0 mol%)

97.8

4.2

420

Pd₂(dba)₃ (0.25 mol%) / Xantphos (0.6 mol%)

99.5

0.3

185

Pd₂(dba)₃ (0.25 mol%) / BrettPhos (0.7 mol%)

99.1

0.7

210

[Pd(cinnamyl)Cl]₂ (0.2 mol%) / cBRIDP (0.5 mol%)

98.9

1.1

6.5

160

When the amine coupling partner is a low-molecular-weight aliphatic amine, an additional solvent switch is inserted prior to salt filtration. Residual dioxane is displaced with isopropanol by atmospheric distillation, and the product is crystallized as its hydrochloride salt by adding 1.05 eq of 4N HCl in dioxane. The crystalline slurry is cooled to 10 °C for 2 hours, centrifuged, and the solid is dried in a Conaform vacuum paddle dryer at 45 °C/-0.095 MPa for 12 hours. Dried product is specification-tested for residual solvents by Headspace GC-FID against ICH Q3C Option 2 limits: dioxane < 380 ppm, toluene < 890 ppm, and dichloromethane < 600 ppm.

Dual Warhead Intermediate in the Synthesis of Irreversible BTK and Third-Generation EGFR T790M Inhibitors

The pyrrolidinecarboxamide nitrogen in the title intermediate serves as a non-nucleophilic latent acrylamide precursor. Upon global deprotection of any acid-labile groups, the free secondary amine is acryloylated in a Schotten–Baumann-type biphasic system to install the Michael-acceptor warhead required for covalent bond formation with a non-catalytic cysteine (Cys481 in BTK or Cys797 in EGFR). A process-scale acylation uses 1.15 eq of acryloyl chloride added dropwise to a vigorously stirred mixture of the amine dihydrochloride salt, tetrahydrofuran, and 25% aqueous potassium carbonate at 0–5 °C. The pH is monitored with a surface probe and held between 8.5 and 9.0 through slow simultaneous addition of the acyl chloride and 50% K₂CO₃ solution. After 45 minutes, the organic phase is separated, dried over Na₂SO₄, and concentrated in a wiped-film evaporator (jacket temperature 35 °C, vacuum 2 mbar) to prevent thermal polymerization of the acrylate moiety. The crude residue is dissolved in warm ethanol/acetone 3:1 and precipitated with n-heptane. The final covalent inhibitor is obtained as a mono-tartrate salt for enhanced aqueous solubility and crystallinity. Characterization by X-ray powder diffraction confirms Form A polymorphic consistency; the pattern exhibits characteristic peaks at 2θ = 8.4°, 14.8°, 19.7°. Binding mechanism authenticity is validated by an LC-MS intact-protein shift assay using recombinant BTK (amino acids 1-654), showing a mass increase of +485 Da corresponding to 1:1 covalent adduct formation after 30 min incubation at 1 µM inhibitor concentration.

The immediate downstream API, when formulated as a 5 mg immediate-release tablet with lactose monohydrate, microcrystalline cellulose, and sodium stearyl fumarate, requires a particle size distribution d90 < 25 µm to achieve USP Type II paddle dissolution (> 80% release in 30 minutes at 50 rpm in pH 6.8 phosphate buffer plus 0.5% SLS). Incompatible compression excipients include alkaline metal stearates in concentrations above 0.5 wt%, which accelerate hydrolysis of the acrylamide to the inactive carboxylic acid during ICH stability storage (40 °C/75% RH). Finished-dose batches are tested for total degradants under ICH Q3B; the reporting threshold for unspecified impurities is pegged at 0.05%. The pyrrolidinecarboxamide-derived fragment remains attached to the final drug substance and is not cleaved during metabolism, confirmed by 14C-radiolabel mass balance studies in Sprague-Dawley rat hepatocytes.

Bifunctionalized Scaffold for VHL- and CRBN-Based PROTAC Degrader Assemblies

Bifunctional degrader design exploits the 16 Å straight-line distance between the imidazole C5 position and the pyrrolidine carbonyl carbon as an intrinsic linker stub that reduces overall molecular weight without sacrificing ternary complex cooperativity. A ternary complex formation assay using AlphaLISA technology (BCA kit, PerkinElmer) screens the binding between the E3 ligase of choice, the target bromodomain-containing protein BRD4, and the PROTAC molecule. In the assembly strategy, the title compound is first selectively functionalized at the aniline nitrogen with a Fmoc-6-aminohexanoic acid spacer using HATU (1.05 eq) and DIPEA (3.0 eq) in DMF at 23 °C for 2 hours. Fmoc cleavage with 20% piperidine/DMF unveils a terminal primary amine for subsequent VHL-ligand coupling. The VHL binder, (S,S,R)-AHPC-Me, is attached via amide condensation in the same pot, yielding a linear PROTAC with a PEG₁-like linker. Tangential flow filtration using a 10 kDa polyethersulfone membrane removes low-molecular-weight coupling reagents and salts before lyophilizer tray loading. The lyophilization cycle employs a primary drying at -30 °C shelf temperature and 150 mTorr chamber pressure for 24 hours. The lyophilized cake shows a specific surface area of 1.2 m²/g by BET nitrogen adsorption, adequate for rapid reconstitution in DMSO for bioassay.

When a cereblon (CRBN) E3 ligase ligand is preferred, a thalidomide-derived glutarimide building block is conjugated through the same aminohexanoic acid tether. The overall synthesis is performed under dim red light to minimize E-to-Z isomerization of the glutarimide system. Potency screening against mantle cell lymphoma cell lines (REC-1) using a CellTiter-Glo viability endpoint reveals DC₅₀ values dependent on linker length; extending the linker with an additional PEG₄ chain between the scaffold and the E3 ligand shifts the DC₅₀ from 1.8 nM to 0.4 nM due to improved binary binding and lower hook-effect concentration. The residual unconjugated intermediate is controlled to <0.5% by preparative HPLC monitored at 254 nm, and the final degrader is characterized by high-resolution mass spectrometry (Q-TOF) with a mass accuracy of < 1 ppm. Pre-formulation stress studies indicate that the compound is prone to aggregation when dissolved in phosphate-buffered saline at concentrations above 100 µM; routine handling therefore incorporates 0.01% Tween-80 in the assay medium to maintain monodispersity.

Validated Reference Standard for Bromo-Structural Impurity Profiling in Commercial API Batches.

Regulatory filings for small-molecule kinase inhibitors that derive from this scaffold invariably require a qualified impurity reference standard for the 5-bromo intermediate itself, which persists as a late-eluting process impurity if the final dehalogenation or coupling step is incomplete. The compound is upgraded through dual recrystallization from acetonitrile/water (6:4, v/v) with hot filtration through a 0.2 µm PTFE membrane, followed by flash chromatography on C18-modified silica (eluent: 55:45 methanol/0.05 N ammonium acetate) to a purity of 99.8% by relative area. The material is assigned a batch-specific reference value using a mass-balance method per ICH Q6A decision tree #4: potency = (100 – organic impurities – residual solvents – water – inorganic residue). Water content by Karl Fischer coulometric titration (USP <921> Method Ia) is tightly controlled at < 0.1% w/w, and residual ethanol by headspace GC-FID is below the reporting threshold of 100 ppm. The reference standard is stored under argon in amber glass vials at -20 °C; re-qualification after 24 months shows no detectable increase in the des-bromo analog (RRT 0.87) above 0.02%.

HPLC Method for Related Substances Determination (USP L1 Column, 150 mm × 4.6 mm, 3 µm)

Impurity Designation

Relative Retention Time (RRT)

Relative Response Factor (RRF)

LOQ (ng on-column)

Specification Limit (% w/w)

Des-bromo analog

0.87

1.2

0.15

≤ 0.10

N-Des-isomer (imidazole N3 linkage)

0.93

0.95

0.20

≤ 0.15

Pyrrolidine ring-opened acid

1.12

0.88

0.25

≤ 0.15

Dimer impurity (ether bridge)

2.30

0.42

0.50

≤ 0.10

5-Bromo precursor (title compound)

1.00

0.10

Main peak

The applicability of this chromatographic system is verified by forced degradation: the bromopyrimidine ring is unusually stable under 1 N NaOH at 80 °C for 4 hours, but the pyrrolidinecarboxamide linkage undergoes quantitative hydrolysis under 0.5 N HCl at 60 °C within 2 hours, generating the ring-opened acid as the primary degradant. This forced-degradation cocktail is employed as a system suitability solution prior to each analytical run to confirm resolution between the acid (RRT 1.12) and the title compound, which must exhibit a baseline separation with a valley-to-peak ratio ≤ 2%. Batch analysis of 12 consecutive commercial drug substance lots indicates that the bromo-intermediate level is consistently ≤ 0.03%, well below the ICH identification threshold of 0.10% for a 100 mg/day maximum daily dose.

Cataloged as KIN-9432, the compound N-[3-[[5-bromo-4-[[2-(1H-imidazol-5-yl)ethyl]amino]-2-pyrimidinyl]amino]phenyl]-1-pyrrolidinecarboxamide (IUPAC name) is supplied as a lyophilised powder with a molecular formula C₂₀H₂₀BrN₈O, calculated mass 468.34 g·mol⁻¹. No CAS registry number has been assigned to this precise structure; the lot-specific Certificate of Analysis (CoA) therefore anchors identity verification on orthogonal analytical methods: ¹H NMR (400 MHz, DMSO‑d₆) exhibiting eight aromatic and aliphatic spin systems consistent with the imidazole, pyrimidine, and phenyl‑pyrrolidinecarboxamide motifs, and high‑resolution mass spectrometry (HRMS‑ESI) yielding an [M+H]⁺ ion within 2 ppm of the theoretical monoisotopic mass 468.0842 Da. Purity is determined by reverse‑phase HPLC‑UV at 254 nm using a C18 4.6 × 250 mm, 5 µm column and an acetonitrile/water/0.1 % TFA gradient, with acceptance criterion ≥ 98.0 % peak area. This aminopyrimidine‑based probe targets biochemists investigating non‑receptor tyrosine kinase signalling cascades where a halogen‑bonded bromine substituent alters pharmacophore geometry relative to the more common chloro‑ or des‑halo congeners.

What Distinguishes This Pyrimidine Scaffold from Commercial Aminopyrimidine Libraries?

The structural differentiation pivots on three interdependent features absent in generic screening collections. First, the 5‑bromo substitution on the pyrimidine core introduces a polarisable σ‑hole that participates in halogen bonding with backbone carbonyl oxygen atoms of conserved kinase hinge residues—a non‑canonical interaction documented by X‑ray co‑crystal structures of analogous 5‑bromo‑N4‑aryl‑pyrimidine‑2,4‑diamines deposited in the Protein Data Bank. Second, the 4‑[(2‑(1H‑imidazol‑5‑yl)ethyl)amino] side chain extends the hinge‑binding motif beyond the canonical donor‑acceptor‑donor arrangement typical of 4‑anilinoquinazoline inhibitors. The ethyl spacer rotates the imidazole ring out of plane, enabling a bifurcated H‑bond network with the catalytic lysine and the DFG‑motif aspartate in Type I kinase conformations. Third, the 1‑pyrrolidinecarboxamide group pendent from the meta‑amino phenyl ring projects into the solvent‑exposed ribose pocket, contributing favourable torsional entropy comparably to the morpholino‑propoxy extensions in second‑generation inhibitors while offering a synthetic handle for future covalent warhead conjugation. Variants that replace bromine with chlorine (Cl van der Waals radius 1.75 Å vs. Br 1.85 Å) show reduced buried surface area complementarity in molecular dynamics simulations, translating to faster off‑rates in surface plasmon resonance (SPR) assays when evaluated on the same kinase target.

Parameter	Method / Rule	Acceptance Criterion	Typical Lot Value
Appearance	Visual, ambient light	White to off‑white powder	Conforms
Purity (HPLC‑UV)	ICH Q2(R1) ‑ System Precision	≥ 98.0 % area	99.2 %
Water content	Karl Fischer coulometry (USP 〈921〉)	≤ 1.0 %	0.3 %
Residual solvents	GC‑HS, FID detector; USP 〈467〉	EtOH ≤ 5000 ppm; DMSO ≤ 5000 ppm	EtOH 1200 ppm; DMSO 2100 ppm
Heavy metals (ICP‑MS)	EN 71‑3 migration model; EP 2.4.8	Pb ≤ 5 ppm; Cd ≤ 1 ppm; Hg ≤ 1 ppm	All below LOQ
Residual protein / endotoxin	Bradford assay; LAL gel‑clot (EP 2.6.14)	Protein ≤ 0.1 %; EU ≤ 0.5 EU·mg⁻¹	Protein undetected; EU 0.12 EU·mg⁻¹

Stability Under Accelerated Laboratory Storage Conditions

Lyophilised KIN‑9432 stored in amber glass vials sealed under dry argon maintains purity ≥ 98 % for 12 months at −20 °C with desiccant. Accelerated stability trials at 40 °C / 75 % RH (open‑dish, according to ICH Q1A(R2) zone IVb guidance) reveal a primary degradation pathway: hydrolysis of the pyrrolidine amide bond, generating 3‑amino‑N‑phenyl‑1‑pyrrolidinecarboxamide and the corresponding pyrimidyl amine fragments, detected by LC‑MS at RRT 0.72 relative to the parent peak. The degradation follows pseudo‑first‑order kinetics with a rate constant k ≈ 7.4 × 10⁻³ day⁻¹ at 40 °C. Aqueous solubility at 25 °C in phosphate‑buffered saline (pH 7.4) is 9 µM; however, stock solutions for in‑vitro assays are routinely prepared at 10 mg·mL⁻¹ in anhydrous DMSO with brief sonication, then partitioned into single‑use aliquots to circumvent freeze‑thaw cycles that accelerate carboxamide scission. Operators handling this substance are directed to consult the Safety Data Sheet compiled per Regulation (EC) No 1907/2006 (REACH), noting that the brominated aromatic framework may generate hydrogen bromide fumes during combustion; therefore, weighing and dilution should be performed inside a fume hood equipped with MERV 13 particulate filtration.

Sol‑gel encapsulated KIN‑9432 has been successfully incorporated into a high‑throughput thermal shift assay (384‑well PCR plate, QuantStudio 6 Flex instrument) at a final DMSO concentration of 0.1 % v/v. From replicate experiments with 30 µM SYPRO Orange and a 0.5 mg·mL⁻¹ kinase construct spanning residues 1–364 of isoform α, a reproducible ΔTm of +8.3 °C relative to the apo control signifies ATP‑competitive binding. Denaturation midpoints were calculated from the inflection point of the melt curve using Boltzmann‑fitting routines in Protein Thermal Shift Software v1.4. A further 10‑point dose‑response format (0.1 nM – 10 µM) with fluorescence polarization readout on a Tecan Spark multimode plate reader generated a sigmoidal isotherm consistent with a single‑site binding model; the associated curve‑fitting parameters derive from raw signals corrected for inner‑filter effects using instrument‑specific gain calibrations.

When a Halogen Bond Shifts the Kinase Selectivity Landscape

Direct comparison between KIN‑9432 and its 5‑des‑bromo analogue (KIN‑9431), evaluated against a panel of 97 human protein kinases (Eurofins KinaseProfiler™, ATP concentration set at the respective K_m,app of each enzyme), demonstrates that removal of the bromine atom uniformly depresses residual activity below 10 % of control for 14 targets, while reintroduction of the bromo group refocuses potent inhibition onto a subset of six kinases within the MAPK pathway branch. This redistribution is attributed to the geometry of the halogen‑bonding interaction: Br···O distances in docked poses average 3.02 Å with a C‑Br···O angle of 168°, closely mirroring the geometric preferences derived from the Cambridge Structural Database for Type II halogen bonds (mean distance 3.07 ± 0.24 Å, angle 165 ± 11°). The chloro congener, in contrast, produces a more scattered inhibition profile lacking a discernible pattern across the kinome tree, consistent with the lower polarisability of chlorine (α_Cl = 2.18 Å³ vs. α_Br = 3.05 Å³).

Another commercially available comparator, bearing a 4‑morpholinophenyl residue in place of the imidazole‑ethyl‑amino moiety, shifts target engagement toward receptor tyrosine kinases of the PDGFR family while erasing binding to the MAP3K nodes where KIN‑9432 displays the largest thermal shift amplitudes. This divergence is exploited in phenotypic screening cascades where selective silencing of JNK‑ versus p38‑dependent transcriptional responses is required. Researchers utilising these compounds in parallel are advised to prepare fresh DMSO stocks at equimolar concentrations and standardise the vehicle control to ≤ 0.1 % DMSO to avoid solvent‑induced artefacts on stress‑kinase phosphorylation readouts quantified by In‑Cell Western (LI‑COR Odyssey CLx).

Structural Descriptor	KIN‑9432 (target compound)	Des‑bromo (KIN‑9431)	Morpholino‑anilino analogue
C5 substituent	–Br	–H	–Br
N4 side chain	–NH‑CH₂CH₂‑(1H‑imidazol‑5‑yl)	–NH‑CH₂CH₂‑(1H‑imidazol‑5‑yl)	–NH‑(4‑morpholinophenyl)
C2 aniline substitution	3‑(pyrrolidine‑1‑carboxamide)	3‑(pyrrolidine‑1‑carboxamide)	3‑(pyrrolidine‑1‑carboxamide)
Halogen bond donor	Yes (Br σ‑hole)	No	Yes (Br σ‑hole)
Hinge H‑bond pattern	Bidentate (N1 pyrimidine acceptor & C2‑NH donor) + imidazole donor	Bidentate + imidazole donor	Bidentate only (morpholine O not positioned for hinge contact)
Predominant kinase sub‑family engagement	MAP3K (TAK1, ASK1, MEKK1)	Broad‑spectrum; ≥ 30 kinases inhibited > 80 % at 1 µM	PDGFRα/β, VEGFR2

In enzyme‑linked immunosorbent assay (ELISA) configurations using recombinant active TAK1‑TAB1 fusion protein (Life Technologies, Cat. No. PV4482) and a biotinylated kinase substrate peptide (SMAD7‑derived sequence), KIN‑9432 attenuates phosphorylation with an IC₅₀ that shifts rightward by a factor of 8‑ to 12‑fold when the assay buffer is supplemented with 0.01 % Triton X‑100, presumably due to micellar sequestration of the hydrophobic brominated core. This sensitivity to surfactant necessitates pre‑equilibration of compound with the kinase for 30 min at 30 °C prior to ATP addition, as outlined in the product application note. For cell‑based assays, the compound penetrates HEK293T monolayers with a cellular‑to‑medium partition coefficient (log C_cell/C_medium) of 0.85 ± 0.07 at 2 h, determined by LC‑MS/MS quantitation of lysates versus supernatant. Washout experiments reveal a target‑residence half‑life on endogenous TAK1 of > 4 h, measured by recovery of phospho‑MKK6 signal in Western blot using a rabbit monoclonal antibody (Cell Signaling Technology, clone D46D3).

HS Code	985515
Chemical Name	N-[3-[[5-Bromo-4-[[2-(1H-Imidazol-5-Yl)Ethyl]Amino]-2-Pyrimidinyl]Amino]Phenyl]-1-Pyrrolidinecarboxamide

Packing	100g of N - [3 - [[5 - Bromo - 4 - [[2 - (1H - Imidazol - 5 - Yl)Ethyl]Amino] - 2 - Pyrimidinyl]Amino]Phenyl] - 1 - Pyrrolidinecarboxamide in sealed vial.
Shipping	The chemical "N-[3-[[5-Bromo-4-[[2-(1H-Imidazol-5-Yl)Ethyl]Amino]-2-Pyrimidinyl]Amino]Phenyl]-1-Pyrrolidinecarboxamide" will be shipped in accordance with strict chemical safety regulations, likely in sealed, protective containers via approved carriers.
Storage	Store "N-[3-[[5-Bromo-4-[[2-(1H-Imidazol-5-Yl)Ethyl]Amino]-2-Pyrimidinyl]Amino]Phenyl]-1-Pyrrolidinecarboxamide" in a cool, dry place, away from direct sunlight and heat sources. Keep it in a tightly - sealed container to prevent exposure to moisture and air, which could potentially degrade the chemical. Ensure storage areas are well - ventilated.

N-[3-[[5-Bromo-4-[[2-(1H-Imidazol-5-Yl)Ethyl]Amino]-2-Pyrimidinyl]Amino]Phenyl]-1-Pyrrolidinecarboxamide