2-[(6-Chloro-2-Methylpyrimidin-4-Yl)Amino]-N-(2-Chloro-6-Methylphenyl)Thiazole-5-Carboxamide

Designated under catalogue identifier RCC-8294 within the supplier’s heterocyclic reference library, the molecule 2-[(6-chloro-2-methylpyrimidin-4-yl)amino]-N-(2-chloro-6-methylphenyl)thiazole-5-carboxamide (empirical formula C₁₆H₁₃Cl₂N₅OS, formula weight 394.28 g/mol) is supplied as a non-GMP research reagent for in‑vitro pharmacological profiling and structure–activity relationship (SAR) campaigns. Batch‑release documentation conforms to the supplier’s internal quality management system audited against ISO 9001:2015; each shipment is accompanied by a lot‑specific certificate of analysis listing retention time, mass‑ion confirmation, and chromatographic purity determined with a Agilent 1290 Infinity II UHPLC system employing a diode‑array detector set to 254 nm acquisition wavelength. The lyophilized powder exhibits an off‑white to pale‑yellow appearance and, when stored in sealed borosilicate vials under argon headspace at ‑20 ± 5 °C, maintains purity within ±0.3% of the released value for a documented shelf‑life of 24 months, as verified by accelerated stability protocols conducted at 25 °C/60 % RH over 6 months in parallel with ‑20 °C control arms.

What Molecular Features Distinguish This Pyrimidinyl–Thiazole Carboxamide Scaffold?

The core architecture couples a 2‑aminothiazole‑5‑carboxamide hinge‑binding motif with a 6‑chloro‑2‑methylpyrimidin‑4‑yl substituent at the exocyclic amine and a 2‑chloro‑6‑methylphenyl group at the carboxamide nitrogen. This specific arrangement places an electron‑withdrawing chlorine atom at the pyrimidine 6‑position, which exerts a mesomeric influence on the adjacent amino linker, and a ortho‑chloro,ortho‑methyl substitution pattern on the terminal phenyl ring that introduces both steric bulk and a modulation of the aryl ring’s torsional angle relative to the carboxamide plane. Single‑crystal X‑ray diffraction data obtained from a structurally analogous des‑chloro congener (CCDC deposition number 2214583) indicate a dihedral angle of 72.4° between the thiazole and the N‑phenyl carboxamide plane; the presence of the ortho‑chloro substituent in the current compound is predicted by density‑functional calculations at the B3LYP‑D3/6‑311+G(d,p) level to increase that angle to approximately 81°, reducing amide resonance and altering the compound’s hydrogen‑bond‑acceptor capacity. Such geometric perturbations have been correlated with differential binding poses against the ATP‑binding pocket of cyclin‑dependent kinases in published co‑crystal structures of close analogs (PDB IDs 5L2W, 6Q4R), though direct crystallographic data for the title compound remain unavailable at this time.

From a medicinal‑chemistry vantage, the combination of the 6‑chloro‑2‑methylpyrimidine and the ortho,ortho‑disubstituted phenyl ring creates a unique pharmacophoric signature. The pyrimidine chlorine atom contributes a +0.12 e partial charge (Mulliken population analysis) relative to the unsubstituted pyrimidine, while the methyl group at position‑2 generates a minor positive inductive effect that partially offsets the electron deficiency of the ring, effectively tuning its π‑stacking capability. The thiazole sulfur atom participates in a long‑range contact (3.61 Å) with the pyrimidine C‑5 hydrogen, a pre‑organized intramolecular feature that restrains the accessible conformational space of the hinge‑binding region to fewer than 3 low‑energy rotameric states at 310 K. These physicochemical attributes collectively differentiate the scaffold from related 2‑(pyrimidin‑4‑ylamino)thiazole‑5‑carboxamides that bear unsubstituted phenylamides or that position the halogen at the pyrimidine 2‑position rather than 6‑position, where the chlorine would exert only an inductive effect without direct conjugation to the amino linker.

Chromatographic Purity and Trace‑Metal Specifications

The compound is released only when reversed‑phase UHPLC‑UV analysis (column: Waters ACQUITY UPLC BEH C18, 1.7 µm, 2.1 × 50 mm; mobile phase gradient: 5–95 % acetonitrile in 0.1 % formic acid over 8 minutes; flow rate 0.6 mL/min) demonstrates area‑% purity not less than 98.0 %. A typical lot records a dominant peak at retention time 4.72 ± 0.05 min with a symmetry factor between 0.90 and 1.15. Any batch exhibiting an impurity with relative retention time 1.23 (tentatively identified by LC‑HRMS as the des‑chloro phenyl analog) at an integrated intensity exceeding 0.5 % is reprocessed by preparative HPLC (column: Waters XBridge Prep C18 OBD, 5 µm, 19 × 150 mm) until the target specification is met.

Quantitative ¹H NMR spectroscopy at 600 MHz in DMSO‑d₆ with 1,3,5‑trimethoxybenzene as internal standard confirms a mass‑balance purity within ±1.5 % of the chromatographic value. Characteristic resonances include a singlet at δ 2.28 ppm (3H, pyrimidine 2‑CH₃), a singlet at δ 2.32 ppm (3H, phenyl 6‑CH₃), and a broad signal at δ 10.82 ppm (1H, carboxamide NH) that vanishes upon D₂O exchange, together with aromatic proton patterns integrating for three and one protons, respectively, consistent with the stated structure. High‑resolution mass spectrometry (ESI‑Q‑TOF) in positive‑ion mode exhibits a monoisotopic [M+H]⁺ peak at m/z 394.0497 (calculated 394.0501, mass error ‑1.0 ppm). Trace‑metal analysis by inductively coupled plasma mass spectrometry after closed‑vessel microwave digestion confirms palladium content below 10 ppm and iron below 25 ppm, ensuring compliance with the supplier’s standard for kinase‑assay‑grade reagents. These analytical release criteria are validated against ICH Q2(R1) guidelines for specificity, linearity, and precision.

When the lyophilized powder is reconstituted in anhydrous DMSO for high‑throughput screening, the recommended protocol calls for preparing a 10 mM stock solution and dispensing it within 30 minutes into single‑use, low‑retention polypropylene vials to avoid freeze‑thaw‑induced aggregation. Repeated 10 freeze‑thaw cycles of a 10 mM DMSO stock stored at ‑20 °C resulted in a purity decline of 2.1 % per cycle (n=3) as determined by post‑thaw UHPLC, presumably due to water ingress‑promoted hydrolysis of the thiazole carboxamide bond. Accordingly, aliquoting is mandatory for users intending to extend stock solution utility beyond a single screening campaign.

Direct exposure to ambient laboratory humidity (relative humidity ≥ 60 %) for periods exceeding 4 hours leads to visible deliquescence and a concomitant mass increase of 2.8–3.5 % (gravimetric analysis, n=5), consistent with the uptake of approximately 0.6 equivalents of water. This water absorption accelerates amide hydrolysis; stressed samples stored at 40 °C/75 % RH in open vials show 7.8 % degradation after 14 days, with the major degradant identified as the free 2‑[(6‑chloro‑2‑methylpyrimidin‑4‑yl)amino]thiazole‑5‑carboxylic acid based on retention time matching and mass spectral fragmentation. Therefore, pre‑drying of the bulk powder under vacuum (< 1 mbar) at 25 °C for 2 hours is advised if the container has been opened outside a dry‑nitrogen glovebox.

The higher lipophilicity and more extensive protein binding of the title compound relative to its des‑chloro or des‑methyl counterparts render it more suitable for probing intracellular targets where membrane partitioning is rate‑limiting, yet it introduces a narrower therapeutic window in whole‑cell viability assays due to concomitantly reduced free fraction. Published data for this specific configuration is limited; the numerical ranges in the table are drawn from internal head‑to‑head profiling performed using identical assay protocols in a single contract research laboratory (Eurofins Discovery Services, study numbers ADME‑22309‑01 through ‑03).

When This Compound Is Deployed in a High‑Content Kinase‑Selectivity Panel

A commercial panel of 468 human kinases (DiscoveRx KINOMEscan, scanMAX format) was probed at a fixed compound concentration of 1 µM in duplicate, with results expressed as percent of control (PoC) binding relative to DMSO‑treated wells. The compound exhibited PoC values ≤ 10 %—indicating strong binding—for 7 kinases, all of which belong to the CMGC and CAMK families. The most potently engaged targets were CDK9/Cyclin T1 (PoC = 0.2 %), CDK2/Cyclin E1 (0.7 %), and DYRK1A (1.4 %). Dose‑response follow‑up for these three kinases generated dissociation constants (K_d) of 12 nM, 38 nM, and 67 nM, respectively, as calculated from an 11‑point, 3‑fold dilution series starting at 10 µM. Residual kinase activities in the presence of 1 mM ATP confirmed the ATP‑competitive nature of inhibition. In contrast, analog A (des‑chloro pyrimidine, des‑chloro phenyl, see table) displayed significantly attenuated binding to CDK9 (K_d = 490 nM), highlighting the critical contribution of the ortho‑chlorine atoms to potency. Selectivity score S(10)—defined as the number of kinases inhibited by ≥ 90 % at 1 µM divided by total kinases tested—equaled 0.015, placing the compound in the lower quartile of selectivity among thiazole‑based ATP‑competitive inhibitors, yet its narrow target spectrum reduces off‑target‑mediated cytotoxicity in certain cell‑line models.

For cell‑based mechanistic studies, a molar equivalent of the compound has been co‑crystallized with a construct of CDK2 (residues 1–298) expressed in E. coli BL21(DE3) and purified via Ni‑NTA and size‑exclusion chromatography. Crystals grown by hanging‑drop vapour diffusion at 4 °C with a reservoir solution comprising 0.2 M ammonium sulfate, 0.1 M BIS‑TRIS pH 6.5, and 23 % PEG 3350 diffracted to 2.10 Å resolution at a synchrotron source (Diamond Light Source beamline I04‑1). The resulting electron density map unambiguously placed the pyrimidine nitrogen and the anilino NH as hydrogen‑bonding partners to Leu83 backbone carbonyl and Glu81 side‑chain carboxylate, respectively, while the ortho‑chlorophenyl ring occupies a hydrophobic cleft lined by Ile10, Val18, and Leu134. These structural details are made available to collaborators through the supplier’s co‑crystallization service and are not deposited in the PDB at the time of writing.

Solubilisation and Formulation Constraints in Animal Models

For pharmacokinetic studies in male Sprague‑Dawley rats (n=3 per route), a clear solution for intravenous bolus administration (1 mg/kg) was achieved by formulating the compound in a vehicle consisting of 10 % DMSO, 40 % PEG 400, and 50 % saline, with a final pH of 4.2 adjusted with 0.1 N HCl. Oral gavage (5 mg/kg) employed a suspension in 0.5 % methylcellulose containing 0.1 % Tween 80. Plasma concentrations were quantified by LC‑MS/MS (LLOQ 1 ng/mL) using a deuterated internal standard. The mean oral bioavailability was 18.3 ± 5.7 %, consistent with the compound’s moderate hepatic extraction ratio inferred from microsomal stability data. Bile‑duct cannulated animals revealed that approximately 22 % of the administered dose was excreted unchanged in bile over 24 hours, suggesting the parent compound is a substrate for hepatic uptake transporters OATP1B1/1B3; rosuvastatin co‑administration (10 mg/kg oral) increased plasma AUC_0–24 by 1.8‑fold, corroborating transporter involvement. Avoid formulation with amine‑based solubilizers such as meglumine or lysine salts, which accelerate carboxamide bond cleavage under accelerated conditions (40 °C/75 % RH) with a degradation rate constant of 0.023 day⁻¹ versus 0.007 day⁻¹ in the recommended acidic vehicle.

Incompatibilities with Nucleophilic Buffers and Redox‑Active Excipients

The thiazole ring undergoes ring‑opening when the compound is incubated at 37 °C in phosphate‑buffered saline containing 5 mM glutathione at pH 7.4, as confirmed by the appearance of a new peak with m/z 527.12 (glutathione adduct) in the extracted ion chromatogram within 2 hours. Therefore, cellular washout experiments that rely on thiol‑containing media must include a pre‑quench step with 10 mM iodoacetamide. Similarly, contact with strong reducing agents such as tris(2‑carboxyethyl)phosphine (TCEP) in protein‑binding assays leads to rapid dechlorination at the pyrimidine 6‑position, generating the des‑chloro analog within 15 minutes at neutral pH. Laboratories utilizing reducing agents in sample preparation should substitute dithiothreitol at concentrations not exceeding 0.1 mM, where the reaction half‑life extends to > 6 hours.

The lyophilized powder has been classified as non‑hygroscopic by dynamic vapour sorption analysis (surface area 3.2 m²/g, < 0.2 % mass increase up to 70 % RH), yet the micronized form (particle size D₉₀ < 10 µm) prepared for inhalation studies exhibits a moisture uptake of 1.1 % at 60 % RH due to increased specific surface area. For aerosol generation in a nose‑only exposure system using a Palas AGK‑2000 dry powder generator, the powder must be blended with α‑lactose monohydrate (inhalation grade, D₅₀ 65 µm) at a ratio of 1:9 (w/w) to achieve a delivered dose uniformity within ±15 % across five serial sampling points as per USP <601> Apparatus 5. Attempts to aerosolize the neat micronized compound resulted in severe agglomeration and a fine particle fraction (≤ 5 µm) below 12 % of the nominal dose, attributed to triboelectric charging measured at ‑45 nC/g on a Kleindoiss Type 892 electrometer.

The pronounced sensitivity to alkaline pH precludes its use in assays requiring pH ≥ 8.0 for extended incubation periods; the degradation rate follows a pseudo‑first‑order dependence on hydroxide ion concentration with an activation energy of 72.4 kJ/mol determined from an Arrhenius plot spanning 25–50 °C. These stability boundaries must be integrated into any experimental design that involves multi‑day cell‑culture exposure, where the medium pH can drift due to metabolic acidification. Twice‑daily medium replacement effectively maintains compound integrity, as verified by LC‑HRMS analysis of spent medium aliquots.