2-((Tert-Butoxycarbonyl) Amino) Thiazole-5-Carboxylic Acid

How Does This Building Block Enable the Construction of Subnanomolar FXa Inhibitors?

The synthesis of orally active factor Xa inhibitors incorporating a 2-aminothiazole-5-carboxamide core begins with the controlled activation of 2-((tert-butoxycarbonyl)amino)thiazole-5-carboxylic acid. In a typical batch process executed in a 20 L glass-lined reactor equipped with a retreat-curve impeller and a nitrogen sweep, the Boc-protected acid (1.05 mol equivalents) is dissolved in anhydrous N,N-dimethylformamide (5.0 L/kg substrate). The solution is cooled to 0–5°C using an external jacket circulator, after which N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl, 1.20 eq) and 1-hydroxybenzotriazole hydrate (HOBt·H₂O, 1.20 eq) are added in sequence. Stirring is maintained for 45 min to generate the active HOBt ester. A pre-dissolved solution of the specific aromatic amine partner—often a substituted indol-7-amine or a 1-aryl-1H-pyrazole-4-amine—in the same solvent (1.00 eq, 1.5 L/kg) is then introduced over 20 min via a dosing pump while the internal temperature is kept below 8°C. 4-Methylmorpholine (NMM, 2.50 eq) is subsequently added dropwise to scavenge liberated HCl, and the cooling is discontinued to allow the reaction mass to reach 20–25°C. HPLC monitoring (Agilent 1260 Infinity, Inertsil ODS-3 column, 254 nm) after 16 h typically indicates consumption of the amine to below 0.5 area%, with the desired amide eluting at a relative retention time of 1.42 ± 0.03 against the amine. The reaction is quenched with purified water (15 volumes) and extracted with ethyl acetate (3 × 5 volumes). The combined organic phase is washed with 5% w/w aqueous sodium hydrogen carbonate (2 × 3 volumes), 1 N hydrochloric acid (2 × 3 volumes), and brine, before drying over anhydrous sodium sulfate. After filtration and vacuum distillation to a residual DMF content below 880 ppm (ICH Q3C Class 2), the crude protected amide is purified by flash chromatography (silica gel 60 Å, gradient elution from 20% to 60% ethyl acetate in n-heptane) to afford a white crystalline solid with isolated yield consistently in the range of 82–89%.

Subsequent removal of the tert-butoxycarbonyl group employs a mixture of trifluoroacetic acid and dichloromethane (1:1 v/v, 10 volumes per kg) at 20–25°C for 2–3 h. The volatile acids are stripped at 40°C under reduced pressure (50 mbar) using a rotary evaporator with a Teflon diaphragm pump, and the residue is azeotroped with toluene (3 × 3 volumes) to remove residual TFA. The resulting 2-aminothiazole-5-carboxamide trifluoroacetate salt is directly taken to the next sulfonylation step without further characterization. In a sulfonylation protocol validated for multi-kilogram scale, the amine salt is suspended in dichloromethane (12 volumes) and pyridine (3.0 eq) at 0°C, and the chosen sulfonyl chloride (methanesulfonyl chloride or cyclopropylsulfonyl chloride, 1.5 eq) is slowly added. After 1 h of stirring at 0–5°C, the reaction is complete as judged by the absence of the primary amine signal in the in-process TLC (Merck silica gel 60 F₂₅₄, eluent hexane:EtOAc 1:1). The terminal product, a 2-sulfonamidothiazole-5-carboxamide, is crystallized from ethanol/water (4:1) to yield the final drug substance candidate in >99.5% chromatographic purity (HPLC, area normalization).

Compliance for these intermediates rests on several intertwined guidelines. Residual metal content is verified by inductively coupled plasma mass spectrometry (ICP-MS) in accordance with USP <233>. Palladium is maintained below 10 µg/g, and hexavalent chromium below 5 µg/g, consistent with Permitted Daily Exposure values for a 10 g/day oral dosage form as outlined in ICH Q3D. Genotoxic impurities arising from the sulfonate ester of HOBt are controlled below the Threshold of Toxicological Concern (1.5 µg/day) per ICH M7, option 4 control. A residual solvent panel run on an Agilent 7890B GC headspace sampler coupled to a 5977A MSD demonstrates compliance with ICH Q3C limits: DMF not more than 880 ppm, TFA not more than 5000 ppm, dichloromethane below 600 ppm. The manufacturing process description is anchored to ISO 9001:2015 and ICH Q7 Q& attributes for active pharmaceutical ingredients, with dedicated batch records demonstrating consistency across 15 consecutive validation runs.

p38α Mitogen-Activated Protein Kinase Inhibitor Lead Optimization Using a Solid-Supported Coupling Strategy

In a parallel medicinal chemistry campaign targeting cytokine-release inhibition, a resin-bound library of 2-aminothiazole-5-carboxamides is constructed from the Boc-protected acid to accelerate SAR expansion. Commercially available 4-hydroxymethylphenoxymethyl polystyrene resin (Wang resin, loading 1.1 mmol/g) is swollen in dichloromethane (12 mL/g) and treated with the Boc-acid (2.5 eq), diisopropylcarbodiimide (2.5 eq), and a catalytic amount of 4-dimethylaminopyridine (0.1 eq) in DMF at 25°C for 18 h. Unreacted hydroxymethyl sites are capped with acetic anhydride/pyridine (1:1 v/v, 30 min). The resin-bound Boc-amino-thiazole is then subjected to TFA/CH₂Cl₂ (20% v/v, 2 × 20 min) to liberate the free amine. After sequential washes with CH₂Cl₂, 10% triethylamine in CH₂Cl₂, and N-methylpyrrolidone (NMP), the resin is split into 96-well microtiter filter plates, each well containing approximately 50 mg of resin. Diverse electrophiles—predominantly aryl sulfonyl chlorides, isocyanates, and α-halo ketones—are introduced in NMP (0.2 M) together with N,N-diisopropylethylamine (4.0 eq) and allowed to react at 50°C for 12 h. The final cleavage step employs TFA/triisopropylsilane/water (95/2.5/2.5 v/v/v, 2 h), which simultaneously detaches the compounds from the solid support and deprotects any side-chain labile groups. Filtrates are evaporated in vacuo (Genevac HT-4X centrifugal evaporator, 40°C) and the crude library members purified by reverse-phase mass-directed preparative HPLC (Waters AutoPurification system, XBridge C18 OBD column, 30 × 150 mm, 5 μm, gradient 10–90% MeCN in 0.1% aq. formic acid). Each isolated compound is characterized by UPLC-MS and ¹H NMR, and only fractions exceeding 95% purity at 214 nm (Waters ACQUITY UPLC, BEH C18 1.7 μm, 2.1 × 50 mm) are progressed to enzyme inhibition assays. The quality of the analytical method is validated according to ICH Q2(R1) parameters; the limit of quantification for the lead scaffold is established at 0.05 µg/mL. Published biochemical data for this structural series show p38α MAP kinase IC₅₀ values descending into the single-digit nanomolar range when a 3-trifluoromethoxybenzyl sulfonamide substituent is attached to the thiazole 2-NH₂ position.

The HCV NS5B polymerase allosteric site, specifically the thumb pocket II, accommodates a 2-aminothiazole-5-carbonyl fragment as a hydrogen-bonding anchor for novel non-nucleoside inhibitors. A convergent solution-phase route relying on 2-((tert-butoxycarbonyl)amino)thiazole-5-carboxylic acid begins with its activation by benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate (BOP, 1.15 eq) in dry acetonitrile at −10°C in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 1.30 eq). A secondary amine surrogate—typically a 1-(2,4-difluorophenyl)piperazine derivative—is added as a single portion (1.00 eq), and the cooling bath is removed. After stirring for 6 h at ambient temperature, the reaction mixture is diluted with ethyl acetate and washed with 0.5 N aqueous citric acid, saturated NaHCO₃, and water. The organic phase is dried over MgSO₄, filtered, and concentrated. The Boc group is subsequently removed with 4.0 M hydrogen chloride in 1,4-dioxane (8 eq acid relative to substrate) at 20°C for 45 min. The precipitated HCl salt of the free amine is collected by filtration under nitrogen pressure, washed with anhydrous diethyl ether, and dried to constant weight in a vacuum oven (45°C, 15 mbar). The amine hydrochloride is then re-dissolved in DMF and acylated with 4-chloro-2-fluoro-3-methoxybenzoic acid using HATU (1.10 eq) and NMM (2.50 eq) to secure the final amide target in 70–78% overall yield after silica gel chromatography. Specifications for the resulting drug candidate intermediate require compliance with Genotoxic Impurity (GTI) control according to ICH M7; any residual phosphonium impurities from BOP are monitored by ³¹P NMR and must be below the 1.5 µg/day Threshold of Toxicological Concern. Purity analysis on a Waters Empower 3 network reports peak area homogeneity > 99.2% at 235 nm with a photodiode array detector spanning 210–400 nm. In HCV genotype 1b replicon assays, representatives of this chemotype have demonstrated EC₅₀ values in the low nanomolar window when combined with a C5-linked pyrazine-2-carbonyl moiety; these biological outcomes are regularly benchmarked against the reference interferon-α pegylated standard as per the EU GMP Annex 2 biological guidelines.

When a Boc-protected 2-aminothiazole-5-carboxylic acid replaces the 2-chloro congener in exploratory SDHI scaffolds, resistance profile shifts are observed

Succinate dehydrogenase inhibitor (SDHI) fungicide discovery programs exploit the latent reactivity of the Boc-protected amino group to introduce N-acyl modifications that are incompatible with the classical thiazole-5-carboxylic acid chlorides. In one such process stream, the Boc-protected acid is refluxed with thionyl chloride (2.5 eq) in toluene containing a catalytic quantity of DMF (0.05 eq) for 3 h to generate the corresponding acid chloride, which is isolated after solvent evaporation as a moisture-sensitive beige solid. This activated intermediate is immediately taken up in dry dichloromethane and added dropwise to a pre-cooled (0°C) solution of the tailored lipophilic amine—often a 2-(1,3-dimethylbutyl)-aniline analog—in the presence of triethylamine (1.2 eq). The amide bond formation proceeds with minimal epimerisation and reaches completion after 2 h as monitored by FT-IR spectroscopy (disappearance of the acid chloride νC=O at 1795 cm⁻¹ and emergence of the amide I band at 1648 cm⁻¹). The Boc group is then selectively cleaved with anhydrous 4.0 M HCl in ethyl acetate (10 eq, 1 h) to furnish the amine hydrochloride. After free-base liberation with aqueous NaOH and phase separation, the primary 2-amino group is acetylated with acetic anhydride (1.05 eq) in acetic acid at 50°C to deliver the final acetamide derivative. The overall sequence from acid to target compound achieves a throughput of 64–71% on a 500 g scale using a Mettler-Toledo EasyMax 402 Advanced Synthesis Workstation with real-time reaction calorimetry for safety screening. Technical-grade active ingredient purity must exceed 98.0% w/w as determined by CIPAC method MT 184 reversed-phase HPLC. Quality control further requires a residue on ignition below 0.1% w/w (ASTM D482) and chloride content (as Cl⁻) not exceeding 200 ppm by potentiometric titration. In vitro bioassays against Alternaria solani and Sclerotinia sclerotiorum reveal that the N-acetyl-substituted 2-aminothiazole-5-carboxamides maintain EC₅₀ values below 2 mg/L even in isolates bearing the C-SdhB-H277Y mutation. Regulatory compliance for the experimental material intended for field trials references EPA 40 CFR Part 180 for residue chemistry data requirements and the OECD 501 hydrolysis guideline for environmental fate assessment.

Pre-blending of 2-((tert-butoxycarbonyl)amino)thiazole-5-carboxylic acid as a thermal latent amine source into a standard DGEBA epoxide formulation alters the curing exotherm profile markedly. At a loading of 7.5 wt% together with 3.5 wt% dicyandiamide in a bisphenol-A diglycidyl ether resin (epoxy equivalent weight 186–190 g/eq), the three-component powder premix is homogenized on a two-roll mill (Bühler SDY-200, gap 0.1 mm, roll temperature 25°C) and degassed at 50°C under 5 mbar vacuum. Differential scanning calorimetry (PerkinElmer DSC 8500, heating rate 10 K/min, nitrogen purge 50 mL/min) shows an onset of the thermolytic deprotection at 162°C with a peak deprotection enthalpy of 145 J/g₍Boc-compound₎. The liberation of CO₂ and isobutylene creates a porous micro-channel network that subsequently facilitates epoxy-amine crosslinking with a peak polymerization exotherm at 214°C. Post-cure dynamic mechanical analysis (TA Instruments DMA Q800, three-point bending, 1 Hz, 2 K/min) of a plaque cured at 180°C for 90 min records a storage modulus glass transition temperature, Tꞌꞌ_g^peak, of 158°C per ASTM D7028. Water absorption after immersion in deionized water at 85°C for 7 days rises only to 1.2% by mass (ASTM D570-98), which indicates that the free thiazole carboxylic acid moieties formed during deprotection do not compromise the network hydrophobicity beyond acceptable limits for electronic encapsulation. The formulation is screened for outgassing during reflow soldering by thermogravimetric analysis-mass spectrometry (TA Instruments Discovery SDT 650 coupled to a Pfeiffer ThermoStar), confirming that isobutylene release plateaus below 260°C and that total volatiles remain under 1.0% of the coating weight. ISO 11357-2 calibration protocols are applied regularly to compensate for thermal resistance corrections. Batch-to-batch variance in the latency window is evaluated by industrial end users against IPC-TM-650 method 2.4.25; the typical shelf life of the premixed powder when stored in vacuum-sealed moisture-barrier bags at 10°C exceeds 6 months.

Post-Synthetic Amine Liberation in Zn(II)-based Pillared-Layer Frameworks Constructed with This Ditopic Linker

Coordination-driven assembly of the Boc-protected acid with zinc(II) nitrate hexahydrate (Zn(NO₃)₂·6H₂O) and 4,4′-bipyridine (bpy) yields a crystalline pillared-layer metal-organic framework after solvothermal treatment. A screw-cap pressure tube is charged with Zn(NO₃)₂·6H₂O (0.50 mmol, 148.6 mg), 2-((tert-butoxycarbonyl)amino)thiazole-5-carboxylic acid (0.50 mmol, 136.1 mg), bpy (0.25 mmol, 39.0 mg), and a mixed solvent of DMF/ethanol/water (5:3:2 v/v/v, total 10 mL). After ultrasonic treatment for 15 min, the sealed vessel is heated in a temperature-controlled oven at 85°C for 24 h. Block-shaped pale yellow crystals are harvested by vacuum filtration, washed with DMF (3 × 5 mL) and methanol (3 × 5 mL), and immersed in methanol for 48 h with three solvent exchanges to remove guest molecules. The as-synthesized MOF exhibits a BET surface area of approximately 1250 m²/g when measured by nitrogen physisorption at 77 K on a Micromeritics ASAP 2020 analyzer (degassing at 120°C for 8 h, IUPAC technical report guidelines for microporous materials). Thermogravimetric analysis reveals a sharp mass loss of 12.4 wt% centered at 195°C, corresponding to the deprotection of the Boc group and release of CO₂ and isobutylene. Inspired by this thermal signature, a controlled post-synthetic modification is performed by suspending the crystals in a methanol/water (1:1 v/v) mixture and heating at 60°C for 24 h in the presence of catalytic 0.1 M HCl—conditions that selectively cleave the tert-butoxycarbonyl group while preserving framework crystallinity as verified by powder X-ray diffraction. After neutralization and reactivation at 150°C under dynamic vacuum, the resulting amine-lined MOF shows a slight reduction in nitrogen-accessible surface area to roughly 1180 m²/g. Single-component gas sorption isotherms collected at 273 K and pressures up to 1 bar demonstrate that the free –NH₂ groups enhance CO₂ uptake by approximately 25% compared to the Boc-protected precursor, a finding consistent with an increased isosteric heat of adsorption in the low-coverage regime (Q_st ≈ 32 kJ/mol for the deprotected version, estimated by the Clausius-Clapeyron equation). The pore size distribution calculated by non-local density functional theory (NLDFT) with a carbon slit-pore model confirms a median pore width of 1.14 nm. ISO 9277:2010 is employed as the reference standard for BET surface area determination, and the CO₂ capture capacity is benchmarked against the requirements set forth in the U.S. Department of Energy MCFB-20 target specifications for point-source carbon capture sorbents.