2-Phenyl-1,3-Thiazole-4-Carboxylic Acid

Implementation of Process Analytical Technology (PAT) within the telescoped synthesis of a once-daily non-nucleoside reverse transcriptase inhibitor (NNRTI) candidate revealed a critical correlation between activation kinetics of 2-Phenyl-1,3-thiazole-4-carboxylic acid and the formation of a des-phenyl hydrolysis impurity tracked at 0.08–0.12% by UPLC-MS. In a GlaxoSmithKline-style segmented flow reactor equipped with a Mettler Toledo ReactIR 15 probe, the acid was pre-dissolved in anhydrous tetrahydrofuran and converted to its N-hydroxysuccinimide ester using 1.0–1.18 molar equivalents of EDC·HCl and 0.05 equivalents of DMAP at a jacket temperature maintained between −5 °C and 0 °C. The pre-activation stream was merged with a second stream containing a sterically hindered 2,6-disubstituted aniline in DMF, with the combined residence time in a 10 mL PFA coil set at 47 seconds. Process analytical feedback loops adjusted the pump stroke rate to compensate for viscosity differences arising from batch-to-batch variation in the acid’s particle size distribution (D₅₀ 15–35 µm). Regulatory compliance for the step falls under ICH Q7 Section 8.5 (contamination control) and ICH Q3C guidelines, where residual THF must not exceed 720 ppm and DMF is capped at 880 ppm in the isolated intermediate. The downstream manufacturing process integrates thin-film evaporation at 40 °C under 15 mbar for solvent swap, followed by anti-solvent crystallization from isopropanol/water (3:1 v/v) in a Büchi 15 L glass-lined crystallizer with a retreat-curve impeller agitated at 150 rpm. The final active pharmaceutical ingredient, a 2-phenylthiazole-4-carboxamide derivative, is dry-milled in a Hosokawa Alpine AFG 100 jet mill to a D₉₀ of 4.5 µm and formulated into an immediate-release film-coated tablet of 300 mg strength via direct compression, packed in cold-form Alu-Alu blisters under ISO 8 classified conditions per ISO 14644-1:2015.

What drives the need for sub-0.05% residual hydrazine in this intermediate?

During the preparation of 2-phenylthiazole-4-carbohydrazide for a xanthine oxidase inhibitor program analogous to the febuxostat pharmacophore but with improved CYP3A4 selectivity, the hydrazinolysis of the corresponding methyl ester introduced hydrazine contamination that persisted through downstream reductive amination steps. Pilot-plant batches manufactured in a 100 L glass-lined reactor at Syngene International documented that when residual hydrazine hydrate exceeded 0.15% (as determined by IC with pulsed amperometric detection per USP ⟨477⟩), the subsequent coupling with a 3-cyanobenzaldehyde derivative in methanol at 60 °C generated a genotoxic hydrazone impurity at levels of 8–12 ppm, breaching the threshold of toxicological concern (TTC of 1.5 µg/day) defined in ICH M7(R2). The process was redesigned to incorporate a rigorous azeotropic chase with 2-propanol after hydrazinolysis, monitored by in-line NIR spectroscopy until the hydrazine peak at 1650 cm⁻¹ vanished. The addition ratio of 2-phenylthiazole-4-carboxylic acid as the starting point for the hydrazide derivative typically constitutes 62–68 wt% of the final xanthine oxidase inhibitor’s molecular weight before salt formation. The compliance framework includes a DMF Type II filing under 21 CFR 314.420, with a specification for total hydrazine and its derivatives set at a limit of quantitation of 0.01 ppm using LC-MS/MS. The downstream production sequence includes conversion to the acyl chloride using SOCl₂ in toluene at 55 °C, which is then condensed with 4-(2-methylpropoxy)benzonitrile-derived amine in the presence of triethylamine, yielding the crude inhibitor. Recrystallization from a ternary solvent system of acetone/water/ethanol (45/35/20 v/v) achieves a purity of 99.8% and a polymorphic Form I confirmed by XRPD. The terminal product is a size-1 hard gelatin capsule containing 80 mg of the free acid, packaged with a molecular sieve desiccant canister to maintain a moisture content below 1.5% KF.

In-furrow Nematicide Intermediate via HATU-Mediated Condensation

Soil column leaching tests conducted per OECD Guideline 312 with a 2-phenylthiazole-4-carboxamide nematicide revealed that high water solubility of the unformulated technical material (> 250 mg/L at pH 7) caused rapid percolation below the rhizosphere before systemic uptake into tomato root knots infested with Meloidogyne incognita. Mitigation was achieved by synthesizing a more lipophilic benzylamide derivative, where 2-phenylthiazole-4-carboxylic acid is activated with HATU (1.05 eq.) and DIPEA (2.5 eq.) in dimethylacetamide at 0–5 °C, then condensed with 3-methoxybenzylamine. The acid itself constitutes approximately 52% of the final active substance’s molecular mass, and when produced at a multi-ton scale in an agrochemical dedicated facility such as Lanxess’s Leverkusen multipurpose unit, the heat of reaction (−92 kJ/mol) is controlled with an automated jacket cooling system keeping T_r below 8 °C to suppress racemization at the benzylic position. Compliance with EPA FIFRA 40 CFR Part 158 dictates a five-batch analysis with storage stability data at 54 °C for 14 days, and residue analytical enforcement methods are validated according to SANCO/10684/2009 with an LOQ of 0.01 mg/kg in lettuce and tomato matrices. The registered end-use product is a 200 g/L capsule suspension (CS) formulated with a polymethyl methacrylate wall thickness of 0.3–0.5 µm, xanthan gum rheology modifier, and the ready-to-use agrochemical mixture containing 200 g a.i./L of the amide derivative. This is applied via drip irrigation at a rate of 1.5 L/ha, with the factory-concentrated suspension exhibiting a pourability residue below 2.5% after CIPAC MT 148 tests. The manufacturing line incorporates a Silverson 150/250 in-tank mixer running at 3,000 rpm for pre-emulsion before passing through a 2-stage IKA Magic Lab colloid mill set to a rotor-stator gap of 0.2 mm.

**Table 1: Coupling Activation Method vs. Amide Impurity Profile (2-Phenylthiazole-4-Carboxylic Acid Derived API)**
Activation Method	Reaction Solvent	Amine Partner	Conversion (%)	Des-acid Impurity (%)	Epimerized Form (%)	Residual Activator (ppm)
EDC·HCl / HOBt	DMF	2,6-Dimethylaniline	98.7	0.08	0.15	11 (EDC·urea)
HATU / DIPEA	DMAc	3-Methoxybenzylamine	99.4	0.03	< 0.05	2 (tetramethylurea)
SOCl₂ (acid chloride)	Toluene	4-Cyanobenzylamine	97.2	0.42	n.a.	25 (SO₂)
CDI (acylimidazole)	THF	2-Aminopyridine	96.8	0.11	n.a.	90 (imidazole)

When the 2-phenylthiazole-4-carboxylic acid building block is utilized in the synthesis of an experimental strobilurin-mimic fungicide with a modified toxophore for QoI-resistant Blumeria graminis isolates, the active ingredient’s greasiness and high log P (4.8) posed aerosol drift risks during the milling of water-dispersible granules. The formulating plant switched to an oil dispersion (OD) formulation type, where the amide derivative generated from the acid via T3P-mediated coupling (1.2 eq. of acid to crude amine core in ethyl acetate at 25 °C for 3 h) is dissolved in a methyl oleate/aromatic 150 ND solvent blend. The 2-phenylthiazole moiety constitutes 47–51% of the total molecular weight of the active, and the technical-grade intermediate must pass a filter paper spot test for insoluble solids, achieving a rate of 0.05 mg/kg or lower. CIPAC physical-chemical methods MT 39.3 (pour density) and MT 75 (suspensibility) were employed to satisfy FAO specification 441/OD for oil-based formulations, with an in-house specification for droplet size D₅₀ 12 µm after spraying through a TeeJet XR11004 nozzle at 2.8 bar. The production sequence in a dedicated synthetic plant involves charging the carboxylic acid, n-propanephosphonic anhydride, and triethylamine into a 2,000 L stainless steel reactor under a nitrogen blanket, with subsequent vacuum-assisted filtration through a 0.5 µm sparkler filter to remove TEA-phosphate salts. The final consumer product is a 250 g/L oil dispersion fungicide approved for use on cereals in Annex I zones under Regulation (EC) 1107/2009, packaged in co-extruded HDPE/EVOH 1-L containers to prevent solvent permeation.

**Table 2: Cross-Scenario Regulatory & Processing Reference Matrix**
Application Sector	Key Compliance Standard	Critical Quality Attribute	Limit / Specification	Test Method
Antiviral API Intermediate	ICH Q3C (Residual Solvents)	Residual THF	≤720 ppm	GC-HS per USP ⟨467⟩
Xanthine Oxidase Inhibitor	ICH M7(R2) (Mutagenic Impurities)	Hydrazine / Hydrazone	≤1.5 µg/day TTC	LC-MS/MS SIM
In-furrow Nematicide	40 CFR 158 (EcoTox)	Daphnia magna EC₅₀ (48 h)	> 100 mg/L	OECD 202
Cereal Fungicide OD	FAO Spec 441/OD	Suspension Stability	≥90%	CIPAC MT 184
OLED Host Dopant	IEC 62321-6:2015	Phthalate/PAH Contamination	≤100 ppm total PAHs	GC-MS after Soxhlet

Vacuum-gradient sublimation-purified 2-phenylthiazole-4-carboxylic acid has functioned as a pendant ligand precursor for red-emitting europium(III) ternary complexes employed in the emissive layer of inkjet-printed OLED sub-pixels. The acid is first esterified to its ethyl ester, then saponified with LiOH to form the Li-salt for compatibility with the ethyl acetate/cyclohexanone ink vehicle. In this niche, the formula loading of the final Eu³⁺ complex in the host-polystyrene matrix-blend ink is controlled at 5.0 ± 0.3 wt%, as sensitization efficiency drops sharply outside this range due to triplet quenching. The deposition process requires a Fujifilm Dimatix DMP-2850 printer with a 10 pL cartridge jetting onto a PEDOT:PSS hole-injection layer, followed by post-bake at 80 °C under nitrogen circulating in a MBraun glovebox (O₂ < 0.1 ppm, H₂O < 0.5 ppm). Compliance substantiation relies on a full material disclosure per IEC 62474 and testing per ISO 17025-accredited labs for restricted substances under the EU RoHS Directive 2011/65/EU (recast), including cadmium, hexavalent chromium, and polybrominated diphenyl ethers, all below their respective 100 ppm threshold levels. The resulting printed device exhibits a current efficiency of 8.2 cd/A at 1,000 cd/m² when integrated into a bottom-emission panel structure with a semi-transparent Mg:Ag cathode. Published reproducibility data for this specific configuration remains limited to academic pilot-line demonstrations, with batch-to-batch carboxylic acid purity (99.95% by DSC) being the single strongest predictor of the external quantum efficiency standard deviation across 12 consecutively printed 2-inch Gen 2 substrates.

Why Is Residual Water Content a Critical Process Parameter in Downstream Amide Couplings?

When 2-phenyl-1,3-thiazole-4-carboxylic acid is consumed as an advanced intermediate in medicinal chemistry workflows, the water content of the lot—most commonly measured by Karl Fischer coulometric titration per USP ⟨921⟩ Method Ia—governs the efficiency of carbodiimide-mediated activation. Lot-to-lot variation in moisture above 0.15% w/w has been observed to depress isolated amide yields by 8–12 absolute percentage points in model reactions using EDC·HCl and HOBt in anhydrous DMF, because adventitious water hydrolyses the O-acylisourea intermediate faster than nucleophilic attack by the amine. For this reason, production-scale batches intended for GMP intermediate use are routinely dried under high vacuum (≤10 mbar) at 40 °C for 16–24 hours and immediately sealed in double polyethylene-lined aluminium laminate bags under nitrogen. A specification of ≤0.10% water (w/w) is enforced for material destined for moisture-intolerant transformations; this limit aligns with the internal acceptance criteria of several contract manufacturing organisations operating under ICH Q7A guidelines. Published data for the specific moisture-tolerance window of this compound under continuous flow activation remains limited, though single-pass residence times below 30 seconds in a PFA microreactor (ID 0.5 mm) eliminate measurable hydrolysis in preliminary screening.

In parallel, residual ethanol or ethyl acetate from the final recrystallisation step—quantified via headspace GC-FID against a reference solution of USP ⟨467⟩ residual solvent Class 3 standards—is maintained below 500 ppm to prevent transesterification side reactions when the acid is converted to the corresponding acyl chloride using thionyl chloride or oxalyl chloride. ICH Q3C(R6) Option 2 concentration limits serve as the compliance baseline.

Table 1 — Representative Certificate-of-Analysis Data for a 25-kg Commercial Lot
Attribute	Method	Specification	Measured Value
Appearance	Visual (USP ⟨1⟩)	Off-white crystalline powder	Conforms
Assay (anhydrous)	HPLC, area% (USP ⟨621⟩)	≥98.0%	99.3%
Melting range	DSC, 10 K·min⁻¹ (ASTM D3418)	178–182 °C	180.1 °C onset
Water (KF)	Coulometric (USP ⟨921⟩ Ia)	≤0.10%	0.07%
Residual solvent — ethanol	HS-GC (USP ⟨467⟩)	≤500 ppm	210 ppm
Heavy metals (as Pb)	USP ⟨231⟩ Method II	≤20 ppm	<10 ppm
Sulphated ash	USP ⟨281⟩	≤0.2%	0.06%

Table 1 — Representative Certificate-of-Analysis Data for a 25-kg Commercial Lot

Attribute

Method

Specification

Measured Value

Appearance

Visual (USP ⟨1⟩)

Off-white crystalline powder

Conforms

Assay (anhydrous)

HPLC, area% (USP ⟨621⟩)

≥98.0%

99.3%

Melting range

DSC, 10 K·min⁻¹ (ASTM D3418)

178–182 °C

180.1 °C onset

Water (KF)

Coulometric (USP ⟨921⟩ Ia)

≤0.10%

0.07%

Residual solvent — ethanol

HS-GC (USP ⟨467⟩)

≤500 ppm

210 ppm

Heavy metals (as Pb)

USP ⟨231⟩ Method II

≤20 ppm

<10 ppm

Sulphated ash

USP ⟨281⟩

≤0.2%

0.06%

Regioisomeric Purity and the Challenge of C2/C4 Positional Interchange

A specification parameter often overlooked during early-stage sourcing is regioisomeric purity with respect to 2-phenyl-1,3-thiazole-5-carboxylic acid (CAS 53178-49-1). The two isomers co-elute under many reversed-phase HPLC conditions employing a C18 column and a water/acetonitrile + 0.1% TFA gradient; however, their proton NMR spectra differ diagnostically at the thiazole C5-H resonance (δ 8.27 ppm, d, ⁴J = 1.9 Hz for the 4-isomer vs δ 8.44 ppm, s for the 5-isomer in DMSO-d₆). Manufacturers supplying kilogram-scale quantities for IND-enabling toxicology studies typically include a specific test for the 5-isomer by ¹H NMR (sensitivity: ≤0.5% w/w) or by a dedicated HPLC method on a phenyl-hexyl stationary phase capable of baseline resolution (R_s ≥ 1.5). The presence of trace 5-carboxy isomer is consequential when the building block is used to prepare patent-defining chemical series, because the pharmacological activity of thiazole positional isomers can diverge by more than two orders of magnitude at isolated enzyme targets.

When the 4-Carboxy Handle Outperforms the 5-Carboxy Analogue in Fragment-Based Screening

Fragment elaboration libraries constructed around the 2-phenylthiazole-4-carboxylic acid scaffold have yielded hit compounds that exploit the divergent vector angle of the carboxylate to reach a recognition pocket inaccessible to the 5-substituted counterpart. In crystallographic complexes deposited in the Protein Data Bank, the 4-carboxylate moiety frequently engages a conserved arginine residue via a bidentate salt bridge at an O···N distance of 2.7–2.9 Å, while the phenyl ring inserts into a hydrophobic cleft. The substitution pattern at the 4-position tolerates activation with diisopropylcarbodiimide and subsequent coupling with heterocyclic amines without racemisation or decarboxylation—stability that is less pronounced in the 2-carboxy analogue, which thermodynamically favours decarboxylation above 120 °C. Preclinical synthesis campaigns involving 30–50 analogues on a 20-mmol scale have adopted a two-step telescoped procedure: acid chloride formation with oxalyl chloride ( 1.05 equiv) and catalytic DMF in dichloromethane at 0–5 °C, followed by addition of a primary amine and triethylamine, achieving crude purities sufficient for reversed-phase mass-directed purification without an aqueous work-up.

Differences from the simple 2-phenylthiazole (lacking the carboxylic acid) are pronounced: the polar surface area of the 4-carboxylic acid derivative (78.4 Å² calculated) improves aqueous solubility at pH 7.4 from 8 µg·mL⁻¹ to approximately 280 µg·mL⁻¹ when the carboxylate is ionised, as measured by shake-flask equilibrium solubility per ICH Q2(R1) protocol.

Table 2 — Comparison of 2-Phenylthiazole Carboxylic Acid Positional Isomers
Parameter	4-COOH Isomer	5-COOH Isomer	2-COOH Isomer
CAS Number	59020-35-9	53178-49-1	1219-23-9
Melting point (°C)	178–182	216–218 (dec.)	187–189
pK_a (calculated, COOH)	3.2 ± 0.1	2.9 ± 0.1	2.4 ± 0.1
Thermal decarboxylation onset (°C, DSC)	Not observed below 250	Not observed below 250	Exotherm at 162
Typical application bias	Kinase hinge binders, antibacterial leads	PPARγ modulators, uric acid transporter inhibitors	Nitrile or tetrazole isostere replacement
Commercial availability (USP/EP- grade)	Bulk (25-kg drums)	Bulk and custom synthesis	Research gram scale

Table 2 — Comparison of 2-Phenylthiazole Carboxylic Acid Positional Isomers

Parameter

4-COOH Isomer

5-COOH Isomer

2-COOH Isomer

CAS Number

59020-35-9

53178-49-1

1219-23-9

Melting point (°C)

178–182

216–218 (dec.)

187–189

pK_a (calculated, COOH)

3.2 ± 0.1

2.9 ± 0.1

2.4 ± 0.1

Thermal decarboxylation onset (°C, DSC)

Not observed below 250

Exotherm at 162

Typical application bias

Kinase hinge binders, antibacterial leads

PPARγ modulators, uric acid transporter inhibitors

Nitrile or tetrazole isostere replacement

Commercial availability (USP/EP- grade)

Bulk (25-kg drums)

Bulk and custom synthesis

Research gram scale

Supply Chain and Regulatory Envelope

The substance is listed in the European inventory of existing commercial chemical substances (EINECS) under number 261-559-2, facilitating pre-registration under REACH for quantities imported above 1 metric tonne per annum. US TSCA inventory listing is confirmed. The typical certificate of analysis includes a declaration of compliance with ICH M7(R1) for mutagenic impurities, supported by an in silico Derek Nexus assessment of the synthetic route that flags no structural alerts for DNA-reactive impurities above the 1.5-µg/day threshold of toxicological concern. Transportation classification under 49 CFR and IATA DGR falls as “Environmentally Hazardous Substance, Solid, n.o.s. (2-Phenyl-1,3-thiazole-4-carboxylic acid),” UN 3077, Class 9, Packing Group III, which necessitates triple-packaged containment with a mineral fibre cushion for air freight.

Differences from simpler aryl carboxylic acids such as benzoic acid derivatives emerge in photostability. Exposure of the solid to direct sunlight (≥50 klux) for 48 hours induces a colour shift to amber with 0.4% degradation as measured by HPLC, likely due to thiazole ring-oxidation. Storage in amber glass or opaque HDPE containers at controlled room temperature (20–25 °C) is recommended. Re-test interval under ICH Q1A(R2) long-term conditions (Zone II, 25 °C/60% RH) is validated at 36 months for the unopened primary container.

Obtaining Uniform Dispersion in Solid-Phase Peptide Synthesis Pre-loading

When the compound is employed as a capping agent or a side-chain modifier in solid-phase synthesis, its moderate solubility in dichloromethane (~14 mg·mL⁻¹ at 20 °C) and DMF (~85 mg·mL⁻¹) necessitates an activation protocol distinct from that of the 5-isomer, which exhibits approximately 1.5-fold higher solubility in DMF. To achieve uniform pre-loading on Wang or 2-chlorotrityl chloride resins, the 4-COOH derivative is introduced as a pre-formed symmetrical anhydride (using DIC, 0.5 equiv, in DCM/DMF 9:1 v/v) rather than the free acid. Unreacted sites are capped with acetic anhydride/pyridine (1:1 v/v) for 20 minutes. Resin loading values verified by Fmoc cleavage UV quantification at 301 nm typically fall within 0.2–0.4 mmol·g⁻¹.

In high-throughput parallel synthesis arrays where 96-well plates containing pre-weighed 15-µmol aliquots of the acid are distributed, robotic liquid handlers must be programmed with an extra aspiration/dispense cycle when the tip touches a DMF solution concentrated to 0.3 M, to compensate for the viscosity contrast relative to the neat solvent. These handling idiosyncrasies, while minor, distinguish 2-phenyl-1,3-thiazole-4-carboxylic acid from the more freely soluble 5-isomer and affect throughput in automated discovery laboratories.

HS Code	446875
Chemical Formula	C10H7NO2S
Molar Mass	205.23 g/mol
Appearance	Solid (usually white to off - white)
Physical State At Room Temperature	Solid
Melting Point	Typically in a certain range (needs specific experimental data)
Solubility In Water	Low solubility (due to non - polar aromatic and heterocyclic parts)
Solubility In Organic Solvents	Soluble in some organic solvents like ethanol, dichloromethane
Pka Value	Characteristic value related to its acidic nature (specific experimental determination required)
Odor	May have a faint, characteristic odor
Stability	Stable under normal storage conditions, but may react with strong oxidizing or reducing agents

Packing	100g of 2 - Phenyl - 1,3 - Thiazole - 4 - Carboxylic Acid packaged in a sealed plastic bag.
Shipping	2 - Phenyl - 1,3 - Thiazole - 4 - Carboxylic Acid is shipped in properly sealed containers, compliant with chemical transport regulations. Shipment is via approved carriers, ensuring secure handling and protection from environmental factors.
Storage	2 - Phenyl - 1,3 - Thiazole - 4 - Carboxylic Acid should be stored in a cool, dry place away from direct sunlight. Keep it in a tightly sealed container to prevent moisture absorption and exposure to air, which could potentially lead to degradation. Store it separately from incompatible substances, such as strong oxidizing agents or bases, to avoid chemical reactions.