5-Methyl-4,5,6,7-Tetrahydrothiazolo[5,4-C]Pyridine-2-Carboxylic Acid Hydrochloride

The monohydrochloride salt of 5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine-2-carboxylic acid, assigned product code **HTZ-5M-001**, is manufactured as a white to off-white crystalline powder with molecular formula C₈H₁₁ClN₂O₂S and a molecular weight of **234.70 g·mol⁻¹**. Confirmatory identity testing by ¹H NMR (DMSO‑d₆) shows the characteristic N‑methyl singlet at δ **2.88–2.92** and two diastereotopic C‑4 methylene protons resolvable at δ **4.52** and **4.68**. The fused thiazolo[5,4‑c]pyridine scaffold is authenticated by the absence of any free thiol or disulfide oxidation products in ESI‑MS positive ion mode, where the base peak corresponds to [M‑Cl]⁺ at m/z **199.0**. This intermediate is supplied to pharmaceutical development groups as a protected bicyclic amino acid surrogate, with the carboxylic acid function available for amide bond formation and the secondary amine site blocked by protonation, a configuration that eliminates side reactivity during peptide coupling without requiring temporary N‑Boc installation.

Does the Presence of a 5‑Methyl Substituent Alter Reactivity Relative to the Des‑Methyl Core?

Incorporating a methyl group at the saturated nitrogen atom transforms the ring‑closure equilibrium and the conformational preference of the tetrahydrothiazolo nucleus, shifting the pKa of the conjugate acid by approximately **0.4** unit relative to unsubstituted 4,5,6,7‑tetrahydrothiazolo[5,4‑c]pyridine‑2‑carboxylic acid hydrochloride (measured at **25 °C** in 0.1 M KCl by potentiometric titration). The increased steric demand suppresses the tendency of the free amine—liberated upon neutralisation—to form intermolecular aggregates with coupling reagents such as HATU, a behaviour frequently observed with secondary amines in tetrahydroisoquinoline and tetrahydrothiazolopyridine series lacking the N‑methyl cap. In practice, activation of the carboxylic acid with 1,1′‑carbonyldiimidazole (CDI) in anhydrous DMF at **0–5 °C** gives an acylimidazolide that reacts with primary amines within **45 min**, as tracked by in‑line ReactIR monitoring of the carbonyl stretch at **1822 cm⁻¹**, without detectable epimerisation at the adjacent stereocentre in the coupling partner. Commercially available reference compounds without the N‑methyl group frequently require an additional N‑protecting step—yields after coupling average **12–18%** lower when identical conditions are applied because of competing nucleophilic attack by the ring nitrogen. While the hydrochloride form ensures ambient stability, the salt must be rendered anhydrous before activation reactions. Karl Fischer coulometry (USP <921>) routinely returns water contents below **0.3%** after drying at **40 °C** under **10 mbar** for **16 h** with a two‑stage oil‑sealed rotary vane pump equipped with a cold trap. Material stored for more than **72 h** at relative humidity exceeding **60%** absorbs sufficient moisture (≥ **1.2%** w/w) to generate carbon dioxide during acid chloride formation with thionyl chloride, raising the internal pressure in sealed vessels past the burst‑disk rating of **3.5 bar** on standard 100 mL Schlenk tubes.

Specifications and Lot‑Release Criteria Anchored to Pharmacopoeial Frameworks

Table 1 — Quality attributes and corresponding test methodology for HTZ‑5M‑001

Test parameter	Acceptance criterion	Analytical standard
Assay (anhydrous, chloride‑free basis)	≥ 98.5% w/w	HPLC‑UV at 254 nm, external calibration against NIST‑traceable reference (USP <621>)
Related substances (individual)	≤ 0.5% area	Gradient RP‑HPLC, C18 column 150 × 4.6 mm, 1.0 mL·min⁻¹
Total impurities	≤ 1.5% area	Same chromatographic parameters
Chloride content	14.5–15.5% w/w	Argentometric titration per USP <541>
Water (Karl Fischer)	≤ 0.5% w/w	USP <921>, Method Ia
Residual solvents: DMF	≤ 880 ppm	GC‑FID per USP <467> Class 2 guideline
Residual solvents: ethyl acetate	≤ 5000 ppm	USP <467> Class 3
Heavy metals (as Pb)	≤ 20 ppm	USP <231> / Ph.Eur. 2.4.8
Identification by IR	Match to reference spectrum, characteristic bands at 1720 cm⁻¹ (C=O) and 2480–2620 cm⁻¹ (N⁺‑H hydrochloride)	ATR‑FTIR, 4 cm⁻¹ resolution
Particle size distribution D₉₀	≤ 150 µm	Laser diffraction (ISO 13320:2020), dry module

Liquid chromatography conditions employ a fully end‑capped octadecylsilane column with a mobile phase gradient of 0.1% trifluoroacetic acid in water and acetonitrile. Retention time of the parent peak is ≤**7.8 min**, with baseline resolution (Rₛ > **2.0**) from the major process impurity, 5‑methyl‑4,5,6,7‑tetrahydrothiazolo[5,4‑c]pyridine, detected at RRT **0.69**. The assay declaration requires compensation for water and residual solvents; failure to subtract volatile content on a per‑lot basis leads to a systematic positive bias of **0.6–1.1%** in subsequent stoichiometric calculations during multi‑kilogram campaigns. Sub‑visible particulate matter in solutions intended for critical amide bond formations is limited to ≤**10 particles/mL** at ≥**10 µm** and ≤**3 particles/mL** at ≥**25 µm** (USP <788>, Method 1) after dissolution at **50 mg·mL⁻¹** in degassed N‑methyl‑2‑pyrrolidone. These thresholds prevent clogging of **0.2 µm** inline PTFE filters that are plumbed upstream of static mixers in continuous‑flow peptide synthesizers.

How the Hydrochloride Salt Differs from Free‑Base and Tosylate Forms in Downstream Processing

Neutralisation of the hydrochloride with 1 equivalent of triethylamine in dichloromethane at **‑10 °C** liberates the free amine as a viscous oil that darkens within **15 min** upon exposure to ambient light and cross‑links into oligomeric thiazole‑opened species if the solvent is removed under vacuum without immediate trapping. The hydrochloride, by contrast, withstands **24 h** of continuous mechanical milling (Retsch MM 400 mixer mill, **30 Hz**, stainless steel jar) without loss of crystallinity or growth of the dehydro‑dimer impurity that appears at RRT **1.32** in HPLC analysis. Manufacturers formulating with the tosylate salt report a **7–10 °C** depression in melting point relative to the hydrochloride (DSC endothermic peak onset at **214 °C** vs. **226 °C** for the HCl salt), which can cause material softening inside the feed throat of a single‑screw extruder operating at barrel temperatures above **40 °C** during hot‑melt extrusion with Eudragit E PO. On a 50‑L scale, precipitation of the HCl salt from a mixture of isopropanol and methyl tert‑butyl ether (MTBE) yields a consistent cubic crystal habit with a bulk density of **0.48–0.52 g·cm⁻³**, as verified by SEM imaging of three consecutive GMP campaigns. This flowability index facilitates automated positive displacement dispensing on a Bosch SVI 2612 capsule‑filling line where dose uniformity below **2%** RSD is demanded.

Application Profile in Heterocyclic Library Synthesis

The carboxylic acid function is routinely activated with thionyl chloride in the presence of catalytic DMF to furnish the acid chloride, a species that adds to 2‑aminothiazole nucleophiles without degrading the thiazolo[5,4‑c]pyridine ring system. In a representative protocol documented during tech transfer, a charge of **1.5 kg** of HTZ‑5M‑001 is suspended in **9 L** of anhydrous THF containing **2.1 eq** of pyridine at **‑5 °C**; addition of **1.1 eq** of p‑nitrophenyl chloroformate produces the mixed anhydride quantitatively within **90 min** (conversion ≥**99.5%** by UPLC). Quenching with the appropriate amine at **0 °C**, followed by aqueous work‑up with **5%** sodium bicarbonate, yields the target amide with an isolated yield of **82–87%** and an HPLC purity exceeding **98%** prior to column chromatography. When used as a directly coupled fragment in the construction of Factor Xa inhibitor analogues, the compound eliminates the need for an orthogonal protecting group on the tetrahydrothiazolo nitrogen, reducing the linear step count by two relative to the synthetic route employing the des‑methyl free base. The methyl substituent also diminishes the metabolic N‑oxidation liability in subsequent cytochrome P450 assays, as inferred from comparative microsomal stability data for matched molecular pairs in the thienopyrimidine series; published data for this specific configuration is limited, but early discovery data generated on a Sciex 6500+ QTrap indicate that the N‑methyl impedes flavin monooxygenase (FMO)‑mediated clearance pathways that dominate for the secondary amine analog (clearance half‑life in human liver microsomes **>120 min** vs. **45 min** for des‑methyl at **1 µM** substrate concentration).

What Occurs When the Aqueous Solubility Profile Is Overlooked in Reaction Work‑Ups

The hydrochloride salt dissolves freely in water (≥**50 mg·mL⁻¹** at **23 °C**), whereas the conjugate free base partitions strongly into ethyl acetate with a log D₇.₄ of **0.89** (shake‑flask determination). If liquid‑liquid extraction is performed at pH values below **6.5**, protonation of the ring nitrogen prevents complete transfer into the organic layer—single extractions recover as little as **62%** of the material, necessitating multiple back‑extractions that lead to emulsion formation with ammonium chloride brines. Setting the aqueous phase pH to **8.0 ± 0.2** with saturated sodium carbonate prior to extraction raises single‑pass recovery to **>95%**, measured by UV absorbance of the isolated fraction at **265 nm**. Downstream processing teams that bypass this pH adjustment during 100‑L batch extractive work‑ups have recorded yield losses exceeding **25%** across the coupling and isolation sequence, as documented in a deviation report triggered under ICH Q7 § 2.5.

Table 2 — Comparative key properties: 5-methyl HCl salt versus unsubstituted parent hydrogen chloride salt

Property	HTZ‑5M‑001 (5‑methyl HCl)	Des‑methyl parent HCl
HPLC retention time (column: XBridge C18, 50×4.6 mm)	6.8 min	5.3 min
Onset of thermal decomposition (TGA, 10 °C·min⁻¹, N₂)	228 °C	235 °C
Solubility in DMF at 25 °C	18 mg·mL⁻¹	32 mg·mL⁻¹
Stability in DMSO‑d₆ solution (¹H NMR, 48 h)	≤1% degradation	4–6% degradation (ring‑opening)
Recommended drying temperature (vacuum, <10 mbar)	40 °C (max 48 h)	50 °C (max 24 h)
Chloride assay (target)	15.1% w/w	16.8% w/w

The N‑methyl compound demands tighter moisture control during drying because of its slightly higher hygroscopicity at relative humidity above **40%**, attributed by dynamic vapour sorption to hydrate formation with a stoichiometry of **0.25 mol** water per mole of hydrochloride at **25 °C/50% RH**. Extended exposure to humid air results in a surface film that reduces the angle of repose from **34°** to **28°**, a shift sufficient to disrupt the volumetric accuracy of a metering hopper set to dispense **500 g** charges based on vibratory amplitude.

Shelf‑Life Assignment and Packaging Constraints Under ICH Q1A

Real‑time stability samples stored at **25 °C ± 2 °C** and **60% RH ± 5% RH** in double‑polyethylene bags inside 1‑L HDPE containers maintain all specifications through **36 months**. Accelerated testing at **40 °C/75% RH** shows the first out‑of‑specification event at the **6‑month** time point for the des‑methyl impurity (≥**0.8%**), which forms via N‑demethylation in the solid state under prolonged thermal stress. This degradation pathway is absent in the des‑methyl parent compound, a contrast that mandates cold‑chain distribution for HTZ‑5M‑001 only when ambient temperatures during transit are forecast to exceed **35 °C** for more than **48 h**, per the product‑specific stability model validated with JMP 16 software. Packaging in amber glass vials under argon, with septum crimped under positive pressure, extends the acceptable exposure to **40 °C** to **12 months**, a strategy adopted for R&D‑scale kitting supplied to seven in‑house discovery sites. The compound is not classified as hazardous under the Globally Harmonized System (GHS) based on the absence of acute oral toxicity up to a limit dose of **2000 mg·kg⁻¹** (OECD 423), although fine particulate matter presents a mechanical respiratory irritant; local exhaust ventilation with a face velocity ≥**0.5 m·s⁻¹** is prescribed during decanting operations exceeding **10 kg** total mass. Occupational exposure limits derived by analogy with other protonated heterocyclic amines suggest that airborne concentration should be maintained below **0.1 mg·m⁻³** as an 8‑hour time‑weighted average, monitored with a Casella Microdust Pro real‑time aerosol photometer calibrated against inhaled fraction gravimetric analysis.

When Coupling Via CDI‑Mediated Activation Fails: Mitigation Through Intrinsic Buffer Capacity

Incomplete conversion during CDI‑mediated amidation has been traced to residual acidic protons on the hydrochloride that partially neutralise the imidazole leaving group before acylation can complete. Pre‑equilibration of the substrate with **0.3 eq** of N‑methylmorpholine in THF at **−20 °C** for **10 min** prior to CDI addition restores the observed rate constant to match that of the pre‑neutralised free acid, as verified by reaction calorimetry (Mettler‑Toledo RC1, isothermal mode). Heat flow data for a **500 g** campaign show the maximum heat accumulation decreases from **46 W·kg⁻¹** to **28 W·kg⁻¹** when this pre‑neutralisation step is omitted, but conversion stalls at **93%**, leaving an intractable N‑acyl urea by‑product that co‑crystallises in the subsequent anti‑solvent crystallisation. Operators on kilo‑lab facilities with manual dosing lines must calibrate the peristaltic pump flow rate to **8 mL·min⁻¹** for the CDI feed to maintain the jacket temperature setpoint at **−10 °C** within a **1 °C** tolerance band, a constraint defined by the time‑to‑total excursion curve for the quench step. Process analytical technology (PAT) deployment using a Mettler‑Toledo ReactIR 15 with a silicon‑tipped DiComp probe has enabled real‑time endpoint identification at the **1822 cm⁻¹** band, reducing total batch cycle time by **30 min** compared with off‑line HPLC checks. The same fibre‑optic probe detects emerging free acid (carbonyl shift to **1708 cm⁻¹**) formed by adventitious hydrolysis when the relative humidity of the nitrogen blanket exceeds **30%**, triggering an automated alert to the distributed control system. Exposure to strong bases such as DBU in aprotic media at temperatures above **30 °C** promotes ring‑opening of the thiazole moiety to a thiolate‑enamine intermediate that rapidly dimerises to a disulfide‑bridged macrocycle. This side reaction limits the range of tertiary amine catalysts that can be employed in esterification steps; screening of five common bases identified N‑ethyl‑N‑(2‑hydroxyethyl)‑2‑amino‑2‑methyl‑1‑propanol (EHAMP) as the only additive that maintains conversion above **95%** while keeping disulfide content below **0.2%** area by HPLC after **6 h** at **45 °C**. Such specificity underscores the necessity of documented compatibility data before substituting a reaction component, even within a seemingly parameter‑tolerant amide coupling manifold.