Dl-6-Phenyl-2,3,5,6-Tetrahydroimidazole(2,1-B)Thiazole-Hydrochloride

Chemical Identity and Specification Parameters

The compound identified as Dl-6-Phenyl-2,3,5,6-Tetrahydroimidazole(2,1-B)Thiazole-Hydrochloride corresponds to the racemic hydrochloride salt of tetramisole, systematically named (RS)-6-phenyl-2,3,5,6-tetrahydroimidazo[2,1-b][1,3]thiazole hydrochloride. The product carries CAS Registry Number 5086-74-8 and molecular formula C₁₁H₁₂N₂S·HCl with a formula weight of 240.75 g·mol⁻¹. Commercial lots intended for research or veterinary active pharmaceutical ingredient (API) synthesis are typically supplied as a white to off-white crystalline powder with a melting point range of 264–266 °C (decomposition) when determined by differential scanning calorimetry at a heating rate of 10 K·min⁻¹ under nitrogen purge. A representative certificate of analysis for a synthesis-grade batch (product code DLTZ-HCl-P) specifies minimum purity of 98.5% by HPLC (area normalization, UV detection at 254 nm) with the L-isomer (levamisole) content not exceeding 2.0% and total related substances below 0.8% as per the monograph for Tetramisole Hydrochloride in the European Pharmacopoeia (Ph. Eur. 10.3, 01/2008:0298). Loss on drying at 105 °C for 2 hours is controlled to ≤0.5%. Residual solvents are tested according to ICH Q3C guidelines, with limits for ethanol and isopropanol set at ≤5000 ppm and ≤5000 ppm respectively. Sulfated ash remains below 0.1%.

Typical batch-release parameters for Dl-Tetramisole Hydrochloride

Parameter	Method	Acceptance Criterion
Appearance	Visual inspection	White to off-white crystalline powder
Assay (anhydrous basis)	Potentiometric titration (HClO4)	99.0–101.0%
Purity (HPLC)	Ph. Eur. 2.2.29, C18 column, acetonitrile/phosphate buffer pH 3.0	≥98.5%
L-Isomer (levamisole)	Chiral HPLC (Chiralpak IA, hexane/ethanol/DEA)	≤2.0%
Water (Karl Fischer)	Ph. Eur. 2.5.12	0.2–0.8%
Heavy metals	Ph. Eur. method A, 2.4.8	≤20 ppm

The racemic nature of the product has direct consequences for biological activity: only the L-enantiomer (levamisole) provides the primary cholinergic anthelmintic effect, while the D-isomer contributes to the compound’s immunomodulatory properties through distinct mechanisms. In analytical laboratories, the product serves as a reference standard for chiral method validation, where separation of D- and L-enantiomers on a polysaccharide-based chiral stationary phase achieves baseline resolution with a separation factor α ≥ 1.15 under optimized conditions (mobile phase: n-hexane/ethanol 80:20 v/v with 0.1% diethylamine).

What Distinguishes the Racemic Hydrochloride from the Pure L-Isomer Base?

Practitioners unfamiliar with the subtle pharmacology of the imidazothiazole class may assume interchangeability between tetramisole hydrochloride and levamisole base or levamisole hydrochloride. The distinctions are non-trivial. Tetramisole hydrochloride (CAS 5086-74-8) is the DL-racemic hydrochloride salt, whereas levamisole hydrochloride (CAS 16595-80-5) corresponds to the L-(−)-isomer. The free base forms differ further in solubility profiles: DL-tetramisole free base (CAS 5036-02-2) exhibits a water solubility of ~4 g·L⁻¹ at 20 °C, while levamisole base is slightly less soluble. The hydrochloride salts, however, both show drastically improved aqueous solubility exceeding 200 g·L⁻¹, enabling high-concentration drench formulations for ruminants. In anthelmintic applications governed by VICH GL7 efficacy guidelines, the racemic hydrochloride retains roughly half the nematocidal potency of an equivalent mass of levamisole hydrochloride, because only the L-enantiomer acts as a nicotinic acetylcholine receptor agonist in nematode somatic muscle. Consequently, formulators targeting a dose of 7.5 mg levamisole·kg⁻¹ body weight must account for a 1.8–2.0 times higher input mass of the racemate. In ovine trials conducted under GCP conditions, fecal egg count reduction (FECR) at 14 days post-treatment with tetramisole HCl at 15 mg·kg⁻¹ reached 97.3% (95% CI: 94.8–99.8%), comparable to levamisole HCl at 7.5 mg·kg⁻¹, provided Ostertagia circumcincta and Haemonchus contortus populations remain susceptible. Published data for benzimidazole-resistant isolates show cross-resistance to imidazothiazoles is not automatic but has been documented in multi-drug-resistant Teladorsagia strains in the UK, where levamisole efficacy dropped below 80%, necessitating careful regional monitoring.

Comparative properties: DL-Tetramisole HCl vs. Levamisole HCl

Property	DL-Tetramisole Hydrochloride	Levamisole Hydrochloride
CAS Number	5086-74-8	16595-80-5
Optical rotation [α]D20	0° (racemate)	−124° ± 2° (c = 1, H2O)
Typical anthelmintic dose (sheep, oral)	15 mg·kg−1	7.5 mg·kg−1
Solubility in water at 25 °C	≥210 g·L−1	≥200 g·L−1
Immunomodulatory effect (in vitro)	Present, attributed to D-isomer	Weak; requires racemic mixture or pure D-isomer for full effect
Regulatory status (Veterinary)	VICH GL7, MRP/DCP in EU	Same; included in Ph. Eur. monographs

When Alkaline Phosphatase Inhibition Is the Primary Screening Target

Beyond parasitic control, imidazothiazole derivatives have found a distinct niche in biochemical research as tissue-nonspecific alkaline phosphatase (TNAP) inhibitors. Levamisole, and by extension the racemic tetramisole hydrochloride, acts as a noncompetitive inhibitor of TNAP with a reported IC₅₀ value of 19 µM in human bone-derived cell lysates under standard assay conditions (pH 9.8, diethanolamine buffer, 1 mM MgCl₂, substrate p-nitrophenyl phosphate at 10 mM). The racemate is frequently employed as an exogenous inhibitor in flow cytometry protocols to confirm the identity of TNAP-positive cell populations, because its cost per gram is typically 40–60% lower than that of pure levamisole hydrochloride without sacrificing specificity in this application. A working concentration of 1–5 mM in isotonic buffer is common; however, lot-to-lot variation in purity must be monitored through control experiments with heat-denatured enzyme aliquots. For laboratories performing in situ hybridization or immunohistochemistry on paraffin-embedded tissue sections, inclusion of tetramisole HCl at 0.24 mg·mL⁻¹ in the substrate-chromogen mixture (NBT/BCIP) suppresses endogenous alkaline phosphatase activity that would otherwise produce diffuse background staining. The protocol, adapted from the manufacturer’s instructions for the DIG RNA Labeling Kit (Roche, Cat. No. 11175025910), requires that the inhibitor be added freshly to the detection buffer upon each run because hydrolytic degradation of the imidazothiazoline ring proceeds with a half-life of approximately 18 hours at room temperature in alkaline media (pH 9.5). Stock solutions at 100× concentration (24 mg·mL⁻¹ in deionized water) can be stored at −20 °C in single-use aliquots for up to 12 months without loss of potency.

Navigating Incompatibilities During Aqueous Formulation

A critical operational boundary emerges when tetramisole hydrochloride is blended with alkaline buffer systems in multi-component oral drenches. The hydrochloride salt is freely soluble in neutral water but undergoes deprotonation and subsequent precipitation as the free base when the pH of the final formulation exceeds 7.5. At pH 8.0, the free base’s solubility limit of ~4 g·L⁻¹ is rapidly breached in typical drench concentrations of 80–120 g·L⁻¹, leading to crystal formation and nozzle clogging during administration via 12.5-mm internal-diameter drench guns. Production-scale compounding in stainless-steel vessels (grade 316L) should therefore maintain a pH endpoint of 4.5–6.0 using hydrochloric acid or citric acid buffers. No precipitation is observed in accelerated stability studies (40 °C / 75% RH) for 3 months when the pH is held at 5.2. Incompatibility also manifests with certain anthelmintic mixtures. When tetramisole hydrochloride is co-formulated with thiabendazole in an aqueous slurry, a slow interaction between the imidazothiazole ring and the benzimidazole carbamate results in the formation of an insoluble complex detectable by HPLC as a new peak at relative retention time 1.27 relative to tetramisole. The reaction rate is temperature-dependent, with 7.8% conversion observed after 72 hours at 30 °C versus 1.2% at 4 °C, as reported in a stability-indicating method published by the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) Q1A(R2) guidance context. Hence, combination drenches are generally formulated as suspensions of the benzimidazole with the imidazothiazole dissolved in the liquid phase, or they are packaged as two-component systems separated until point of use. Direct contact with strong oxidizing agents—including hypochlorite-based sanitation solutions used for line clean-in-place (CIP) cycles—must be avoided. Spills on equipment should be removed with a 5% aqueous sodium metabisulfite solution prior to CIP, because oxidative cleavage of the thiazole sulfur yields sulfonic acid derivatives with unknown toxicological profiles.

Storage Stability Under Tropical Climate Conditions

Long-term storage of bulk tetramisole hydrochloride in non-climate-controlled warehouses, particularly in ICH Zone IVb regions (e.g., Southeast Asia, Northern Australia), requires pre-drying of the double polyethylene-lined fiber drums if the ambient relative humidity exceeds 60% at the time of packing. The product’s moisture sorption isotherm, measured by dynamic vapor sorption (DVS) at 25 °C, shows a mass increase of 1.8% when RH reaches 70%, at which point caking and reduced flowability become noticeable in a powder rheometer test (Freeman FT4). Desiccant pouches containing silica gel (minimum 50 g per 25-kg drum) are standard practice. Storage at 2–8 °C is not mandatory but prolongs the time-to-failure for chiral purity from 36 months (at 25 °C/60% RH) to 60 months based on long-term stability data generated in accordance with ICH Q1E. Photostability testing per ICH Q1B Option 2 (cool white fluorescent and near-UV light) demonstrates no significant degradation over the illuminance requirement, confirming that amber glass or opaque HDPE containers are not essential for light protection, though they remain adopted throughout the supply chain as a conservative measure.

Industrial Synthesis Route and Chiral Resolution Considerations

The racemic product is typically manufactured via condensation of 2-imino-3-(2-chloroethyl)thiazolidine with styrene oxide or 2-amino-1-phenylethanol derivatives under acidic catalysis, followed by ring closure in refluxing butanol. Without a chiral auxiliary or asymmetric catalyst, the process yields a 1:1 enantiomeric mixture. Patent literature (US 3,274,209) describes the resolution using (+)-O,O′-dibenzoyltartaric acid, which preferentially crystallizes the levamisole salt while leaving the D-isomer in the mother liquor. The racemic hydrochloride offered as catalog item DLTZ-HCl-P thus represents the unresolved intermediate before this resolution step—hence its lower per-kilogram cost relative to levamisole hydrochloride, a factor of importance in large-animal veterinary markets where price sensitivity dictates formulary choices. Particle size distribution for the unmicronized powder, measured by laser diffraction (Malvern Mastersizer 3000, dry dispersion at 2 bar), shows D₅₀ values typically between 85 and 120 µm. The material is classified as “readily friable” in a ball mill with hardened steel balls (10 mm diameter, 60 rpm), reaching a D₅₀ of 12 µm after 45 minutes of milling, a property that aids suspension homogeneity in oral paste formulations for equine applications. Handling during manufacturing should follow standard industrial hygiene practices: engineering controls include local exhaust ventilation at blending and packing stations to keep airborne dust below the occupational exposure limit of 5 mg·m⁻³ (respirable fraction, 8-hour TWA) as referenced in the supplier safety data sheet. Eye irritation studies in rabbits (OECD Test Guideline 405) classify the material as a moderate irritant (GHS Category 2A). Personnel are advised to wear chemically resistant gloves (nitrile, assessed to EN 374-1:2016) and tight-fitting safety goggles (EN 166).

Regulatory Status and Residue Monitoring

Tetramisole hydrochloride is approved as a veterinary anthelmintic in multiple jurisdictions. In the European Union, maximum residue limits (MRLs) for the sum of tetramisole and its metabolites, expressed as levamisole, are established in Commission Regulation (EU) No 37/2010 (Table 1, All food-producing species): 10 µg·kg⁻¹ in muscle, 10 µg·kg⁻¹ in fat, 100 µg·kg⁻¹ in liver, and 100 µg·kg⁻¹ in kidney. The withdrawal period for ovine and bovine animals administered the racemic hydrochloride orally at the recommended dose is 14 days. The marker residue for official controls is levamisole itself, and confirmation must be performed by LC-MS/MS with a decision limit (CCα) ≤ 10.5 µg·kg⁻¹ and detection capability (CCβ) ≤ 12.0 µg·kg⁻¹, as required by Commission Decision 2002/657/EC. From a trade perspective, residue violations occasionally arise when producers inadvertently substitute racemic tetramisole for levamisole at equivalent mass doses, resulting in larger amounts of the D-isomer being excreted and cross-reacting in immunoassay screening tests. Confirmatory methods using chiral columns can distinguish the two forms, but national residue surveillance programs generally report the total levamisole-equivalent value.

Differences from Piperazine and Macrocyclic Lactone Anthelmintics

In the anthelmintic pharmacopeia, tetramisole hydrochloride occupies a distinct niche separate from piperazine salts and avermectin/milbemycin macrocyclic lactones. Piperazine dihydrochloride acts as a reversible GABA receptor agonist causing flaccid paralysis of ascarids, whereas tetramisole produces sustained contraction through nicotinic receptor depolarization and is more effective against a broader spectrum that includes lungworm (Dictyocaulus spp.) and strongyles—targets not addressed by piperazine alone. Comparative dose-finding studies in swine have shown tetramisole HCl at 10 mg·kg⁻¹ achieves 99.2% clearance of Ascaris suum adult worms versus 89.5% for piperazine citrate at 250 mg·kg⁻¹, with the advantage persisting in mixed-infection scenarios. Unlike macrocyclic lactones such as ivermectin, which induce pharyngeal pump inhibition in nematodes via glutamate-gated chloride channels, tetramisole does not confer activity against ectoparasites. The absence of a withdrawal period impact on milk production (tetramisole is not permitted in lactating dairy cattle in many jurisdictions due to incomplete residue depletion data) contrasts with moxidectin, which is specifically formulated as a pour-on for lactating cows with zero milk discard time under certain labels. These differences underscore the importance of accurate species- and production-stage targeting when selecting the racemic hydrochloride. For companion animal practice, the racemic compound sees limited use compared to the purer L-isomer because the D-isomer contribution to emetic side effects in dogs at doses above 5 mg·kg⁻¹ subcutaneous route has been documented in a clinical observation series (n=47), with 23% of animals showing vomiting within 30 minutes. This adverse effect profile, while manageable in a research setting with antiemetic premedication, reduces its suitability for routine small-animal deworming protocols relative to alternative nicotinic agents with a greater therapeutic index. Therefore, the primary commercial destination of the racemic hydrochloride remains large-animal veterinary medicine and laboratory reagent markets.