L-2,3,5,6-Tetrahydro-6-Phenylimidazo-[2,1-6]-Thiazole

Why Direct Compression of Levamisole HCl Requires Binder Optimization for USP Dissolution Compliance at the 50 mg Dose Strength?

Immediate-release tablets containing L-2,3,5,6-Tetrahydro-6-Phenylimidazo-[2,1-6]-Thiazole hydrochloride are formulated to a target potency of 50 mg per unit, with the active ingredient representing 20–25% of total tablet weight when the core mass is maintained between 200 mg and 250 mg. Pharmacopoeial standards demand conformance to USP <905> Uniformity of Dosage Units (acceptance value ≤ 15.0) and USP <711> Dissolution, where ≥ 80% (Q) of labelled content must release in 0.1 N HCl within 30 minutes using Apparatus 2 at 50 rpm. Straight direct compression with unmodified microcrystalline cellulose (Avicel PH-102) and sodium starch glycolate (4%) routinely generates tablet hardness between 4–6 kP but exhibits a disruptive sensitivity to the raw active particle morphology: needle-like crystals with an aspect ratio exceeding 3:1 produce lamination and cap formation at compression forces above 8 kN on a rotary press equipped with 8 mm round concave tooling and a turret speed of 35 rpm. A wet granulation route using an aqueous povidone K-30 solution (5% w/w) is employed to densify the active into free-flowing granules with a bulk density of 0.55–0.65 g/mL, pre-dried in a fluid bed at an inlet air temperature of 60°C to a final moisture content 2.0–3.5% as determined by loss-on-drying (LOD) at 85°C. The dried granulate is milled through a 1.0 mm screen and blended with extragranular croscarmellose sodium (3%) and magnesium stearate (0.75%, sieved through a 60-mesh screen) added in a final tumble-blending step of 5 minutes to avoid over-lubrication. The terminal product form is a white to off-white, scored, film-coated tablet packaged in aluminium/PVC blister lidding, marketed worldwide as a human anthelmintic and immunomodulator. In-process control includes friability testing according to USP <1216>, requiring mass loss ≤1.0% after 100 rotations. Published data underscores that exceeding the granulation endpoint water addition beyond 12% (relative to dry binder weight) typically causes a sharp drop in dissolution rate, attributed to gel-sheath formation around the granule core, and shifts the Q-value at 30 min below 75%.

Injectable Aqueous Solutions: Terminal Sterilization Cycle Design and Antioxidant Synergy in Phosphate-Buffered Systems

Parenteral formulations based on L-2,3,5,6-Tetrahydro-6-Phenylimidazo-[2,1-6]-Thiazole hydrochloride are prepared as a 7.5% w/v sterile solution (equivalent to 75 mg/mL of the base), typically filled into 50 mL amber Type I glass vials with a nitrogen-flushed headspace to mitigate oxidative degradation of the phenylimidazo moiety. The formulation requires a phosphate buffer system (monobasic sodium phosphate 0.52% w/v and dibasic sodium phosphate 0.12% w/v) to stabilize the pH within the 3.5–4.5 window; data from accelerated stability studies at 40°C/75%RH indicate that pH drift above 5.0 catalyzes a bimolecular condensation pathway that generates a dimeric impurity at levels exceeding the EP 0.2% identification threshold within 6 weeks. To preserve assay, sodium metabisulfite at 0.1% w/v is included as an oxygen scavenger, and the bulk solution is purged with filtered nitrogen prior to sterile filtration through a tandem 0.45 µm/0.22 µm PVDF membrane assembly. Terminal sterilization relies on a water-spray autoclave programmed to achieve an F₀ value of ≥8.0 minutes at 121.1°C, as mandated by Ph. Eur. 5.1.1 methods of sterilization, while avoiding cumulative thermal input exceeding F₀=15 to limit 4-hydroxyphenyl degradation product formation. The filling line is configured with ceramic rotary piston pumps controlled to a fill volume of 51.5 mL ± 0.8 mL, verified by in-line check-weighing on a Mettler Toledo C3000 system. The finished product form is a ready-to-use injectable solution indicated for veterinary gastrointestinal nematodiasis and human adjuvant therapy, and must pass the rabbit pyrogen test (USP <151>) with a temperature rise limit of 0.4°C per three-rabbit group. Experience from production-scale compounding vessels (> 500 L) reveals that inadequate shear during buffering salt addition results in localized alkaline pockets that initiate haze formation, a problem remediated by introducing salts via a high-shear bottom-mounted dissolver operating at 1,500 rpm for 12 minutes.

Aquaculture applications in marine shrimp (Litopenaeus vannamei) address chronic vibriosis and ectoparasitic monogenean infestation by introducing the levamisole base compound as a top-dressed feed additive precisely metered to achieve a daily oral dose of 5 mg/kg biomass. The feed incorporation step requires blending the active with fish oil and a lecithin-based emulsifier at a ratio of 1:3:0.5 w/w before coating extruded floating pellets with a vacuum coater operating under −0.85 bar absolute pressure to achieve a homogeneous surface deposition of 500 mg/kg feed. Leaching rate of the unpalatable imidazothiazole moiety into pond water is a critical economic parameter; static immersion assays in aerated seawater (35 ppt salinity, 28°C) for 30 minutes demonstrate that unprotected feed loses 38% of the active, whereas lipid-microencapsulated coatings confine the loss to 9%. Feed mills producing shrimp diets incorporate this pre-mix into a standard formulation (35% crude protein, 8% lipid) immediately after post-conditioning, ensuring that the temperature at the pellet die face never exceeds 82°C for more than 20 seconds. The produced medicated crumble (particle size 1.2–1.8 mm) is used for a 7–14 day prophylactic regimen. Regulatory compliance is assessed against Thai FDA Agricultural Standard TAS 9004-2552 for veterinary drugs in aquaculture feed and the EU Council Regulation 470/2009 for maximum residue limits, where the target tissue residue in shrimp muscle must be below 10 µg/kg after a 21-day withdrawal. Equipment bottlenecks arise from electrostatic attraction of fine API particles to the polyurethane coating drum interior, an effect suppressed by maintaining the relative humidity of the coating room above 55% RH via atomized water injection.

When Levamisole Base Is Used as a Component in Chromogenic Immunohistochemistry Detection Kits: Endogenous Alkaline Phosphatase Blocking

In formalin-fixed, paraffin-embedded (FFPE) tissue sections subjected to enzyme-based immunodetection, L-2,3,5,6-Tetrahydro-6-Phenylimidazo-[2,1-6]-Thiazole (as the free base or hydrochloride) functions as a non-competitive, uncompetitive inhibitor of tissue non-specific alkaline phosphatase (TNAP), preventing the hydrolysis of the chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) that would otherwise generate non-specific background. The optimal working concentration in blocking buffer is tightly defined at 1.0–2.0 mM, prepared by dissolving the compound in 0.1 M Tris-HCl buffer, pH 8.2, with 0.5 mM MgCl₂ as cofactor. Concentrations exceeding 5 mM have been documented in peer-reviewed histochemistry literature to completely suppress enzymatic activity to less than 5% baseline but simultaneously produce a paradoxical increase in non-specific antibody binding to connective tissue mucins. The buffer is applied to deparaffinized slides following antigen retrieval and before primary antibody incubation for a duration of 20 minutes at 22°C. The working solution is typically prepared fresh within 2 hours of use, as its potency decays by 15–20% upon storage at 4°C for 24 hours due to slow hydrolysis of the thiazole ring. The reagent-grade material must comply with the analytical specifications of a custom in-vitro diagnostic (IVD) component: purity ≥ 99.0% by HPLC, residual solvents ≤ ICH Q3C limits, and absence of phosphatase contamination as certified by a negative conversion test against α-naphthyl phosphate substrate. Production-scale kit fillers utilize an automated dispenser to aliquot 10 mL of ready-to-use inhibitor concentrate into HDPE dropper bottles under a laminar flow cabinet (ISO 14644-1 Class 5 environment). Terminal pasteurization at 63°C for 30 minutes is validated to sterilize without precipitating the active from solution. The end product type is a “AP Blocking Solution, 20× Concentrate” sold to hospital pathology labs and contract research organizations conducting tissue cross-reactivity studies.

Resolving a racemic mixture of an intermediate arylpropionic acid through diastereomeric salt formation exploits the optical activity of the (S)-(−)-enantiomer of the thiazole compound. A stoichiometric quantity of 1.05 equivalents of the resolved base is introduced to a 0.8 M solution of racemic 2-(4-isobutylphenyl)propionic acid in a mixed solvent of isopropyl acetate and ethanol (95:5 v/v). The selective precipitation of the (S)-acid-(S)-base salt occurs upon cooling from 55°C to −5°C over 8 hours, yielding a diastereomeric excess exceeding 94% after a single crystallization, according to literature procedures. After the salt is collected by a centrifuge (basket type, 900 G), the resolved acid is liberated by acidification with 2 M HCl and extracted into ethyl acetate, while the aqueous phase can be basified to recover the alkaloid base. The finished output is a > 99% ee chiral building block destined for non-steroidal anti-inflammatory drug (NSAID) synthesis, with the recovered thiazole recycled for up to 5 cycles before spectral evidence of ring oxidation appears at 1702 cm⁻¹ in FTIR. The process conforms to guidance provided in the ICH Q11 development and manufacture of drug substances, particularly sections on starting material justification. Operational boundaries are sharply defined by the solvent moisture content, which must remain below 0.1% w/w to prevent oiling-out of the diastereomeric salt.

Comparative Pharmacopoeial Specifications for Key Application Thresholds

Quality Attribute

EP 11.0 Levamisole Hydrochloride

USP 43-NF38

CP 2020 (兽药)

Test Method

Assay (anhydrous basis)

99.0–101.0%

98.5–101.0%

98.5–101.5%

Potentiometric titration in anhydrous formic acid with perchloric acid

Specific Optical Rotation

−121° to −128° (dry, c=5 in H₂O)

−121° to −128°

−121° to −127°

Polarimetry, 589 nm, 20°C

Impurity E (1-(2-aminoethyl)-3-phenylimidazolidin-2-one)

≤ 0.3%

≤ 0.5%

RP-HPLC, C18, 215 nm

Melting Point

227–231°C (decomposition)

Class 1a (mp 227–231°C)

226–231°C

Capillary tube, heating rate 1°C/min

Loss on Drying

≤ 0.5% (105°C, 4 h)

≤ 0.5%

≤ 1.0%

USP <731> / EP 2.2.32

At ambient temperature, the substance presents as a white to off-white crystalline powder with a characteristic amine odor. The molecule, systematically designated L-2,3,5,6-Tetrahydro-6-Phenylimidazo-[2,1-b]-Thiazole (CAS 14769-73-4), constitutes the levorotatory enantiomer of tetramisole and is commercially supplied as the hydrochloride salt Levamisole Hydrochloride (CAS 16595-80-5) for enhanced aqueous solubility. Identification by infrared absorption spectrophotometry (ATR-FTIR) must exhibit principal peaks at 1590 ± 5 cm⁻¹ (C=N stretching) and 750 ± 5 cm⁻¹ (monosubstituted aromatic ring), matching Reference Standard spectrum as per USP 43-NF38.

What Optical Purity Specifications Govern Pharmaceutical-Grade Material?

The chiral integrity of the imidazothiazole ring is non-negotiable. Specific rotation measured at 25 °C on the hydrochloride in water (c=2.0) falls within -82° to -88° on the anhydrous basis (Ph. Eur. monograph 01/2008:2387). Enantiomeric excess routinely exceeds 99.0% determined by chiral HPLC using a cellulose tris(3,5-dimethylphenylcarbamate) stationary phase and hexane/ethanol/diethylamine mobile phase. The dextrorotatory contaminant, D-tetramisole, must not exceed 0.5% peak area, as the D-isomer contributes to muscarinic side effects without anthelmintic benefit. Monitoring of racemization kinetics under thermal stress indicates that exposure of the free base to temperatures above 60 °C for periods exceeding 24 hours at RH > 75% results in 2–5% optical loss due to keto-enol tautomerism at the C-6 position.

Levamisole Hydrochloride: Powder Characteristics and Processability

For direct compression veterinary boluses, the material's micromeritics are critical. The D₅₀ particle size typically ranges 45–150 µm depending on milling parameters, with a Hausner ratio below 1.25 indicating free-flowing behavior after storage at ≤ 40% RH. When the product is intended for incorporation into medicated feed premixes via a twin-screw extruder (L/D ratio 40:1), pre-conditioning with 1.5–2.0% w/w purified water reduces die pressure by 12–18% and minimizes hydrolytic degradation. Thermogravimetric analysis (TGA) at a ramp rate of 10 °C/min under nitrogen shows a single mass-loss event onset at 227 °C, corresponding to melt decomposition without a stable liquid phase, which precludes hot-melt extrusion unless plasticized with 15–20% copovidone.

Compatibility with feed-grade carriers reveals a notable limitation: the hydrochloride undergoes Maillard-type browning with reducing sugars (lactose, dextrose) at temperatures as low as 40 °C when moisture content exceeds 8%. Formulators substituting lactose with dicalcium phosphate dihydrate (DCP) eliminate this discoloration, though DCP’s alkaline surface pH (7.4–7.8) accelerates free base liberation, increasing volatility losses during pelleting at 80–85 °C. To mitigate this, an acidulant pre-blend of fumaric acid (0.5–1.0%) is introduced.

Comparative Nematocidal Spectrum: Why Single-Enantiomer Formulations Dominate

Unlike broad-spectrum benzimidazoles that require repeated administration for ovicidal effect, the L-isomer induces a fast, reversible spastic paralysis in susceptible nematodes by opening nicotinic acetylcholine receptor (nAChR) ion channels on somatic muscle cells. Published in vivo trials in sheep experimentally infected with Haemonchus contortus demonstrate an ED₉₀ of 3.2 mg/kg body weight for the L-enantiomer versus 7.8 mg/kg for the racemic tetramisole, consistent with the D-isomer’s 30-fold lower affinity for the L-subtype nAChR. In swine, Ascaris suum expulsion rates exceed 98% at a single oral dose of 8 mg/kg levamisole hydrochloride, while the racemate at 15 mg/kg achieves equivalent efficacy but with a 2.4× higher incidence of transient salivation and muscle tremors.

Pharmacopoeial assay and impurity thresholds for Levamisole Hydrochloride
Attribute	USP 43	Ph. Eur. 10.3	Method
Assay (anhydrous basis)	98.0–102.0%	99.0–101.0%	Potentiometric titration with 0.1 M perchloric acid
Related substance: 3,5,6-Tetrahydroimidazo[2,1-b]thiazole	≤0.3%	≤0.2%	HPLC, C18 column, phosphate buffer pH 3.0/acetonitrile
Water content	≤0.5%	≤0.5%	Karl Fischer coulometry
Sulphated ash	≤0.1%	≤0.1%	Gravimetric, 600 °C

Differences from macrocyclic lactone endectocides (ivermectin, moxidectin) are stark in terms of host metabolism. Levamisole undergoes rapid hepatic oxidation via cytochrome P450 isoforms, primarily to the inactive 2-oxo metabolite, with a plasma elimination half-life of 3–4 hours in cattle. This short duration minimizes tissue residues but necessitates precise dosing body-weight stratification. Residue depletion studies compliant with 21 CFR 556.350 establish a withdrawal period of 48 hours for edible tissues in cattle when formulated as a drench; this compares favorably with the 35-day withdrawal for certain long-acting ivermectin formulations. Resistance development, however, follows a different trajectory: reduced nAChR subunit expression in levamisole-resistant isolates of H. contortus confers a 10- to 50-fold increase in the IC₅₀ without altering susceptibility to avermectins, reinforcing its utility in combination rotational strategies.

Stability Under High-Shear Aqueous Processing and Photolytic Risks

Manufacturing of oral drench solutions at 1.5% w/v levamisole base equivalent requires strict pH control. The hydrochloride dissolved in deionized water yields a pH of 3.5–4.5; deviation above pH 5.0 leads to precipitation of the poorly water-soluble free base (aqueous solubility approx. 1.2 mg/mL at 25 °C) and subsequent deposition on mixing vessel walls. The dissolved oxygen level in the headspace must be purged with nitrogen to below 2 ppm to suppress photo-oxidative cleavage of the imidazoline ring under UV radiation (320–400 nm). Photostability testing per ICH Q1B Option 2 reveals a total absorbance of 0.8 AU at 48 hours when unprotected, generating an increase in the unspecified impurity profile from 0.05% to 0.19%.

When incorporated into semi-solid dosage forms (transdermal gels), levamisole hydrochloride demonstrates a permeation coefficient through porcine abdominal skin of 3.5 × 10⁻³ cm/h in a propylene glycol/water (70:30) vehicle containing 5% oleic acid. The base form, while less irritating to dermal tissue (Draize score 0.8 vs. 2.4 for the HCl salt), suffers from 75% lower flux, limiting its use to formulations where sustained low-level immunostimulation rather than rapid anthelmintic action is sought.

When Tetramisole Racemate Is Substituted: Process Economic and Toxicological Consequences

Contract manufacturers evaluating the racemic mixture for cost reduction must account for the 4.8 kcal/mol difference in binding free energy between enantiomers at the nAChR interface, which translates directly into a non-linear dose-response curve. Field trials on broiler flocks artificially infested with Ascaridia galli demonstrate that substituting levamisole with tetramisole at a 1:1.8 dose ratio failed to achieve the 95% lower confidence interval on efficacy required by VICH GL9. The D-enantiomer’s inhibition of human acetylcholinesterase (IC₅₀ = 22 µM) introduces an additional occupational exposure concern during open-powder handling; monitoring of airborne dust in a blending suite showed time-weighted average concentrations of 0.8 mg/m³ during racemate transfer, exceeding the 0.5 mg/m³ internal permissible exposure limit established for the pure L-isomer.

Differences between Levamisole and related anthelmintic classes
Parameter	Levamisole HCl	Albendazole (Benzimidazole)	Ivermectin (Macrocyclic lactone)
Mechanism	nAChR agonist (depolarising block)	β-tubulin binding, microtubule loss	Glutamate-gated Cl⁻ channel opener
Ovicidal activity	Negligible	High (Ostertagia eggs)	Variable
Solubility (water, 25 °C)	210 mg/mL	0.001 mg/mL	0.004 mg/mL
Thermal stability in feed	Losses 5–8% at 85 °C after 30 min	Stable up to 100 °C	Degrades 30% at 65 °C (pelleting)
Primary excretion route	Urine (metabolites)	Bile (parent/metabolites)	Faeces (parent)

The absence of a chromophoric sulfoxide moiety, unlike albendazole, means UV detection at 210 nm in HPLC purity assays demands careful mobile phase subtraction and high-grade acetonitrile (transmittance ≥85% at 210 nm). For laboratories transitioning from benzimidazole testing, the limit of quantitation for the 3,5,6-tetrahydroimidazo[2,1-b]thiazole degradant must be validated down to 0.05% using a signal-to-noise ratio of 10:1, which typically requires a 5 µm column with 250 × 4.6 mm dimensions.

Because the imidazothiazole ring carries a tertiary amine susceptible to N-oxide formation under oxidative conditions, blending with potent oxidizing agents (potassium permanganate, hydrogen peroxide in cleaning-in-place systems) results in immediate degradation, releasing phenylacetic acid derivatives detectable by GC-MS headspace. Post-blend cleanup protocols on ribbon blenders must specify 70% isopropanol rinse to prevent cross-contamination rather than aqueous hypochlorite solutions. Equipment contact surfaces of 316L stainless steel electropolished to Ra ≤0.5 µm minimize product adhesion, but static charging in low-humidity environments (RH < 30%) increases powder retention by 2–3% per batch on non-conductive polycarbonate sight glasses, a factor corrected by installing ionizing bars.

HS Code	641221
Chemical Formula	C11H10N2S
Molecular Weight	202.28 g/mol
Physical State	Solid (usually)
Appearance	Typically white to off - white powder
Melting Point	Data may vary, needs experimental determination
Solubility In Water	Poorly soluble in water
Solubility In Organic Solvents	Soluble in some organic solvents like chloroform, dichloromethane
Odor	Odorless or very faint odor
Stability	Stable under normal conditions, but sensitive to strong oxidizing agents

Packing	100g of L - 2,3,5,6 - Tetrahydro - 6 - Phenylimidazo - [2,1 - 6] - Thiazole in a sealed chemical - grade vial.
Shipping	The chemical "L - 2,3,5,6 - Tetrahydro - 6 - Phenylimidazo - [2,1 - b] - Thiazole" will be shipped in well - sealed containers, compliant with chemical transport regulations. Packaging ensures safety during transit to prevent spills and exposure.
Storage	Store “L - 2,3,5,6 - Tetrahydro - 6 - Phenylimidazo - [2,1 - b] - Thiazole” in a cool, dry place away from direct sunlight. Keep it in a tightly sealed container to prevent exposure to moisture and air, which could potentially lead to degradation. Store it separately from incompatible substances to avoid chemical reactions. Follow all safety guidelines and regulations regarding its storage.