Technical Applications of 3-(1-Piperazinyl)-1,2-Benzisothiazole Hydrochloride in Downstream Manufacturing and Research
Anhydrous dimethylformamide (DMF, water content <0.05% by Karl Fischer) is charged into a 2000 L glass-lined reactor under nitrogen sweep at 20°C, and 235 kg (1.0 kmol) of 3-(1-piperazinyl)-1,2-benzisothiazole hydrochloride—pre-dried in a conical vacuum dryer at 55–60°C until the loss-on-drying value falls below 0.15% w/w—is introduced through a nitrogen-blanketed manway. The suspension is agitated at a tip speed of 1.8 m/s while potassium carbonate (152 kg, 1.10 kmol, milled to d50<45 µm) is added portionwise over 45 minutes; the batch is then heated to 78–82°C and held for 14 hours as a 1.05 molar-equivalent stream of 5-[2-chloroethyl]-6-chloro-1,3-dihydro-2H-indol-2-one (moisture <0.1%) dissolved in 1.5 volumes of DMF is metered in via a peristaltic pump at a rate not exceeding 12 L/h to maintain the internal temperature within ±2°C of the setpoint and to suppress the formation of the quaternary ammonium dimer impurity (monitored at RRT 1.34 by HPLC). Compliant with ICH Q7 Section 8.3 (reaction steps) and ICH Q3A (impurity control), the crude ziprasidone free base is precipitated by transferring the cooled mixture into 3500 L of purified water at 2–5°C under high-shear dispersion (3000 rpm rotor-stator, Silverson-type); the filter cake is washed with water and subjected to two sequential re-slurry washes with n-propanol at 75°C to reduce the des-chloro impurity below the 0.10% threshold, then vacuum-dried at 50°C and 5 mbar. The free base is subsequently converted to ziprasidone hydrochloride monohydrate by dissolution in 3 volumes of tetrahydrofuran and addition of 37% hydrochloric acid (1.02 equivalents) at 25°C, seeded with micronized crystals (d50<10 µm) and cooled linearly to -5°C at 0.15°C/min; the crystalline product exhibits a residual DMF content below 880 ppm (conforming to ICH Q3C Class 2 solvent limits) and an individual unspecified impurity profile <0.10% when analyzed by USP 〈621〉 compliant HPLC with detection at 229 nm. This intermediate is warehoused in double LDPE liners within fibre drums at ≤25°C and <60% RH before being tableted with excipients (pregelatinized starch, lactose monohydrate) to produce oral ziprasidone hydrochloride capsules in 20 mg, 40 mg, 60 mg and 80 mg strengths, or lyophilized for the intramuscular injection formulation containing 20 mg/mL of ziprasidone mesylate adjusted to pH 7.2–7.8.
Does in situ generation of the free base alter the amide-coupling impurity profile in lurasidone synthesis?
In the convergent route to lurasidone hydrochloride—an atypical antipsychotic active in the 20–120 mg oral tablet range—3-(1-piperazinyl)-1,2-benzisothiazole hydrochloride is most reliably deployed after a controlled neutralization step rather than as a direct charged salt. The hydrochloride is partitioned between dichloromethane (8 volumes) and 2M aqueous sodium hydroxide (1.05 equivalents), mixed for 30 minutes at 20°C in a multi-plate counter-current extraction column (rotor speed 600 rpm), and the organic layer is azeotropically dried with a Dean-Stark trap until the water content falls below 50 ppm. The resultant 3-(1-piperazinyl)-1,2-benzisothiazole free base in DCM is then reacted with (1R,2R)-cyclohexane-1,2-dicarboxylic acid monoamide activated ester (prepared using 1.10 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.05 equivalents of hydroxybenzotriazole) at a strict 1.00:1.00 molar ratio of amine to activated acid, with 0.15 equivalents of N,N-diisopropylethylamine added as a proton scavenger. The batch is held at 0–5°C for the first 4 hours to suppress O-acylisourea rearrangement, then allowed to reach 22°C over 12 hours; real-time HPLC monitoring (column: C18 150×4.6 mm, 5 µm; eluent: phosphate buffer pH 6.5/acetonitrile 60:40 at 1.0 mL/min) detects the transient formation of the bis-acylated impurity (<0.15% by peak area) which is held below the ICH M7 threshold-of-toxicological-concern intake of 1.5 µg/day by a terminal silica-gel plug filtration (flash column, 40–63 µm silica, 1.5 kg per 100 g of product). Post-workup, the lurasidone base is precipitated from isopropanol, and the hydrochloride salt is formed in acetone with 1.05 equivalents of 37% HCl, controlling the seed bed temperature at 48°C to obtain the desired polymorph (Form I, identified by XRPD peaks at 6.2°, 12.5°, and 18.9° 2θ) compliant with the monograph in the European Pharmacopoeia 10.8. The finished drug product—lurasidone hydrochloride tablets 20 mg, 40 mg, 80 mg—is manufactured under FDA 21 CFR Part 211 and requires a batch-wise demonstration that the piperazinyl-benzisothiazole-related process impurities (specifically the des-piperazine byproduct and the N-oxide derivative) do not exceed 0.15% in the API release specification, as confirmed by LC-MS/MS (Q-TOF, resolution >30,000) following a ICH Q3B qualification threshold of 0.15% for a 2 g/day maximum dose.
In the manufacturing sequence for perospirone hydrochloride—a serotonin-dopamine antagonist dispensed in 4 mg and 8 mg tablets—3-(1-piperazinyl)-1,2-benzisothiazole hydrochloride is first neutralized to its free base by partition between toluene and 10% aqueous sodium bicarbonate under a nitrogen atmosphere; the toluene layer is azeotropically dried (Dean-Stark, target water <80 ppm) and the free amine is alkylated with 2-(thiophen-2-yl)ethyl methanesulfonate (1.18 molar equivalents) in the presence of finely powdered cesium carbonate (1.25 equiv., d50<20 µm) at 65°C over 18 hours in a baffled 500 L glass-lined reactor. The heterogeneous reaction mixture is then filtered through a 0.5 µm polyethylene sinter to remove inorganic salts, and the toluene solution is charged with 5% palladium-on-charcoal (0.5% by weight of substrate, Johnson Matthey type 87L) for a debenzylation hydrogenolysis conducted at 2.5 bar hydrogen pressure and 35°C; the endpoint is monitored by the disappearance of the benzyl protecting group at HPLC retention time 12.1 min. Careful control of the catalyst loading and filtration temperature (<40°C) is essential to prevent Pd leaching, which is verified by inductively coupled plasma mass spectrometry (ICH Q3D) to remain below the peroral PDE of 100 µg/day for elemental palladium. The crude perospirone is crystallized from ethanol/water (7:3 v/v) with a cooling profile of 0.3°C/min from 60°C to 2°C to afford the free base, which is subsequently dissolved in acetone and reacted with 1.02 equivalents of concentrated hydrochloric acid at 25°C to obtain perospirone hydrochloride dihydrate (confirmed by TGA mass loss of 8.9% observed between 80–120°C). Each batch is evaluated per ICH Q1A(R2) accelerated stability conditions (40°C/75% RH, 6 months) in the intended immediate-release tablet formulation containing carmellose calcium and crystalline lactose, with strict monitoring of the thiophene-related oxidative degradation product (limit 0.5%).
| Parameter | Ziprasidone Hydrochloride | Lurasidone Hydrochloride | Perospirone Hydrochloride |
|---|---|---|---|
| Reaction type | N-alkylation (chloroethyl indolone) | Amide coupling (cyclohexane diacid monoamide) | N-alkylation (thienylethyl mesylate) |
| Molar input ratio (HCl salt/electrophile) | 1.0 : 1.05 | 1.00 : 1.00 (after neutralization) | 1.0 : 1.18 |
| Base/scavenger | K2CO3 (1.10 equiv.) | N,N-diisopropylethylamine (0.15 equiv.) | Cs2CO3 (1.25 equiv.) |
| Critical impurity specification | Quaternary ammonium dimer (RRT 1.34) <0.15% | Bis-acylated adduct <0.15% | Pd residue <10 ppm; oxidative degradant <0.5% |
| Drying endpoint for salt | Moisture <0.15% before alkylation | Organic layer <50 ppm water before coupling | Toluene layer <80 ppm water before alkylation |
| Relevant ICH guideline | ICH Q3C (DMF limit 880 ppm) | ICH M7 (mutagenic impurity control), ICH Q3B | ICH Q3D (Pd PDE 100 µg/day) |
When 3-(1-piperazinyl)-1,2-benzisothiazole hydrochloride is integrated into in-vitro receptor pharmacology profiling campaigns—typically outsourced to contract research organizations operating under OECD Good Laboratory Practice principles—the compound is prepared as a 10 mM stock solution in anhydrous dimethyl sulfoxide (DMSO, dried over molecular sieve 3Å), aliquoted into single-use glass vials, and stored at -20°C under argon to preserve the free-base form from moisture-induced disproportionation upon thawing. For competitive radioligand binding assays at recombinant human 5-HT1A and 5-HT2A receptor subtypes expressed in CHO-K1 cell membranes, the stock is serially diluted in assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 0.1% ascorbic acid) to yield final incubation concentrations spanning 0.1 nM to 10 µM; a fixed concentration of [3H]-8-OH-DPAT (1.0 nM) or [3H]-ketanserin (2.0 nM) serves as the hot ligand. The reaction is terminated by rapid vacuum filtration through 0.5% polyethyleneimine-soaked GF/B glass-fiber filters using a Brandel harvester, and the retained radioactivity is quantified by liquid scintillation counting after 8 hours of extraction in Ultima Gold cocktail. The resultant inhibition constant (Ki) values and the dose-response curves generated on a 96-well plate layout provide the primary endpoint for the structure-activity relationship reports delivered to the sponsor as a PDF/A-2 compliant document; these data guide the selection of back-up series within a CNS discovery program without ever constituting a pharmaceutical preparation per se.
When the hydrochloride serves as a system-suitability reference material for ZIP hydrochloride monograph testing
In the quality control analytical laboratory, 3-(1-piperazinyl)-1,2-benzisothiazole hydrochloride of specified purity (99.85% on the anhydrous basis, certified against a USP reference standard) is employed as a process-specific impurity marker in the chromatographic system suitability mixture described in the United States Pharmacopeia monograph for Ziprasidone Hydrochloride. A precisely weighed 25.0 mg portion of the hydrochloride is dissolved in 50.0 mL of mobile phase A (0.02 M pH 6.8 ammonium acetate buffer:acetonitrile 80:20) to yield a stock standard of 500 µg/mL, which is further diluted to a working concentration of 5.0 µg/mL (representing 0.10% of the API test concentration of 5.0 mg/mL); a 10 µL injection onto a 4.6×150 mm column packed with 3.5 µm octadecylsilane material (endcapped, carbon load 12%) at a flow rate of 1.0 mL/min and a column temperature of 35°C yields a retention time of 6.3 minutes, with resolution between the compound and the nearest eluting ziprasidone peak of greater than 2.0. The analytical procedure—fully validated per ICH Q2(R1) over a linear range of 0.05–10.0% of the nominal API concentration—delivers a relative standard deviation of peak area <1.0% across six replicate injections. This standardisation protocol, documented in the electronically archived audit trail (21 CFR Part 11 compliant), directly supports the release of finished dosage forms (capsules, injection vials) by establishing that the analytical system can reliably separate and detect potential residual starting material below the reporting threshold of 0.05%.
| Standard / Guideline | Scope of Application | Critical Parameter |
|---|---|---|
| ICH Q7 (GMP for Active Pharmaceutical Ingredients) | Chemical synthesis steps from intermediate to API | Section 8.3 control of reaction completeness and impurity formation |
| ICH Q3A (Impurities in New Drug Substances) | Ziprasidone and perospirone API | Reporting threshold 0.05%; identification threshold 0.10% for a 2 g/day dose |
| ICH Q3C (Residual Solvents) | Ziprasidone HCl monohydrate manufacture | Class 2 solvent DMF: permissible daily exposure 8.8 mg/day; concentration limit 880 ppm |
| ICH M7 (Assessment and Control of DNA Reactive Impurities) | Lurasidone amide coupling process | Threshold of Toxicological Concern intake: 1.5 µg/day for lifetime exposure |
| ICH Q3D (Elemental Impurities) | Perospirone hydrogenolysis step | Palladium oral PDE: 100 µg/day |
| USP 〈621〉 Chromatography | HPLC system suitability evaluation | Resolution >2.0; RSD <1.0% |
Parallel reductive amination libraries in early-stage CNS drug discovery
The use of 3-(1-piperazinyl)-1,2-benzisothiazole hydrochloride as a common amine building block in automated parallel synthesis platforms relies on its sequential deprotonation and reaction with diverse aldehyde sets. In a typical library construction run performed on a 48-well MiniBlock reaction station under positive argon pressure, each well is charged with 0.12 mmol of the hydrochloride salt (dried for 2 hours at 40°C in vacuo) and 0.10 mmol of a discrete aldehyde dissolved in 1.0 mL of anhydrous 1,2-dichloroethane containing 0.5% acetic acid; after 1 hour of orbital shaking at 250 rpm, 0.25 mmol of sodium triacetoxyborohydride is added portionwise, and the plate is sealed and agitated for 16 hours at 22°C. Quenching with 7% aqueous sodium bicarbonate and liquid-liquid extraction is avoided in favour of parallel solid-phase extraction on propylsulfonic acid (PSA) cartridges (bed weight 200 mg), wherein the crude product is loaded, the stationary phase is washed with 2 volumes of methanol, and the tertiary amine target is released with 5% ammonia in methanol. The eluent is evaporated in a Genevac HT-4X centrifugal evaporator at 34°C and 8 mbar, yielding dry films that are reconstituted in DMSO-d6 for 1H NMR purity assessment (integration standard: 2,5-dimethylfuran added at 0.05% v/v) and parallel LC-ELSD quantification before being registered into the corporate compound collection with a unique barcode linked to the well position, aldehyde cassette number, and operator ID. The resulting library provides 10–50 mg per compound, sufficient for a primary dose-response screen in a fluorescence-based plate reader assay at 10 µM single concentration; hits are cherry-picked and re-arrayed for Ki determination. The process adheres to internal standard operating procedures aligned with ISO/IEC 17025:2017 general requirements for the competence of testing and calibration laboratories, although the compounds are classified as research chemicals and are neither manufactured under cGMP nor intended for in-vivo administration without further confirmatory batch analysis for purity and counter-ion content.
