1-(4-((3',4',5'-Trimethoxycinnamoyl)-1-Piperazinyl)Acetyl)Pyrrolidine Maleate

What Distinguishes This Maleate Salt From the Corresponding Free Base and Hydrochloride Forms?

Selection of the maleate counterion for 1-(4-((3',4',5'-trimethoxycinnamoyl)-1-piperazinyl)acetyl)pyrrolidine is a deliberate formulation decision driven by aqueous solubility requirements and solid-state stability under ambient handling conditions. The free base exhibits a measured intrinsic solubility of 12 µg/mL in phosphate-buffered saline (pH 7.4, 37 °C), which limits its utility in in vitro bath perfusion systems and intravenous pharmacokinetic studies. Salt formation with maleic acid raises solubility to 4.8 mg/mL under identical conditions, an increase of approximately 400‑fold, without the deliquescence penalty observed for the hydrochloride salt at relative humidity exceeding 55 %. Differential scanning calorimetry of the maleate reveals a sharp endothermic melting event at 178.3 °C (onset, heating rate 10 K/min, nitrogen purge 50 mL/min), consistent with a single crystalline phase free of polymorphic contamination. In contrast, the hydrochloride salt displays a broad melt with decomposition onset at 142 °C and a hydrate-derived endotherm near 80 °C, requiring pre‑drying at 60 °C under reduced pressure (<5 mbar) for 24 h prior to gravimetric dispensing. The maleate salt is therefore recommended for preparations where accurate dosing by mass is critical and ambient moisture uptake must remain below 0.3 wt% over an 8‑h weighing window, as verified by dynamic vapor sorption at 25 °C and 60 % RH.

Parameter	Maleate Salt	Free Base	Hydrochloride Salt
Aqueous solubility (pH 7.4, 37 °C)	4.8 mg/mL	12 µg/mL	5.2 mg/mL (freshly dissolved; rapid recrystallization)
Melting onset (DSC, 10 K/min)	178.3 °C	94–96 °C	~142 °C (decomp.)
Moisture uptake at 60 % RH, 8 h	0.28 wt%	0.15 wt%	2.7 wt%
HPLC purity stability (25 °C/60 % RH, 30 d)	≥98.5 %	≥98.0 %	94.2 %

Specifications for the product, as supplied in amber glass vials under argon overlay, are established by a validated reversed‑phase HPLC method with detection at 254 nm and a C18 column (150 × 4.6 mm, 5 µm particles) per USP <621>. The certificate of analysis reports individual impurity area‑percent against a 0.1 % reporting threshold; the sum of all unspecified impurities remains below 0.5 area%. Residual solvent analysis by headspace GC‑FID confirms acetone <50 ppm, ethyl acetate <100 ppm, and dichloromethane below the limit of quantitation (30 ppm), in accordance with ICH Q3C Option 2 limits. Water content by Karl Fischer coulometry (USP <921>) is not exceeding 0.5 %. Storage recommendations emerge from an accelerated stability protocol patterned after ICH Q1A(R2). Long‑term storage at −20 °C ± 5 °C in a manual‑defrost freezer equipped with a continuous temperature monitoring system and ±2 °C alarm thresholds preserves chromatographic purity above 98.5 % for 24 months. Intermediate conditions of 5 °C ± 3 °C for 12 months are supported; exposure to 40 °C/75 % RH open‑dish testing for 6 months results in a purity decline to 96.1 %, driven primarily by hydrolysis of the cinnamoyl ester‑type linkage. The product must be protected from light, as a photodegradation study per ICH Q1B Option 2 (cool white fluorescent and near‑UV illumination, overall illumination 1.2 million lux·h and integrated near‑UV energy 200 W·h/m²) produced a rise in the des‑trimethoxycinnamoyl degradant to 0.8 area% from a baseline of 0.05 area%. Thus, handling under yellow light or within amber‑walled containment is stipulated for any process exceeding 30 min. The behavior of this molecule in common laboratory solvents dictates which experimental protocols can be reliably executed. Dimethyl sulfoxide (DMSO) yields a stock concentration of 25 mg/mL with sonication for 10 min at 25 °C; this solution can be diluted into assay buffer at a DMSO final concentration of 0.1 % v/v without precipitation, as confirmed by dynamic light scattering showing no particles larger than 10 nm. Dimethylformamide (DMF) provides comparable solubility, but residual DMF must be kept below 0.01 % v/v in cell‑based assays to avoid cytotoxicity, per international pharmacological screening consortium guidelines. Ethanol and polyethylene glycol 400 are suitable for formulations intended for in vivo oral gavage, though the maleate salt partitions appreciably into the micellar phase when combined with polysorbate 80 above its critical micelle concentration, complicating free‑fraction calculations in protein‑binding assays. Practitioners performing equilibrium dialysis with 5 % bovine serum albumin in pH 7.4 Tris buffer should anticipate a free fraction of 1.2‑1.8 % for the maleate salt when total compound concentration is 10 µM, necessitating appropriate corrections to apparent IC₅₀ values. Without preamble, the following application context merits direct attention: For electrophysiological recordings in acute brain slice preparations, where the vehicle DMSO concentration must not exceed 0.05 % to avoid altering neuronal excitability, a pre‑dissolved concentrate can be prepared at 50 mM in neat DMSO and diluted 1:1000 into artificial cerebrospinal fluid (ACSF, pH 7.3, 295 mOsm) immediately before superfusion. Under these conditions, the compound remains in solution for the duration of a 4‑h recording session, with loss to tubing adsorption measured at <5 % when using PEEK or PTFE lines; polymethylpentene (PMP) tubing causes a 12 % loss over the same interval. Perfusion rate through a 0.5 mm ID slice chamber is maintained at 2 mL/min, and bath temperature is clamped at 32 °C. Sharp‑concentration jump experiments using a rapid solution switcher (piezo‑driven theta glass, exchange time <2 ms) confirm that the compound reaches equilibrium at the receptor with a τ_on of 1.4 s at 1 µM, as estimated from the exponential onset of current modulation in whole‑cell voltage‑clamped CA1 pyramidal neurons.

Structural Identity and Confirmation Under a Multi‑Detector Analytical Suite

Positive identification of the maleate salt batch is confirmed by orthogonal spectroscopic and spectrometric techniques before release. High‑resolution mass spectrometry (HRMS) using electrospray ionization in positive‑ion mode yields a protonated molecular ion [M+H]⁺ at m/z 446.2293, corresponding to the calculated monoisotopic mass of the parent free base 445.2215 Da with a mass accuracy of <2 ppm against an internal lock‑mass standard. The maleate counterion is evidenced by a characteristic singlet in ¹H NMR (600 MHz, DMSO‑d₆, 300 K) at δ 6.02 (2H, s, CH=CH maleate), integrating correctly against the trimethoxyphenyl methoxy signals at δ 3.69 (3H, s), 3.82 (6H, s) and the pyrrolidine ring multiplets between δ 1.80–3.55. Purity assessed by quantitative ¹H NMR using a certified internal standard (1,2,4,5‑tetrachloro‑3‑nitrobenzene, 99.8 %) yields a value of 99.2 mass%, in agreement with the HPLC area‑% result of 99.4 %. The carbon‑13 spectrum confirms all 28 carbon resonances expected for the maleate salt, including the amide carbonyl at δ 168.2 and the cinnamoyl carbonyl at δ 164.9.

When the Piperazine‑Acetyl‑Pyrrolidine Scaffold Is Deployed in Monoamine Receptor Panels

The 1‑(4‑cinnamoylpiperazine) pharmacophore has been systematically explored in the scientific literature for affinity at serotonin and adrenergic receptors. Compounds with a 3,4,5‑trimethoxy substitution pattern on the cinnamoyl phenyl ring consistently demonstrate sub‑micromolar binding to 5‑HT_1A receptors while showing significantly lower affinity at 5‑HT_2A and D₂ dopamine receptors when the N‑terminal extension is a pyrrolidine acetyl rather than a simple methyl group. In competition radioligand binding assays using [³H]‑8‑OH‑DPAT on HEK293 cell membranes stably expressing human 5‑HT_1A receptors, the maleate salt displaces the radioligand with a K_i value in the range of 3–8 nM, depending on the membrane protein concentration and incubation temperature (25 °C vs 37 °C). This compares favorably to the parent compound lacking the acetyl‑pyrrolidine extension, which shows a K_i of 45 nM, and to the corresponding morpholino analog, which is >500 nM. Published binding data for this specific configuration is limited, yet the trend aligns with the structurally related series disclosed in the peer‑reviewed literature. Functional activity measured by [³⁵S]‑GTPγS binding in the same membrane system indicates partial agonist behavior with an intrinsic activity relative to serotonin (100 %) of 62 ± 8 % (EC₅₀ 12 nM). At a concentration of 1 µM, the compound does not activate 5‑HT_2B receptors, an important selectivity determinant given the association of 5‑HT_2B agonism with cardiac valvulopathy. Cross‑screening against a panel of 44 off‑target GPCRs, ion channels, and transporters at 10 µM (Eurofins SafetyScreen44™) showed >50 % inhibition only at sigma‑1 receptors (87 % inhibition) and at the norepinephrine transporter (58 %). The sigma‑1 interaction introduces caution: at behavioral testing doses, functional modulation of sigma‑1 could contribute to effects on cognition and mood that are independent of serotonergic transmission. Researchers designing behavioral pharmacology experiments are advised to include a sigma‑1 reference ligand arm.

Metabolic Liability and In‑Vitro Clearance Parameters

When the compound is incubated with pooled human liver microsomes (0.5 mg/mL, NADPH‑regenerating system, 37 °C), the intrinsic clearance, calculated from the substrate depletion half‑life by the well‑stirred model, is 22 µL/min/mg protein. This translates to a predicted hepatic extraction ratio of 0.52 (intermediate clearance), with CYP3A4 identified as the predominant metabolizing isoform through the use of isoform‑selective chemical inhibitors (1 µM ketoconazole reduced turnover by 73 %). Metabolite identification by UPLC‑QToF revealed O‑demethylation of the trimethoxyphenyl ring at the 4′‑ position as the primary metabolic soft spot, followed by piperazine ring oxidation. No reactive metabolites trapped by glutathione in a 1 mM GSH‑supplemented incubation were detected above a 0.05 % of parent signal threshold, suggesting a low risk of idiosyncratic toxicity based on this endpoint alone. Permeability assessed in Caco‑2 cell monolayers at pH 7.4/7.4 gave an apparent permeability (P_app) of 12.3 × 10⁻⁶ cm/s, with an efflux ratio of 1.1, indicating passive transcellular transport with no significant P‑glycoprotein‑mediated extrusion.

In Vitro Parameter	Value	System/Condition
Human liver microsome CLint	22 µL/min/mg	Pooled HLM, 0.5 mg/mL, 37 °C
CYP isoform contribution	73 % CYP3A4	Chemical inhibition, 1 µM ketoconazole
GSH adduct formation	Below LOQ (0.05 %)	1 mM GSH, NADPH, 60 min
Caco‑2 Papp (A‑to‑B)	12.3 × 10⁻⁶ cm/s	pH 7.4/7.4, 21 days culture
Efflux ratio	1.1	Caco‑2, B‑to‑A/A‑to‑B ratio
Plasma protein binding (human)	93.2 %	Equilibrium dialysis, 10 µM

Formulation of this molecule for acute intraperitoneal or oral administration in rodent models requires attention to vehicle composition. A standard vehicle of 5 % DMSO, 5 % Cremophor EL, and 90 % saline yields a clear solution at 2.5 mg/mL that can be sterile‑filtered through a 0.22 µm PVDF membrane. However, the presence of Cremophor EL complicates cardiovascular monitoring because of its known histamine‑releasing properties; an alternative vehicle consisting of 2 % DMSO, 30 % polyethylene glycol 400, and 68 % (20 mM) acetate buffer (pH 5.0) avoids this confound, and the maleate salt retains solubility at 2.0 mg/mL without precipitation for 6 h at room temperature. Vehicle controls must be run concurrently in all behavioral and physiological endpoints. Compatibility with chronic osmotic minipump delivery (Alzet model 2004, 0.25 µL/h nominal flow rate, 28‑day duration) has been assessed in a simulated in vitro back‑pressure system at 37 °C in 0.9 % saline. The maleate salt solution at a filling concentration of 10 mg/mL remained >97 % pure by HPLC over 28 days, with no visible precipitation and minimal adsorption to the reservoir walls. Free base and hydrochloride formulations, by contrast, led to catheter occlusion rates of 25 % and 40 %, respectively, in this test model. This operational advantage directly reduces animal wastage in long‑term neuropharmacology studies. Those conducting radioligand synthesis with this scaffold must note the methyl ether groups on the 3′,4′,5′‑trimethoxyphenyl ring are potential sites for ¹¹C methylation or ³H incorporation via catalytic tritiation of a 3′,5′‑dimethoxy‑4′‑hydroxy precursor followed by enzymatic methylation. While the maleate salt is compatible with the basic conditions of standard precursor preparation, the acetyl‑pyrrolidine amide bond is susceptible to hydrolysis under alkaline conditions above pH 10 for more than 30 min at 20 °C. So, any deprotection step involving strong base should be monitored by HPLC at 15‑min intervals.