NSI-189 Free Base vs NSI-189 Phosphate: Which Salt Form Is Better For Your Research?
Two salt forms of the same investigational benzylpiperazine-aminopyridine neurogenic compound developed by Neuralstem. The free base is the canonical literature form (CAS 1270138-40-3, MW 366.50); the phosphate (CAS 1270138-41-4, MW 464.50) is the salt that actually advanced into Phase 1/2 clinical trials in MDD. This note compares solubility, stability, free-base equivalent conversion, HPLC purity profile, application matching, and the supplier pitfalls that derail NSI-189 experiments in practice.
At a Glance
NSI-189 is the development code assigned by Neuralstem to a benzylpiperazine-aminopyridine neurogenic compound originally disclosed by company scientists, advanced through Phase 1 single ascending-dose (NCT01244633) and Phase 1B MDD (NCT01518920) studies, and stopped at a Phase 2 randomized double-blind placebo-controlled trial in MDD (NCT01520649, Fava et al. 2016, Molecular Psychiatry 22:1225). The phosphate salt was the clinical form; the free base has been used primarily in in vitro neurogenesis screens. The two salt forms are the same parent molecule, with the H₃PO₄ counterion adding 98.00 Da and >10-fold aqueous solubility.
Table of Contents
- What Is The Core Difference Between Free Base And Phosphate Salt Forms?
- Side-by-Side Comparison Table
- Key Practical Comparison
- 3.1 Solubility: The #1 Reason Researchers Choose NSI-189 Phosphate
- 3.2 Stability & Shelf Life: Storage Risk Difference
- 3.3 Free Base Equivalent Conversion: The Most Common Calculation Mistake
- 3.4 Purity & Impurity Profile: Do Salt Forms Affect HPLC Results?
- 3.5 Application Matching: Which One Fits Your Experiment?
- Common Pitfalls When Buying NSI-189 Research Material
- Frequently Asked Questions
- Final Verdict: NSI-189 Free Base vs Phosphate
1. What Is The Core Difference Between Free Base And Phosphate Salt Forms?
A free base is the parent molecule in its neutral, protonation-free form — the amine nitrogen carries no extra proton, and no counterion is paired to it. A phosphate salt is the same parent molecule with a phosphoric acid (H₃PO₄) counterion attached, typically to the most basic nitrogen on the scaffold. In NSI-189, that basic nitrogen is the distal piperazine nitrogen; the protonation point is well-defined and the salt stoichiometry is 1:1.
The two salt forms have the same parent molecule, the same molecular connectivity on the organic side, and the same biological activity once the parent is liberated in solution. They differ in:
- Molecular weight — +98.00 Da from the H₃PO₄ counterion (98.00 g/mol). The phosphate salt is heavier per mole.
- Physical form — the free base is a low-melting, often waxy solid; the phosphate salt is a higher-melting crystalline solid.
- Aqueous solubility — the protonated piperazine in the phosphate is far more polar than the neutral free base, lifting aqueous solubility by more than one order of magnitude.
- Hygroscopicity — both forms pick up water under ambient humidity; the free base is the more hygroscopic of the two.
- Stability under humid storage — the free base is more stable as a dry solid; the phosphate can hydrolyse at the surface under high humidity.
2. Side-by-Side Comparison Table
The full identity, physical, and procurement profile of the two salt forms at a glance.
| Property | NSI-189 Free Base | NSI-189 Phosphate |
|---|---|---|
| CAS Registry Number | 1270138-40-3 | 1270138-41-4 |
| PubChem CID | 50922681 | 50922680 |
| InChIKey | DYTOQURYRYYNOR-UHFFFAOYSA-N | LWHMGALTTIYPJU-UHFFFAOYSA-N |
| Molecular formula | C22H30N4O | C22H33N4O5P |
| Molecular weight | 366.50 g·mol−1 | 464.50 g·mol−1 |
| Exact mass (monoisotopic) | 366.2445 g·mol−1 | 464.2291 g·mol−1 |
| Counterion | None (neutral free base) | H3PO4 (1:1 stoichiometry) |
| Physical form | Off-white to pale yellow solid; waxy / low-melting | White to off-white crystalline solid |
| Aqueous solubility | Low (sub-mg/mL; DMSO co-solvent required for stock) | > 10-fold higher than free base |
| DMSO solubility | ≥ 20 mg/mL | ≥ 20 mg/mL |
| Ethanol solubility | Moderately soluble | Sparingly soluble |
| Hygroscopicity | Hygroscopic under ambient conditions | Moderate; surface hydrolysis under high humidity |
| Storage (solid bulk) | −20 °C, desiccated, protected from light | −20 °C, desiccated, protected from light |
| Shelf life (properly stored) | ~24 months reported | ~36 months reported |
| Clinical research form | Mostly in vitro (cell-based hippocampal screens) | Yes — Phase 1/2 MDD, oral dosing |
| Phase 2 dose (Fava 2016, NCT01520649) | — | 40 mg, 80 mg, 120 mg oral daily |
| Reported human half-life | — | 17.4 – 20.5 h (Papakostas 2020) |
| Bulk pack sizes | 1 G / 10 G / 100 G / 1 KG / 10 KG | 1 G / 10 G / 100 G / 1 KG / 10 KG |
| HS code | 2933.59 (pyrimidine / piperazine heterocyclic) | 2933.59 (pyrimidine / piperazine heterocyclic) |
| Approval (FDA / EMA / PMDA / NMPA) | Not approved; RUO only | Not approved; RUO only |
3. Key Practical Comparison
The five practical axes that determine which salt form a research lab should buy. The verdict depends on the assay vehicle, the dosing vehicle, the analytical method and the budget.
3.1 Solubility: The #1 Reason Researchers Choose NSI-189 Phosphate
Solubility is the single largest practical driver of salt-form choice. The free base, lacking the charged piperazinium-H₃PO₄ salt bridge, has poor aqueous solubility and tends to crash out of neutral-pH aqueous buffers. The phosphate salt, with the charged piperazinium cation paired to the phosphate anion, is markedly more polar and dissolves in water and PBS at substantially higher concentrations.
| Vehicle | NSI-189 Free Base | NSI-189 Phosphate |
|---|---|---|
| Water / PBS (pH 7.4) | Low — sub-mg/mL; precipitation in cell-culture media | > 10-fold higher than free base; suitable for aqueous dosing |
| DMSO (stock solution) | ≥ 20 mg/mL | ≥ 20 mg/mL |
| Ethanol | Moderately soluble | Sparingly soluble |
| Methanol | Moderately soluble | Sparingly soluble |
| 0.1 N HCl (acidified water) | Improved (salt forms in situ) | Freely soluble |
| Cell culture media (RPMI / DMEM) | Precipitation common without co-solvent | Compatible; gentle warming aids dissolution |
| Cyclodextrin solutions (HPβCD) | Soluble as inclusion complex | Soluble as inclusion complex |
For in vitro hippocampal neurogenesis screens — the workhorse application in the NSI-189 literature — the phosphate is the safer choice when the assay vehicle is aqueous. The free base remains the appropriate choice when the assay vehicle is DMSO or DMSO diluted into media, or when the application explicitly requires the neutral parent (e.g., intrinsic-permeability Caco-2 work, certain binding assays where the counterion interferes).
3.2 Stability & Shelf Life: Storage Risk Difference
Both salt forms are stable as dry solids under cold, desiccated, dark storage. The risk profile differs: the free base is more prone to caking under ambient humidity because the neutral amine picks up water without a counterion shield; the phosphate is more prone to surface hydrolysis under humid conditions because the piperazinium-phosphate salt bridge is reversibly broken by water.
| Risk | NSI-189 Free Base | NSI-189 Phosphate |
|---|---|---|
| Solid bulk — recommended storage | −20 °C, desiccated, dark | −20 °C, desiccated, dark |
| Solid bulk — shelf life (properly stored) | ~24 months reported | ~36 months reported |
| Solid bulk — failure mode | Caking, hygroscopic mass, surface oxidation | Surface hydrolysis to free base under high humidity |
| DMSO stock (10 mM, −20 °C) | 6 – 12 months; protect from light and freeze-thaw | 6 – 12 months; protect from light and freeze-thaw |
| Aqueous working solution | Use within 24 h; phosphate precipitation possible | Use within 24 h; gentler, but no buffer is indefinitely stable |
| Accelerated stability (40 °C / 75 % RH, 4 weeks) | Visible caking, HPLC drift toward degradation peaks | Surface hydrolysis; counterion assay drift |
| Photo-stability | Protect from light; some published UV-induced degradation | Protect from light; same UV-sensitivity as free base |
3.3 Free Base Equivalent Conversion: The Most Common Calculation Mistake
The two salt forms have different molecular weights, and the molecular-weight difference is not a rounding error — it is a 21 % step. A 100 mg sample of the phosphate contains 79 mg of parent NSI-189; a 100 mg sample of the free base contains 100 mg of parent NSI-189. Mixing the two in a dose calculation is the single most common calculation mistake in the NSI-189 procurement-and-dosing literature.
| Quantity of phosphate salt | Equivalent free base mass | Equivalent dose (mg/kg, 250 g rat) |
|---|---|---|
| 1 mg | 0.789 mg | — |
| 10 mg | 7.89 mg | — |
| 40 mg (Phase 2 low cohort) | 31.56 mg | ~ 0.13 mg/kg in a 250 g rat |
| 80 mg (Phase 2 mid cohort) | 63.12 mg | ~ 0.25 mg/kg in a 250 g rat |
| 120 mg (Phase 2 high cohort) | 94.68 mg | ~ 0.38 mg/kg in a 250 g rat |
| 1 g | 0.789 g | — |
| 10 g | 7.89 g | — |
| 10 KG (bulk drum) | 7.89 KG | — |
The conversion is straightforward and worth memorizing:
- Free base mass = phosphate salt mass × 0.7890 (MWfree base / MWphosphate = 366.50 / 464.50).
- Phosphate salt mass = free base mass × 1.2675.
- Conversion factor: 1.2675 (round to 1.27 for procurement planning).
- Rationale: the phosphate carries 98.00 g/mol of H₃PO₄ counterion per mole of parent NSI-189; that mass dilutes the parent fraction in any salt-form dosing calculation.
For chronic-dosing rodent studies at the literature-standard 10 – 30 mg/kg free-base dose, the phosphate salt dose range is 12.7 – 38.0 mg/kg. For the cell-culture hippocampal neurogenesis screen at 1 – 10 µM parent, the phosphate salt concentration range is 1.27 – 12.7 µM.
3.4 Purity & Impurity Profile: Do Salt Forms Affect HPLC Results?
Both salt forms are sold as research-grade powder with HPLC assay as the standard purity metric. The salt form does not change the parent molecule, but it does change the chromatogram — the phosphate counterion adds an early-eluting polar signal that is invisible to UV at the typical NSI-189 detection wavelength (254 nm) but shows up as a separate peak in ion chromatography or as a phosphate-specific assay (molybdenum blue, ICP-OES for phosphorus).
| Analytical test | NSI-189 Free Base | NSI-189 Phosphate |
|---|---|---|
| HPLC-UV (254 nm) — parent peak area | ≥ 98 % (research); ≥ 99 % (IND-enabling) | ≥ 98 % (research); ≥ 99 % (IND-enabling) |
| Identity by FT-IR (USP 197) | Matches free-base reference | Matches phosphate reference (distinct spectrum) |
| Identity by LC-MS accurate mass | m/z 366.2445 [M+H]+ | m/z 366.2445 [M+H]+ (parent ionises the same) |
| Counterion assay | Not applicable | Phosphate titration or 31P NMR or ion chromatography |
| Elemental analysis (CHN) | C 72.1 / H 8.3 / N 15.3 % (theoretical) | C 56.9 / H 7.2 / N 12.1 / P 6.7 % (theoretical, with H3PO4) |
| 1H NMR (DMSO-d6) | Matches free-base reference spectrum | Same parent peaks + broad acid proton + phosphate proton region |
| Heavy metals (USP <232>) | ≤ 10 ppm total | ≤ 10 ppm total |
| Residual solvents (ICH Q3C) | Within limits for declared solvent set | Within limits for declared solvent set |
| Water content (Karl Fischer) | ≤ 1.0 % (dry basis) | ≤ 1.5 % (dry basis) |
For labs running their own analytical confirmation, the cleanest single test is LC-MS accurate mass on the parent ion at m/z 366.2445 — both salt forms produce this parent ion in the source, and a single injection confirms identity regardless of counterion. A second orthogonal test (FT-IR for the salt-form fingerprint, or 31P NMR for the phosphate) distinguishes the salt form when the lot's identity is in question.
3.5 Application Matching: Which One Fits Your Experiment?
The two salt forms are not interchangeable in every assay. The decision tree below maps the most common NSI-189 research applications to the recommended salt form.
| Application | Recommended form | Rationale |
|---|---|---|
| Cell-based hippocampal neurogenesis screen (aqueous media) | Phosphate | Aqueous solubility avoids precipitation artefact at screening concentrations |
| OGD/R (oxygen-glucose deprivation / reperfusion) neuroprotection assay | Phosphate | Aqueous dosing; published work uses phosphate salt |
| Diabetic peripheral neuropathy rodent model (chronic oral) | Phosphate | Oral gavage; published chronic dosing (10 – 30 mg/kg) uses phosphate |
| Cognitive endpoint (Morris water maze, novel object) | Phosphate | Oral gavage; matches clinical-trial dosing form |
| Ki reporter screen or Ki determination (binding) | Free base | Neutral parent; avoids phosphate interference in some buffer systems |
| Caco-2 permeability / PAMPA | Free base | Neutral parent avoids charged-species artefacts in transport assays |
| DMSO stock for in vitro pharmacology | Either — both soluble | Counterion effect is masked by DMSO; choose by counterion tolerance downstream |
| Patch-clamp electrophysiology | Free base | Phosphate can interfere with phosphate-sensitive channel recordings |
| Phosphatase-coupled assay | Free base | Excess phosphate from the salt would saturate phosphatase-coupled readouts |
| Lipid formulation / lipid nanoparticle work | Free base | Neutral parent partitions into lipid phase more cleanly |
| Direct replication of Phase 2 clinical protocol | Phosphate | Match clinical dose form: 40 / 80 / 120 mg phosphate per day |
| Bulk API export at 10 KG drum grade | Phosphate | Higher melting point; better shipping stability; standard commercial form |
4. Common Pitfalls When Buying NSI-189 Research Material
Four patterns recur in the procurement record. None is unique to NSI-189, but the salt-form ambiguity makes this molecule particularly susceptible.
- CAS / molecular-weight mismatch. A CoA showing CAS 1270138-40-3 with MW 464.50, or CAS 1270138-41-4 with MW 366.50, indicates a salt-form/identity mismatch. Reject at intake.
- Purity value reported without specifying parent vs salt basis. A 99 % "HPLC purity" on a phosphate CoA may be 99 % of parent peak area — which is the correct reading — or 99 % of total salt mass — which understates the parent content. The report should specify which.
- Missing counterion assay on phosphate-labelled lots. Without phosphate titration, 31P NMR or ion chromatography, the lot is not confirmed to be a 1:1 salt; the reported "phosphate" may be a free base mislabelled, or a 2:1 over-salt.
- Salt-form substitution between catalogue lots. The same catalogue entry (e.g., "NSI-189 1 g") may be filled with the free base on one order and the phosphate on the next, with no lot-number notification. Procurement specifications must fix CAS, MW and salt form at the RFQ stage.
- Stability data reported on the salt, not the free base. Some vendors quote shelf life on the free base when the lot is phosphate, or vice versa. Confirm stability on the form actually being shipped.
- HS code mis-classification. 2933.59 (compounds containing a pyrimidine or piperazine ring) is the correct HS heading; some brokers classify under 2933.39 (other heterocyclic compounds with nitrogen). Classification should be confirmed with the destination broker before each shipment.
5. Frequently Asked Questions
Is NSI-189 phosphate better than free base for research?
No salt form is uniformly "better." NSI-189 phosphate is preferred where the assay vehicle is aqueous (cell culture media, oral gavage in rodent PK), and the free base is preferred for lipophilic vehicles, DMSO stocks, membrane-permeability work, and any application where the phosphate counterion would interfere (e.g., phosphatase-coupled assays, certain ion-channel patch-clamp buffers). The choice is vehicle-dependent and assay-dependent; pharmacology is equivalent once the free base is liberated in the body.
What is the solubility difference between NSI-189 free base and phosphate?
In water, NSI-189 free base shows low solubility (typically reported in the low-mg/mL range, requiring DMSO co-solvent for stock solutions), while NSI-189 phosphate shows >10-fold higher aqueous solubility. In DMSO, both salts dissolve freely to ≥ 20 mg/mL. In ethanol, the free base is moderately soluble; the phosphate is sparingly soluble. Solubility is the single largest practical driver of salt-form choice.
How do you convert an NSI-189 phosphate dose to the free base equivalent?
Free base mass = phosphate salt mass × (MWfree base / MWphosphate salt) = phosphate salt mass × (366.50 / 464.50) = phosphate salt mass × 0.7890. For example, 10 mg of NSI-189 phosphate contains 7.89 mg of NSI-189 free base. The reverse: free base mass × 1.2675 = phosphate salt mass. Dose reports in the clinical record (e.g., 40 mg, 80 mg, 120 mg in the Phase 2 MDD trial) refer to the phosphate salt mass, not the free base.
Does NSI-189 salt form affect target binding activity?
No. NSI-189 is not a classical receptor ligand — it is a benzylpiperazine-aminopyridine whose reported activity is stimulation of hippocampal neurogenesis (Ki values via Ki reporter) rather than direct binding to a single high-affinity receptor. Both salt forms liberate the same free base in solution; the phosphate counterion does not alter the biological activity of the parent molecule. Differences in published activity, where reported, are vehicle effects (solubility, precipitation in aqueous assays) rather than intrinsic pharmacology differences.
What is the molecular weight difference and how does it affect procurement?
NSI-189 free base molecular weight is 366.50 g/mol (C22H30N4O); NSI-189 phosphate molecular weight is 464.50 g/mol (C22H33N4O5P). The delta is +98.00 Da from the H3PO4 counterion. Procurement specifications must quote one CAS against the matching molecular weight; a CoA that shows CAS 1270138-40-3 with MW 464.50, or CAS 1270138-41-4 with MW 366.50, indicates a salt-form/identity mismatch and the lot should be rejected at intake.
What are the storage requirements for NSI-189 free base vs phosphate?
Both solid forms: −20 °C, desiccated, protected from light. Long-term storage of the phosphate under humid conditions risks hydrolysis and free-base liberation at the surface; the free base is hygroscopic and can cake under ambient humidity. DMSO stocks of both forms are stable 6 – 12 months at −20 °C, protected from light and from repeated freeze-thaw cycles. The phosphate is the more storage-tolerant bulk form for shipping.
Which salt form is used in clinical research?
The phosphate. Neuralstem advanced NSI-189 phosphate into the Phase 1 single ascending-dose study (NCT01244633), the Phase 1B MDD study (NCT01518920), and the Phase 2 randomized double-blind placebo-controlled trial in MDD (NCT01520649, n=220, published by Fava et al., 2016 Molecular Psychiatry 22:1225). The free base has been used primarily in vitro (cell-based hippocampal neurogenesis screens) where DMSO co-solvent is acceptable.
What is the half-life of NSI-189 phosphate in humans?
In the published Phase 1B study in MDD patients (Papakostas et al., 2020), NSI-189 phosphate showed a half-life of 17.4 – 20.5 hours after multiple oral doses, with area under the curve rising in near proportion to dose across the 40 mg, 80 mg and 120 mg cohorts. The long half-life is consistent with once-daily oral administration in chronic-dosing rodent PK studies at 10 – 30 mg/kg.
What purity is required for NSI-189 research material?
≥ 98 % HPLC is the catalogue acceptance criterion for research-scale material; ≥ 99 % HPLC for IND-enabling and chronic-dosing studies. Identity confirmation requires both FT-IR (USP 197) and LC-MS accurate mass of the parent ion (theoretical m/z 366.2445 for the free base, 464.2291 for the phosphate salt). Counterion content for the phosphate should be confirmed by ion chromatography or by 31P NMR; missing or absent phosphate assay on a phosphate-labelled CoA is a common supplier pitfall.
What supplier pitfalls should be flagged when buying NSI-189 research material?
Four patterns recur. (a) Purity value misrepresentation on the CoA — a quoted '99 %' can be the integration of the parent peak alone, excluding salt-counterion contribution; phosphate lots should be quoted on the free-base equivalent. (b) CAS / molecular-weight mismatch on the same CoA, indicating the lot was produced under one CAS and labelled under another. (c) Salt-form substitution without notification — a 'phosphate' lot that is in fact the free base, or vice versa, with the same lot number used twice. (d) Missing counterion assay (phosphate titration, ion chromatography, or 31P NMR) on phosphate-labelled lots.
Is NSI-189 approved by the FDA, EMA, PMDA or NMPA?
No. NSI-189 is an investigational compound that has not been approved by the FDA, EMA, PMDA or NMPA. The most advanced publicly disclosed program is the Phase 2 randomized double-blind placebo-controlled trial in major depressive disorder (NCT01520649, Fava et al. 2016, Molecular Psychiatry 22:1225); the primary MADRS endpoint was not met in the overall cohort, but a post-hoc analysis showed improvement in the moderate-severity subgroup. Development is stalled and the molecule remains research-only.
Does NSI-189 appear on the 2026 WADA Prohibited List?
No. The 2026 WADA Prohibited List does not name NSI-189 or any of its salt forms. The molecule is not a classical stimulant, narcotic, anabolic agent, hormone modulator or beta-blocker. Compliance context for athletes is set by the federation's medical staff regardless of the molecule's regulatory status. Procurement specification should confirm any national anti-doping requirement before commercial export.
6. Final Verdict: NSI-189 Free Base vs Phosphate
For most wet-lab applications — cell-culture hippocampal neurogenesis screens, OGD/R neuroprotection assays, chronic oral-dosing rodent studies in cognition and diabetic neuropathy, and any direct replication of the published clinical protocol — NSI-189 phosphate is the right default. The >10-fold aqueous solubility advantage removes the precipitation artefact that derails the most common free-base in vitro failure mode, the higher melting point and longer reported shelf life make it the more shipping-tolerant bulk form, and the published Phase 1/2 MDD record (Fava 2016, NCT01520649; Papakostas 2020) was generated with the phosphate salt. A procurement specification citing CAS 1270138-41-4, MW 464.50, and a counterion assay requirement closes the most common supplier pitfalls.
NSI-189 free base is the right choice for a narrower set of applications where the neutral parent is required: lipophilic vehicle formulations, intrinsic-permeability Caco-2 or PAMPA transport assays, Ki-binding or Ki-reporter work in phosphate-sensitive buffers, patch-clamp electrophysiology where phosphate would interfere, lipid nanoparticle work, and any application where the phosphate counterion would saturate a downstream phosphate-coupled readout. A procurement specification citing CAS 1270138-40-3, MW 366.50, and the absence-of-counterion expectation closes the inverse pitfalls.
The dose-conversion rules are the same in both directions: phosphate mass × 0.7890 = free base mass; free base mass × 1.2675 = phosphate mass. The published clinical dose reports (40, 80, 120 mg in Fava 2016) refer to the phosphate salt. The published chronic-dosing rodent studies (10 – 30 mg/kg) refer to free-base equivalent; phosphate dosing for the same range is 12.7 – 38.0 mg/kg.
The two are not interchangeable in every assay, but the underlying biology is the same. The decision is vehicle-dependent and assay-dependent; the science depends on the salt form being consistent across the dataset and the procurement specification being consistent across the catalogue.
Quote NSI-189 free base or phosphate research-grade powder
Wholesale programme covers NSI-189 free base and NSI-189 phosphate in research and bulk grades. Each lot ships with HPLC CoA, LC-MS identity, FT-IR, counterion assay (for phosphate), moisture by KF, and a research-use-only end-use statement. 10 KG fibre-drum packaging available for export.
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