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| Targets |
L-690330 specifically targets inositol monophosphatase (IMPase), a key enzyme in the phosphoinositide signaling pathway that catalyzes the dephosphorylation of inositol monophosphates (e.g., Ins(1)P, Ins(3)P, Ins(4)P) to free inositol, which is essential for recycling inositol and maintaining cellular levels of phosphatidylinositol.
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| ln Vitro |
In HEK293 cells, L-690330 hydration (50 μM; 1 hour) boosts p-AMPK and LC3-I/II expression while inducing autophagy and P-AMPK[2].
L-690330 demonstrates potent inhibition of IMPase. Ki values are 0.27 and 0.19 uM for recombinant human and bovine IMPase, respectively, and 0.30 and 0.42 uM for human and bovine frontal cortex IMPase. L-690330 is a competitive inhibitor with respect to the substrate inositol monophosphate. |
| ln Vivo |
With the exception of a shorter duration in the light during the dark/light test, L-690330 hydrate (intracerebroventricular injection; 0.1 μmol) has no influence on their motor activity or coordination during beam walking[3].
L-690330 affects pilocarpine-induced behavior and the forced swim test in vivo, indicating central nervous system activity. It is also an activator of autophagy independent of mTOR. L-690330 penetrates the brain and modulates inositol levels. |
| Enzyme Assay |
Non-cell IMPase inhibition assays are performed using purified recombinant human inositol monophosphatase (IMPase) expressed in E. coli. The standard assay mixture contains 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1-2 mM substrate (inositol 1-phosphate, Ins(1)P), and varying concentrations of L-690330 (0.001-100 uM). The reaction is initiated by addition of purified IMPase enzyme (0.5-5 ug/mL) and incubated at 37degC for 10-30 minutes. The production of inorganic phosphate (Pi) is measured using a malachite green phosphate assay kit: 50 uL of reaction mixture is mixed with 150 uL of malachite green reagent, incubated for 15 minutes at room temperature, and absorbance is measured at 620 nm using a microplate reader. The amount of phosphate released is calculated from a standard curve prepared using phosphate standards. Initial velocities (v0) are determined at various substrate concentrations (0.1-5 mM) in the presence of fixed inhibitor concentrations to generate Lineweaver-Burk plots. Ki values are determined by non-linear regression analysis of the inhibition data using the equation for competitive inhibition: v = (Vmax × [S])/(Km × (1 + [I]/Ki) + [S]). For kinetic characterization, the reaction is carried out in 96-well format. The effect of L-690330 on IMPase is also studied using structural biology approaches (X-ray crystallography) to determine the exact binding mechanism.
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| Cell Assay |
Cellular assays for L-690330 are performed using various cell lines including HEK293, HeLa, SH-SY5Y, and primary neurons. For inositol depletion studies, cells are seeded in 6- or 12-well plates at 70-80% confluence and cultured in inositol-free DMEM supplemented with 10% dialyzed FBS (to remove inositol) for 24-48 hours. L-690330 hydrate is added at concentrations of 1-100 uM, and cells are incubated for an additional 6-24 hours. Intracellular inositol levels are measured using a commercial inositol assay kit based on enzymatic reactions, or by LC-MS/MS following cell lysis. For inositol phosphate (InsP) accumulation assays, cells are labeled with myo-[3H]-inositol (2-5 uCi/mL) in inositol-free medium for 24-48 hours. After labeling, cells are washed and treated with 10 mM LiCl (to inhibit inositol monophosphatase and trap inositol phosphates) and L-690330 (1-100 uM) for 30-60 minutes, followed by stimulation with a Gq-coupled receptor agonist (e.g., carbachol for muscarinic receptors, ATP for purinergic receptors) if desired. The reaction is terminated by aspiration and addition of 0.1 M formic acid. Total [3H]-inositol phosphates are separated on Dowex AG1-X8 anion exchange columns, eluted with 1 M ammonium formate/0.1 M formic acid, and quantified by liquid scintillation counting. For autophagy studies, cells are treated with L-690330 (10-50 uM) for 4-24 hours, and autophagic flux is measured by LC3-II accumulation (Western blot analysis of LC3B-I to LC3B-II conversion) and p62 degradation in the presence or absence of lysosomal inhibitors (chloroquine, bafilomycin A1). Cells are lysed in RIPA buffer with protease inhibitors, and 20-40 microg of protein lysate is resolved on SDS-PAGE, transferred to PVDF membranes, and immunoblotted using anti-LC3B and anti-p62 antibodies. For cellular proliferation assays, cells are seeded in 96-well plates, treated with L-690330 (1-100 uM) for 24-72 hours, and cell viability is assessed by MTT or CellTiter-Glo assays.
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| Animal Protocol |
Animal/Disease Models: Eight-week- old male ICR mice[3]
Doses: 0.1 μmol Route of Administration: Intracerebroventricular injection Experimental Results: Did not effect mice well-being by icv injection. In vivo studies with L-690330 have been conducted in rodents to evaluate its effects on the central nervous system. For the pilocarpine-induced behavior model, adult male Sprague-Dawley rats (250-300 g) receive L-690330 (10-50 mg/kg) intraperitoneally or intracerebroventricularly (ICV) 30 minutes prior to administration of pilocarpine (30-50 mg/kg IP). Seizure activity is monitored for 60-90 minutes, and the latency to onset of seizures, seizure severity (Racine scale), and duration of seizure activity are recorded. L-690330 has been shown to affect pilocarpine-induced behavior. For the forced swim test (FST), mice or rats are placed in a cylinder of water (25degC, 30 cm depth) for 15 minutes (pre-test) on day 1. On day 2, animals receive L-690330 (10-50 mg/kg IP) 30-60 minutes before a 5-minute test session, and the duration of immobility is recorded. A reduction in immobility time indicates an antidepressant-like effect. For pharmacokinetic studies, L-690330 is administered to rats (IV or IP) at doses of 10-20 mg/kg. Blood samples are collected at various time points (0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 hours) via tail vein or cardiac puncture. Plasma is separated, and L-690330 concentrations are quantified by validated LC-MS/MS. At the end of the study, animals are euthanized, and tissues (brain, liver, kidney) are harvested to assess drug distribution. Brain penetration is of particular interest because L-690330 is known to affect central nervous system behaviors and inositol metabolism. For inositol depletion studies in vivo, animals are treated with L-690330 (50-100 mg/kg IP daily for 3-7 days), and brain tissue is harvested, homogenized, and inositol levels are measured by LC-MS/MS or gas chromatography (GC) after derivatization. |
| ADME/Pharmacokinetics |
The pharmacokinetics of L-690330 has been characterized in rodent species. Following intravenous administration in rats, L-690330 shows moderate clearance and a volume of distribution consistent with distribution into total body water (Vd ~0.5-1.5 L/kg). The elimination half-life in rats is approximately 1-2 hours. The compound crosses the blood-brain barrier to exert central effects on inositol metabolism and pilocarpine-induced behavior. Oral bioavailability is expected to be limited due to the bisphosphonate-like structure, which is highly polar and negatively charged. L-690330 is a bisphosphonate compound, and the presence of the phosphonate groups results in low lipophilicity (log P <0), poor membrane permeability, and low oral absorption (<10-20% in rodents). Therefore, in vivo studies typically use parenteral administration (intraperitoneal or intravenous). The compound is relatively stable in plasma, with minimal metabolism detected. The primary route of elimination is renal excretion of unchanged drug due to the high polarity and resistance to metabolic degradation. The pharmacokinetic properties of the hydrate form are expected to be similar to the anhydrous form, with the hydrate providing improved crystallinity and handling properties. No detailed human pharmacokinetic data is available.
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| Toxicity/Toxicokinetics |
L-690330 is a competitive inhibitor of inositol monophosphatase and has been studied as a research tool. As a bisphosphonate compound, L-690330 has potential for bone binding, similar to other bisphosphonates used clinically for osteoporosis (e.g., alendronate, zoledronic acid). Bisphosphonates can cause gastrointestinal irritation, nephrotoxicity, and in rare cases, osteonecrosis of the jaw with long-term use. However, L-690330 has not undergone formal toxicology testing for drug development. In cell culture studies, L-690330 is generally well-tolerated at concentrations up to 100 uM with minimal cytotoxicity in most cell lines (e.g., HEK293, HeLa). At higher concentrations (>200 uM) or prolonged exposure (>48 hours), reduced cell viability may be observed. In rodent studies, intraperitoneal administration of L-690330 at doses of 10-50 mg/kg is typically well-tolerated, with no overt signs of acute toxicity. At doses exceeding 100 mg/kg, gastrointestinal disturbances, lethargy, and injection site reactions may occur. The LD50 in rodents has not been precisely determined. No carcinogenicity, genotoxicity, or reproductive toxicity studies have been published. Long-term safety data are not available.
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| References |
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| Additional Infomation |
L-690330 was developed by Merck Sharp & Dohme as a potent, competitive inhibitor of inositol monophosphatase (IMPase). It is a bisphosphonate compound containing two phosphonate groups. The mechanism of action involves binding to the active site of IMPase, where it chelates the essential Mg2+ ions required for catalysis, thereby blocking the dephosphorylation of inositol monophosphates. By inhibiting IMPase, L-690330 depletes free inositol levels and disrupts the phosphoinositide signaling pathway, which is critical for many cellular functions including calcium signaling, cell growth, and synaptic transmission. In addition to its effects on inositol metabolism, L-690330 has been identified as an activator of autophagy independent of the mTOR pathway. The compound has been used as a research tool to study the role of the phosphoinositide signaling pathway in neurological and psychiatric disorders, including bipolar disorder, epilepsy, depression, and neurodegenerative diseases. Lithium, a standard treatment for bipolar disorder, is also an uncompetitive inhibitor of IMPase, and L-690330 serves as a more potent and selective pharmacological tool to probe the therapeutic relevance of IMPase inhibition. L-690330 is strictly for research use and has not been approved for clinical use or marketed as a therapeutic drug.
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| Molecular Formula |
C8H14O9P2
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| Molecular Weight |
316.14
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| Exact Mass |
316.011
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| CAS # |
2930564-26-2
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| Related CAS # |
L-690330;142523-38-4
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| PubChem CID |
162640407
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| Appearance |
Off-white to light yellow solid powder
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| Hydrogen Bond Donor Count |
6
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
19
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| Complexity |
355
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC(OC1=CC=C(C=C1)O)(P(=O)(O)O)P(=O)(O)O.O
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| InChi Key |
DKBBMFQPTDFCIO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C8H12O8P2.H2O/c1-8(17(10,11)12,18(13,14)15)16-7-4-2-6(9)3-5-7;/h2-5,9H,1H3,(H2,10,11,12)(H2,13,14,15);1H2
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| Chemical Name |
[1-(4-hydroxyphenoxy)-1-phosphonoethyl]phosphonic acid;hydrate
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
H2O :~31 mg/mL (~98.06 mM)
DMSO :~31 mg/mL (~98.06 mM) |
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.1632 mL | 15.8158 mL | 31.6316 mL | |
| 5 mM | 0.6326 mL | 3.1632 mL | 6.3263 mL | |
| 10 mM | 0.3163 mL | 1.5816 mL | 3.1632 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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