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| Other Sizes |
Purity: ≥98%
| Targets |
Phospholipase D1 (PLD1) and Phospholipase D2 (PLD2). In vitro IC50: approximately 25 nM for both PLD1 and PLD2 (measured using head-group release assay with recombinant enzymes) [2]. In vivo IC50 (cellular): PLD2 (CHO overexpressing cells) IC50 = 10 nM; PMA-stimulated PLD1 (CHO overexpressing cells) IC50 < 1 nM; endogenous PLD in parental CHO cells stimulated with PMA IC50 = 0.5 nM [2]. No effect on MitoPLD, yeast Spo14, or autotaxin (LysoPLD) [2].
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| ln Vitro |
FIPI has a half-life of more than five hours, a Cmax of more than ten times 50, and a bioavailability of 18%, despite clearance exceeding 2 L/h/kg [1]. A 50% loss of activity is seen at about 25 nM in the dose-dependent inhibition of PLD1 and PLD2 by FIPI. Although FIPI does not prevent suicide completely, it also does not cause it to happen quickly when the medication is stopped. IPI has no effect on actin stress fibers, PIP2 access, PLD subcellular localization, or upstream signaling events. PLD2-inhibited membrane ruffling and cell spreading are saved by FIPI [2]. In MAECs, FIPI reduced PLD activation induced by thimerosal in a dose-dependent manner, as well as PLD activation induced by mercury, agonists, and oxidants in a dose-dependent manner [3].
In vitro enzyme inhibition: FIPI inhibited recombinant PLD1 and PLD2 in a dose-dependent manner with approximately 25 nM causing 50% loss of activity (IC50 ~25 nM) [2]. In vitro PLD2 inhibition from biochemical assay (unpublished method) gave IC50 = 20 nM [1]. FIPI did not inhibit the catalytic activity of MitoPLD (a divergent mammalian PLD superfamily member) in a cell-based assay (mitochondrial clustering), nor did it inhibit yeast sporulation dependent on Spo14 (yeast PLD) [2]. FIPI did not affect autotaxin (LysoPLD) activity in vitro [2]. |
| Enzyme Assay |
The in vitro PLD activity assay (head-group release) was performed as described (Morris et al., 1997). Recombinant human PLD1 and mouse PLD2 were expressed in Sf9 insect cells using baculovirus. Membrane fractions containing PLD protein were incubated with increasing concentrations of FIPI (diluted from 7.5 mM stock in DMSO) in the presence of [3H]phosphatidylcholine substrate. For PLD1, ADP-ribosylation factor was added as a stimulator. The reaction was carried out at 37°C, and the released [3H]choline was quantified by scintillation counting. Control wells contained matching amounts of DMSO or no DMSO. Assays were performed in duplicate with variance <5% [2].
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| Cell Assay |
PLD activity assay in cells (transphosphatidylation): CHO cells inducibly expressing PLD1 or PLD2 were treated with doxycycline (1 μg/ml) for 24 h to induce expression. Cells were preincubated with FIPI (diluted from 7.5 mM stock in DMSO) or vehicle for 30 min, then 0.3% 1-butanol was added for 30 min. Lipids were extracted, and [3H]phosphatidylbutanol (Ptd-But) was quantified by scintillation counting. IC50 values were determined from dose-response curves [2].
PA sensor assay: Cells were transfected with GFP-Spo20-PABD (PA-binding domain). After 24 h, cells were pretreated with FIPI (750 nM) or vehicle for 1 h, then stimulated with high glucose (20 mM) or PMA. Cells were fixed and imaged by confocal microscopy. Translocation of the sensor to the plasma membrane was quantified [2]. Cell spreading assay: CHO cells were suspended by trypsinization, rested for 2 h, then preincubated with FIPI (750 nM) or vehicle for 30 min. Cells were plated on fibronectin-coated coverslips for 15 min, fixed, and stained with phalloidin to visualize F-actin [2]. Neutrophil chemotaxis assay: Differentiated HL-60 cells (dHL-60) were treated with FIPI (750 nM) or vehicle for 1 h, then resuspended in chemotaxis buffer (RPMI 1640 +0.5% BSA). Cells (200 μl at 10^6/ml) were placed in transwell inserts (5 μm pore membrane) over lower wells containing 10 nM fMLP or buffer. After 1 h at 37°C, migrated cells in lower wells were counted. Each experiment repeated at least four times [2]. Glucose-stimulated insulin secretion: Min6 cells were preincubated in low-glucose KRBH buffer (2.5 mM glucose) for 60 min, then treated with FIPI (75 or 750 nM) for 30 min, followed by stimulation with high-glucose KRBH (20 mM glucose) for 60 min. Secreted and total insulin were measured by ELISA [2]. Western blotting for signaling: Cells were lysed, resolved by SDS-PAGE, transferred, and blotted with antibodies against phospho-AKT, total AKT, phospho-ERK, total ERK, phospho-p38, total p38. Detection was performed with fluorescent secondary antibodies and an Odyssey infrared imaging system [2]. |
| Animal Protocol |
Rat pharmacokinetic study: Male Harlan Sprague Dawley rats (not further specified) were dosed with FIPI at 5 mg/kg orally (p.o.) or 1 mg/kg intravenously (i.v.) in a vehicle of 20% PEG/80% CMC/0.025% Tween 80. Blood samples were collected at various time points (not detailed). Plasma concentrations were analyzed (method not described). The half-life (T1/2) = 5.57 h, clearance (Cl) = 2.18 L h⁻¹ kg⁻¹, Cmax = 0.363 μM, AUC = 1.03 μM h L⁻¹, and oral bioavailability (%F) = 18.5% [1].
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| ADME/Pharmacokinetics |
FIPI (5-fluoro-2-indolyl des-chlorohalopemide, compound 4k) was synthesized as described (Monovich et al., 2007) and purified by preparative HPLC. In a rat pharmacokinetic study (single dose), oral administration at 5 mg/kg gave a Cmax of 0.363 μM (approximately 10-fold above its PLD2 IC50) and half-life >5 h. The compound showed moderate oral bioavailability (18.5%) and high clearance (>2 L h⁻¹ kg⁻¹) [1].
In aqueous media, FIPI has a half-life greater than 5 h [2]. No other ADME parameters (e.g., volume of distribution, protein binding, metabolism) are reported in these manuscripts. |
| References |
[1]. Monovich L, et al. Optimization of halopemide for phospholipase D2 inhibition. Bioorg Med Chem Lett. 2007 Apr 15;17(8):2310-1.
[2]. Su W, et al. 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), a phospholipase D pharmacological inhibitor that alters cell spreading and inhibits chemotaxis. Mol Pharmacol. 2009 Mar;75(3):437-46. [3]. Secor JD, et al. Novel lipid-soluble thiol-redox antioxidant and heavy metal chelator, N,N'-bis(2-mercaptoethyl)isophthalamide (NBMI) and phospholipase D-specific inhibitor, 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) attenuate mercury-induced lipid si |
| Additional Infomation |
FIPI belongs to the benzimidazole class of compounds.
FIPI is a potent inhibitor of both PLD1 and PLD2 with subnanomolar to nanomolar potency in cells. It does not affect PLD subcellular localization, PIP2 availability, actin stress fibers in resting cells, or AKT/ERK/p38 phosphorylation in response to various stimuli (serum, LPS, fMLP). FIPI blocks PLD2-mediated suppression of membrane ruffling and promotes cell spreading, phenocopying PLD2 knockdown. It inhibits fMLP-directed chemotaxis of differentiated HL-60 cells. Notably, FIPI does not block glucose-stimulated insulin secretion, in contrast to 1-butanol, suggesting that 1-butanol may exert PLD-independent effects. FIPI is derived from halopemide and has potential utility as an in vivo tool to study PLD in inflammation and cancer metastasis [1,2]. |
| Molecular Formula |
C23H24N5O2F
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|---|---|
| Molecular Weight |
421.46736
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| Exact Mass |
421.191
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| Elemental Analysis |
C, 65.54; H, 5.74; F, 4.51; N, 16.62; O, 7.59
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| CAS # |
939055-18-2
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| Related CAS # |
1781834-93-2 (HCl);939055-18-2;
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| PubChem CID |
16739265
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| Appearance |
Light yellow to khaki solid powder
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| Density |
1.4±0.1 g/cm3
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| Index of Refraction |
1.663
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| LogP |
3.97
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
31
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| Complexity |
670
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(C1NC2C(=CC(=CC=2)F)C=1)NCCN1CCC(N2C3C(=CC=CC=3)NC2=O)CC1
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| InChi Key |
LHABRXRGDLASIH-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H24FN5O2/c24-16-5-6-18-15(13-16)14-20(26-18)22(30)25-9-12-28-10-7-17(8-11-28)29-21-4-2-1-3-19(21)27-23(29)31/h1-6,13-14,17,26H,7-12H2,(H,25,30)(H,27,31)
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| Chemical Name |
5-fluoro-N-[2-[4-(2-oxo-3H-benzimidazol-1-yl)piperidin-1-yl]ethyl]-1H-indole-2-carboxamide
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| Synonyms |
FIPI free base, FIPI; 5-Fluoro-2-indolyl des-chlorohalopemide;
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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 |
| 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) |
DMSO : ~14.67 mg/mL (~34.81 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 | 2.3726 mL | 11.8632 mL | 23.7265 mL | |
| 5 mM | 0.4745 mL | 2.3726 mL | 4.7453 mL | |
| 10 mM | 0.2373 mL | 1.1863 mL | 2.3726 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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