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].
PLD1 and PLD2 (phospholipase D 1 and 2). FIPI is a potent and selective phospholipase D (PLD) inhibitor with IC₅₀ values of 20 nM for PLD2 and 25 nM for PLD1. PLD enzymes hydrolyze phosphatidylcholine to generate phosphatidic acid (PA) and choline. PA is a key lipid signaling molecule involved in cell proliferation, migration, survival, and vesicle trafficking. Inhibition of PLD by FIPI disrupts these processes.

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In vitro PLD2 inhibition from biochemical assay (unpublished method) gave IC50 = 20 nM [1].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].
In vitro, FIPI inhibits PLD1 and PLD2 with IC₅₀ values of 25 nM and 20 nM, respectively. The compound is a potent and selective PLD inhibitor. FIPI is used to study PLD-mediated signaling pathways in various cellular contexts. The compound's high potency makes it a valuable tool for PLD research.

Detailed in vivo activity data for FIPI are limited in publicly available sources. As a PLD inhibitor, FIPI is expected to have effects on PLD-mediated signaling pathways in vivo, including effects on cell migration, inflammation, and cancer progression. The compound has been used in preclinical studies to investigate the role of PLD in various disease models.

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].
The PLD enzyme inhibition assay for FIPI typically involves recombinant PLD1 or PLD2 incubated with a substrate (e.g., phosphatidylcholine or a fluorescent PLD substrate) in the presence of varying concentrations of the compound. PLD activity is measured by quantifying the production of phosphatidic acid or choline using methods such as thin-layer chromatography, mass spectrometry, or fluorescent assays. IC₅₀ values are calculated from dose-response curves.

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].
Cells (e.g., cancer cell lines, primary cells) are cultured in appropriate medium. Cells are treated with varying concentrations of FIPI (typically ranging from 1 nM to 10 μM) for various time points. PLD activity is assessed by measuring phosphatidic acid production using lipidomics or by using fluorescent PLD activity assays. Downstream signaling (e.g., mTOR, AKT, ERK) is assessed by Western blotting.

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].
In vivo studies of FIPI likely involve mouse models of cancer, inflammation, or other diseases where PLD plays a role. FIPI is typically administered via intraperitoneal injection at various doses. Disease progression is monitored, and tissues are collected for analysis of PLD activity and downstream signaling. The compound has been used to study the role of PLD in various pathological conditions.

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.
FIPI has a molecular weight of 421.47 g/mol and a molecular formula of C₂₃H₂₄FN₅O₂. Detailed pharmacokinetic parameters are not extensively published. As a small molecule, FIPI is expected to be orally bioavailable. The compound is typically stored as a powder at -20°C. Pharmacokinetic studies would be required to support further development.

Detailed toxicology data for FIPI are limited in publicly available sources. As a PLD inhibitor, the compound's toxicity profile would be related to the inhibition of PLD-mediated signaling in normal tissues. Standard preclinical safety evaluation would be required for further development. The compound is used in research applications.

[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

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].
FIPI (5-Fluoro-2-indolyl deschlorohalopemide) is a small-molecule, direct-acting inhibitor of PLD1 and PLD2 with IC₅₀ values of 25 nM and 20 nM, respectively. It is a derivative of halopemide. FIPI has a molecular formula of C₂₃H₂₄FN₅O₂ and a molecular weight of 421.47 g/mol. The compound is used in research to study PLD-mediated signaling pathways involved in cell proliferation, migration, and survival.

C23H24N5O2F

421.46736

421.191

C, 65.54; H, 5.74; F, 4.51; N, 16.62; O, 7.59

939055-18-2

1781834-93-2 (HCl);939055-18-2;

16739265

Light yellow to khaki solid powder

1.4±0.1 g/cm3

1.663

3.97

3

4

5

31

670

0

O=C(C1NC2C(=CC(=CC=2)F)C=1)NCCN1CCC(N2C3C(=CC=CC=3)NC2=O)CC1

LHABRXRGDLASIH-UHFFFAOYSA-N

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)

5-fluoro-N-[2-[4-(2-oxo-3H-benzimidazol-1-yl)piperidin-1-yl]ethyl]-1H-indole-2-carboxamide

FIPI free base, FIPI; 5-Fluoro-2-indolyl des-chlorohalopemide;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~14.67 mg/mL (~34.81 mM)

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 Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

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
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)