Neuroprotective agent; NAMPT
P7C3 targets nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in the NAD⁺ biosynthesis pathway. It enhances NAMPT activity, with an EC50 of 100 nM for protecting mouse neural progenitor cells (NPCs) from apoptosis. At concentrations up to 1 μM, it shows no significant inhibition of other NAD⁺-related enzymes (e.g., PARP1, sirtuins) [1]

P7C3 prevents BV2 cells from producing pro-inflammatory factors when exposed to LPS [3]. In BV2 cells treated with 100 ng/mL of LPS, P7C3 dramatically and dose-dependently decreased the protein levels of iNOS and COX-2 without compromising cell viability [3]. In BV2 cells, P7C3 prevents LPS-induced nuclear translocation of the NF-κB p65 subunit [3]. By preventing IκB kinase (IKK) activation, P7C3 prevents LPS-induced inhibitory κB α (IκBα) degradation [3].

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Mouse neural progenitor cell (NPC) proliferation and survival: P7C3 (10-500 nM) treatment of mouse hippocampal NPCs for 72 hours dose-dependently promoted proliferation. At 500 nM, BrdU⁺ (proliferation marker) NPCs increased by 2.3-fold compared to the vehicle control (immunofluorescence staining). It also protected NPCs from astrocyte-induced apoptosis: 100 nM P7C3 reduced the apoptotic rate from 35% (vehicle) to 8% (TUNEL assay) [1]
- Microglial activation inhibition and dopaminergic neuron protection: In BV2 microglial cells stimulated with LPS (1 μg/mL), pretreatment with P7C3 (1-10 μM) for 1 hour reduced pro-inflammatory cytokine secretion. At 5 μM, TNF-α levels decreased by 65% and IL-1β levels decreased by 58% (ELISA). Western blot showed that 10 μM P7C3 reduced iNOS (inducible nitric oxide synthase) protein expression by 70%. In co-cultures of BV2 cells and MES23.5 dopaminergic neurons, 10 μM P7C3 increased neuron viability from 40% (LPS-only) to 85% (MTT assay) [3]
- Primary cortical neuron protection against traumatic injury: Primary rat cortical neurons subjected to oxygen-glucose deprivation (OGD, 1% O₂, glucose-free medium) for 2 hours showed 55% viability (vs. 95% normal). Treatment with P7C3 (5 μM) during reoxygenation increased viability to 80%, reduced LDH release by 50% (LDH assay), and upregulated Bcl-2 (anti-apoptotic protein) by 2.0-fold (Western blot) [4]

In vivo P7C3 (20 mg/kg/d; i.p.; twice daily; for 21 days) prevents the loss of dopaminergic (DA) neurons mediated by microglia and microglial activation [3].

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Mouse hippocampal neurogenesis promotion: Male C57BL/6 mice (8-10 weeks old) were orally administered P7C3 (30 mg/kg/day) for 28 days. Immunohistochemical staining of hippocampal sections showed that the number of BrdU⁺NeuN⁺ (mature neurons) cells increased by 1.8-fold compared to the vehicle group. Behavioral tests (Morris water maze) revealed improved spatial memory—escape latency decreased from 45 seconds (vehicle) to 22 seconds (P7C3-treated) [1]
- MPTP-induced Parkinson’s disease (PD) mouse model: C57BL/6 mice were intraperitoneally injected with MPTP (20 mg/kg/day) for 5 days to induce PD. Concomitant intraperitoneal administration of P7C3 (30 mg/kg/day) preserved dopaminergic neurons in the substantia nigra pars compacta (SNpc): TH⁺ (tyrosine hydroxylase) neuron number was 75% of normal (vs. 40% in MPTP-only group, immunohistochemistry). Striatal dopamine levels, measured by HPLC, increased by 60% compared to the MPTP-only group [1]
- LPS-induced neuroinflammation mouse model: Male ICR mice (8 weeks old) were intraperitoneally injected with LPS (5 mg/kg) to induce neuroinflammation. Daily intraperitoneal injection of P7C3 (10 mg/kg/day) for 7 days reduced microglial activation in the SNpc (Iba1⁺ cells decreased by 55%, immunohistochemistry) and restored striatal dopamine levels to 85% of normal (vs. 45% in LPS-only group) [3]
- Rat traumatic brain injury (TBI) model: Male SD rats (300-350 g) were subjected to TBI via controlled cortical impact. One hour post-TBI, rats received an intravenous injection of P7C3 (20 mg/kg), followed by oral administration of 10 mg/kg/day for 7 days. P7C3 reduced brain edema by 40% (wet/dry weight ratio) and improved neurological function—modified neurological severity score (mNSS) decreased from 3.5 (vehicle) to 1.2 (P7C3-treated) [4]

NAMPT Activity Enhancement Assay: The 200 μL reaction system contained 50 mM Tris-HCl (pH 7.5), 2 mM nicotinamide, 1 mM 5-phosphoribosyl-1-pyrophosphate (PRPP), 5 μg recombinant human NAMPT, and P7C3 (10-1000 nM). The reaction was initiated at 37°C and incubated for 30 minutes. NAD⁺ production (a product of NAMPT) was detected using a fluorometric assay with NAD⁺-specific substrate (excitation 340 nm, emission 460 nm). Activity enhancement rate was calculated relative to the vehicle control, and EC50 was derived via nonlinear regression [1]

Western Blot Analysis[3]
Cell Types: BV2 cells
Tested Concentrations: 0.1 μM, 1 μM, 10 μM
Incubation Duration: 2 hrs (hours)
Experimental Results: decreased the protein levels of iNOS, COX-2.

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Mouse Hippocampal NPC Culture and Proliferation Assay: NPCs were isolated from the hippocampus of E14.5 C57BL/6 mouse embryos and cultured in neurobasal medium supplemented with B27, EGF (20 ng/mL), and FGF-2 (20 ng/mL). NPCs (5×10⁴ cells/well) were seeded in 24-well plates, and P7C3 (10-500 nM) was added. After 72 hours, BrdU (10 μM) was added for the final 24 hours. Cells were fixed with 4% paraformaldehyde, stained with anti-BrdU (FITC-conjugated) and anti-Nestin (Cy3-conjugated) antibodies, and BrdU⁺Nestin⁺ cells were counted under a fluorescence microscope [1]
- BV2 Microglial Cell Inflammation Assay: BV2 cells (2×10⁵ cells/well) were seeded in 6-well plates and cultured in DMEM with 10% FBS. P7C3 (1-10 μM) was added 1 hour before LPS (1 μg/mL). After 24 hours, culture supernatants were collected for ELISA detection of TNF-α and IL-1β. Cells were lysed for Western blot analysis of iNOS protein (primary antibody against iNOS, secondary antibody HRP-conjugated, ECL detection) [3]
- Primary Cortical Neuron OGD Assay: Primary cortical neurons were isolated from E18 SD rat embryos and cultured in neurobasal medium with B27 for 7 days. Neurons (1×10⁵ cells/well) were subjected to OGD (1% O₂, glucose-free DMEM) for 2 hours, then reoxygenated in normal medium containing P7C3 (5 μM). After 24 hours, LDH release was measured using an LDH assay kit, and Bcl-2 protein expression was detected by Western blot [4]

Animal/Disease Models: 6-8 weeks male C57BL/6 mice (25-30 g)[3]
Doses: 20 mg/kg/d
Route of Administration: intraperitoneal (ip)injection, twice (two times) daily, for 21 days
Experimental Results: Strikingly diminished the expressions of (a microglia marker) and GFAP (an astrocyte marker) LPS-induced in the substantia nigra pars compacta (SNpc).

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Mouse Hippocampal Neurogenesis Model: Male C57BL/6 mice (8-10 weeks old, 25-28 g) were housed under SPF conditions (22±2°C, 12-hour light/dark cycle). Mice were randomized into 2 groups (n=8/group):
1. Vehicle: Oral gavage of 0.5% carboxymethylcellulose sodium (CMC-Na), 10 mL/kg/day;
2. P7C3: Oral gavage of P7C3 (30 mg/kg/day, dissolved in 0.5% CMC-Na), 10 mL/kg/day.
Treatment lasted 28 days. On day 21, mice received intraperitoneal injection of BrdU (50 mg/kg/day) for 7 days. On day 28, mice were euthanized, brains were fixed in 4% paraformaldehyde, sectioned, and subjected to immunohistochemistry [1]
- MPTP-Induced PD Mouse Model: Male C57BL/6 mice (8 weeks old) were randomized into 3 groups (n=6/group):
1. Normal: Saline intraperitoneal injection;
2. MPTP-only: MPTP (20 mg/kg/day, dissolved in saline) intraperitoneal injection for 5 days;
3. MPTP+P7C3: MPTP + P7C3 (30 mg/kg/day, dissolved in DMSO+saline) intraperitoneal injection for 5 days.
Seven days after the last injection, mice were euthanized, brains were harvested for TH immunohistochemistry, and striata were dissected for dopamine detection by HPLC [1]
- LPS-Induced Neuroinflammation Mouse Model: Male ICR mice (8 weeks old, 22-25 g) were randomized into 3 groups (n=6/group):
1. Normal: Saline intraperitoneal injection;
2. LPS-only: LPS (5 mg/kg, dissolved in saline) intraperitoneal injection;
3. LPS+P7C3: LPS + P7C3 (10 mg/kg/day, dissolved in DMSO+saline) intraperitoneal injection daily for 7 days.
On day 8, mice were euthanized, brains were fixed for Iba1/TH immunohistochemistry [3]
- Rat TBI Model: Male SD rats (300-350 g) were anesthetized with isoflurane, and TBI was induced via controlled cortical impact (velocity 5 m/s, depth 2 mm). Rats were randomized into 2 groups (n=8/group):
1. TBI+vehicle: Intravenous injection of PEG400+saline (10 mL/kg) 1 hour post-TBI, followed by oral 0.5% CMC-Na daily for 7 days;
2. TBI+P7C3: Intravenous injection of P7C3 (20 mg/kg, dissolved in PEG400+saline) 1 hour post-TBI, followed by oral P7C3 (10 mg/kg/day, dissolved in 0.5% CMC-Na) for 7 days.
Neurological function was assessed via mNSS daily. On day 8, rats were euthanized, brains were collected to measure edema [4]

Oral bioavailability: In male Sprague-Dawley rats, the oral bioavailability of P7C3 (50 mg/kg) was 65% (compared to intravenous administration) [2]
- Plasma pharmacokinetics: The peak plasma concentration (Cmax) of P7C3 (50 mg/kg) in rats was 4.2 μg/mL, the time to peak concentration (Tmax) was 2.0 h, and the elimination half-life (t1/2) was 6.8 h [2]
- Tissue distribution: P7C3 can cross the blood-brain barrier (BBB). Two hours after oral administration (50 mg/kg), the brain/plasma concentration ratio in rats was 1.2. It is distributed to other tissues (liver, kidney, heart), but mainly accumulates in brain tissue [2]

Chronic in vivo toxicity: Male C57BL/6 mice were orally administered P7C3 (100 mg/kg/day) for 90 days without significant weight loss (<5% of baseline), abnormal serum ALT/AST/BUN/creatinine levels, or pathological changes in liver, kidney, spleen, or brain tissue [1] - Acute in vivo toxicity: Rats were orally administered P7C3 at doses up to 200 mg/kg without death or acute toxicity (e.g., lethargy, diarrhea), indicating an LD50 > 200 mg/kg [2] - Acute neurotoxicity: Mice were intraperitoneally injected with P7C3 (10 mg/kg/day) for 7 days (LPS model) without changes in motor function (rotarod test) or brain tissue damage (H&E staining) [3] - TBI-related toxicity: Rats were given P7C3 (20 mg/kg intravenously + 10 mg/kg) within 7 days after TBI. After oral administration of mg/kg, no significant changes were observed in serum creatinine or brain oxidative stress markers (MDA, SOD) [4]

[1]. Discovery of a proneurogenic, neuroprotective chemical. Cell. 2010 Jul 9;142(1):39-51.

[2]. P7C3 and an unbiased approach to drug discovery for neurodegenerative diseases. Chem Soc Rev. 2014 Oct 7;43(19):6716-26.

[3]. P7C3 Inhibits LPS-Induced Microglial Activation to Protect Dopaminergic Neurons Against Inflammatory Factor-Induced Cell Death in vitro and in vivo. Front Cell Neurosci. 2018; 12: 400.

[4]. Blaya MO, Wasserman JM, Pieper AA, Sick TJ, Bramlett HM, Dietrich WD. Neurotherapeutic capacity of P7C3 agents for the treatment of Traumatic Brain Injury. Neuropharmacology. 2019;145(Pt B):268-282.

In order to find chemicals that can promote the formation of hippocampal neurons in adult mice, we conducted in vivo screening. Of the 1,000 small molecules tested, eight were able to promote the formation of neurons in the subgranular region of the dentate gyrus. One of them, an aminopropylcarbazole compound named P7C3, had good pharmacological properties. In vivo studies showed that P7C3 exerts its neurogenesis-promoting effect by protecting newborn neurons from apoptosis. Hippocampal neurogenesis was absent in mice lacking the gene encoding the neuronal PAS domain protein 3 (NPAS3), and they exhibited dentate gyrus malformation and electrophysiological dysfunction. Long-term administration of P7C3 to npas3(-/-) mice could correct these defects by restoring the apoptosis level of newborn hippocampal neurons to normal. Long-term administration of P7C3 to aged rats enhanced dentate gyrus neurogenesis, inhibited neuronal death, and maintained cognitive abilities during terminal aging. [1]

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Researchers used a target-independent in vivo screening method to discover a novel neuroprotective small molecule in live mice. This aminopropylcarbazole compound, named P7C3, has good oral bioavailability, can cross the blood-brain barrier, and is non-toxic at doses far above the effective dose. Researchers have optimized the potency and drug-like properties of P7C3 through medicinal chemistry methods and obtained analogs for detailed studies. Improved analogs, such as (-)-P7C3-S243 and P7C3-A20, have shown neuroprotective effects in rodent models of Parkinson's disease, amyotrophic lateral sclerosis, traumatic brain injury, and age-related cognitive decline. Derivatives with immobilized groups may reveal the protein targets of P7C3-like neuroprotective compounds. Our results suggest that unbiased in vivo screening may provide a starting point for developing treatments for neurodegenerative diseases and for studying the biological mechanisms of these diseases. [2] Parkinson's disease (PD) is the second most common neurodegenerative disease. Although its pathogenesis is not well understood, increasing evidence suggests that microglia-mediated neuroinflammation plays an important role in the progression of PD. P7C3 is an aminopropylcarbazole compound that has shown significant neuroprotective effects in animal models of various neurodegenerative diseases, including neurodegenerative diseases (PD). In this study, we aimed to investigate the effects of P7C3 on neuroinflammation. We found that P7C3 specifically inhibited the expression of pro-inflammatory factors in microglia induced by lipopolysaccharide (LPS), but had no effect on anti-inflammatory factors. The anti-inflammatory mechanism of P7C3 involves the inhibition of the nuclear factor κB (NF-κB) signaling pathway. In microglia, P7C3 pretreatment attenuated LPS-induced IκB kinase (IKK) activation, inhibitory κBα (IκBα) degradation, and NF-κB nuclear translocation. Furthermore, in LPS-treated microglia, P7C3 pretreatment reduced the toxicity of conditioned medium to MES23.5 cells (a dopaminergic (DA) cell line). More importantly, the anti-inflammatory effect of P7C3 was also observed in an LPS-stimulated mouse model. In summary, our study shows that P7C3 inhibits LPS-induced microglial activation both in vivo and in vitro by inhibiting the NF-κB pathway, providing a theoretical basis for the anti-inflammatory effect of P7C3. [3]

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Traumatic brain injury (TBI) is a significant public health problem worldwide. A promising area of research is the identification of small molecule drugs with strong clinical activity. One such drug, an aminopropylcarbazole called P7C3, was discovered through in vivo screening aimed at finding drugs that can enhance net neurogenesis in the adult hippocampus. P7C3 significantly enhances neurogenesis by increasing the survival rate of immature neurons. The potent neuroprotective effect of P7C3 may be attributed to its enhanced nicotinamide phosphoribosyltransferase (NAMPT) activity, which supports key cellular processes. Studies have found that the P7C3 scaffold has good pharmacokinetic properties, bioavailability, and is non-toxic. Preclinical studies have shown that administration of the P7C3 series of neuroprotective compounds after traumatic brain injury (TBI) can rescue and reverse harmful cellular events, thereby improving functional recovery. In various TBI models and different species, P7C3 and its analogues have shown significant neuroprotective effects, including axonal protection, a significant increase in net adult neurogenesis, prevention of injury-induced long-term enhancement (LTP) deficiency, and improvement of neurological function. This review will elucidate the exciting and diverse therapeutic outcomes achieved by P7C3 administration in the face of the complex and multifactorial cellular and molecular challenges of experimental TBI. The clinical potential and broad therapeutic prospects of P7C3 warrant further investigation to verify whether these therapeutic effects can be replicated in clinical practice. P7C3 is expected to be an important step in the design, understanding, and implementation of drug therapy for TBI patients. This article is part of a special issue entitled “New Therapies for Traumatic Brain Injury”. [4] Mechanism of action: P7C3 exerts its neuroprotective and neurogenic effects by activating NAMPT, the rate-limiting enzyme in the NAD⁺ biosynthetic salvage pathway. Enhanced NAMPT activity can increase intracellular NAD⁺ levels, thereby promoting ATP production, inhibiting neuronal apoptosis (by activating SIRT1), and alleviating neuroinflammation (by inhibiting microglia activation) [1,3]. Research Applications: P7C3 is a preclinical tool compound for studying neurodegenerative diseases (Parkinson's disease, Alzheimer's disease), neuroinflammation, and traumatic brain injury (TBI). In preclinical models, P7C3 has shown therapeutic potential by protecting neurons, promoting neurogenesis, and alleviating neurological deficits [1,3,4]. Current Status: P7C3 is currently in the preclinical research stage and has not yet undergone clinical trial evaluation or received FDA approval for therapeutic use. Its good oral bioavailability and blood-brain barrier penetration make it a potential lead compound for the treatment of central nervous system (CNS) diseases [2].
- Chemical properties: P7C3 is a small molecule compound with good chemical stability (stable for 72 hours in aqueous solution at 37°C) and solubility in common solvents (DMSO, PEG400, 0.5% CMC-Na) [2].

C21H18BR2N2O

474.19

471.978

C, 53.19; H, 3.83; Br, 33.70; N, 5.91; O, 3.37

301353-96-8

P7C3-A20;1235481-90-9

2836187

White to off-white solid powder

1.6±0.1 g/cm3

656.4±55.0 °C at 760 mmHg

350.8±31.5 °C

0.0±2.1 mmHg at 25°C

1.687

6.6

2

2

5

26

433

0

BrC1C([H])=C([H])C2=C(C=1[H])C1C([H])=C(C([H])=C([H])C=1N2C([H])([H])C([H])(C([H])([H])N([H])C1C([H])=C([H])C([H])=C([H])C=1[H])O[H])Br

FZHHRERIIVOATI-UHFFFAOYSA-N

InChI=1S/C21H18Br2N2O/c22-14-6-8-20-18(10-14)19-11-15(23)7-9-21(19)25(20)13-17(26)12-24-16-4-2-1-3-5-16/h1-11,17,24,26H,12-13H2

1-anilino-3-(3,6-dibromocarbazol-9-yl)propan-2-ol

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.39 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.39 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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