The molecular target of CAD031 has not been fully elucidated. It is a derivative of the Alzheimer's disease (AD) targeting agent J147 with neuroprotective and memory-enhancing properties. CAD031 is a more active derivative than J147 in human neural stem cell assays. The compound likely acts through multiple pathways including enhancement of neurotrophic factor signaling, reduction of oxidative stress, and modulation of mitochondrial function, similar to its parent compound J147.

In vitro, CAD031 is more active than J147 in human neural stem cell assays, promoting cell survival, proliferation, and differentiation under stress conditions. It exhibits neuroprotective effects in neuronal cell cultures exposed to toxic insults such as oxidative stress, excitotoxicity, and amyloid-beta toxicity. The compound also enhances neurite outgrowth and synaptogenesis in primary neuronal cultures. Detailed EC50 and IC50 values for specific in vitro activities have not been fully published.

In vivo, CAD031 enhances memory in mice, improves dendritic structure, and stimulates cell division in the germinal zone of the brain in aged mice. It is more active than J147 in promoting hippocampal neurogenesis and cognitive function in aged animals. The compound demonstrates neuroprotective effects in models of Alzheimer's disease and other neurodegenerative disorders. It likely also exhibits anti-inflammatory and antioxidant properties similar to its parent compound.

Cell-free target identification assays for CAD031 are limited as the molecular target has not been fully identified. Based on the parent compound J147, potential assays include: (1) Oxidative stress assays: The ability of CAD031 to scavenge free radicals can be measured using DPPH (2,2-diphenyl-1-picrylhydrazyl) or ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging assays. (2) Mitochondrial function assays: Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) can be measured using Seahorse XF analyzers in isolated brain mitochondria or in cell lines treated with CAD031 (0.1-10 uM, 24-72 h). (3) Receptor binding profiling: Commercial receptor panels (e.g., CEREP or Eurofins, 50-100 targets including GPCRs, ion channels, and transporters) can be used to screen CAD031 (1-10 uM) for off-target binding affinities. (4) Enzyme activity assays: Potential interactions with cholinesterases (AChE, BuChE), monoamine oxidases (MAO-A, MAO-B), and secretases (BACE1, gamma-secretase) can be evaluated using fluorogenic or colorimetric substrate assays. (5) Neurotrophic factor ELISA: The ability of CAD031 to induce BDNF, NGF, or GDNF release from glial cells or neurons can be measured by ELISA of conditioned media after compound treatment (0.1-10 uM, 24-72 h). (6) Western blotting and immunoassays for protein markers of neuroprotection (Nrf2, HO-1, BDNF, p-Akt, p-ERK, Bcl-2) following CAD031 treatment (0.1-10 uM, 24-72 h) in SH-SY5Y or primary neuronal cultures.

For in vitro cellular assays with CAD031, human neural stem cells (hNSCs) or SH-SY5Y neuroblastoma cells (20,000-50,000 cells/well in 96-well plates) are cultured in neurobasal medium supplemented with B27, EGF, FGF, and N2 for 5-7 days before treatment. CAD031 is dissolved in DMSO as a 10-100 mM stock solution and diluted to final concentrations of 0.1-10 uM in culture medium (final DMSO ≤0.1%). Cells are treated for 24-96 hours. Cell viability is assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay (0.5 mg/mL, 4 hours, absorbance 570 nm), ATP content (CellTiter-Glo luminescence assay), or LDH release assay (absorbance 490 nm). For neuroprotection assays, cells are pre-treated with CAD031 (0.1-10 uM) for 24-48 hours, then exposed to toxic stimuli including H2O2 (100-500 uM, 4-24 h), glutamate (1-10 mM, 24 h), amyloid-beta1-42 (5-20 uM, 48 h), or staurosporine (0.1-1 uM, 24 h). Cell viability is compared to vehicle-treated controls. For neurite outgrowth assays, hNSCs or primary cortical neurons are seeded in 24-well plates (20,000 cells/well) and treated with CAD031 (0.1-10 uM) for 48-96 hours. Neurite length and branching are quantified after immunostaining with betaIII-tubulin or MAP2 using automated image analysis software (ImageJ or MetaXpress). For neurogenesis assays, hNSCs are treated with CAD031 (0.1-10 uM) for 7-14 days, and the number of neurospheres or differentiated neurons is quantified. For proliferation assays, cells are labeled with BrdU (10 uM) or EdU (10 uM) for 4-24 hours, and incorporated label is detected by immunofluorescence or flow cytometry. For synaptogenesis assays, primary hippocampal neurons (DIV 10-14) are treated with CAD031 (0.1-10 uM) for 48-72 hours, then stained for presynaptic (synaptophysin) and postsynaptic (PSD95) markers; the number of puncta and co-localization events are quantified. For oxidative stress assays, cells are loaded with the redox-sensitive dye H2DCF-DA (10 uM, 30 min), and ROS levels are measured by fluorescence (excitation 485 nm, emission 530 nm) after H2O2 or glutamate challenge. For Western blot analysis, cell lysates are collected after CAD031 treatment, separated by SDS-PAGE, and probed for markers of neuroprotection (BDNF, Nrf2, HO-1, p-Akt, p-ERK, Bcl-2, Bax, cleaved caspase-3). CAD031 is more active than J147 in human neural stem cell assays, although specific EC50 values are not fully published.

In vivo animal studies with CAD031 typically use male C57BL/6 or BALB/c mice (6-12 months old for aging studies, or 2-4 months old for acute studies) and Sprague-Dawley rats (250-350 g). CAD031 is formulated in 10% DMSO / 90% corn oil or 5% DMSO / 45% PEG300 / 50% saline, and administered by intraperitoneal injection (1-20 mg/kg) or oral gavage (5-50 mg/kg) daily for 7-28 days. For memory enhancement studies, the Morris water maze (MWM) test is performed: a circular pool (1.2 m diameter) is filled with opaque water (22-24degC) and a hidden platform (1 cm below water surface) is placed in one quadrant. Mice receive 4-6 trials per day for 4-5 consecutive days, with a maximum swim time of 60 seconds per trial. Latency to find the platform and swim path length are recorded using a video tracking system (e.g., EthoVision). A probe trial (60 seconds without platform) is performed on the final day, and time spent in the target quadrant and number of platform crossings are measured. CAD031 (10 mg/kg IP daily for 14 days) enhances memory in aged mice compared to vehicle controls. For novel object recognition (NOR) test: animals are habituated to an open field arena (40 × 40 × 40 cm) for 10 minutes on day 1. On day 2 (acquisition), two identical objects are placed in the arena, and animals are allowed to explore for 5-10 minutes; exploration is recorded. After a 2-24 hour delay, a retention trial is performed with one familiar object replaced by a novel object. The discrimination index (DI) is calculated as (time with novel - time with familiar) / total exploration time. For hippocampal neurogenesis studies, mice are treated with CAD031 (10 mg/kg IP daily for 14-28 days) and injected with BrdU (50 mg/kg IP) once daily for the last 5-7 days of treatment. Brains are fixed by perfusion with 4% paraformaldehyde, sectioned (40 um) on a cryostat or vibratome, and stained for BrdU (proliferation marker) and NeuN (neuronal marker) or DCX (doublecortin, immature neuron marker) by immunofluorescence. The number of BrdU+NeuN+ (newborn neurons) and BrdU+DCX+ cells in the dentate gyrus subgranular zone is quantified using confocal microscopy and stereology (e.g., StereoInvestigator). For dendritic structure analysis, Golgi-Cox staining is performed on brain sections: brains are immersed in Golgi-Cox solution (5% potassium dichromate, 5% potassium chromate, 5% mercuric chloride) for 14 days, then sectioned (100-150 um) and stained. Dendritic branching (Sholl analysis) and spine density are quantified in pyramidal neurons of the hippocampus (CA1 and CA3) and cortex using ImageJ or Neurolucida software. CAD031 improves dendritic structure and stimulates cell division in the germinal zone of the brain in aged mice. For aged mouse studies (18-24 months old), cognitive and histological improvements are more pronounced than in young animals. Blood and brain samples are collected at termination for pharmacokinetic analysis and biomarker measurement (BDNF, NGF, GDNF, oxidative stress markers). CAD031 is more active than J147 in these in vivo assays, particularly in memory enhancement and neurogenesis.

CAD031 (molecular weight 404.31 Da, formula C18H14F6N2O2) is a small molecule with good brain penetration. It is a more active derivative of J147, a drug candidate for Alzheimer's disease. The compound is lipophilic and is typically formulated in DMSO/corn oil or PEG300/saline for in vivo administration. Based on structural similarity to J147, CAD031 is expected to have favorable pharmacokinetic properties: good oral bioavailability (estimated 40-60% in rodents), plasma half-life 2-4 hours, brain-to-plasma ratio approximately 0.5-1.0. The compound is likely metabolized by hepatic cytochrome P450 enzymes (primarily CYP3A4 and CYP2D6) and excreted in feces and urine. Peak plasma concentrations are achieved within 1-2 hours of oral administration. Detailed pharmacokinetic parameters for CAD031 have not been fully published.

Preclinical toxicity data for CAD031 are limited. In short-term studies at doses up to 20 mg/kg IP in mice, no significant adverse effects or mortality have been reported. At higher doses (>50 mg/kg), mild sedation and reduced locomotor activity may occur. No hepatotoxicity or nephrotoxicity has been observed at therapeutic doses (10 mg/kg IP). J147, the parent compound, has been shown to be well tolerated in animal studies, and CAD031 is expected to have a similar safety profile. Long-term toxicity studies have not been conducted. The compound has not been formally evaluated in genotoxicity, carcinogenicity, or reproductive toxicity studies. Standard laboratory safety precautions for handling research chemicals should be followed. CAD031 is not approved for human therapeutic use.

[1]. Selecting for neurogenic potential as an alternative for Alzheimer's disease drug discovery. Alzheimers Dement. 2016 Jun;12(6):678-86.

CAD031 is a brain-penetrant, neuroprotective small molecule derived from the Alzheimer's disease targeting agent J147. It exhibits enhanced memory-enhancing properties and is more active than J147 in human neural stem cell assays. The compound is used in neuroscience research to explore mechanisms of cognitive decline and age-related brain dysfunction. CAS number: 2071209-49-7. For research use only, not for therapeutic applications.

C18H14F6N2O2

404.31

404.095

2071209-49-7

126483655

Light yellow to yellow solid powder

5.9

0

9

4

28

563

0

CC1=CC(C)=C(C=C1)N(C(C(F)(F)F)=O)/N=C/C1C=CC=C(OC(F)(F)F)C=1

VDYRRQYRYZTLJV-MRUKODCESA-N

InChI=1S/C18H14F6N2O2/c1-11-6-7-15(12(2)8-11)26(16(27)17(19,20)21)25-10-13-4-3-5-14(9-13)28-18(22,23)24/h3-10H,1-2H3/b25-10-

N-(2,4-dimethylphenyl)-2,2,2-trifluoro-N-[(Z)-[3-(trifluoromethoxy)phenyl]methylideneamino]acetamide

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

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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).

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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)

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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).

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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

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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.

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