Melatonin MT2 receptor (Ki = 1.58 nM for human recombinant Mel1b receptor) [2]
Melatonin MT1 receptor (Ki = 466 nM for human recombinant Mel1a receptor) [2]

Aminotetrahydroethylene 4-P-PDOT (10 mM) in the absence or presence of melatonin did not alter the levels of cyclic AMP induced by forskolin in CHO-mt1 cells. On the other hand, 4-P-PDOT is an agonist that, in the presence of -MT2 cells in CHO, results in concentration-dependent suppression of cyclic AMP induced by forskolin, with a pEC50 value of 8.72 and an actual activity of 0.86 [1]. Test for Cell Viability [5] Cell

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It competitively antagonizes the inhibitory effect of melatonin on the calcium-dependent release of [³H]dopamine from rabbit retina, with a KB value of 9.3 nM and a pA2 value of 8.03. [2]
It has no intrinsic efficacy (no agonistic activity) in the [³H]dopamine release assay in rabbit retina at the concentrations tested (100, 1000, 10000 nM). [2]
In human colorectal cancer HT-29 cells and cervical cancer HeLa cells, pre-treatment with 50 µM 4P-PDOT for 30 minutes does not reverse the potentiating effects of melatonin on chemotherapy-induced (cisplatin or 5-fluorouracil) cytotoxicity, caspase-3 activation, or apoptotic cell death. [4]
Pre-treatment with 1.0 mg/kg 4P-PDOT counteracts the melatonin-mediated attenuation of the decrease in ERK phosphorylation in the hippocampus of klotho mutant mice. [5]
Pre-treatment with 0.5 or 1.0 mg/kg 4P-PDOT counteracts the melatonin-mediated attenuation of decreased Nrf2 nuclear translocation, Nrf2 DNA-binding activity, and GCLc/GCLm mRNA expression in the hippocampus of klotho mutant mice. [5]

The administration of 4-P-PDOT (0.5-1.0 mg/kg; intravenous; klotho mutation) markedly counteracted the antioxidant benefits mediated by melatonin. and dramatically undid the variations in these GSH-related parameter values. 4-P-PDOT therapy dramatically improved the memory function of klotho mutants treated with melatonin. 4-P-PDOT can also control Nrf2, the expression of phosphorylated ERK in the klotho mutant hippocampal region, which is known as melatonin-mediated signal attenuation.

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In klotho mutant mice, which exhibit memory impairment, treatment with melatonin (20 mg/kg, i.p.) significantly attenuates memory deficits. This memory-enhancing effect of melatonin is significantly reversed by pre-treatment with 1.0 mg/kg 4P-PDOT (i.v.) in both the novel object recognition test and the passive avoidance test. [5]
In klotho mutant mice, the protective effects of melatonin (20 mg/kg, i.p.) against increases in hippocampal oxidative stress markers (synaptosomal ROS, malondialdehyde, protein carbonyl) and homeostatic imbalance of the glutathione system (decreases in GSH level and GSH/GSSG ratio) are significantly counteracted by pre-treatment with 1.0 mg/kg 4P-PDOT (i.v.). [5]

Competition binding assays for the human recombinant Mel1a and Mel1b melatonin receptors are performed. COS-7 cell lysates expressing the receptors are incubated with 80 pM of the radioligand 2-[¹²⁵I]-iodomelatonin and various concentrations of test compounds (0.1 pM - 100 µM) for 1.5 hours at 25°C. The incubation is terminated by rapid vacuum filtration through glass fiber filters. Specific binding is defined using 1 µM melatonin. The equilibrium dissociation constants (Ki values) are calculated from IC50 values obtained from competition curves using the method of Cheng and Prusoff. Under these conditions, 4P-PDOT shows a Ki of 466 nM for the Mel1a receptor and 1.58 nM for the Mel1b receptor. [2]

Cell Viability Assay[5] Cell
Cell Types: HT-29 and HeLa cells.
Tested Concentrations: 50 µM
Incubation Duration: 30 minutes
Experimental Results: Negligible effect on melatonin-induced cell viability.

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Cell viability is evaluated using the MTT assay. HT-29 and HeLa cells are seeded in 12-well plates at a density of 0.1 x 10⁶ cells per well. Cells are pre-treated with 50 µM 4P-PDOT for 30 minutes, then incubated with chemotherapeutic agents (20 µM cisplatin or 1 mM 5-fluorouracil) in the absence or presence of 1 mM melatonin for 48 hours. After treatments, MTT is added to each well and incubated for 60 minutes at 37°C. The supernatant is discarded, and DMSO is added to dissolve the formazan crystals. Optical density is measured at 490 nm and 650 nm (reference). Data are presented as percentage of control (untreated cells). [4]
Apoptosis is determined using an Annexin V-FITC Apoptosis Detection Kit. HT-29 and HeLa cells (1.2 x 10⁶ cells/mL) are pre-treated with drugs, harvested by trypsinization, washed, and centrifuged. The pellet is resuspended in binding buffer containing annexin V-FITC, incubated for 10 minutes, washed, and resuspended in binding buffer containing propidium iodide (PI). Cells are immediately analyzed by flow cytometry, with 10,000 events analyzed using FL-1 (annexin V-FITC) and FL-3 (PI) detectors. [4]
Caspase-3 activity is measured. Stimulated or resting cells (1.2 x 10⁶ cells/mL) are sonicated, and cell lysates are incubated with a substrate solution (20 mM HEPES, pH 7.4, 2 mM EDTA, 0.1% CHAPS, 5 mM DTT, and 8.25 µM caspase-3 substrate AC-DEVD-AMC) for 1 hour at 37°C. Activity is calculated from the cleavage of the fluorogenic substrate, measured with a microplate reader at excitation 360 nm and emission 460 nm. [4]
Western blot analysis is performed on hippocampal tissues from mice. Tissues are homogenized in lysis buffer containing Tris-HCl, SDS, EGTA, EDTA, glycerol, and phosphatase/protease inhibitor cocktails. Lysates are centrifuged, and the supernatant is used for SDS-PAGE and immunoblotting with anti-phospho-ERK and anti-ERK antibodies. [5]
Nuclear translocation of Nrf2 is analyzed. Nuclear and cytosolic fractions of hippocampal lysates are extracted using a commercial kit. The fractions are subjected to SDS-PAGE and immunoblotted with anti-Nrf2 antibody. Anti-histone H4 and anti-β-actin are used as loading controls for nuclear and cytosolic fractions, respectively. [5]
Nrf2 DNA-binding activity is measured using a commercial TransAM Nrf2 transcription factor ELISA kit. Nuclear protein extracts (10 µg) are added to wells coated with an oligonucleotide containing an ARE consensus binding site. After incubation and washing, the plate is incubated with an anti-Nrf2 antibody, followed by a secondary antibody. A colorimetric reaction is initiated, and absorbance is measured at 450 nm. [5]
RT-PCR is performed to assess mRNA expression of GCLc and GCLm. Total RNA is isolated from hippocampal tissues, and reverse transcription is carried out. PCR products are separated on 2% agarose gels containing ethidium bromide and visualized under ultraviolet light. [5]

Animal/Disease Models: treating Klotho mutant mice with melatonin [2]
Doses: 0.5 mg/ kg or 1.0 mg/kg.
Route of Administration: intravenous (iv) (iv)injection.
Experimental Results: Dramatically reversed nuclear translocation, Nrf2 DNA binding activity and diminished GCL mRNA expression [2]. Antioxidant effects mediated by melatonin. Dramatically reversed the changes in the levels of these GSH-related parameters. Dramatically reverses memory function in melatonin-treated klotho mutant mice.

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For studies in klotho mutant mice (a genetic model of aging derived from a C3H/Hej background), 4P-PDOT is dissolved in 20% DMSO and then diluted in sterile saline. It is administered intravenously (i.v.) at doses of 0.5 or 1.0 mg/kg, 5 minutes before the memory trial in behavioral tests (novel object recognition test and passive avoidance test). [5]
Mice receive melatonin (10, 20, or 30 mg/kg, i.p.) twice a day for 17 days from postnatal day (PND) 35 to 51. On behavioral testing days (PND 52, 53 for NORT; PND 54, 55 for PAT), mice receive an additional dose of melatonin 45 minutes prior to the test. 4P-PDOT is administered 5 minutes before the memory trial. Mice are sacrificed 30 minutes after the PAT retention trial on PND 55 for neurochemical assays. [5]
For the in vitro cell studies, HT-29 and HeLa cells are pre-treated with 50 µM 4P-PDOT for 30 minutes before being incubated with melatonin and/or chemotherapeutic agents (cisplatin or 5-fluorouracil) for 48 hours. [4]

[1]. Dubocovich ML. Melatonin receptors: are there multiple subtypes? Trends Pharmacol Sci. 1995 Feb;16(2):50-6.

[2]. Melatonin attenuates memory impairment induced by Klotho gene deficiency via interactive signaling between MT2 receptor, ERK, and Nrf2-related antioxidant potential. Int J Neuropsychopharmacol. 2014 Dec 30;18(6). pii: pyu105.

[3]. Pharmacological characterization of human recombinant melatonin mt1 and MT2 receptors. British Journal of Pharmacology (2000) 129, 877-886.

[4]. Melatonin receptor antagonists that differentiate between the human Mel1a and Mel1b recombinant subtypes are used to assess the pharmacological profile of the rabbit retina ML1 presynaptic heteroreceptor. Naunyn Schmiedebergs Arch Pharmacol. 1997 Mar;355(3):365-75.

[5]. Participation of MT3 melatonin receptors in the synergistic effect of melatonin on cytotoxic and apoptotic actions evoked by chemotherapeutics. Cancer Chemother Pharmacol. 2017 Nov;80(5):985-998.

N-(4-phenyl-1,2,3,4-tetrahydronaphth-2-yl)propionamide is a member of the tetrahydronaphthyl group of compounds.

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4P-PDOT (4-phenyl-2-propionamidotetralin) is a non-indolic competitive melatonin receptor antagonist. It is a selective antagonist for the MT2 (Mel1b) melatonin receptor subtype, showing high selectivity (295-fold higher affinity for Mel1b over Mel1a based on Ki values: 466 nM vs 1.58 nM). [2]
The selectivity affinity ratio (Mel1a/Mel1b) for 4P-PDOT is greater than 100. [2]
In the context of cancer research, pre-treatment with 4P-PDOT (an MT2 antagonist) does not block the synergistic pro-apoptotic and cytotoxic effects of melatonin with chemotherapeutic agents (cisplatin, 5-fluorouracil) in HT-29 and HeLa cells, suggesting these actions are not mediated via MT2 receptors. [4]
In a study on cognitive impairment, the protective effects of melatonin against memory deficits and oxidative stress in klotho mutant mice are mediated through the MT2 receptor, as these effects are counteracted by the selective MT2 antagonist 4P-PDOT. [5]
The molecular signaling pathway proposed involves melatonin activating the MT2 receptor, leading to ERK phosphorylation, which in turn promotes Nrf2 nuclear translocation and DNA-binding activity. This upregulates the expression of GCLc and GCLm, restoring GSH levels and reducing oxidative stress, ultimately improving memory function. [5]

C19H21NO

279.383

279.162

134865-74-0

3976006

Off-white to yellow solid powder

1.11g/cm3

480.1ºC at 760 mmHg

292ºC

2.22E-09mmHg at 25°C

1.595

4.05

1

1

3

21

348

0

RCYLUNPFECYGDW-UHFFFAOYSA-N

InChI=1S/C19H21NO/c1-2-19(21)20-16-12-15-10-6-7-11-17(15)18(13-16)14-8-4-3-5-9-14/h3-11,16,18H,2,12-13H2,1H3,(H,20,21)

cis-N-(4-phenyl-1,2,3,4-tetrahydronaphthalen-2-yl)propanamide

4P-PDOT 4PPDOTAH-0244-P-PDOT 4-phenyl-2- propionamidotetralin

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 : ~41.67 mg/mL (~149.15 mM)

Solubility in Formulation 1: ≥ 4.17 mg/mL (14.93 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 41.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 2: ≥ 4.17 mg/mL (14.93 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 41.7 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.)