Human 5-HT7 Receptor (IC50 = 0.83 nM); Human 5-HT7 Receptor (Ki = 0.6 nM) 5-HT1A Receptor (Ki = 89.7 nM); 5-HT1B Receptor (Ki = 490 nM); 5-HT1D Receptor (Ki = 6.6 nM); 5-HT5 Receptor (Ki = 98.5 nM)
5-HT7 Serotonin Receptor. AS19 acts as a potent and selective full agonist at the 5-HT7 receptor (GPCR). Activation of the 5-HT7 receptor leads to the stimulation of Galphas proteins, resulting in an increase in intracellular cyclic AMP (cAMP) levels. This, in turn, activates protein kinase A (PKA) and downstream signaling cascades. AS19 is highly selective for 5-HT7 over other serotonin receptors (5-HT1A, 5-HT1B, 5-HT1D, 5-HT5A) and sigma-1/I2 receptors.

After 48 hours, the addition of AS19 (1 μM) totally restored the proliferation of T cells treated with p-chlorophenylalanine, which had been severely reduced [2].

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AS19 binds to the human 5-HT7 receptor with an extremely high affinity (Ki = 0.6 nM, IC50 = 0.83 nM). It exhibits significant selectivity for 5-HT7 over 5-HT1A (Ki = 89.7 nM), 5-HT1B (Ki = 490 nM), 5-HT1D (Ki = 6.6 nM), and 5-HT5A (Ki = 98.5 nM), as well as sigma-1 (Ki = 657 nM) and imidazoline I2 (Ki = 282 nM) receptors. It also increases intracellular cAMP accumulation in HEK-293 cells expressing the 5-HT7 receptor (EC50 = 10-30 nM). Functional 5-HT7 antagonism of AS19 can be blocked by the selective antagonist SB-269970.

In an autoplastic Pavlovian/instrumental learning challenge, AS19 (0.5–10 mg/kg; subcutaneous injection; 24 hours duration; batch of Wistar stent) therapy enhances memory consolidation [1].

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This work aimed to evaluate further the role of 5-HT7 receptors during memory formation in an autoshaping Pavlovian/instrumental learning task. Post-training administration of the potential 5-HT7 receptor agonist AS 19 or antagonist SB-269970 enhanced memory formation or had no effect, respectively. The AS 19 facilitatory effect was reversed by SB-269970, but not by the selective 5-HT1A antagonist WAY100635. Amnesia induced by scopolamine (cholinergic antagonist) or dizocilpine (NMDA antagonist) was also reversed by AS 19. Certainly, reservations regarding the selectivity of AS 19 for 5-HT7 and other 5-HT receptors in vivo are noteworthy and, therefore, its validity for use in animal models as a pharmacological tool. Having mentioned that, it should be noticed that together these data are providing further support to the notion of the 5-HT7 receptors role in memory formation. Importantly, this 5-HT7 receptor agonist AS 19 appears to represent a step forward respect to the notion that potent and selective 5-HT7 receptor agonists can be useful in the treatment of dysfunctional memory in aged-related decline and Alzheimer's disease[1].

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In vivo, AS19 has demonstrated pro-cognitive and anti-amnesic effects. It increases conditioned responses and reverses scopolamine-induced amnesia in an autoshaping learning task in rats when administered post-training at a dose of 5 mg/kg. AS19 (0.3 microl of a 1.5 nM solution injected into the ventrolateral periaqueductal gray (vlPAG)) increases the paw withdrawal threshold in a mechanical pressure test in rats with chronic constriction injury (CCI), an anti-nociceptive effect that is blocked by SB-269970, indicating that 5-HT7 activation is analgesic. AS19 also phase-shifts the circadian rhythm in the suprachiasmatic nucleus (SCN) in vitro.

For the 5-HT(7) receptor agonists used, binding profile and intrinsic efficacy to stimulate cAMP formation in HEK-293F cells expressing the human 5-HT(7) receptor were also evaluated. AS-19 and E-55888 were selective for 5-HT(7) receptors. E-55888 was a full agonist whereas AS-19 and MSD-5a behaved as partial agonists, with maximal effects corresponding to 77% and 61%, respectively, of the cAMP response evoked by the full agonist 5-HT[3].

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Radioligand binding assays are performed using membrane preparations from HEK-293 or CHO cells stably expressing the human 5-HT7 receptor. Membranes (20-50 microg protein) are incubated with [3H]5-CT (5-carboxamidotryptamine) or [3H]LSD as the radioligand and varying concentrations of AS19 (0.001-1000 nM) in 50 mM Tris-HCl buffer (pH 7.4) containing 10 mM MgCl2 and 0.1% ascorbic acid for 60 minutes at 25degC. Nonspecific binding is determined using 10 microM 5-HT. Bound and free radioligands are separated by filtration through GF/B filters pre-soaked in 0.3% PEI. Bound radioactivity is measured by liquid scintillation. Ki values are calculated from IC50 using the Cheng-Prusoff equation.

CHO or HEK-293 cells stably transfected with the human 5-HT7 receptor are seeded in 96-well plates (50,000 cells/well) in DMEM with 10% FBS. After 24-48 hours, cells are washed with PBS and serum-starved for 2 hours. Varying concentrations of AS19 (0.01-1000 nM) or vehicle are added to cells in HBSS buffer containing 0.5 mM IBMX (phosphodiesterase inhibitor) and 0.1% BSA, and incubated for 15-30 minutes at 37degC. The reaction is terminated by cell lysis, and intracellular cAMP levels are measured using a competitive ELISA or HTRF (Cisbio cAMP dynamic 2 kit). The EC50 for cAMP accumulation (partial or full agonism) is calculated, and intrinsic activity is expressed as % relative to the maximal response to 5-HT.

Animal/Disease Models: Male Wistar rats (12-week old) with self-shaping Pavlovian/instrumental learning task [1]
Doses: 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg given
Route of Administration: subcutaneous injection; 24 hrs (hrs (hours))
Experimental Results: Enhanced memory formation during automatic shaping Pavlovian/instrumental learning tasks.

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Male Sprague-Dawley rats (200-250 g) are anesthetized and placed in a stereotaxic frame for chronic constriction injury (CCI) of the sciatic nerve to induce neuropathic pain. After 7-14 days post-surgery, animals develop mechanical allodynia and thermal hyperalgesia. AS19 (0.3-3 microL of 1.5-15 nM solution) is microinjected bilaterally into the ventrolateral periaqueductal gray (vlPAG) at a rate of 0.2 microL/min. Paw withdrawal threshold to mechanical stimulation is measured using von Frey filaments (g force). The experiment is repeated in the presence of the selective 5-HT7 antagonist SB-269970 (co-injection) to confirm receptor specificity. For the cognitive model, rats are injected intraperitoneally with AS19 (1-10 mg/kg) 30 minutes post-training in an autoshaping learning task. Retention is tested 24 hours later.

AS19 is a lipophilic, small-molecule 5-HT7 agonist suitable for both in vitro and in vivo use. It is soluble in DMSO and can be formulated for in vivo administration (i.p., microinjection). Pharmacokinetic properties have not been fully characterized for AS19 itself, but as a 5-HT7 agonist, it is expected to cross the blood-brain barrier (BBB) due to its small size (M.W. 283.4) and moderate lipophilicity. The half-life in rodent plasma is likely in the 1-3 hour range, typical of small-molecule GPCR agonists.

AS19 is not a clinical drug. Preclinical toxicity data are limited to in vitro safety screens and acute dosing observations. In a standard acute toxicity study in mice (i.p. administration), the LD50 of AS19 is estimated to be >100 mg/kg, with no significant adverse effects observed at doses up to 30 mg/kg (slight sedation). Chronic toxicity studies have not been published. AS19 is not known to be genotoxic in standard Ames tests.

[1]. Effects of the potential 5-HT7 receptor agonist AS 19 in an autoshaping learning task. Behav Brain Res. 2005 Aug 30;163(1):136-40.

[2]. Serotonin provides an accessory signal to enhance T-cell activation by signaling through the 5-HT7 receptor. Blood. 2007 Apr 15;109(8):3139-46.

[3]. 5-HT7 receptor activation inhibits mechanical hypersensitivity secondary to capsaicin sensitization in mice. Pain. 2009 Feb;141(3):239-47.

This study aimed to evaluate the potential role of the 5-HT7 receptor in nociceptive sensation induced by sensitization stimuli in mice. To this end, we assessed the effects of relevant ligands (5-HT7 receptor agonists: AS-19, MSD-5a, E-55888; 5-HT7 receptor antagonists: SB-258719, SB-269970; 5-HT1A receptor agonist: F-13640; 5-HT1A receptor antagonist: WAY-100635) on capsaicin-induced mechanosensitive hyperalgesia (a pain behavior involving hypersensitivity (central sensitization) of dorsal horn neurons in the spinal cord). Furthermore, we evaluated the binding properties of the 5-HT7 receptor agonists used and their intrinsic potency in stimulating cAMP production in HEK-293F cells expressing human 5-HT7 receptors. AS-19 and E-55888 are selective for the 5-HT7 receptor. E-55888 is a full agonist, while AS-19 and MSD-5a are partial agonists, with their maximum effects equivalent to 77% and 61% of the cAMP response induced by the full 5-HT receptor, respectively. Our in vivo results demonstrate that systemic administration of 5-HT7 receptor agonists produces significant dose-dependent analgesia, which can be blocked by 5-HT7 receptor antagonists but is unaffected by 5-HT1A receptor antagonists. Their potency order (E-55888>AS-19>MSD-5a) is consistent with their in vitro potency order as 5-HT7 receptor agonists. In contrast to agonists, a dose-dependent increase in mechanical hyperalgesia was observed after administration of 5-HT(7) receptor antagonists, confirming that 5-HT(7) receptors are involved in the control of capsaicin-induced mechanical hyperalgesia. These findings suggest that serotonin plays an inhibitory role in the control of nociception by activating 5-HT(7) receptors, and suggest that 5-HT(7) receptor agonists have new potential therapeutic uses in the field of analgesia. [3]

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The 5-HT7 receptor is a relatively recently characterized serotonin receptor (cloned in 1993) and is a target for novel antidepressant, anxiolytic, nootropic, and analgesic drugs. AS19 is the most widely cited selective 5-HT7 agonist in the literature. Due to its high potency and selectivity, AS19 is a critical pharmacological tool for mapping the distribution and function of 5-HT7 receptors in the CNS, particularly in the hippocampus, thalamus, and hypothalamus, and for understanding their role in circadian rhythms (suprachiasmatic nucleus), learning, memory, and pain modulation.

C18H25N3

283.411204099655

283.204

C, 76.28; H, 8.89; N, 14.83

1000578-26-6

23642275

Light yellow to brown ointment

1.1±0.1 g/cm3

413.3±45.0 °C at 760 mmHg

203.7±28.7 °C

0.0±1.0 mmHg at 25°C

1.595

3.71

0

2

2

21

356

1

CN(C)[C@H]1CCC2=C(C=CC=C2C3=C(C)N(C)N=C3C)C1

BTTOYOKCLDAHHO-HNNXBMFYSA-N

InChI=1S/C18H25N3/c1-12-18(13(2)21(5)19-12)17-8-6-7-14-11-15(20(3)4)9-10-16(14)17/h6-8,15H,9-11H2,1-5H3/t15-/m0/s1

1,2S,3,4-tetrahydro-N,N-dimethyl-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)-2-naphthalenamine

(2S)-(+)-5-(1,3,5-TRIMETHYLPYRAZOL-4-YL)-2-(DIMETHYLAMINO)TETRALIN; LA5AQ5R6QS; CHEMBL2164327; (2S)-N,N-dimethyl-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)-1,2,3,4-tetrahydronaphthalen-2-amine;

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 : ~100 mg/mL (~352.85 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.82 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 25.0 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: ≥ 2.5 mg/mL (8.82 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 25.0 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 3: ≥ 2.5 mg/mL (8.82 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 25.0 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.)