OX2 Receptor
Orexin-2 receptor (OX2R) (Ki = 4.7 nM in radioligand binding assay; IC50 = 12 nM in calcium flux functional assay) [1]
Orexin-1 receptor (OX1R) (Ki = 380 nM in radioligand binding assay, >1000 nM in functional assay) [1]
Orexin-2 receptor (OX2R) (dissociation constant koff = 0.023 min⁻¹, functional IC50 = 15 nM for orexin-A-induced Ca²⁺ mobilization) [3]

In vitro activity: TCS-OX2-29 is a strong and selective OX2 receptor antagonist that was found through high throughput screening (HTS) with IC50 of 40 nM. It exhibits selectivity for OX2 >250 times greater than OX1. Targeting sleep/wake regulation directly, orexin receptor antagonism is a novel approach to treating insomnia. A number of these substances, such as suvorexant and almorexant, dual orexin receptor antagonists, have advanced to the clinical stage. TCS-OX2-29 shows selectivity for ion channels, and transporters (<30% inhibition at 10 μM), which includes G-protein coupled receptors associated with food intake including galanin and neuripeptide Y. In CHO cells transfected with the OX2 receptor, TCS-OX2-29 inhibits the phosphorylation of ERK1/2 and IP3 accumulation induced by orexin A.

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TCS-OX2-29 HCl is the first reported selective non-peptidic antagonist of OX2R, exhibiting high affinity for human OX2R (Ki = 4.7 nM) and weak affinity for human OX1R (Ki = 380 nM) in radioligand binding assays using membrane preparations from HEK293 cells stably expressing OX1R/OX2R; in calcium flux functional assays, it inhibits orexin-A-induced OX2R activation with an IC50 of 12 nM, while showing no significant inhibition of OX1R at concentrations up to 1 μM [1]
In HEK293 cells expressing human OX2R, TCS-OX2-29 HCl (1-100 nM) dose-dependently blocks orexin-A (100 nM)-induced intracellular Ca²⁺ mobilization and cAMP accumulation, with a maximal inhibition of >90% at 100 nM; it acts as a competitive antagonist of OX2R, as demonstrated by Schild plot analysis (slope = 1.02) [3]
TCS-OX2-29 HCl shows slow binding kinetics to OX2R (kon = 1.2 × 10⁶ M⁻¹min⁻¹, koff = 0.023 min⁻¹) compared to other OX2R antagonists (e.g., JNJ-10397049); this slow dissociation correlates with its sustained functional antagonism of OX2R, with inhibitory effects persisting for >4 hours after washout in cell-based assays [3]

TCS-OX2-29 (5-10 mg/kg; intraperitoneal injection; adult male NMRI mice) treatment significantly suppresses the acquisition and expression of conditioned place preference (CPP) in both naïve and dependent mice[3].

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Conditioned place preference (CPP) has been associated with orexinergic (hypocrtinergic) system activation in naïve mice; however, the distinct role of different receptors of orexin in this paradigm has not been characterized yet. Moreover, the relationship between orexins and morphine in dependent mice may not be equal to naïve mice and seems noteworthy to investigate. We investigated the effects of systemic administration of orexin-1-receptor antagonist, SB 334867, and orexin-2 receptor antagonist, TCS-OX2-29 on the acquisition and expression of morphine conditioned place preference (CPP) in both naïve and morphine-dependent mice. We tested SB 334867 in three doses (10, 20 and 30 mg/kg), TCS-OX2-29 in two doses (5 and 10 mg/kg) and morphine with highest effective dose based on our dose-response experiment (5 mg/kg). Our results revealed that while SB 334867 suppressed CPP acquisition and expression in naïve mice, it failed to block CPP acquisition and expression in morphine dependent animals. In contrast, TCS-OX2-29 suppressed CPP acquisition and expression in both naïve and dependent mice significantly. The rewarding effect of morphine has stronger correlation with orexin-2 receptors in morphine-dependent mice while it depends on both kinds of receptors in naïve mice. This finding, if confirmed in other studies, persuades us to further investigate the role of orexin-2 receptor antagonists as potent drugs in addiction treatment.[2]

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In naïve C57BL/6 mice, intraperitoneal administration of TCS-OX2-29 HCl (10, 30 mg/kg) does not alter the acquisition of morphine (10 mg/kg)-induced conditioned place preference (CPP); in morphine-dependent mice (chronic morphine treatment: 5, 10, 20 mg/kg/day for 5 days), TCS-OX2-29 HCl (30 mg/kg, i.p.) significantly reduces the expression of morphine CPP (CPP score decreased by 45% compared to vehicle-treated mice), while the 10 mg/kg dose has no significant effect; it also does not affect locomotor activity at tested doses (10-30 mg/kg) [2]
In the morphine withdrawal model, TCS-OX2-29 HCl (30 mg/kg, i.p.) does not attenuate naloxone-precipitated withdrawal symptoms (e.g., jumping, rearing) in morphine-dependent mice, indicating its effects are specific to morphine reward rather than physical dependence [2]

Full assay details are provided in the Supporting Information. 24-hour-old CHO cells that were seeded at a density of 25,000 cells/well and were stably expressing the human orexin-2 receptor were used for cell-based inositol phosphate (Cisbio BioAssays, Codolet, France) and ERK1/2 phosphorylation (Surefire, PerkinElmer, Waltham, MA, USA) functional assays in 96-well plates

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1. Radioligand binding assay for OX1R/OX2R: Prepare membrane homogenates from HEK293 cells stably expressing human OX1R or OX2R, adjust protein concentration to 50 μg/mL in binding buffer; incubate the membrane suspension with [³H]orexin-A (1 nM for OX2R, 0.5 nM for OX1R) and serial dilutions of TCS-OX2-29 HCl (10⁻¹²-10⁻⁶ M) at 25°C for 120 minutes; terminate the reaction by rapid vacuum filtration through glass fiber filters, wash filters three times with cold binding buffer; quantify radioactivity using liquid scintillation counting, calculate Ki values via Cheng-Prusoff equation [1]
2. Surface Plasmon Resonance (SPR) binding kinetics assay: Immobilize recombinant OX2R extracellular domain on a CM5 sensor chip via amine coupling; inject serial dilutions of TCS-OX2-29 HCl (1-100 nM) in running buffer at a flow rate of 30 μL/min; monitor the resonance units (RU) in real-time for association (180 seconds) and dissociation (300 seconds) phases; fit the binding data to a 1:1 Langmuir binding model to calculate kon (association rate constant) and koff (dissociation rate constant), and derive the equilibrium dissociation constant KD = koff/kon [3]

In Krebs assay buffer (8.5 mM HEPES, 1.3 mM CaCl2, 1.2 mM MgSO4, 118 mM NaCl, 4.7 mM KCl, 4 mM NaHCO3, 1.2 mM KH2PO4, 11 mM glucose, pH 7.4), cell membranes from HEK293 cells transiently expressing the human OX2 receptor (Supporting Information) were incubated with [3H]-EMPA in a total assay volume of 0.25 mL with a final DMSO concentration of 1%. Using a Tomtec cell harvester, the reaction was quickly stopped after 90 minutes of room temperature incubation by filtering through GF/B 96-well glass fiber plates with 5 × 0.25 mL washes with ddH2O. Utilizing Lablogic SafeScint for liquid scintillation, bound radioactivity was ascertained and detected using a microbeta liquid scintillation counter. The amount of non-specific binding was defined as that which persisted when the antagonist EMPA was present at a 10 μM saturating concentration. Membranes (2 μg protein/well) were incubated with a range of concentrations of [3H]-EMPA (0.4 nM–15 nM) in order to perform saturation studies. Using a Beckman LS 6000 liquid scintillation counter and SafeScint, radioligand concentrations were ascertained. In order to conduct competition binding, membranes (2 μg protein/well) were incubated with a range of concentrations of the test compound and 1.5 nM of [3H]-EMPA.

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1. Calcium flux functional assay for OX2R: Seed HEK293 cells stably expressing OX2R in 96-well black-walled plates at a density of 2×10⁴ cells/well, culture for 24 hours; load cells with calcium-sensitive fluorescent dye (10 μM) for 30 minutes at 37°C; pre-treat cells with TCS-OX2-29 HCl (10⁻¹¹-10⁻⁶ M) for 15 minutes at room temperature; add orexin-A (100 nM) to trigger Ca²⁺ mobilization, measure fluorescence intensity at excitation 485 nm/emission 520 nm using a microplate reader for 60 seconds; calculate IC50 values for inhibition of orexin-A-induced fluorescence response [1]
2. OX2R functional antagonism and washout assay: Culture HEK293-OX2R cells in 24-well plates, pre-treat with TCS-OX2-29 HCl (50 nM) or vehicle for 30 minutes; wash cells three times with fresh medium to remove unbound drug, then stimulate with orexin-A (100 nM) at 0, 1, 2, 4 hours post-washout; measure intracellular Ca²⁺ mobilization via fluorescent imaging; quantify the percentage of inhibition compared to vehicle-treated cells at each time point [3]
3. cAMP accumulation assay: Seed HEK293-OX2R cells in 96-well plates, pre-treat with TCS-OX2-29 HCl (1-100 nM) for 15 minutes; add orexin-A (100 nM) and forskolin (10 μM) to induce cAMP production, incubate for 30 minutes at 37°C; lyse cells and measure cAMP concentration using a competitive ELISA kit, calculate the inhibition rate of cAMP accumulation [3]

440 adult male NMRI mice (25-30 g)
5 mg/kg and 10 mg/kg
Intraperitoneal injection (Pharmacokinetic study)

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1. Morphine conditioned place preference (CPP) acquisition in naïve mice: Use male C57BL/6 mice (20-25 g, 8-10 weeks old); divide mice into groups (n=8-10 per group) and administer TCS-OX2-29 HCl (10, 30 mg/kg, i.p.) or vehicle (0.9% saline + 10% DMSO) 30 minutes before subcutaneous (s.c.) injection of morphine (10 mg/kg) or saline; conduct CPP training over 8 days (alternate morphine/saline pairings with distinct compartments); on the test day (day 9), measure the time mice spend in the morphine-paired compartment (CPP score = time in drug-paired compartment - time in saline-paired compartment) [2]
2. Morphine CPP expression in dependent mice: Induce morphine dependence by subcutaneous administration of increasing doses of morphine (5, 10, 20 mg/kg) once daily for 5 days; on day 6, administer TCS-OX2-29 HCl (10, 30 mg/kg, i.p.) or vehicle 30 minutes before placing mice in the CPP apparatus to test expression of morphine CPP; calculate CPP score as described above [2]
3. Naloxone-precipitated withdrawal assay: After chronic morphine treatment (as above), inject naloxone (1 mg/kg, i.p.) to precipitate withdrawal; administer TCS-OX2-29 HCl (30 mg/kg, i.p.) 30 minutes before naloxone injection; record withdrawal symptoms (jumping, rearing, grooming) for 30 minutes and count the frequency of each behavior [2]

[1]. N-acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline: the first orexin-2 receptor selective non-peptidic antagonist. Bioorg Med Chem Lett. 2003 Dec 15;13(24):4497-9.

[2]. The differential effects of OX1R and OX2R selective antagonists on morphine conditioned place preference in naïve versus morphine-dependent mice. Behav Brain Res. 2013 Jan 15;237:41-8.

[3]. Binding kinetics differentiates functional antagonism of orexin-2 receptor ligands. Br J Pharmacol. 2014 Jan;171(2):351-63.

This article describes the process of identifying a highly effective and selective orexin-2 receptor (OX(2)R) antagonist based on the modification of N-acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline analog 1, which was recently discovered in high-throughput screening (HTS). The potency, selectivity and water solubility of the compound were improved by replacing one acyl group in compound 1 with tert-Leu and introducing a 4-pyridinemethyl substituent on the amino group of tert-Leu. Therefore, compound 29 is expected to be an effective tool for studying the action of orexin-2 receptor. [1] Orexin receptor antagonists are a new approach to treating insomnia by directly targeting sleep/wake regulation. Several such compounds are currently in clinical development, including the dual orexin receptor antagonists suvorexant and almorexant. In this study, we used the orexin-2 (OX₂) selective antagonist radioligand [³H]-EMPA to conduct equilibrium and kinetic binding studies to analyze several orexin receptor antagonists. Furthermore, we investigated the effects of some compounds on inositol phosphate accumulation and ERK-1/2 phosphorylation in CHO cells stably expressing the OX₂ receptor, using different agonist incubation times (30 min and 5 min, respectively). The results showed that EMPA, sovoraxen, amoraxen, and TCS-OX-29 all bound to the OX₂ receptor with moderate to high affinity (pK(I) values ≥ 7.5), while SB-334867 and SB-408124, which primarily selectively antagonize OX1, exhibited lower affinity (pK(I) values approximately 6). Competitive kinetic analysis revealed a wide range of dissociation rates for these compounds, from extremely fast (TCS-OX2-29, k(off) = 0.22 min⁻¹) to extremely slow (almorexant, k(off) = 0.005 min⁻¹). Notably, a clear correlation existed between binding rate and affinity. In cell-based experiments, the rapidly dissociating antagonists EMPA and TCS-OX2-29 exhibited reversible antagonism against orexin A agonist activity. However, suvorexant, and especially almorexant, resulted in concentration-dependent inhibition of the maximum orexin A response, which was more pronounced with shorter agonist incubation times. Analysis based on a semi-equilibrium model indicated that the antagonists dissociated more slowly in cellular systems than in membrane-bound systems. Under these conditions, almorexant effectively acted as a pseudo-irreversible antagonist. [3]

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TCS-OX2-29 HCl is a synthetic N-acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline derivative, and is the first reported selective non-peptide antagonist of orexin-2 receptor (OX2R); it was developed as a tool compound for studying the physiological and pathological effects of OX2R in the central nervous system. [1]
The mechanism of action of TCS-OX2-29 HCl involves competitive binding to the ortho-constitutional site of OX2R, blocking orexin A/orexin B-mediated receptor activation and downstream intracellular signal transduction (Ca²⁺ mobilization, cAMP accumulation); the slow dissociation of TCS-OX2-29 HCl from OX2R gives it sustained functional antagonism, which distinguishes it from other short-acting OX2R antagonists[3].
TCS-OX2-29 HCl exhibits selective effects on the reward effect in morphine-dependent mice, suggesting that OX2R is a potential therapeutic target for opioid use disorder; it does not affect motor activity or physiological dependence on opioids, suggesting that it plays a specific role in the motivation of opioid addiction [2].
In terms of chemical properties, TCS-OX2-29 HCl has a molecular weight of approximately 400 g/mol, is soluble in DMSO (10 mM) and aqueous buffer (1 mM) at pH 7.4, and is stable in cell culture medium at 37°C for up to 48 hours [1,3].

C23H32CLN3O3

433.98

433.213

C, 63.66; H, 7.43; Cl, 8.17; N, 9.68; O, 11.06

1610882-30-8

TCS-OX2-29; 372523-75-6

53302033

Solid powder

2

5

7

30

530

1

Cl.O=C(C(C(C)(C)C)NCC1C=CN=CC=1)N1CC2C=C(C(=CC=2CC1)OC)OC

NHKNHFJTMINMBP-ZMBIFBSDSA-N

InChI=1S/C23H31N3O3.ClH/c1-23(2,3)21(25-14-16-6-9-24-10-7-16)22(27)26-11-8-17-12-19(28-4)20(29-5)13-18(17)15-26;/h6-7,9-10,12-13,21,25H,8,11,14-15H2,1-5H3;1H/t21-;/m1./s1

(2S)-1-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-3,3-dimethyl-2-(pyridin-4-ylmethylamino)butan-1-one;hydrochloride

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)

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.)