kappa-opioid receptor/KOR( Kd = 2.2 nM); μ-opioid receptor/MOR (Kd = 430 nM)

In acutely infected blood monocyte-derived macrophages (MDM), (-)-U-50488 hydrochloride (1 pM-100 nM; 7 days) shows a concentration-dependent decrease of HIV-1 expression, with the greatest inhibition occurring at 10−13 M (about 73% suppression)[2]. (-)-U-50488 hydrochloride (10-13 M; 7-14 days) (10−13 M) has a prolonged inhibitory impact on HIV-1 infection in MDM, and it significantly suppresses HIV-1 expression at 7 and 14 days after infection[3].

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Effect of U50488 on HIV-1 expression in acutely infected MDM [3]
Treatment of MDM with U50488 resulted in marked inhibition of HIV-1 expression at day 7 post-infection (Fig. 1A). The inhibitory effect of U50488 was concentration-dependent with a U-shaped dose–response curve showing maximal inhibition at 10−13 M (approximately 73% suppression). To further confirm this anti-HIV-1 effect of U50488, culture supernatants harvested at 14 days post-infection were also examined. Again, U50488 (10−13 M) markedly inhibited (P<0.01) HIV-1 expression both at 7 and 14 days after infection (Fig. 1B), suggesting that U50488 had a sustained inhibitory effect on HIV-1 infection in MDM. Pretreatment of microglial cell cultures with 10−12-M Nor-BNI antagonized (P<0.01) the inhibitory effect of U50488 (10−13 M) on HIV-1 expression by >65% (Fig. 2), indicating that the anti-HIV-1 effect of U50488 was mediated by KOR. We also found that pretreatment of MDM with U50488 was not necessary for its anti-viral effect (percent inhibition of p24 Ag production, 65, 68, and 64% in MDM treated simultaneously or for 6 and 24 h prior to infection, respectively).

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Involvement of cytokines or chemokines [3]
Because certain proinflammatory cytokines (i.e. IL-1β, IL-6, and TNF-α) and the chemokine RANTES have potent anti-HIV-1 activity (Herbein and Gordon, 1997, Lokensgard et al., 1997), we next evaluated whether the anti-HIV-1 effect of U50488 involved release of these mediators. To test this hypothesis, antibodies specific to these molecules were added to MDM cultures prior to treatment with U50488. While treatment of acutely infected MDM with 1 μg/ml of antibodies specific to IL-1β, IL-6, or TNF-α failed to block the anti-HIV-1 effect of U50488, treatment with antibody specific to RANTES attenuated by 57±6% U50488's anti-HIV-1 effect (Fig. 5). This finding suggests that stimulation of RANTES production plays a role in the anti-HIV-1 effect of U50488.

In the nucleus accumbens (NAc), (-)-U-50488 hydrochloride (intraperitoneal injection; 5 mg/kg; 2 hours before 4% paraformaldehyde (PFA)) selectively and acutely induced pMeCP2-S421 (phosphorylation of the methyl-DNA binding protein MeCP2 at Ser421) and Fos, but did not change the levels of MeCP2 in any other area of the brain[2].

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U50488 administration acutely induced pMeCP2-S421 and Fos selectively in the NAc but did not alter MeCP2 levels in any brain region. U50488-induced CPA was associated with decreased pMeCP2-S421 in the ILC and BLA and induced Fos in the BLA. MeCP2 KI mice showed CPA indistinguishable from their WT littermates, but they also showed less BLA Fos induction upon CPA. Conclusion: These data are the first to show that pMeCP2-S421 is induced in the brain acutely after U50488 administration but not upon U50488-induced CPA. Although pMeCP2-S421 is not required for U50488-induced CPA, this phosphorylation event may contribute to molecular plasticities in brain regions that govern aversive behaviors. [2]

Treatment and infection of MDM [3]
To evaluate the direct effect of KOR activation on HIV-1 expression in acutely infected MDM, we first pretreated these cells with the indicated concentrations of U50488 for 24 h followed by infection with HIV-1SF162 at a multiplicity of infection of 0.02 for an additional 24 h, as described previously (Lokensgard et al., 1997). After extensive washing, U50488 was added back to the culture medium and supernatants were harvested on day 7 or 14 post-infection for assays for HIV-1 p24 antigen (Ag) levels. HIV-1 p24 Ag levels were measured using an enzyme-linked immunosorbent assay as described previously (Chao et al., 1996). The sensitivity of this assay is 30 pg/ml.

Animal/Disease Models: C57BL/6J mice[2]
Doses: 5 mg/kg
Route of Administration: intraperitoneal (ip) injection; single dose; 2 hrs before 4% PFA
Experimental Results: Induced pMeCP2-S421 in the brain acutely.

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Acute U50488 Exposure [2]
C57BL6 male mice were placed in the open field for 30 min prior to injection of Vehicle (saline) or 5mg/kg i.p. U50488. 2 hrs later mice were transcardially perfused with 4% paraformaldehyde (PFA) and 0.1 M PBS. Brains were postfixed overnight in PBS with 4% PFA and then transferred to 20% sucrose in PBS with 4% PFA.

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Vehicle-treated (control) mice received saline in alternative chambers on each day. Drug-treated mice were treated with U50488 (5mg/kg i.p.) on days 2,4, and 6 in the baseline more preferred chamber and with saline on days 3,5, and 7 in the alternate chamber. Finally on day 8, the challenge day, all mice were placed in the center chamber and allowed free access to the entire apparatus for 30 min with no drug exposures. On challenge day, total time spent in each chamber was measured and preference scores were determined as above. By comparing the time spent in the two chambers (drug/saline) and how their difference changes during conditioning, the preference score captures the animal’s true change in place preference, irrespective for example of shifts in time spent in the neutral (gray) chamber of the apparatus. 2 hours following behavioral analysis on challenge day, mice were perfused for brain immunostaining or were killed with CO2 and brain regions were harvested for western blotting. [2]

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Objectives: The goal was to establish the role and regulation of pMeCP2-S421 in corticolimbic brain regions of mice upon acute treatment with the kappa opioid receptor agonist U50488 and during the expression of U50488-induced conditioned place aversion (CPA). Methods: pMeCP2-S421 levels were measured in the nucleus accumbens (NAc), prelimbic cortex, infralimbic cortex (ILC), and basolateral amygdala (BLA) of male mice after intraperitoneal administration of U50488 and upon the expression of U50488-induced CPA. Fos was measured as marker of neural activity in the same brain regions. U50488-induced CPA and Fos levels were compared between knockin (KI) mice that lack pMeCP2-S421 and their wild-type (WT) littermates. [2]

[1]. The search for the "next" euphoric non-fentanil novel synthetic opioids on the illicit drugs market: current status and horizon scanning. Forensic Toxicol. 2019;37(1):1-16.

[2]. Regulation and function of MeCP2 Ser421 phosphorylation in U50488-induced conditioned place aversion in mice. Psychopharmacology (Berl). 2017 Mar;234(6):913-923.

[3]. U50488 inhibits HIV-1 expression in acutely infected monocyte-derived macrophages. Drug Alcohol Depend. 2001 Apr 1;62(2):149-54.

Opioids may play an immunomodulatory role in the pathogenesis of human immunodeficiency virus-1 (HIV-1) infection. Recent studies have found that synthetic κ-opioid receptor (KOR) ligands possess anti-HIV-1 activity in acutely infected brain macrophages. This study investigated whether the selective KOR ligand U50488 exerts an anti-HIV-1 effect in acutely infected blood monocyte-derived macrophages (MDM). Treatment of acutely infected MDM with U50488 induced a concentration-dependent inhibition of HIV-1 expression. The dose-response relationship of U50488 was U-shaped, reaching a peak effect at a concentration of 10⁻¹³ M, which was visible at 7 and 14 days post-infection. The κ-opioid receptor (KOR) antagonist nor-binaltorphimine blocked 73% of the anti-HIV-1 effect of U50488, indicating that a KOR-mediated mechanism is involved. In addition, the expression of KOR mRNA and its binding activity with fluorescently labeled KOR ligands also support the presence of KOR on MDM. Antibodies against β-chemokine RANTES (a regulator of expression and secretion by normal T cells after activation), rather than antibodies against other cytokines, blocked the inhibitory effect of U50488 by 56%, suggesting that the anti-HIV-1 effect of U50488 is partly involved in the production of RANTES by MDM. In summary, these in vitro results support the anti-HIV-1 properties of U50488 and suggest that KOR ligands may have the potential to treat patients with acquired immunodeficiency syndrome (AIDS). [3]

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Objective: This article reviews in detail the chemical and pharmacological properties of novel non-fentanyl synthetic opioid receptor agonists, especially N-substituted benzamides and acetamides (commonly known as U-class drugs) and 4-aminocyclohexanol drugs developed by Upjohn in the 1970s and 1980s.
Methods: This study tracked the emergence of these drugs as substances of abuse using peer-reviewed literature, patents, professional literature, data from international early warning systems, and discussion posts on drug user forums.
Results: In terms of impact on the drug market, prevalence, and harm, the most influential compound to date is U-47700 (trans-3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methylbenzamide), which users report producing transient euphoria and inducing cravings for re-drug use. Since U-47700 was added to the international control list in 2017, a number of structurally similar compounds (derived from the original patented compound) have appeared on the illicit drug market. Interest in a structurally unrelated opioid, developed by Upjohn and now known as BDPC/bromadol, appears to be increasing and should be closely monitored.
Conclusions: International early warning systems are an important component in tracking emerging psychoactive substances, aiding in countermeasures and collecting relevant data for detailed risk assessments. It is recommended that preliminary studies be conducted on the most likely novel compounds to provide drug metabolism and pharmacokinetic data to ensure early detection of new substances in toxicological samples. Since these compounds are chiral, stereochemistry has a significant impact on their potency, so configuration determination should be considered in the detection methods. [1] A detailed description of U-50488H was first published in a peer-reviewed literature in 1982. It does not cause respiratory depression or addiction, but has been found to cause sedation, diuresis and agitation, the latter two of which are undesirable in drugs, and its action is indicated to be mediated by the κ opioid receptor (KOR). (−)trans-(1S,2S)U-50488 (U-50488H) and (±)trans racemic mixtures are available as reagents from specialty chemical suppliers such as Merck, Tocris Bioscience, Axon Medchem, etc., and are widely used as κ opioid receptor agonist models in experimental studies. The discovery and structure-activity relationship of U-50488H and its related compounds, including the important role of stereochemistry in agonist activity, were succinctly described by Jacob Szmuszkovicz, the Upjohn chemist responsible for its development [15]. The general structures of N-substituted benzamides and acetamides are shown in Figure 2a. The structural requirements for potent KOR agonists appear to be a combination of the following conditions: (1) (−) trans-(1S,2S) configuration; (2) the presence of an N-methyl group on the nitrogen atom adjacent to the carbonyl group; and (3) the presence of a methylene (CH2) "spacer group". Modifications to the structure of U-50488H further illustrate this point, as these modifications synthesize two other highly selective KOR agonists with (−) trans-(1S,2S) configurations. U-62066 (spiradoline, 2-(3,4-dichlorophenyl)-N-methyl-N-[(5R,7S,8S)-7-pyrrolidine-1-yl-1-oxaspiro[4.5]decane-8-yl]acetamide) and U-69593 (N-methyl-2-phenyl-N-[(5R,7S,8S)-7-pyrrolidine-1-yl-1-oxaspiro[4.5]decane-8-yl]acetamide) both possess heterocyclic structures fused with the cyclohexyl moiety at the R3 position (Figure 2a, Table 1). The properties of U-50488H were compared with those of the hypothetical novel μ-opioid receptor (MOR) agonist U-47109 (3,4-dichloro-N-(2-(dimethylamino)cyclohexyl)benzamide). This study used U-47700 and U-51754 (2-(3,4-dichlorophenyl)-N-[2-(dimethylamino)cyclohexyl]-N-methylacetamide) (Figure 2) to demonstrate the drug's selectivity for the κ-opioid receptor (KOR). The study found that U-47700, with its (+)-trans-(1R,2R) configuration (assuming U-47109 and U-51754 also have the same configuration), exhibited analgesic effects 7.5 times, 8 times, and 3 times greater than morphine, respectively, in analgesic tests (tail flick test, tail pinch test, and hydrochloric acid writhing test). U-50488H, U-51754, and U-47109 were less potent than morphine but still provided measurable analgesic effects. When mice were treated with U-47700, U-51754, and U-47109, typical MOR-mediated effects (Straub tail, arched back, and increased kinetic activity) were observed, but not with U-50488H treatment. [1]

C19H27CL3N2O

405.79

422.129

114528-79-9

(1R,2R)-U-50488 hydrochloride;109620-49-7;(±)-U-50488 hydrochloride;67197-96-0;(±)-U-50488 hydrate hydrochloride;(+)-U-50488;67198-17-8;(+)-U-50488 hydrochloride;114528-81-3

9931141

White to off-white solid powder

5.076

1

2

4

25

428

2

CN([C@H]1CCCC[C@@H]1N2CCCC2)C(=O)CC3=CC(=C(C=C3)Cl)Cl.Cl

KGMMGVIYOHGOKQ-APTPAJQOSA-N

InChI=1S/C19H26Cl2N2O.ClH/c1-22(19(24)13-14-8-9-15(20)16(21)12-14)17-6-2-3-7-18(17)23-10-4-5-11-23;/h8-9,12,17-18H,2-7,10-11,13H2,1H3;1H/t17-,18-;/m0./s1

2-(3,4-dichlorophenyl)-N-methyl-N-[(1S,2S)-2-pyrrolidin-1-ylcyclohexyl]acetamide;hydrochloride

(-)-U-50488 HYDROCHLORIDE; 114528-79-9; (-)-trans-(1S,2S)-U-50488 hydrochloride; CHEMBL593781; (-)-trans-(1S,2S)-u-50488 Hydrochloride potent k opioid recep; 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S,2S)-2-pyrrolidin-1-ylcyclohexyl]acetamide;hydrochloride; SR-01000075483; SR-01000597740;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: 50 mg/mL (123.22 mM)

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.)