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. Recently, synthetic kappa-opioid receptor (KOR) ligands have been found to have anti-human immunodeficiency virus type 1 activity in acutely infected brain macrophages. In the present study, we investigated whether the selective KOR ligand U50488 would exert such 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 with a peak effect observed at 10(-13) M, which was evident at both 7 and 14 days post-infection. The KOR antagonist nor-binaltorphimine blocked the anti-HIV-1 effect of U50488 by 73%, indicating involvement of a KOR-mediated mechanism. Also, expression of KOR mRNA and binding activity with a fluorescence-labeled KOR ligand supported the existence of KOR on MDM. Antibodies to the beta-chemokine, RANTES (regulated on activation normal T-cell expressed and secreted), but not to various other cytokines, blocked U50488 inhibition by 56% suggesting that the anti-HIV-1 effect of U50488 involved, in part, the production of RANTES by MDM. Taken together, these in vitro findings support the anti-HIV-1 property of U50488, and suggest that KOR ligands may have therapeutic potential for treating patients with acquired immunodeficiency syndrome. [3]

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Purpose: A detailed review on the chemistry and pharmacology of non-fentanil novel synthetic opioid receptor agonists, particularly N-substituted benzamides and acetamides (known colloquially as U-drugs) and 4-aminocyclohexanols, developed at the Upjohn Company in the 1970s and 1980s is presented.
Method: Peer-reviewed literature, patents, professional literature, data from international early warning systems and drug user fora discussion threads have been used to track their emergence as substances of abuse.
Results: In terms of impact on drug markets, prevalence and harm, the most significant compound of this class to date has been U-47700 (trans-3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methylbenzamide), reported by users to give short-lasting euphoric effects and a desire to re-dose. Since U-47700 was internationally controlled in 2017, a range of related compounds with similar chemical structures, adapted from the original patented compounds, have appeared on the illicit drugs market. Interest in a structurally unrelated opioid developed by the Upjohn Company and now known as BDPC/bromadol appears to be increasing and should be closely monitored.
Conclusions: International early warning systems are an essential part of tracking emerging psychoactive substances and allow responsive action to be taken to facilitate the gathering of relevant data for detailed risk assessments. Pre-emptive research on the most likely compounds to emerge next, so providing drug metabolism and pharmacokinetic data to ensure that new substances are detected early in toxicological samples is recommended. As these compounds are chiral compounds and stereochemistry has a large effect on their potency, it is recommended that detection methods consider the determination of configuration. [1]

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A detailed description of U-50488H was first published in the peer reviewed literature in 1982. It did not cause respiratory depression and was not habit-forming, but was found to cause sedation, diuresis and dysphoria, the latter properties being undesirable in medicinal products and indicative of KOR-mediated effects. The (−) trans-(1S,2S) U-50488 (U-50488H) and the (±) trans-racemic mixture are commercially available as reagents from specialist chemical supply companies (e.g. Merck, Tocris Bioscience, Axon Medchem etc.) and are widely used as model KOR agonists in experimental studies. The discovery and deconvolution of the structure activity relationships of U-50488H, and its related compounds, including the important role of stereochemistry in agonist activity, was elegantly described by the Upjohn chemist responsible for its development, Jacob Szmuszkovicz [15]. The generic structure of the N-substituted benzamides and acetamides is shown in Fig. 2a. The structural requirements for a potent KOR agonist appear to be a combination of the following: (1) a (−) trans-(1S,2S) configuration; (2) the presence of an N-methyl group on the nitrogen adjacent to the carbonyl and (3) the presence of a methylene (CH2) “spacer”. This is further illustrated by the modifications of the U-50488H structure which lead to the synthesis of two further highly selective KOR agonists with a (−) trans-(1S,2S) configuration; U-62066 (spiradoline, 2-(3,4-dichlorophenyl)-N-methyl-N-[(5R,7S,8S)-7-pyrrolidin-1-yl-1-oxaspiro[4.5]decan-8-yl]acetamide) and U-69593 (N-methyl-2-phenyl-N-[(5R,7S,8S)-7-pyrrolidin-1-yl-1-oxaspiro[4.5]decan-8-yl]acetamide) both of which have a heterocyclic ring structure fused to the cyclohexyl moiety in the R3 position (Fig. 2a, Table 1).
The properties of U-50488H were compared to those of the assumed novel mu-opioid (MOR) agonists U-47109 (3,4-dichloro-N-(2-(dimethylamino)cyclohexyl)benzamide), U-47700 and U-51754 (2-(3,4-dichlorophenyl)-N-[2-(dimethylamino)cyclohexyl]-N-methyl-acetamide) (Fig. 2) to demonstrate the drug’s selectivity for the KOR. In this study, U-47700, which has a (+) trans-(1R,2R) configuration (and it is assumed U-47109 and U-51754 also have the same configuration), was found to have 7.5, eight and three times greater analgesic effect than morphine in antinociceptive tests (tail flick, tail pinch and hydrochloric acid writhing respectively). U-50488H, U-51754 and U-47109 were found to be less potent than morphine, but still had measurable analgesic effects. Characteristic MOR mediated effects (Straub tail, arched back, increased locomotor activity) were observed when mice were treated with U-47700, U-51754 and U-47109 but were not observed for U-50488H. [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.)