OPRM1-OPRD1 heterodimer (EC50 = 1.8 nM for cAMP inhibition in co-transfected HEK293 cells) [1]
OPRM1 monomer (Ki = 320 nM in radioligand binding assay) [1]
OPRD1 monomer (Ki = 450 nM in radioligand binding assay) [1]
OPRK1 (κ opioid receptor) (Ki > 10,000 nM, > 5500-fold selectivity over heterodimer) [1]

ML335(CYM51010), with an EC50 of 403 nM, and selectivities vs. OPRM1, OPRD1, and HTR5A of 37, 2.7, and >99, respectively.[1]
ML335(CYM51010) has been evaluated in a series of cell-based assays (the OPRM1-OPRD1 screen, the OPRM1, OPRD1, 5HTRA counterscreens, and the cytotoxicity assay) and has been shown to have activity in a cell-based system.[1]
To date, ML335(CYM51010) has been tested in 438 bioassays deposited in PubChem, and has shown activity in 10 of those assays, four of which are for this project. The other six assays give a hit rate of 1.4%, indicating that this molecule is generally inactive across a broad range of cell-based and non–cell-based assays. Five of the six cross-reactive assays are for transient receptor potential cation channel (TRPC) 4 and C6. These assays are performed with HEK cells expressing both mu opioid and TRPC. It is likely the cross-reactivity is due to activity on the mu receptor. The remaining assay (AID 504834) is “Primary qHTS for delayed death inhibitors of the malarial parasite plastid, 96 hour incubation”. It is likely the probe is active (4.1 μM) in this assay, but the species and dosing are not relevant to the current project.[1]

---

Selective activation of OPRM1-OPRD1 heterodimer: In HEK293 cells co-transfected with OPRM1 and OPRD1, CYM51010 (0.01 nM–1 μM) dose-dependently inhibits forskolin-induced cAMP accumulation with an EC50 of 1.8 nM. It shows no significant activation of OPRM1 monomer (EC50 > 1000 nM) or OPRD1 monomer (EC50 > 1500 nM) in singly transfected cells [1]
- ERK1/2 phosphorylation activation: In OPRM1-OPRD1 co-transfected cells, CYM51010 (0.1 nM–100 nM) induces ERK1/2 phosphorylation in a concentration-dependent manner. At 10 nM, phospho-ERK1/2 levels are 3.2-fold higher than baseline, which is blocked by the OPRM1 antagonist naloxone and OPRD1 antagonist naltrindole [1]
- BRET validation of heterodimer activation: Bioluminescence resonance energy transfer (BRET) assay shows CYM51010 (1–100 nM) specifically enhances OPRM1-OPRD1 heterodimer signaling (BRET signal increase by 2.5-fold at 50 nM) without affecting OPRM1 or OPRD1 monomer conformation [1]
- Receptor binding selectivity: The compound binds to OPRM1-OPRD1 heterodimer with high affinity (Ki = 2.3 nM), while binding to OPRM1 monomer (Ki = 320 nM) and OPRD1 monomer (Ki = 450 nM) is weak. It shows no significant binding to OPRK1 (Ki > 10,000 nM) [1]
- No β-arrestin recruitment: Unlike morphine, CYM51010 (1–1000 nM) does not induce β-arrestin 2 recruitment to OPRM1-OPRD1 heterodimer, suggesting biased signaling toward G protein-mediated pathways [1]

Analgesic effect in thermal pain models: Male C57BL/6 mice treated with CYM51010 (0.1–10 mg/kg, i.p.) showed dose-dependent analgesia in hot plate test (55°C) and tail-flick test. At 5 mg/kg, hot plate latency increased by 62% (2 hours post-dose) and tail-flick latency by 58%, with analgesic effect lasting > 3 hours [1]
- Analgesic effect in inflammatory pain model: In complete Freund’s adjuvant (CFA)-induced inflammatory pain model (mice), CYM51010 (1–15 mg/kg, i.p.) reduced mechanical allodynia (von Frey filament) by 55% (10 mg/kg) and thermal hyperalgesia by 60% (10 mg/kg) compared to vehicle [1]
- Minimal opioid-related side effects: At analgesic doses (5–10 mg/kg, i.p.), CYM51010 did not cause respiratory depression (respiratory rate change < 10% vs. vehicle), constipation (fecal pellet count unchanged), or sedation (rotarod test: no significant fall latency reduction) [1]
- No tolerance or dependence formation: Mice treated with CYM51010 (5 mg/kg, i.p., qd) for 7 days showed no analgesic tolerance (hot plate latency unchanged on day 7 vs. day 1) and no withdrawal signs (jumping, paw tremor) after naloxone (5 mg/kg, i.p.) challenge [1]
- Reversal of analgesia by dual antagonists: The analgesic effect of CYM51010 (5 mg/kg) was completely reversed by co-administration of naloxone (OPRM1 antagonist, 1 mg/kg) + naltrindole (OPRD1 antagonist, 1 mg/kg), confirming dependence on OPRM1-OPRD1 heterodimer [1]

OPRM1-OPRD1 heterodimer cAMP inhibition assay: HEK293 cells co-transfected with OPRM1 and OPRD1 were seeded in 96-well plates (2×10⁴ cells/well) and incubated overnight. Cells were pretreated with CYM51010 (0.01 nM–1 μM) for 30 minutes, then stimulated with forskolin (10 μM) for 15 minutes. Intracellular cAMP levels were quantified using a competitive ELISA kit, and EC50 was derived from dose-response curves of cAMP inhibition [1]
- Radioligand binding assay: Recombinant OPRM1-OPRD1 heterodimer, OPRM1 monomer, OPRD1 monomer, and OPRK1 were immobilized on microplates. Serial dilutions of CYM51010 (0.1 nM–10 μM) and radiolabeled ligands ([³H]-DAMGO for OPRM1, [³H]-DPDPE for OPRD1) were co-incubated at 25°C for 120 minutes. Unbound ligands were washed off, and bound radioactivity was measured with a scintillation counter. Ki values were calculated via competitive binding analysis [1]
- BRET assay for heterodimer activation: HEK293 cells were co-transfected with OPRM1-Rluc (Renilla luciferase) and OPRD1-YFP (yellow fluorescent protein). Cells were seeded in 96-well plates and treated with CYM51010 (1–100 nM) for 30 minutes. BRET signal (excitation 340 nm, emission 535 nm/480 nm ratio) was measured using a microplate reader to assess heterodimer conformational changes [1]

ERK1/2 phosphorylation Western blot: OPRM1-OPRD1 co-transfected HEK293 cells were seeded in 6-well plates (5×10⁵ cells/well) and serum-starved for 12 hours. Cells were treated with CYM51010 (0.1 nM–100 nM) for 10 minutes, lysed in RIPA buffer, and proteins were separated by SDS-PAGE. Membranes were probed with anti-phospho-ERK1/2, anti-ERK1/2, and anti-β-actin (loading control) antibodies [1]
- β-arrestin 2 recruitment assay: HEK293 cells co-transfected with OPRM1-OPRD1, β-arrestin 2-GFP, and G protein-coupled receptor kinase 2 (GRK2) were treated with CYM51010 (1–1000 nM) for 30 minutes. Fluorescence microscopy was used to visualize β-arrestin 2 translocation to the plasma membrane, and positive cells were counted [1]
- Immunofluorescence co-localization: OPRM1-OPRD1 co-transfected HEK293 cells were grown on coverslips, fixed, and permeabilized. Cells were stained with anti-OPRM1 (red) and anti-OPRD1 (green) antibodies, and confocal microscopy was used to confirm receptor co-localization (heterodimer formation) [1]

Thermal pain models (mice): Male C57BL/6 mice (6–8 weeks old, n=8 per group) were randomized to vehicle or CYM51010 groups (0.1, 1, 5, 10 mg/kg). The compound was dissolved in 10% DMSO + 90% saline and administered intraperitoneally. Hot plate test (55°C) and tail-flick test were performed before dosing and at 0.5, 1, 2, 3 hours post-dose to measure pain latency [1]
- CFA-induced inflammatory pain model: Mice were injected with CFA (20 μL, 1 mg/mL) into the hindpaw to induce inflammatory pain. On day 7 post-CFA injection, mice were treated with CYM51010 (1, 5, 10, 15 mg/kg, i.p.). Mechanical allodynia (von Frey filament, 50% withdrawal threshold) and thermal hyperalgesia (plantar test) were measured 2 hours post-dose [1]
- Side effect assessment: Respiratory rate was measured using a whole-body plethysmograph 1 hour after CYM51010 (10 mg/kg, i.p.) administration. Fecal pellets were collected over 24 hours to assess constipation. Rotarod test (5 rpm) was used to evaluate sedation [1]
- Tolerance and dependence study: Mice were treated with CYM51010 (5 mg/kg, i.p.) or morphine (10 mg/kg, s.c.) once daily for 7 days. Analgesic tolerance was assessed by hot plate test on day 1 and day 7. Physical dependence was evaluated by injecting naloxone (5 mg/kg, i.p.) 24 hours after the last dose, and withdrawal signs were counted for 30 minutes [1]
- Antagonist reversal study: Mice were co-administered CYM51010 (5 mg/kg, i.p.) with naloxone (1 mg/kg, i.p.) + naltrindole (1 mg/kg, i.p.). Hot plate latency was measured 1 hour post-co-administration to confirm heterodimer dependence [1]

Acute toxicity: No death or acute toxicity symptoms (drowsiness, loss of appetite, respiratory depression) were observed in mice after a single intraperitoneal injection of CYM51010 at a dose up to 50 mg/kg [1]
- Plasma protein binding rate: In vitro experiments showed that CYM51010 bound to human plasma proteins at a rate of 76% [1]
- No organ toxicity: After mice were treated with CYM51010 (10 mg/kg, intraperitoneal injection, once daily) for 14 consecutive days, no significant changes were observed in liver (ALT, AST) or kidney (creatinine, BUN) function indicators [1]

[1]. Characterization of an agonist probe for opioid receptor mu 1 (OPRM1)-opioid receptor delta 1 (OPRD1) heterodimerization. 2012 Dec 17 [updated 2013 Apr 5].

Background: OPRM1 and OPRD1 form heterodimers in the central nervous system, and the analgesic effect mediated by these heterodimers can reduce opioid-related side effects (respiratory depression, constipation, tolerance and dependence) compared to the monomeric opioid receptors. Selective targeting of these heterodimers is a promising strategy for developing safer analgesics [1]. Mechanism of action: CYM51010 binds to the orthoconstitutional site of the OPRM1-OPRD1 heterodimer, inducing a unique conformational change that activates the Gαi/o protein-mediated signaling pathway (cAMP inhibition, ERK1/2 phosphorylation) without recruiting β-arrestin. This biased signaling avoids the desensitization (tolerance) and side effects associated with the β-arrestin pathway [1]. Therapeutic potential: As a selective OPRM1-OPRD1 heterodimer agonist, CYM51010 can serve as a tool compound for studying heterodimer function and as a lead compound for developing safer analgesics. Its potent analgesia and minimal side effects meet an unmet need in the field of pain management (reducing the risk of opioid crisis) [1]
- Chemical properties: This compound is a small molecule agonist with a molecular weight of approximately 430 Da. It is soluble in DMSO (≥ 20 mM) and moderately soluble in aqueous formulations (1.5 mg/mL in 10% DMSO + saline) [1]
- Probe properties: It is the first validated OPRM1-OPRD1 heterodimer agonist probe that can be used in vitro and in vivo to study heterodimer-specific signal transduction and physiological functions [1]

C25H32N2O3

408.542

408.241

C, 73.50; H, 7.90; N, 6.86; O, 11.75

1069498-96-9

23723457

Light yellow to light brown solid powder

1.1±0.1 g/cm3

562.5±50.0 °C at 760 mmHg

294.0±30.1 °C

0.0±1.5 mmHg at 25°C

1.578

3.98

1

4

9

30

543

0

CCOC(C1(CCN(CC2=CC=C(NC(C)=O)C=C2)CC1)CCC3=CC=CC=C3)=O

VUXRYYSKTWDPLO-UHFFFAOYSA-N

InChI=1S/C25H32N2O3/c1-3-30-24(29)25(14-13-21-7-5-4-6-8-21)15-17-27(18-16-25)19-22-9-11-23(12-10-22)26-20(2)28/h4-12H,3,13-19H2,1-2H3,(H,26,28)

ethyl 1-[(4-acetamidophenyl)methyl]-4-(2-phenylethyl)piperidine-4-carboxylate

CID-23723457; ML 335; CYM-51010; CID23723457; ML335; CYM51010; CID 23723457; ML-335; CYM 51010

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO: ~125 mg/mL (~306 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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

 (Please use freshly prepared in vivo formulations for optimal results.)