 Chemical Structure.png)
Physicochemical Properties
| Molecular Formula | C36H49N9O4 |
| Molecular Weight | 671.83 |
| Exact Mass | 671.391 |
| CAS # | 228546-92-7 |
| PubChem CID | 25030387 |
| Appearance | Typically exists as White to off-white solid at room temperature |
| LogP | 6.67 |
| Hydrogen Bond Donor Count | 7 |
| Hydrogen Bond Acceptor Count | 7 |
| Rotatable Bond Count | 20 |
| Heavy Atom Count | 49 |
| Complexity | 1040 |
| Defined Atom Stereocenter Count | 4 |
| SMILES | O=C([C@@H](CCCC)NC([C@@H](CC1C=CC=CC=1)NC([C@@H](CC1C=CC=CC=1)N)=O)=O)N[C@@H](C(NCC1C=CN=CC=1)=O)CCC/N=C(\N)/N |
| InChi Key | DBOGGOVKHSCMNB-OMRVPHBLSA-N |
| InChi Code | InChI=1S/C36H49N9O4/c1-2-3-15-30(34(48)43-29(16-10-19-41-36(38)39)33(47)42-24-27-17-20-40-21-18-27)44-35(49)31(23-26-13-8-5-9-14-26)45-32(46)28(37)22-25-11-6-4-7-12-25/h4-9,11-14,17-18,20-21,28-31H,2-3,10,15-16,19,22-24,37H2,1H3,(H,42,47)(H,43,48)(H,44,49)(H,45,46)(H4,38,39,41)/t28-,29-,30-,31-/m1/s1 |
| Chemical Name | (2R)-2-[[(2R)-2-[[(2R)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-N-[(2R)-5-(diaminomethylideneamino)-1-oxo-1-(pyridin-4-ylmethylamino)pentan-2-yl]hexanamide |
| Synonyms | CR665; CR 665; CR-665; UNII-3BX9UG06GG; 228546-92-7; 3BX9UG06GG; FE-200665; D-Argininamide, D-phenylalanyl-D-phenylalanyl-D-norleucyl-N-(4-pyridinylmethyl)-; |
| HS Tariff Code | 2934.99.9001 |
| Storage |
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, avoid exposure to moisture. |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | κ-opioid (IC50 = 10.9 nM)[1] |
| ln Vitro | High selectivity from peripheral to central nervous system (CNS) is demonstrated by CR 665[1]. With an EC50 value of 10.9 nM, CR 665 can activate the kappa opioid receptor[1]. |
| ln Vivo |
Although not orally available, CR665 given i.v. exhibits high peripheral to CNS selectivity and benefits patients with visceral and neuropathic pain. In this study we have generated a series of derivatives of CR665 and screened them for oral activity in the acetic acid-induced rat writhing assay for peripheral pain. Five compounds were further screened for specificity of activation of kappa receptors as well as agonism and antagonism at mu and delta receptors, which can lead to off-target effects. All active derivatives engaged the kappa receptor with EC50s in the low nM range while agonist selectivity for kappa over mu or delta was >11,000-200,000-fold. No antagonist activity was detected. One compound was chosen for further analysis (Compound 9). An oral dose response of 9 in rats yielded an EC50 of 4.7 mg/kg, approaching a druggable level for an oral analgesic. To assess the peripheral selectivity of this compound an i.v. dose response in rats was assessed in the writhing assay and hotplate assay (an assay of CNS-mediated pain). The EC50 in the writhing assay was 0.032 mg/kg while no activity was detectable in the hotplate assay at doses as high as 30 mg/kg, indicating a peripheral selectivity of >900-fold. We propose that compound 9 is a candidate for development as an orally-available peripherally-restricted kappa agonist[2]. CR665 kinetics were optimally fitted with a two-compartment model, while oxycodone kinetics were best described by a one-compartment model with transit compartment absorption feeding directly into the central compartment. For both drugs, the plasma concentration effects on VPTT were best fit by a direct linear model, i.e. without the concentration-analgesia delay characteristic of brain-penetrant opioids. The slope of oxycodone (0.089 mL per ng/mL) was steeper than that of CR665 (0.0035 mL per ng/mL) for the plasma drug concentration acting on the VPTT. Conclusion: The results are consistent with the peripheral selectivity of CR665, as well as the possibility that peripheral actions of oxycodone contribute to its visceral analgesic efficacy[1]. |
| Enzyme Assay |
Opioid Receptor Activation Assay[2] PathHunter™ Beta-Arrestin GPCR Functional Profiling and Screening cell lines that express the individual opioid receptors were obtained from DiscoveRx. These cells express the kappa (or mu or delta) opioid receptor fused to a proprietary β-galactosidase enzyme fragment and the β-arrestin gene fused to an enzyme acceptor (the remaining fragment of β-galactosidase). Upon activation of the receptor, β-arrestin is recruited to the GPCR and the β-galactosidase is activated through enzyme complementation. Cells were grown using standard cell culture techniques and then plated at 30,000 cells/well in 90 µl in a 96 well white, clear bottom plate and incubated overnight. Dilutions were prepared in dilution buffer (HBBS, 20 mMHepes, 0.1%BSA) and 10 µl added per well. Plates were then incubated for 90 min at 37oC in a water-saturated atmosphere. Plates were then allowed to come to room temp and DiscoveRx ‘s proprietary developing agent added as recommended by the manufacturer. This agent contains a detergent, 6-O-β-galactopyranosyl-luciferen and luciferase. Plates were then incubated for 1 hr at room temperature before being read on a luminometer. Antagonist studies were performed in an identical manner except after the overnight incubation 10 µl media was removed and replaced with drug or naloxone at the indicated concentration. Cells were then incubated for 30 min at 37oC in a water-saturated atmosphere before addition of 10 µl of the indicated agonist (lys-dermorphin for mu receptors and D-Ala2, D-Leu5-enkephalin (DADLE) for delta receptors). |
| Animal Protocol |
Pain Models[2] Acetic Acid-Induced Writhing Model[2] This protocol is a well-established model of peripheral pain. Following oral or i.v. administration of compound (e.g. CR665), rats are rested for 20 min before i.p. injection with 2 ml/kg of 3% acetic acid. Animals are then rested an additional 10 min before being placed in a 10 x 10 inch chamber. A video of the rats is then recorded for next 20 min. The video is subsequently blindly scored by an investigator who examines each rat every 20 sec for the entire 20 min (60 individual observations). At each observation rats are scored on whether they are writhing or not. Writhing is defined as a constriction of the abdominal area, often with extension of the hind legs. At the end of the experiment the percent of the time the animal was writhing was calculated. The dose-response curves and EC50 values were generated using GraphPad Prism®. Hotplate Model[2] The hotplate model evaluates central pain attenuation in a rodent after applying an acute thermal stimuli. The rat is treated with compound and assessed over time on a hotplate analgesia meter, essentially a flat surface maintained at 53.0+/-0.2oC. The time until the rat lifts, nibbles or shakes one of its hind paws is recorded, which is known as the response latency. Animals remain on the hotplate no longer than 30 sec to avoid tissue damage. Experiments are scored as the percent of maximal possible effect (%MPE) calculated using the following equation: %MPE = [(post-drug latency – pre-drug latency)/(cut-off pre-drug latency)] x 100%. The dose-response curves and EC50 values were generated using GraphPad Prism®. In a randomized, placebo-controlled, double-blind, three-way crossover study, healthy male volunteers were administered CR665 (0.36 mg/kg, intravenous), oxycodone (15 mg, oral) or placebo (intravenous and oral), followed by assessment of visceral pain tolerance thresholds (VPTT) measured as volume of water (mL) in the bag placed on an oesophageal probe. Plasma drug concentration data were used to generate pharmacokinetic models, which were then used to fit the VPTT data using NONMEM(®) VI to generate population pharmacokinetic/pharmacodynamic models.[1] |
| References |
[1]. A population pharmacokinetic and pharmacodynamic study of a peripheral κ-opioid receptor agonist CR665 and oxycodone. Clin Pharmacokinet. [2]. Development of a Peptide-derived orally-active kappa-opioid receptor agonist targeting peripheral pain. Open Med Chem J. 2013 Nov 4;7:16-22. |
| Additional Infomation |
CR665 is the lead clinical development candidate from a series of highly selective peripheral kappa opioid receptor agonists. In preclinical studies, CR665 was highly selective for the peripheral kappa opioid receptor. Preclinical animal studies suggest that CR665 is a potent analgesic compound. In addition, unlike currently marketed opioids, CR665 does not produce inhibition of intestinal transit (ileus), induce respiratory depression, or elicit signs of euphoria or addiction in animal models. Preclinical studies also indicate that CR665 possesses anti-inflammatory activities. Drug Indication Investigated for use/treatment in pain (acute or chronic). Mechanism of Action CR665 was highly selective for the peripheral kappa opioid receptor. It is intrinsically poor at penetrating the blood-brain barrier, which decreases the likelihood of CNS-mediated side effects. By acting with unprecedented selectivity at pain relieving receptors on peripheral nerves, and avoiding receptors in the central nervous system and gastrointestinal tract, CR665 has the potential to provide pain relief with minimal side effects. |
Solubility Data
| Solubility (In Vitro) | May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples |
| Solubility (In Vivo) |
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 Formulations (e.g. IP/IV/IM/SC) 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). 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 Formulations 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). 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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.4885 mL | 7.4424 mL | 14.8847 mL | |
| 5 mM | 0.2977 mL | 1.4885 mL | 2.9769 mL | |
| 10 mM | 0.1488 mL | 0.7442 mL | 1.4885 mL |