## Endpoint to Predict
- Endpoint scope: assay
- Measurement type: EC50
- Label type: continuous
- Binary semantics: pchembl
- Assay description: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1x HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 uM to 0.13 nM cAMP. EC50 values were determined using Activity Base analysis (ID Business Solution, Limited). The EC50 values for a wide range of cannabinoid agonists generated from this assay were in agreement with the values published in the scientific literature. The compounds of the invention are CB2 receptor agonists with EC50 below 1 uM and selectivity versus CB1 in the corresponding assay of at least 10 fold. Particular compound of the invention are CB2 receptor agonists with EC50 below 0.05 uM and selectivity versus CB1 in the corresponding assay of at least 500 fold.
- Assay organism: Homo sapiens
- Target name: Cannabinoid receptor 1
- Target type: SINGLE PROTEIN
- Target organism: Homo sapiens

## Reference Molecule A
- Canonical SMILES: CC(C)(C)c1nc(N2CCC(F)(F)C2)c2ncn(CCc3ccccc3)c2n1
- Properties:
- ChEMBL ID: CHEMBL5773507
- Molecular weight: 385.46
- Heavy atoms: 28
- Hydrogen bond donors: 0
- Hydrogen bond acceptors: 4
- TPSA: 46.84
- LogP: 4.21
- Rotatable bonds: 4
- Ring count: 4
- Aromatic rings: 3
- Formal charge: 0
- Fraction Csp3: 0.48
- QED: 0.67

## Known Reference Result on This Endpoint
- Molecule A clean result: median pChEMBL = 5.83
- This result is provided as context for predicting Molecule B on the same endpoint.

## Reference Molecule A Other ChEMBL Evidence
- type EC50; relation =; value 1497.1; units nM; pChEMBL 5.83; comment 452716

## Query Molecule B
- Canonical SMILES: CC(C)(C)c1nc(N2CCC(F)(F)C2)c2ncn(Cc3ccccc3C(F)(F)F)c2n1
- Properties:
- ChEMBL ID: CHEMBL5850247
- Molecular weight: 439.43
- Heavy atoms: 31
- Hydrogen bond donors: 0
- Hydrogen bond acceptors: 4
- TPSA: 46.84
- LogP: 5.04
- Rotatable bonds: 3
- Ring count: 4
- Aromatic rings: 3
- Formal charge: 0
- Fraction Csp3: 0.48
- QED: 0.54

## Molecule Similarity
- Weighted Tanimoto: 0.69

Answer: 6.13