. 2020 Jun 12;11(8):896–904. doi: 10.1039/d0md00104j

Table 2. Inhibition of forskolin-induced cAMP accumulation: functional activity for cAMP and MOR-mediated β-arrestin2 recruitment.

#			MOR cAMP		KOR cAMP		DOR cAMP		β-Arrestin2 recruitment ^a (PathHunter assay)			β-Arrestin2 recruitment ^b (Tango assay)
#	R₁	R₂	EC₅₀ ^c ^, ^d (nM)	E _MAX %	EC₅₀ (nM)	E _MAX %	EC₅₀ (nM)	E _MAX %	EC₅₀ (nM)	E _MAX %	Bias factor	EC₅₀ (nM)	E _MAX %	Bias factor
11a	CH CH–CO₂Me	H	132 ± 62	52.6 ± 6.5%	>10 000	—	143.0 ± 18.0	27.2 ± 3.7%	>25 000 ^e	—	NC ^f	NT ^g	NT ^g	NT ^g
12	CH₂CH₂CH₃	H	0.95 ± 0.35	63.3 ± 3.9%	>10 000	—	>10 000	—	>25 000 ^e	—	NC ^f	>25 000 ^e	—	NC ^f
13	CH CH–CH₂OH	H	2.31 ± 0.78	33.5 ± 5.9%	>10 000	—	>10 000	—	>25 000 ^e	—	NC ^f	>25 000 ^e	—	NC ^f
14a	CH₂CH₂–CO₂Me	H	0.46 ± 0.09	94 ± 2.3%	>10 000	—	>10 000	—	1.72 ± 0.17	4.5 ± 0.38%	0.55	12.5	123.4	0.15
15a	CH₂CH₂CH₂OH	H	1.2 ± 0.37	65 ± 6%	>10 000	—	80.9 ± 33.9	22.62 ± 6%	>25 000 ^e	—	NC ^f	>25 000 ^e	—	NC ^f
16	CH₂CH₂–CO₂Et	H	0.24 ± 0.09	100.1 ± 0.7%	35.3 ± 19.9	36.5 ± 3.8%	49.1 ± 10.1	74.2 ± 4.9%	11.57 ± 3.1	18.26 ± 1.7%	1.89	>25 000 ^e	—	NC ^f
17	CH₂CH₂–CO₂H	H	31.7 ± 11.4	49 ± 2.5%	>10 000	—	>10 000	—	>25 000 ^e	—	NC ^f	>25 000 ^e	—	NC ^f
22	H	CH₂CH₂–CO₂Me	854 ± 114	63 ± 2.4%	322.8 ± 110.5	55.01 ± 5.0%	>10 000	—	>25 000 ^e	—	NC ^f	NT ^g	NT ^g	NT ^g

			MOR cAMP		KOR cAMP		DOR cAMP		β-Arrestin2 recruitment ^a (PathHunter assay)			β-Arrestin2 recruitment ^b (Tango assay)
	R₁	R₂	EC₅₀ ^c ^, ^d (nM)	E _MAX %	EC₅₀ (nM)	E _MAX %	EC₅₀ (nM)	E _MAX %	EC₅₀ (nM)	E _MAX %	Bias factor	EC₅₀ (nM)	E _MAX %	Bias factor
14b	H	CH₂CH₂–CO₂Me	31.3 ± 4.6	42 ± 4.8%	>10 000	—	>10 000	—	>25 000	—	NC ^f	>25 000 ^e	—	NC ^f
15b	H	CH₂CH₂CH₂–OH	35.2 ± 12.59	48.7 ± 3.7%	>10 000	—	>10 000	—	>25 000 ^e	—	NC ^f	>25 000 ^e	—	NC ^f
23 (ref. 16)	H	OH ^g	0.017 ± 0.004	101%	322.8 ± 110.5	55.01 ± 5.0%	2.6 ± 0.5	74.0 ± 20.8%	6.08 ± 0.74	116 ± 5.4%	2.16	NT ^h	NT ^h	NT ^h

Standards
Morphine	4.7 ± 0.6	103 ± 0.6%	NT ^h	NT ^h	NT ^h	NT ^h	379.6 ± 40.7	25.7 ± 0.6%	2.3	NT ^h	NT ^h	NT ^h
SR 17018 ⁱ (ref. 12)	15.6 ± 5.1	101.2 ± 1.8%	NT ^h	NT ^h	NT ^h	NT ^h	2975 ± 1623	89.2 ± 3.4%	1.96	NT ^h	NT ^h	NT ^h
PZM21 (ref. 11)	2.5 ± 0.4	101 ± 0.6%	NT ^f	NT ^f	NT ^f	NT ^f	57.1 ± 8.5	4.3 ± 0.4%	2.98	NT ^h	NT ^h	NT ^h
Salvinorin A	NT ^h	NT ^h	0.03 ± 0.007	99.8 ± 0.3%	NT ^h	NT ^h	NT ^h	NT ^h	NT ^h	NT ^h	NT ^h	NT ^h
DADLE	NT ^h	NT ^h	NT ^h	NT ^h	0.02 ± 0.004	92.1 ± 0.7%	NT ^h	NT ^h	NT ^h	NT ^h	NT ^h	NT ^h
DAMGO	0.3 ± 0.04	101.5 ± 0.5	NT ^h	NT ^h	NT ^h	NT ^h	44.1 ± 3.9	103 ± 0.4	1.0	NT ^h	NT ^h	NT ^h

^aHitHunter Chinese hamster ovary cells (CHO-K1) that express human μ-opioid receptor (OPRM1), human κ-opioid receptor (OPRK1), and human δ-opioid receptor (OPRD1) were used for the forskolin-induced cAMP accumulation assay to determine potency and efficacy of the compounds. PathHunter CHO cells expressing human μ-opioid receptor β-arrestin-2 EFC cells were used for the β-arrestin-2 EFC recruitment assay. All cell lines were purchased from Eurofins DiscoverX (Fremont, CA). Cell culture was performed as previously described.21

^bThermo Fisher Scientific's SelectScreen™ Profiling Service: 10 point titration agonist results.25

^cEfficacy values were determined by normalization to DAMGO.

^dMean ± standard error of the mean; n ≥ 2.

^e E _MAX = 0% at the concentrations tested.

^fNC = not calculable. Bias factor could not be calculated because recruitment of β-arrestin2 was not observed under any of the conditions tested.

^gThe C9–OH compound 23 has 1R,5R,9S stereochemistry.18

^hNT = not tested.

ⁱPurchased from Cayman Chemical, Ann Arbor, MI (CAS 2134602-45-0; labelled purity ≥98%), HRMS-ESI (m/z): [M + H]⁺ calcd. for C₁₉H₁₉ Cl₃N₃O 410.0594, found 410.0592.