Table 1:
Summary of the Pyrrolidine Bis-Cyclic Guanidine Pharmacology at the Selected Mouse Melanocortin Receptors
| ID | R1 | R2 | R3 | R4 | EC50(nM) |
|||
|---|---|---|---|---|---|---|---|---|
| mMC1R | mMC3R | mMC4R | mMC5R | |||||
| NDP-MSH [(Nle4, DPhe7)α-MSH] | 0.09 ± 0.03 | 0.36 ± 0.04 | 0.12 ± 0.01 | 2.6 ± 0.6 | ||||
| α-MSH | 0.64 ± 0.16 | 0.56 ± 0.06 | 1.6 ± 0.2 | 0.7 ± 0.2 | ||||
| Ac-His-DPhe-Arg-Trp-NH2 | 13.4 ± 3.9 | 31 ± 9 | 5.5 ± 0.8 | 4.2 ± 1 | ||||
| THIQ | 100 ± 20 | 260 ± 20a | 0.9 ± 0.2 | 0.49 ± 0.08 | ||||
| 1 | R-isobutyl | R-cyclohexyl-methyl | (S,R)-1-hydroxyethyl | adamantan-1-yl-methyl | 330 ± 110 | 240 ± 40 | 650 ± 120 (55%) pA2= 5.5 ± 1.1 |
340 ± 140 |
| 2 | R-isobutyl | R-cyclohexyl-methyl | (S,R)-1-hydroxyethyl | 4-tbutyl-cyclohexyl-methyl | 300 ± 90 | 210 ± 50 | 270 ± 70 (45%) pA2 = 5.8 ± 0.1 |
140 ± 70 |
| 3 | R-isobutyl | R-cyclohexyl-methyl | (S,R)-1-hydroxyethyl | 4-methylpentyl | 7300 ± 4400 | 58% @ 10 μM (B) | (A) | 630 ± 50 |
| 4 | R-isobutyl | R-cyclohexyl-methyl | R-propyl | adamantan-1-yl-methyl | 70% @ 10 μM (B) | 67% @ 10 μM (B) | 51% @ 10 μM (B) | 470 ± 90 |
| 5 | R-isobutyl | R-cyclohexyl-methyl | R-propyl | 4-tbutyl-cyclohexyl-methyl | 500 ± 50 | 75% @ 10 μM (B) | 65% @ 10 μM (B) | 530± 50 |
| 6 | R-isobutyl | R-cyclohexyl-methyl | R-propyl | 4-methylpentyl | 2300 ± 90 | 58% @ 10 μM (B) | 6700 ± 2400 | 1800 ± 130 |
| 7 | R-isobutyl | R-cyclohexyl-methyl | R-isopropyl | adamantan-1-yl-methyl | 570 ± 150 | 70% @ 10 μM (B) | 50% @ 10 μM (A) | 500 ± 40 |
| 8 | R-isobutyl | R-cyclohexyl-methyl | R-isopropyl | 4-tbutyl-cyclohexyl-methyl | 54% @ 10 μM (B) | 69% @ 10 μM (B) | >100,000 | 560 ± 50 |
| 9 | R-isobutyl | R-cyclohexyl-methyl | R-isopropyl | 4-methylpentyl | 50% @ 10 μM (B) | 33% @ 10 μM (A) | (B) | 1850 ± 800 |
| 10 | R-isobutyl | R-benzyl | (S,R)-1-hydroxyethyl | adamantan-1-yl-methyl | 41% @ 10 μM (A) | 360 ± 40 | 58% @ 10 μM (B) pA2 = 5.6 ± 0.2 |
810 ± 280 |
| 11 | R-isobutyl | R-benzyl | (S,R)-1-hydroxyethyl | 4-tbutyl-cyclohexyl-methyl | 58% @ 10 μM (B) | 310 ± 60 | (A) pA2 = 7.0 ± 0.7 |
530 ± 50 |
| 12 | R-isobutyl | R-benzyl | (S,R)-1-hydroxyethyl | 4-methylpentyl | 62% @ 10 μM (B) | >100,000 | >100,000 | 77% @ 10 μM (B) |
| 13 | R-isobutyl | R-benzyl | R-propyl | adamantan-1-yl-methyl | 7000 ± 6100 | 74% @ 10 μM (B) | 1330 ± 510 | 610 ± 160 |
| 14 | R-isobutyl | R-benzyl | R-propyl | 4-tbutyl-cyclohexyl-methyl | 38% @ 10 μM (A) | 83% @ 10 μM (B) | 28% @ 10 μM (A) | 480 ± 170 |
| 15 | R-isobutyl | R-benzyl | R-propyl | 4-methylpentyl | >100,000 | >100,000 | (B) | 2180 ± 625 |
| 16 | R-isobutyl | R-benzyl | R-isopropyl | adamantan-1-yl-methyl | 15500 ± 14000 | 67% @ 10 μM (B) | 8200 ± 3550 | 440 ± 110 |
| 17 | R-isobutyl | R-benzyl | R-isopropyl | 4-tbutyl-cyclohexyl-methyl | 6800 ± 600 | 62% @ 10 μM (B) | >100,000 | 560 ± 170 |
| 18 | R-isobutyl | R-benzyl | R-isopropyl | 4-methylpentyl | >100,000 | (A) | (A) | 1260 ± 130 |
| 19 | R-isopropyl | R-cyclohexyl-methyl | (S,R)-1-hydroxyethyl | adamantan-1-yl-methyl | 200 ± 80 | 370 ± 90 | 49% @ 10 μM (A) pA2 = 5.8 ± 0.1 |
600 ± 210 |
| 20 | R-isopropyl | R-cyclohexyl-methyl | (S,R)-1-hydroxyethyl | 4-tbutyl-cyclohexyl-methyl | 300 ± 120 | 220 ± 30 | 41% @ 10 μM (A) pA2 = 5.9 ± 0.1 |
150 ± 20 |
| 21 | R-isopropyl | R-cyclohexyl-methyl | (S,R)-1-hydroxyethyl | 4-methylpentyl | 700 ± 80 | 5200 ± 1540 | >100,000 | 740 ± 270 |
| 22 | R-isopropyl | R-cyclohexyl-methyl | R-propyl | adamantan-1-yl-methyl | 550 ± 170 | 2800 ± 1800 | 45% @ 10 μM (A) | 590 ± 180 |
| 23 | R-isopropyl | R-cyclohexyl-methyl | R-propyl | 4-tbutyl-cyclohexyl-methyl | (B) | 350 ± 20 | 50% @ 10 μM (A) pA2 = 5.8 ± 0.2 |
540 ± 50 |
| 24 | R-isopropyl | R-cyclohexyl-methyl | R-propyl | 4-methylpentyl | (B) | (B) | 40% @ 10 μM (A) | 1040 ± 150 |
| 25 | R-isopropyl | R-cyclohexyl-methyl | R-isopropyl | adamantan-1-yl-methyl | 300 ± 30 | 3700 ± 880 | 40% @ 10 μM (A) | 580 ± 160 |
| 26 | R-isopropyl | R-cyclohexyl-methyl | R-isopropyl | 4-tbutyl-cyclohexyl-methyl | 290 ± 20 | 4300 ± 3300 | 28% @ 10 μM (A) | 510 ± 50 |
| 27 | R-isopropyl | R-cyclohexyl-methyl | R-isopropyl | 4-methylpentyl | 6100 ± 4700 | (B) | (B) | 1700 ± 520 |
| 28 | R-isopropyl | R-benzyl | (S,R)-1-hydroxyethyl | adamantan-1-yl-methyl | 440 ± 140 | 360 ± 10 | 580 ± 130 (75%) pA2 = 5.8 ± 0.1 |
180 ± 90 |
| 29 | R-isopropyl | R-benzyl | (S,R)-1-hydroxyethyl | 4-tbutyl-cyclohexyl-methyl | 600 ± 340 | 420 ± 120 | (A) pA2 = 6.5 ± 0.5 |
490 ± 155 |
| 30 | R-isopropyl | R-benzyl | (S,R)-1-hydroxyethyl | 4-methylpentyl | (B) | >100,000 | >100,000 | 40% @ 10 μM (A) |
| 31 | R-isopropyl | R-benzyl | R-propyl | adamantan-1-yl-methyl | 6700 ± 4700 | 2000 ± 850 | 1400 ± 760 | 6600 ± 5740 |
| 32 | R-isopropyl | R-benzyl | R-propyl | 4-tbutyl-cyclohexyl-methyl | 2300 ± 1000 | 6200 ± 5400 | 27% @ 10 μM (A) | 940 ± 340 |
| 33 | R-isopropyl | R-benzyl | R-propyl | 4-methylpentyl | 22900 ± 10000 | >100,000 | 41% @ 10 μM (A) | 2030 ± 980 |
| 34 | R-isopropyl | R-benzyl | R-isopropyl | adamantan-1-yl-methyl | 47800 ± 23100 | 4000 ± 1900 | 54% @ 10 μM (B) | 660 ± 130 |
| 35 | R-isopropyl | R-benzyl | R-isopropyl | 4-tbutyl-cyclohexyl-methyl | 29100 ± 20000 | 68% @ 10 μM (B) | >100,000 | 600 ± 170 |
| 36 | R-isopropyl | R-benzyl | R-isopropyl | 4-methylpentyl | 10220 ± 6570 | >100,000 | 21% @ 10 μM (A) | 1600 ± 460 |
| 37 | R-2-butyl | R-benzyl | R-4-hydroxybenzyl | 4-tbutyl-cyclohexyl-methyl | 290 ± 20 | 65% @ 10 μM (B) | 23% @ 10 μM (A) | 380 ± 50 |
The compounds were screened at the selected receptor subtypes for agonist activity from 10−4 to 10−10 M and the results are tabulated as the mean EC50 values of duplicate values for at least three independent experiments. For select compounds, the 100 μM concentration resulted in cytotoxicity and was excluded from the sigmoidal dose-response fittings. For these compounds, the activity observed at 10 μM relative to maximal expression was tabulated to aide in the differentiation between partial agonists which have both a reported EC50 and percent activation and compounds that exhibited some receptor activation at 10 μM. These compounds were binned as A (10-50% receptor activation) or B (51-90% receptor activation). Follow up antagonist experiments and corresponding pA2 values, via a Schild analysis,73 were determined for the mMC4R if the compound possessed a nanomolar EC50 value (< 1 μM) at the mMC3R and produced little to no activity at the mMC4R (< 20% of maximal activity). Included as positive controls were α-MSH, the α-MSH analogue NDP-MSH, the tetrapeptide Ac-His-DPhe-Arg-Trp-NH2, and the THIQ small molecule.
The efficacy of THIQ was observed to be 90-95% at the mMC3R compared to the maximal response of α-MSH.