S. Zhao, et al.
BioorganicChemistry96(2020)103596
due to the lack of functionally-selective CTR agonists.
(s, 1H), 6.57 (d, J = 6.0 Hz, 1H), 6.43 (s, 2H), 5.68 (s, 1H), 5.43 (s, 2H),
2.48 (s, 3H), 2.37 (s, 3H). 13C NMR (75 MHz, DMSO‑d6): δ 168.8, 164.3,
158.9, 150.0, 148.6, 137.3, 129.9, 113.0, 112.6, 110.1, 109.8, 98.9,
25.2, 20.7. HRMS (ESI, positive) Calcd for C14H15N3O3H [M+H]+
274.1186, found: 274.1184.
Recently, development of biased agonists with functional se-
lectivity, which signal with different efficacies to a particular receptor’s
multiple downstream pathways (e.g. individual G protein subtype or β-
arrestin pathways) are useful tools for dissection of specific signaling
pathways[29,30,49–52]. These biased ligands may also possess better
tively, compared to “balanced agonists” [46,47]. Therefore, develop-
ment of small molecules eliciting biased CTR functions will be valuable
tools in studying selective CTR downstream functions. However, the β-
arrestin activity of the only known CTR small-molecule agonist SUN-
B8155 has not been fully characterized [20].
S8155-4 (R = 4-NH2): 63% yield, pale yellow solid. Mp 251–253
°C. 1H NMR (300 MHz, DMSO‑d6): δ 14.62 (s, 1H), 10.04 (s, 1H), 6.95
(d, J = 8.7 Hz, 2H), 6.63 (d, J = 8.7 Hz, 2H), 5.63 (s, 1H), 5.42 (s, 2H),
2.43 (s, 3H), 2.34 (s, 3H). 13C NMR (75 MHz, DMSO‑d6): δ 168.9, 164.0,
158.8, 148.5, 148.4, 126.3, 124.5, 114.0, 109.3, 98.7, 25.2, 20.6;
HRMS (ESI, positive) Calcd for C14H15N3NaO3 [M+Na]+ 296.1006,
found: 296.1004.
In the present study, we synthesized a series of compounds (S8155-1
to S8155-9) based on the chemical structure of SUN-B8155, and in-
vestigated the bias property using SUN-B8155 as control. Compound
S8155-7 displayed a Gs biased property and at least five SUN-B8155
derivatives exhibited β-arrestin-2 biased signalling properties. Cellular
transwell studies demonstrated that compounds SUN-B8155, S8155-4
and S8155-7 inhibited breast cancer cells (MCF-7) migration via acti-
vation of CTR.
S8155-5 (R = 2-MeO): 76% yield, pale yellow solid. Mp 238–240
°C. 1H NMR (300 MHz, DMSO‑d6): δ 14.72 (s, 1H), 10.09 (s, 1H),
7.38–7.31 (m, 2H), 7.21–7.19 (m, 1H), 7.08–7.05 (m, 1H), 5.67 (d,
J = 0.6 Hz, 1H), 3.84 (s, 3H), 2.43 (s, 3H), 2.37 (s, 3H). 13C NMR
(75 MHz, DMSO‑d6): δ 169.1, 164.4, 158.9, 153.0, 148.4, 128.9, 127.0,
125.2, 120.6, 112.3, 110.0, 99.3, 55.9, 25.3, 20.7. HRMS (ESI, positive)
Calcd for C15H16N2NaO4 [M+Na]+ 311.1002, found: 311.1004.
S8155-6 (R = 3-Cl): 67% yield, pale yellow solid. Mp 204–206 °C.
1H NMR (300 MHz, CDCl3): δ 14.84 (s, 1H), 9.24 (s, 1H), 7.41–7.31 (m,
2H), 7.21 (s, 1H), 7.11 (d, J = 7.5 Hz, 1H), 5.81 (s, 1H), 2.46 (s, 3H),
2.37 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 168.8, 162.1, 158.1, 148.3,
137.9, 135.4, 130.8, 128.3, 126.2, 124.3, 110.3, 100.0, 26.2, 21.2.
HRMS (ESI, positive) Calcd for C14H13ClN2NaO3 [M+Na]+ 315.0507,
found: 315.0508.
2. Materials and methods
2.1. Chemical synthesis
2.1.1. Synthetic procedure for 5-acetyl-1,6-dihydroxy-4-methyl-2(1H)-
pyridone (1)
S8155-7 (R = 2-Me): 45% yield, pale yellow solid. Mp 197–199 °C.
1H NMR (300 MHz, CDCl3): δ 14.74 (s, 1H), 9.24 (s, 1H), 7.33–7.26 (m,
3H), 7.15–7.12 (m, 1H), 5.85 (s, 1H), 2.42 (s, 3H), 2.41 (s, 3H), 2.30 (s,
3H). 13C NMR (75 MHz, CDCl3): δ 169.7, 161.9, 158.0, 148.3, 135.7,
134.0, 131.4, 128.6, 127.0, 126.7, 109.4, 99.5, 26.2, 21.0, 18.1. HRMS
(ESI, positive) Calcd for C15H16N2NaO3 [M+Na]+ 295.1053, found:
295.1053.
Hydroxylamine hydrochloride (9 g, 130 mmol), water and acet-
onitrile (2:1 v/v, 6 mL) were added to a 250 mL three-necked flask. At 0
°C, triethylamine (60 mL, 260 mL) was slowly added to the reaction
mixture, and then diketene (20 mL, 260 mmol) dissolved in a mixture of
water and acetonitrile (2:1 v/v, 12 mL) was added dropwise to the
reaction solution while the temperature was maintained below 5 °C.
After the completion of addition of diketene solution, the reaction
mixture was stirred at 0 °C for 30 min, and then allowed to stand at
ambient temperature for 1 h. 6 N HCl aqueous solution was slowly
added to the reaction mixture to adjust the pH to 7 that resulted in
formation of a white suspension. The precipitated solid was filtered and
washed with water, and the crude product was recrystallized from
ethanol to obtain pale yellow solid 1 (11.9 g, 50% yield). Mp 195–197
°C. 1H NMR (300 MHz, DMSO‑d6): δ 5.81 (s, 1H), 2.56 (s, 3H), 2.32 (s,
3H). 13C NMR (75 MHz, DMSO‑d6): δ 194.3, 165.1, 157.9, 149.1, 107.6,
104.7, 28.0, 23.3.
S8155-8 (R = 3-Br): 50% yield, pale yellow solid. Mp 227–229 °C.
1H NMR (300 MHz, DMSO‑d6): δ 14.86 (s, 1H), 10.14 (s, 1H), 7.62–7.57
(m, 2H), 7.45 (t, J = 7.8 Hz, 1H), 7.35 (d, J = 7.8 Hz, 1H), 5.71 (s, 1H),
2.47 (s, 3H), 2.36 (s, 3H). 13C NMR (75 MHz, DMSO‑d6): δ 168.6, 164.4,
158.9, 148.5, 138.5, 131.3, 130.2, 128.5, 124.9, 122.0, 110.6, 99.7,
25.1, 20.9. HRMS (ESI, positive) Calcd for C14H13BrN2O3H [M+H]+
337.0182, found: 337.0180.
S8155-9: 71% yield, pale yellow solid. Mp 231–233 °C. 1H NMR
(300 MHz, DMSO‑d6): δ 15.24 (s, 1H), 10.23 (s, 1H), 8.08–7.90 (m, 3H),
7.66–7.58 (m, 4H), 5.75 (s, 1H), 2.42 (s, 3H), 2.39 (s, 3H). 13C NMR
(75 MHz, DMSO‑d6): δ 169.9, 164.8, 158.9, 148.6, 133.8, 132.8, 128.6,
128.4, 128.1, 127.7, 127.0, 125.7, 124.3, 121.9, 110.3, 99.6, 25.2,
2.1.2. General procedure for synthesizing SUN-B8155 and its derivatives
(S8155-1, -2, -4-9)
20.9. HRMS (ESI, positive) Calcd for
331.1053, found: 331.1049.
C
18H16N2NaO3 [M+Na]+
A mixture of 1 (300 mg, 1.6 mmol) and substituted aniline
(1.6 mmol) in EtOH (12 mL) was heated at 80 °C for 3 h. After the
reaction was completed, the reaction solution was cooled to ambient
temperature, resulting in precipitates. The solid was obtained by suc-
tion filtration, washed with cold ethanol and air-dried. SUN-B8155
(R = 2-NH2): 75% yield, pale yellow crystal. Mp 196–198 °C. 1H NMR
(300 MHz, DMSO‑d6): δ 14.36 (s, 1H), 10.06 (s, 1H), 7.12–7.07 (m, 1H),
7.00 (dd, J = 7.8 Hz, 1.5 Hz, 1H), 6.82 (dd, J = 8.1, 1.2 Hz, 1H),
6.65–6.60 (m, 1H), 5.65 (s, 1H), 5.26 (s, 2H), 2.37 (s, 6H). 13C NMR
(75 MHz, DMSO‑d6): δ 170.7, 164.3, 158.8, 148.6, 144.0, 128.8, 127.1,
121.4, 116.3, 115.8, 109.3, 99.2, 25.3, 20.2. HRMS (ESI, positive)
Calcd for C14H15N3NaO3 [M+Na]+ 296.1006, found: 296.1007.
S8155-1 (R = 4-F): 40% yield, pale yellow solid. Mp 215–216 °C.
1H NMR (300 MHz, DMSO‑d6): δ 14.72 (s, 1H), 10.05 (s, 1H), 7.34–7.23
(m, 4H), 5.61 (s, 1H), 2.35 (s, 3H), 2.28 (s, 3H). 13C NMR (75 MHz,
DMSO‑d6): δ 169.1, 164.4, 162.6, 159.3, 159.0, 148.6, 133.2, 128.0,
127.9, 116.6, 116.3, 99.3, 25.2, 20.7. HRMS (ESI, positive) Calcd for
2.1.3. Synthetic procedure for S8155-3
A
50 mL round-bottom flask was charged with 1 (300 mg,
1.6 mmol), Raney nickel (30 mg), and methanol (15 mL), and the re-
action was carried out at ambient temperature and under H2 atmo-
sphere for 12 h. After the reaction was completed, the catalyst was
removed by filtration, and the crude product was recrystallized to af-
ford 5-acetyl-6-hydroxy-4-methyl-2(1H)-pyridone (2) (207 mg, 80%
yield) as white crystal. 1H NMR (300 MHz, DMSO‑d6): δ 12.07 (s, 1H),
5.85 (s, 1H), 2.70 (s, 3H), 2.49 (s, 3H); 13C NMR (75 MHz, DMSO‑d6): δ
195.0, 169.3, 161.5, 151.9, 110.4, 103.8, 28.1, 23.5.
To a 50 mL round bottom flask was added 2 (196 mg, 1.3 mmol), O-
phenylenediamine (138 mg, 1.3 mmol) and ethanol (10 mL), and the
mixture was heated to reflux for 3 h. After the disappearance of the
starting material, the reaction solution was cooled to ambient tem-
perature, and the resulting solid (213 mg, 60% yield) was obtained by
suction filtration, washing and air-drying.
C
14H13FN2O3H [M+H]+ 277.0983, found: 277.0984.
S8155-2 (R = 3-NH2): 63% yield, pale yellow solid. Mp 214–216
S8155-3: Mp 230–232 °C. 1H NMR (300 MHz, DMSO‑d6): δ 14.56 (s,
1H), 10.75 (s, 1H), 7.10–7.04 (m, 1H), 6.98–6.95 (m, 1H), 6.82–6.80
°C. 1H NMR (300 MHz, DMSO‑d6): δ 14.77 (s, 1H), 10.10 (s, 1H), 7.12
2