A potent benzopyran-based inhibitor of Kir3.1 heteromers
Subsequently, the system was heated from 0 K to 303 K using
Langevin dynamics with the collision frequency of 1 ps−1
2,2,9-trimethyl-2H-pyrano[2,3-g]quinoline (4; GAT1572)
.
i) The compound was synthesized starting with the reduc-
tion of compound (Fig. 6A, compound 3) (700 mg, mmol) to 3
2,2-dimethyl-2H-chromen-6-amine by using Tin (II) Chloride
(3.2 g, 17.07 mmol), dissolved in ethanol, and refluxed for 12 h.
Upon completion the reaction was then concentrated to dry to
remove the ethanol and then dissolved in ethyl acetate. The
reaction was then quenched with aqueous sodium bicarbonate
solution and then filtered through silica to remove the metal
tin from the mixture. The layers were then separated after
filtering and dried over sodium sulfate and concentrated to
dry. This compound was taken ahead without further purifi-
cation: ii) compound was synthesized by dissolving 2,2-
dimethyl-2H-chromen-6-amine crude (300 mg), Ferric Chlo-
ride coated silica (300 mg) were dissolved in acetic acid and
stirred at room temperature, and methyꢁl vinyl ketone was
added dropwise (0.2 ml) and stirred at 70 C for 1 h. ii) Next,
Zinc (II) Chloride (300 mg) and Indium (III) Chloride (50 mg,
mmol) were added to the reaction mixture and refluxed for 2 h
under argon. The reaction was then concentrated to dry to
remove acetic acid, then redissolved in ethyl acetate and
quenched with aqueous and solid sodium sulfate until effer-
vescence stopped. The reaction mixture was then separated,
and the organic layer was dried over sodium sulfate, Na2SO4
and evaporated under vacuum. The crude residue was purified
by silica gel column chromatography (EtOAc/hexane) to yield
the desired 120 mg, of a light brown oily solid: 1H NMR
(400 MHz, CDCl3 δ 8.57(d, J = 5.0 Hz, 1H), 7.67 (s, 1H), 7.24
(s, 1H), 7.11 (d, J = 4.5 Hz, 1H), 6.58 (d, J = 10.0 Hz, 1H), 5.90
(d, J = 10.0 Hz, 1H), 2.59(s, 3H), 1.50 (s, 6H). LC/MS: m/z
calculated for C15H15NO [M + H]+, 226.29.
During the heating, the BP-G1-Channel complex, PIP2 was
position-restrained using an initial constant force of
500 kcal/mol/Å2 and weakened to 10 kcal/mol/Å2, allowing
lipid and water molecules to move freely. Then, the system
went through 5 ns equilibrium MD simulations. Finally, a
total of 1μs production MD simulation was conducted, and
coordinates were saved every 50 ps for analysis.
Chemical synthesis
For the experiments presented in this article, some of the BP-
G1 and its inactive isomer were obtained through academic and
industrial partnerships not disclosed here. Related analog
compounds, GAT1572 (compound 4, Fig. 6A), GAT1573
(compound 6, Fig. 6A), and GAT1588, and intermediates were
all synthesized at Northeastern University. BP-G1 was synthe-
sized and confirmed by LCMS; however, final purification
proved challenging with a low yielding reaction. Improvement
on the BP-G1 synthesis yield will be the focus of future ex-
periments. All reagents were purchased from commercial
sources as reagent grade. Reactions were monitored by thin-
layer chromatography (TLC) using commercially prepared sil-
ica gel 60 F254 glass plates. Compounds were visualized under
ultraviolet (UV) light or by staining with iodine. Flash column
chromatography was carried out on an autoflash purification
unit using prepacked columns from Biotage and Buchi. Solvents
used include hexanes and ethyl acetate, methanol, dichloro-
methane for purification. Characterization of compounds and
their purity was established by a combination of LCMS, TLC,
and NMR analyses. NMR spectra were recorded in CDCl3 on a
NMR spectrometer (1H NMR at 400 MHz). Chemical shifts
were recorded in parts per million (δ) relative to tetrame-
thylsilane (TMS; 0.00 ppm) or solvent peaks as the internal
reference. Multiplicities are indicated as br (broadened), s
(singlet), d (doublet), t (triplet), q (quartet), quin (quintet), and
m (multiplet). Coupling constants (J) are reported in hertz (Hz).
All test compounds were greater than 95% pure, as determined
by LC-MS analysis performed with a dual-wavelength UV-
visible detector and quadrupole mass spectrometer.
(3R,4S)-2,2,9-trimethyl-4-(phenethylamino)-3,4-dihydro-2H-
pyrano[2,3-g]quinolin-3-ol (GAT1588)
GAT1572 (compound 4) (200 mg) was dissolved in ethyl
acetate and 1-methylimidazole (0.5 ml) and R-R Mn Salen
catalyst (120 mg). Aqueous sodium hypochlorite (2 ml) was
then added dropwise to the reaction and stirred at RT for 24 h
to yield the epoxide intermediate (compound 5). The reaction
mixture was poured into water and extracted with ethyl acetate
(3 × 50 ml). The combined organic layers were washed with
water (20 ml) and brine (20 ml), dried over Na2SO4, and
evaporated under vacuum. The epoxide crude was taken ahead
without further purification. The Compound 5 (100 mg,
mmol) was dissolved in 1 to 4 dioxane followed by the addition
of lithium perchlorate (170 mg, mmol), 2-pheneythylamine
(2 ml), and refluxed for 12 h to yield mg, % yield, of a solid:
1H NMR (400 MHz, CDCl3) δ 8.57(d, J = 5.0 Hz, 1H), 7.67 (s,
1H), 7.24 (s, 1H), 7.11 (d, J = 4.5 Hz, 1H), 6.58 (d, J = 10.0 Hz,
1H), 5.90 (d, J = 10.0 Hz, 1H), 2.59 (s, 3H), 1.50 (s, 6H). LC/MS:
m/z calculated for C23H26N2O2 [M + H]+, 363.47.
2,2-dimethyl-6-nitro-2H-chromene (Fig. 6A, compound 3)
The compound was synthesized using nitrophenol (com-
pound 2) (1.0 g, mmol), 1,1-diethoxy-3-methylbut-2-ene
(compound 1) (1 g, 7.19 mmol), pyridine (142 mg,
1.79 mmol) and loading in a 100 ml round bottom flask.
Starting material was dissolved in anhydrous toluene and
refluxed for 12 h. The reaction mixture was poured into water
and extracted with ethyl acetate (3 × 50 ml). The combined
organic layers were washed with water (20 ml) and brine
(20 ml), dried over Na2SO4, and evaporated under vacuum. The
crude residue was purified by silica gel column chromatography
(EtOAc/hexane) with the compound eluting in 100% hexane to
yield 700 mg, of light yellow oil: 1H NMR (400 MHz, CDCl3): δ
8.01(d, J = 11.5 Hz, 1H), 7.89 (s, 1H), 6.81 (d, J = 9.0 Hz, 1H),
6.35 (d, J = 12.0 Hz, 1H), 5.75 (d, J = 13.0 Hz, 1H), 1.48 (s, 6H).
(2,2,9-trimethyl-2H-pyrano[2,3-g]quinolin-7-yl)methanol
(compound 6; GAT1573)
Compound 5 (100 mg, mmol), para toluene sulfonic acid
(150 mg,), Na2S2O8 (400 mg) were added to a screwcap vial
J. Biol. Chem. (2021) 296 100535 13
Cui, Meng
