2
K. A. Piechowicz et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
Chemistry: general
Unless otherwise indicated, all reaction solvents were anhydrous
and obtained as such from commercial sources. All other reagents
were used as supplied. Reverse-phase high-pressure liquid
chromatography (RP-HPLC) analysis was performed using a
Dionex Ultimate 3000 system, using a C18 column [3 ꢃ 150 mm].
Low-resolution electrospray ionization (ESI)-liquid chromatogra-
phy mass spectrometry (LCMS) was carried out with an Agilent
1100 HPLC coupled to an Agilent 1956B mass spectrometry
detector (MSD). RP-HPLC runs typically employed gradients of
two solvents: [A] ¼ H2O (0.05% trifluoroacetic acid (TFA)) and
[B] CH3CN (0.05% TFA); RP-LCMS used the same solvent
system with TFA replaced with formic acid (88% aq). The
standard HPLC and LCMS gradients proceeded with
Figure 1. The structure of lead inhibitor T16Ainh-A01.
substituent (aromatic and heteroaromatic). On the pyrimidine, alkyl
and cycloalkyl substituents were chosen to probe a possible
hydrophobic pocket in the binding site, noting the presence of ethyl
and methyl in the lead inhibitor. Fluoroalkyl substituents were
considered given their resistance to oxidative metabolism, and
ability to form electrostatic interactions. We hypothesized a more
substantial interaction with the disubstituted pyrimidine and the
binding site, while assuming the role of the aminothiazole sub-
stituent less important, and as a possible position for solubilizing
groups. To probe this hypothesis, we designed inhibitors replacing
4-methoxyphenyl in T16Ainh-A01 with other aromatic rings,
as well as a small selection of mono- and bicyclic heterocycles.
1
[A:B] ¼ 95:5 to [A:B] ¼ 5:95 over 10 min. H and 13C nuclear
magnetic resonance (NMR) spectra were recorded on either a
1
Bruker 300 or 500 MHz instrument. H NMR chemical shifts are
relative to tetramethylsilane (TMS) (d ¼ 0.00 ppm), CDCl3 (d
7.26), CD3OD (d ¼ 4.87 and 3.31), acetone-d6 (d 2.05), or
DMSO-d6 (d 2.5). 13C NMR chemical shifts are relative to
CD3OD (d 49.2) or CDCl3 (d 77.2). Microwave-assisted organic
synthesis was performed using a Biotage Initiator instrument.
Several compounds were prepared but also had a commercial
supplier or were known: 2a–c (via general procedure 1); 7a, b, e
and f (via general procedure 4); 8a–c, e, h–m, o and p (via general
procedure 6); 9ag, ai, aj and ax (via general procedure 7).
Materials and methods
Cell lines and culture
General procedure 1: 4-aryl-2-aminothiazole bromoacetamides
(2a–c) prepared from 4-aryl-2-aminothiazoles (1a–c)
Fischer rat thyroid (FRT) cells were stably transfected with human
TMEM16A (the abc isoform) and halide sensor YFP-H148Q/
I152L/F46L. Cells were plated in 96-well black-walled microplates
(Corning Inc., Corning, NY) at a density of 20 000 cell/well in
Coon’s modified F-12 medium supplemented with 10% fetal calf
serum, 2 mM L-glutamine, 100 units/ml penicillin and 100mg/ml
Substituted 4-aryl-2-aminothiazole (1.0 eq, 2.5 mmol) (1a–c)
was dissolved in anhydrous methylene chloride (0.3 M), fol-
lowed by treatment with triethylamine (1.2 eq) and placed into
an ice bath. The reaction mixture was stirred under argon until
internal temp was about 0 ꢂC and bromoacetyl bromide (1.05 eq)
dissolved in dichloromethane (DCM) was added dropwise. Next,
the reaction mixture was stirred under argon for 1 h at room
temperature (RT). LCMS indicated consumption of starting
material and formation of a product. The crude product was
treated with HCl (0.1 M aq; 50 ml), transferred to a separatory
funnel and extracted with 1:1 mixture of ethyl acetate and
diethyl ether (50 ml). Then, the organic phase was washed with
additional HCl (0.1 M aq), brine and was then dried over
Na2SO4 and concentrated in vacuo.
streptomycin. Assays were done 24 h after plating20,23
.
TMEM16A functional assay
Each well of a 96-well plate containing the cultured cells was
washed twice with phosphate-buffered saline (PBS) leaving 50 ml.
Test compounds (0.5 ml in dimethyl sulfoxide (DMSO)) were
added to each well at specified concentration. After 10 min, each
well was assayed individually for TMEM16A-mediated Iꢀ influx
by recording fluorescence continuously (400 ms/point) for 2 s
(baseline), then 50 ml of 140 mM Iꢀ solution was added at 2 s, and
then 50 ml of 70 mM Iꢀ solution containing 300 mM adenosine
triphosphate (ATP) was added at 6.4 s. The 70 mM Iꢀ solution
consisted of a 1:1 mixture of PBS and the 140 mM Iꢀ solution.
The initial rate of Iꢀ influx following each of the solution
additions was computed from fluorescence data by non-linear
General procedure 2: 2-amino heteroaryl thiazoles (4b–d)
prepared from heteroaryl methyl ketones (3b–d)
Heteroaryl methyl ketone (1.0 eq; 8 mmol) (3b–d) was dissolved
in EtOAc (0.1 M), followed by the addition of CuBr2 (2.0 eq).
This reaction mixture was refluxed at 100 ꢂC for 1 h. LCMS
indicated consumption of starting material and the formation of
the desired bromoketone intermediate. The reaction mixture was
then left to cool to RT. Upon reaching RT, the reaction mixture
was filtered by using a Buchner funnel, to remove excess
precipitated CuBr2, and the filtrate was then added to a fresh
round bottom flask (RBF). Thiourea (2.0 eq) was then added into
the reaction mixture, which was then heated again for 1 h at
100 ꢂC. Reaction mixtures typically changed from green to orange
during the course of the reaction, with the formation of a
precipitate. The mixture was then allowed to cool to RT. After
reaching RT, the mixture was filtered with a Buchner funnel. The
precipitate was then rinsed with ethyl acetate, in order to remove
excess thiourea, which generated a crude product. The identity
and purity of the product was confirmed by LCMS.
regression20,23,28
.
Short-circuit current assay
FRT-TMEM16A cells were grown on Snapwell inserts as
described20 and mounted in Ussing chambers (Physiologic
Instruments, San Diego, CA). The basolateral membrane was
permeabilized with amphotericin B (250 mg/ml) for 30 min, and a
chloride gradient was applied in which the basolateral membrane
was bathed with the HCO3-buffered solution, and in the apical
solution 120 mM NaCl was replaced by sodium gluconate.
Compounds were added to the apical solution. Cells were
bathed for a 10-min stabilization period and aerated with 95%
O2/5% CO2 at 37 ꢂC before addition of 100 mM ATP. Short-circuit
current was measured using an EVC4000 Multi-Channel V/I
Clamp (World Precision Instruments, Sarasota, FL).