Substituted 2-Acylaminocycloalkylthiophene-3-carboxylic Acid Arylamides as Inhibitors of the Calcium-Activated Chloride Channel Transmembrane Protein 16A (TMEM16A)

Article abstract of DOI:10.1021/acs.jmedchem.7b00020

Transmembrane protein 16A (TMEM16A), also called anoctamin 1 (ANO1), is a calcium-activated chloride channel expressed widely mammalian cells, including epithelia, vascular smooth muscle tissue, electrically excitable cells, and some tumors. TMEM16A inhibitors have been proposed for treatment of disorders of epithelial fluid and mucus secretion, hypertension, asthma, and possibly cancer. Herein we report, by screening, the discovery of 2-acylaminocycloalkylthiophene-3-carboxylic acid arylamides (AACTs) as inhibitors of TMEM16A and analysis of 48 synthesized analogs (10ab-10bw) of the original AACT compound (10aa). Structure-activity studies indicated the importance of benzene substituted as 2- or 4-methyl, or 4-fluoro, and defined the significance of thiophene substituents and size of the cycloalkylthiophene core. The most potent compound (10bm), which contains an unusual bromodifluoroacetamide at the thiophene 2-position, had IC₅₀ of ～30 nM, ～3.6-fold more potent than the most potent previously reported TMEM16A inhibitor 4 (Ani9), and >10-fold improved metabolic stability. Direct and reversible inhibition of TMEM16A by 10bm was demonstrated by patch-clamp analysis. AACTs may be useful as pharmacological tools to study TMEM16A function and as potential drug development candidates.

Full text of DOI:10.1021/acs.jmedchem.7b00020

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Article
Substituted 2-acylamino-cycloalkylthiophene-3-carboxylic
acid arylamides as inhibitors of the calcium-activated
chloride channel transmembrane protein 16A (TMEM16A)
Eric C Truong, Puay-Wah Phuan, Amanda L Reggi, Loretta Ferrera, Luis J.V. Galietta, Sarah E Levy,
Alannah C Moises, Onur Cil, Elena Diez-Cecilia, Sujin Lee, Alan S Verkman, and Marc O. Anderson
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 11 May 2017
Downloaded from http://pubs.acs.org on May 12, 2017
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Substituted 2ꢀacylaminoꢀcycloalkylthiopheneꢀ3ꢀcarboxylic acid arylamides as inhibitors
of the calciumꢀactivated chloride channel transmembrane protein 16A (TMEM16A)
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Eric C. Truong1, Puay W. Phuan2, Amanda L. Reggi1, Loretta Ferrera3, Luis J.V. Galietta4,
Sarah E. Levy1, Alannah C. Moises1, Onur Cil2, Elena DiezꢀCecilia1, Sujin Lee2,
Alan S. Verkman2, and Marc O. Anderson1*
1Department of Chemistry and Biochemistry, San Francisco State University, San Francisco CA,
94132ꢀ4136 USA; 2Departments of Medicine and Physiology, University of California, San Francisco
CA, 94143ꢀ0521 USA; 3U.O.C. Genetica Medica, Istituto Giannina Gaslini, Genova, ITALY;
4Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli (NA), ITALY.
Corresponding author: Marc O. Anderson, Ph.D. Department of Chemistry and Biochemistry, San
Francisco State University, San Francisco CA 94132. Phone 415ꢀ338ꢀ6495; Fax 415ꢀ405ꢀ0377;
email marc@sfsu.edu.
Keywords: TMEM16A, calciumꢀactivated chloride channel, ANO1, hypertension, cancer, anoctamin,
cycloalkylthiophene, 2ꢀacylaminoꢀcycloalkylthiopheneꢀ3ꢀcarboxylic acid arylamide
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ABSTRACT
Transmembrane protein 16A (TMEM16A), also called anoctamin 1 (ANO1), is a calciumꢀ
activated chloride channel expressed widely mammalian cells, including epithelia, vascular smooth
muscle tissue, electrically excitable cells and some tumors. TMEM16A inhibitors have been
proposed for treatment of disorders of epithelial fluid and mucus secretion, hypertension, asthma, and
possibly cancer. Herein we report by screening the discovery of 2ꢀacylaminoꢀcycloalkylthiopheneꢀ3ꢀ
carboxylic acid arylamides (AACTs) as inhibitors of TMEM16A, and analysis of 48 synthesized
analogs (10abꢀ10bw) of the original AACT compound (10aa). Structureꢀactivity studies indicated
the importance of benzene substituted as 2ꢀ or 4ꢀmethyl, or 4ꢀfluoro, and defined the significance of
thiophene substituents and size of the cycloalkylthiophene core. The most potent compound (10bm),
which contains an unusual bromodifluoroacetamide at the thiophene 2ꢀposition, had IC50 ~ 30 nM,
~3.6ꢀfold more potent than the most potent previously reported TMEM16A inhibitor 4 (Ani9), and
>10ꢀfold metabolic stability. Direct and reversible inhibition of TMEM16A by 10bm was
demonstrated by patchꢀclamp analysis. AACTs may be useful as pharmacological tools to study
TMEM16A function and as potential drug development candidates.
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INTRODUCTION
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Transmembrane protein 16A (also known as TMEM16A, ANO1, DOG1, ORAOV2, TAOSꢀ2) is
a calciumꢀactivated chloride channel (CaCC) that is expressed in a variety of mammalian tissues
including smooth muscle cells, airway mucinꢀsecreting cells, secretory epithelia, interstitial cells of
Cajal, and nociceptive neurons.1ꢀ3 TMEM16A is overexpressed in some human cancers,4, 5 and
knockdown of TMEM16A inhibits cancer cell proliferation, cell cycle progression, and apoptosis.6
TMEM16A was reported recently as a biomarker for gastrointestinal stromal and esophageal tumors,7,
8 and TMEM16A expression is associated with good prognosis in PRꢀpositive or HER2ꢀnegative
breast cancer following tamoxifen treatment.9
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Early studies suggested TMEM16A contains eight putative transmembrane domains with
intracellular Nꢀ and Cꢀ termini, and two calmodulin binding domains.10ꢀ12 Multiple TMEM16A splice
variants may explain its variable behavior in different tissues and cell types.13ꢀ16 Recent studies have
questioned the role of calmodulin in TMEM16A activation, and have identified four acidic sideꢀchain
residues involved in Ca2+ binding in mouse TMEM16A: E698, E701, E730 and D734.17, 18 Other
divalent cations (Sr2+ and Ni2+) appear to activate the channel as well.19 Four basic sideꢀchain
residues appear to be critical for anion selectivity in mouse TMEM16A: R511, K599, R617 and
R784.20 The kinetics of TMEM16A and TMEM16B Clꢀ conductance are complex, with distinct fast
and slow gating mechanisms, regulated by membrane voltage and Clꢀ concentration.21 Biophysical
experiments suggest TMEM16A is present in membranes as a homodimer,22, 23 with the dimerization
domain located in the Nꢀterminal sequence.24 Recently, the Cꢀterminal region of TMEM16A was
modified to produce a constitutively active channel, providing a strategy to design TMEM16A
activators.25
A 3.4Å Xꢀray crystal structure was solved recently of a Ca2+ꢀactivated lipid scramblase expressed
in the fungus Nectria haematococca (nhTMEM16), which is ~ 40% homologous to mammalian
TMEM16A, indicating a tenꢀtransmembrane helical structure.26 Using the nhTMEM16 structure, two
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homology models of TMEM16A suggest it contains ten transmembrane helical segments,21, 25
revising earlier eight helix structural models.
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Inhibitors of TMEM16A has been proposed to be of potential utility for treatment of
hypertension, asthma, inflammatory and reactive airways diseases, pain, and possibly cancer.1, 3, 4, 27
Reported inhibitors (Figure 1) include the nonꢀselective CaCC inhibitor CaCCinhꢀA01 (1),28 and
TMEM16Aꢀselective inhibitors such as the thiopyrimidine aryl aminothiazole T16AinhꢀA01 (2),29, 30
Nꢀ((4ꢀmethoxy)ꢀ2ꢀnaphthyl)ꢀ5ꢀnitroanthranilic acid (MONNA) (3),31 and the acyl hydrazone Ani9
(4).32 The use of compound 2 to inhibit TMEM16A in various tissues has been reviewed.1
Compound 2 blocks Ca2+ꢀactivated Clꢀ currents in vascular smooth muscle cells, and relaxes mouse
and human blood vessels,33 This compound also prevents serotoninꢀinduced contractile responses in
pulmonary arteries of chronic hypoxic rats, a model of pulmonary hypertension,34 and reverses EGFꢀ
induced increases in CaCC currents in T84 colonic epithelial cells.35 Recently, 2 was also shown to
attenuate angiotensin IIꢀinduced cerebral vasoconstriction in rat basilar arteries, further supporting
TMEM16A as a target in vascular function, hypertension, and stroke.36 The nonꢀselective CaCC
inhibitor 1 was shown to accelerate the degradation of TMEM16A in cancer cells by the ubiquitinꢀ
proteasome pathway by a mechanism that may not involve channel inhibition.37 Several recent
studies address inhibitor selectivity. Studies of 1, 2, and 3 in isolated resistance arteries suggested
poor TMEM16A selectivity for all three compounds.38 Another study reported 1 as a nonꢀselective
inhibitor of CaCCs TMEM16A and Bestrophin1, while 2 selectively inhibited TMEM16A but with
low potency.39 As such, the discovery of potent and selective TMEM16A inhibitors continues to be a
focus of multiple laboratories.
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Herein, we report the discovery by screening of a 2ꢀacylaminoꢀcycloalkylthiopheneꢀ3ꢀcarboxylic
acid arylamide (AACT) class of TMEM16A inhibitors, exemplified by 10aa. Synthesis and
evaluation of 48 analogs of 10aa has provided compounds with substantially improved TMEM16A
inhibition potency and metabolic stability than the recently reported compound 4.
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Figure 1. Structures of the nonꢀselective CaCC inhibitor 1,28 and TMEM16A inhibitors 2,29, 30 3,31 4,32
and the new class of cycloalkylthiophene inhibitors (10aa) described herein.
RESULTS AND DISCUSSION
A mediumꢀthroughput screening assay was previously developed to identify small molecule
inhibitors of TMEM16A.40 The screen utilized FRT cells that were stably transfected with human
TMEM16A and the iodideꢀsensitive fluorescent protein YFPꢀH148Q/I152L/F46L. The assay
involved addition of test compounds to the cells for 10 min in a physiological chlorideꢀcontaining
solution, followed by addition of an iodide solution containing ATP. ATP is a P2Y2 agonist in FRT
cells used to increase cytosolic Ca2+ and activate TMEM16A channels. TMEM16Aꢀfacilitated iodide
influx was determined from the initial time course of decreasing YFP fluorescence. TMEM16A
inhibitors reduce iodide influx, resulting in a reduced rate of decreasing fluorescence. Here, screening
of 50,000 drugꢀlike synthetic small molecules not previously tested identified 2ꢀacylaminoꢀ
cycloalkylthiopheneꢀ3ꢀcarboxylic acid arylamide (AACT) 10aa with IC50 ~ 0.42 ꢀM (Figure 2). The
structure of 10aa resembles that of the previously identified nonꢀselective CaCC inhibitor 1,28
although the latter molecule is substituted with a tertꢀbutyl group, has a different assortment of
functionality on the thiophene (e.g. lacking the haloacetamide), and was reported to be significantly
less potent (IC50 = 2.1 ꢀM) than 10aa against TMEM16A.29
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Figure 2. Identification of TMEM16A inhibitors. A. Schematic of assay. FRT cells stably expressing
TMEM16A and a halideꢀsensitive YFP sensor were incubated for 10 min with test compound.
Extracellular addition of addition of iodide and ATP results in YFP quenching whose rate is reduced by
TMEM16A inhibition. B. Original fluorescence quenching curves for inhibition of TMEM16A by 10aa.
C. Concentrationꢀdependent inhibition of TMEM16A by 10aa (mean ± S.E., n=4).
Motivated by the submicromolar potency of 10aa and the modularity and synthetic accessibility
of this scaffold, we further optimized this class of compounds. The only other reported biological
activity of AACT compounds is inhibition of the protozoan parasite Leishmania donovani (EC50 = 6.4
ꢀM), with no cytotoxicity seen against human macrophages (CC50 > 50 ꢀM).41 The most potent
inhibitor in the antiꢀparasite study was an analog of 10aa, with 2ꢀmethylanilide replaced with 4ꢀ
methoxyanilide.
Chemistry
AACT compounds were prepared using the modular synthetic strategy shown in Scheme 1. The
synthesis begins with the generation of substituted aryl cyanoacetamides, followed by a twoꢀstep
KnoevenagelꢀGewald sequence to generate 2ꢀaminothiophenes, and coupling with simple
electrophilic acylating agents. Substituted anilines (5aꢀ5k) were coupled with cyanoacetic acid using
EDCIꢀHCl to generate the library of cyanoacetamides (6aꢀ6k). The substituent composition of this
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library, prepared typically in good yields, is reported in Table 1, with some of the cyanoacetamides
also being commercially available.
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Scheme 1. Synthesis of 2ꢀacylaminoꢀcycloalkylthiopheneꢀ3ꢀcarboxylic acid arylamides. Reagents and
conditions: (a) cyanoacetic acid (1.0 eq) and EDCIꢀHCl (1.2 eq) (rt, 6 min); (b) cycloalkyl ketone,
NH4OAc, and AcOH (100 oC, 60 min); (c) S8, morpholine (3.0 eq), EtOH (90 0C, 5h); (d) electrophilic
acylating agent (1.3 eq), Et3N (1.3 eq) (rt, 10 min) or difluoroiodoacetic acid (1.2 eq) and EDCIꢀHCl (1.5
eq) (rt, 60 min).
Table 1. Synthesis yields for EDCIꢀmediated cyanoacetamide generation reactions (5 ꢀ 6);
yields (%) are of the isolated or purified products. Purity of compounds was >95% based on
HPLCꢀLCMS analysis at 254 nm, and absence of impurities was confirmed by 1H NMR spectra.
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Product
Isolated
Yield
(%)
SM
Cyanoꢀ
R2
Aniline
acetamide
9
6a
6b
6c
6d
6e
6f
5a
5b
5c
5d
5e
5f
2ꢀ(CH3)
H
50
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purchased
2ꢀ(CH2CH3)
2ꢀF
76
78
70
82
80
89
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86
87
4ꢀF
2ꢀCl
6g
6h
6i
5g
5h
5i
3ꢀCl
4ꢀCl
4ꢀ(CF3)
4ꢀ(CH3)
2ꢀ(OCF3)
6j
5j
6k
5k
Next, the substituted aryl cyanoacetamides (6aꢀ6k) were condensed with a small collection of
cycloalkyl ketones (7aꢀ7d) under buffered acidꢀcatalyzed aldol conditions (AcOH:NH4OAc) to
generate Knoevenagel adducts (8aꢀ8v). While excess cyclic ketone was useful to obtain high
conversion, we were pleased that this material could be removed by evaporation. The Knoevenagel
adducts were subjected to the Gewald cyclization reaction in the presence of molecular octasulfur
(S8), to yield 2ꢀaminoꢀcycloalkylthiopheneꢀ3ꢀcarboxylic acid arylamides (9aꢀ9v), which were
typically crystalline and easily purified by trituration. The composition of the library and yields for
the Knoevenagel and Gewald reactions are reported in Table 2, separated by the different cycloalkyl
ketones.
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Table 2. Synthesis yields for Knoevenagel reaction / aminothiophene formation (7 + 6 ꢀ [8] ꢀ
9). Yields (%) are of the isolated or purified products. Purity of compounds was >95% based on
HPLCꢀLCMS analysis at 254 nm, and absence of impurities was confirmed by 1H NMR spectra.
Isolated Isolated
Product
Aminoꢀ
SM
Yield
(%)
Yield
(%)
Cyanoꢀ
R1
R2
thiophene acetamide
Intmdt
8aꢀ8v
Product
9aꢀ9v
(based on cycloheptanone 7a)
9a
9b
9c
9d
9e
9f
6a
6b
6c
6d
6e
6f
87
52
48
93
60
70
73
90
76
58
50
90
70
51
69
85
93
67
98
7
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
2ꢀ(CH3)
H
2ꢀ(CH2CH3)
2ꢀF
4ꢀF
2ꢀCl
9g
9h
9i
6g
6h
6i
3ꢀCl
4ꢀCl
4ꢀ(CF3)
2ꢀ(OCF3)
9j
6j
(based on cyclohexanone 7b)
9k
9l
6b
6a
70
90
62
95
ꢀ(CH2)3ꢀ
ꢀ(CH2)3ꢀ
H
2ꢀ(CH3)
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9m
9n
6j
3
4
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6
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37
97
ꢀ(CH2)3ꢀ
ꢀ(CH2)3ꢀ
4ꢀ(CH3)
4ꢀF
6e
(based on tetrahydro-4H-pyran-4-one 7c)
7
8
9
9o
9p
9q
9r
6a
6f
6j
6e
86
66
55
70
92
21
23
26
ꢀCH2OCH2ꢀ
ꢀCH2OCH2ꢀ
ꢀCH2OCH2ꢀ
ꢀCH2OCH2ꢀ
2ꢀ(CH3)
2ꢀCl
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4ꢀ(CH3)
4ꢀF
(based on cyclopentanone 7d)
9s
9t
6a
6j
60
60
84
38
92
55
72
82
ꢀ(CH2)2ꢀ
ꢀ(CH2)2ꢀ
ꢀ(CH2)2ꢀ
ꢀ(CH2)2ꢀ
2ꢀ(CH3)
4ꢀ(CH3)
2ꢀCl
9u
9v
6f
6k
2ꢀ(OCF3)
Finally, coupling of the aminothiophenes (9aꢀ9v) with alkyl and fluoroalkyl acyl chlorides,
anhydrides, or EDCIꢀcoupling was done to generate the final desired AACT compounds (10aaꢀ
10bw), also typically as crystalline solids, in fair to good yields (Table 3). After completion of a 1st
generation of compounds (10aaꢀ10bj) based on simple alkyl and fluoroalkyl groups at the R3
position, we designed a 2nd generation library with halodifluoroalkyl (chloro, bromo, and iodo) and
heptafluorobutyryl at R3, based on the most promising combinations of R1 and R2 (10bkꢀ10bw). The
synthesis of the difluoroiodoacetyl inhibitors (10bn, 10bq, 10bt, and 10bw) was accomplished by
EDCIꢀmediated coupling of aminothiophenes with difluoroiodoacetic acid. In total, 49 inhibitor
candidates were prepared by variations at the R1, R2, and R3 positions. The structure and purity of the
final products were confirmed by 1HꢀNMR, ESIꢀLCMS (UV absorption detection at 254 nm), with
purities estimated to be >95%.
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Table 3. Coupling yields and TMEM16A inhibition of AACT compounds (10aaꢀ10bw). Yields
(%) are of the isolated or purified products. IC50 (µM) for inhibition of TMEM16A anion
conductance using a fluorescence plate reader (FPR) assay (SEM in parentheses; n = 3). Purity of
assayed compounds was >95% based on HPLCꢀLCMS analysis at 254 nm, and absence of
impurities was confirmed by inspection of 1H NMR spectra. aLow solubility in DMSO.
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50
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58
59
60
SM
FPR IC50
TMEM16A
(µM)
Product
Final
Isolated
R1
R2
R3
Amino
Yield (%)
thiophene
(based on cycloheptanone 7a)
10aa
10ab
10ac
10ad
10ae
10af
10ag
10ah
10ai
9a
9a
9a
9a
9b
9b
9b
9b
9c
9d
9e
9f
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
2ꢀ(CH3)
2ꢀ(CH3)
2ꢀ(CH3)
2ꢀ(CH3)
H
CF3
CF2CF3
CH3
61
65
82
70
38
97
80
87
6
0.42 (0.03)
1.3
>10
CH2CH3
CF3
>20
0.3 (0.005)
2.5
H
CF2CF3
CH3
H
>20
H
CH2CH3
CF3
1.2
2ꢀ(CH2CH3)
2ꢀF
1.3 (0.7)
1.3
10aj
10ak
10al
CF3
48
60
87
91
4ꢀF
CF3
0.32 (0.11)
0.66 (0.02)
>20
2ꢀCl
CF3
10am
9f
2ꢀCl
CF2CF3
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10an
10ao
10ap
10aq
10ar
10as
10at
9g
9h
9i
3
4
5
6
7
8
9
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
3ꢀCl
CF3
CF3
23
4
5 (0.2)
3 (0.09)
5 (0.10)
1.3 (0.2)
2.7 (0.07)
>20
4ꢀCl
4ꢀ(CF3)
2ꢀ(OCF3)
2ꢀ(OCF3)
2ꢀ(OCF3)
2ꢀ(OCF3)
CF3
2
9j
9j
9j
9j
CF3
12
30
55
50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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50
51
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55
56
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58
59
60
CF2CF3
CH3
CH2CH3
>20
(based on cyclohexanone 7b)
10au
10av
10aw
10ax
9k
9l
ꢀ(CH2)3ꢀ
ꢀ(CH2)3ꢀ
ꢀ(CH2)3ꢀ
ꢀ(CH2)3ꢀ
H
CF3
CF3
CF3
CF3
93
17
30
48
0.37 (0.01)
0.17 (0.001)
0.22 (0.01)
0.49 (0.02)
2ꢀ(CH3)
4ꢀ(CH3)
4ꢀF
9m
9n
(based on tetrahydro-4H-pyran-4-one 7c)
10ay
10az
10ba
10bb
10bc
9o
9o
9p
9q
9r
ꢀCH2OCH2ꢀ
ꢀCH2OCH2ꢀ
ꢀCH2OCH2ꢀ
ꢀCH2OCH2ꢀ
ꢀCH2OCH2ꢀ
2ꢀ(CH3)
2ꢀ(CH3)
2ꢀCl
CF2CF3
CH2CH3
CF3
6
1.6 (0.09)
3 (0.09)
1.3
40
67
38
15
4ꢀ(CH3)
4ꢀF
CF3
5 (0.1)
CF3
3.8 (0.09)
(based on cyclopentanone 7d)
10bd
10be
10bf
10bg
10bh
10bi
10bj
9s
9s
9s
9s
9t
ꢀ(CH2)2ꢀ
ꢀ(CH2)2ꢀ
ꢀ(CH2)2ꢀ
ꢀ(CH2)2ꢀ
ꢀ(CH2)2ꢀ
ꢀ(CH2)2ꢀ
ꢀ(CH2)2ꢀ
2ꢀ(CH3)
2ꢀ(CH3)
2ꢀ(CH3)
2ꢀ(CH3)
4ꢀ(CH3)
2ꢀCl
CF3
CF2CF3
CH3
10
37
38
33
77
64
56
>20
6.2
>20
CH2CH3
CF3
>20
2.5 (0.04)
1.3
9u
9v
CF3
2ꢀ(OCF3)
CF3
0.37 (0.02)
(2nd-generation inhibitors with novel R3 substituents, including chloro/bromo/iodo difluoroacetyl)
ꢀ(CH2)4ꢀ 2ꢀ(CH3) CF2Cl 27
10bk
9a
0.18 (0.008)
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10bl
10bm
10bn
10bo
10bp
10bq
10br
10bs
10bt
9a
9a
9a
9b
9b
9b
9e
9e
9e
9v
9v
9v
3
4
5
6
7
8
9
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)2ꢀ
ꢀ(CH2)2ꢀ
ꢀ(CH2)2ꢀ
2ꢀ(CH3)
2ꢀ(CH3)
2ꢀ(CH3)
H
CF2CF2CF3
CF2Br
CF2I
61
36
67
16
15
13
19
32
60
81
40
50
0.38 (0.01)
0.083 (0.007)
0.6 (0.02)
CF2Cl
CF2Br
CF2I
0.925 (0.002)
0.23 (0.004)
0.23 (0.004)
0.84 (0.04)
0.45 (0.03)
0.15 (0.002)
>20a
10
11
12
13
14
15
16
17
18
19
20
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H
H
4ꢀF
CF2Cl
CF2Br
CF2I
4ꢀF
4ꢀF
10bu
10bv
10bw
2ꢀ(OCF3)
2ꢀ(OCF3)
2ꢀ(OCF3)
CF2Cl
CF2Br
CF2I
0.70a (0.002)
1.88 (0.04)
Biological characterization
2ꢀAcylaminoꢀcycloalkylthiopheneꢀ3ꢀcarboxylic acid arylamides 10aaꢀ10bw were initially
evaluated for inhibition of TMEM16A anion channel function using a cellꢀbased functional plate
reader assay as reported.29, 30, 40 IC50 values determined from concentrationꢀinhibition measurements
are summarized in Table 3.
The 1st generation library (10aaꢀ10bj) showed several compounds with apparent IC50 of 0.2ꢀ0.3
ꢀM: 10aa (initial inhibitor), 10ae, 10ak, 10au, 10av, 10aw. These results showed that 5ꢀ, 6ꢀ, and 7ꢀ
member rings were tolerated at the R1 position, while compounds based on tetrahydroꢀ4Hꢀpyranꢀ4ꢀ
one (10ayꢀ10bc) were inactive. The best inhibitors contained H, 2ꢀ or 4ꢀ(CH3), or 4ꢀF on the
aromatic ring (R2), and CF3 as the acylamido substituent (R3). Inhibitors with differing groups at R2,
such as 2ꢀF, 2ꢀCl, 3ꢀCl, 4ꢀCl, 4ꢀ(CF3), 2ꢀ(OCF3), were less potent. Likewise, compounds with
alternative substituents at R3, including CF2CF3, CH3, and CH2CH3, also had reduced potency.
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Based on results that favored CF3 at the R3 position, we designed a 2ndꢀgeneration library (10bkꢀ
10bw) that incorporated novel groups such as chlorodifluoro, bromodifluoro, or difluoroiodo, probing
steric and electronic effects at that position. The 2ndꢀgeneration library focused on structural features
seen in the more active compounds from the 1stꢀgeneration library (R1 = ꢀ(CH2)4ꢀ; R2 = 2ꢀ(CH3), H, or
4ꢀF). One 2ndꢀgeneration compound was synthesized that incorporated a heptafluorobutyryl
substituent (10bl) to evaluate the effect of a multiꢀcarbon fluoroalkyl group. We found three 2ndꢀ
generation compounds with lower apparent IC50 of 0.08ꢀ0.18 ꢀM: 10bk, 10bm, and 10bt.
Additionally, the full series of inhibitor candidates (10aaꢀ10bw) was subjected to a computational
screen for panꢀassay interference (PAINS),42 and all 49 compounds passed.
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The most potent TMEM16A inhibitors identified using the semiꢀquantitative plate reader assay
were then studied using a shortꢀcircuit current assay in which measured current is a linear measure of
TMEM16A Clꢀ conductance. Compounds 10aa (original inhibitor from screen), 10ae, 10bk, 10bm,
10bn and 10bt were tested, and compared with reported inhibitors 2, 3 and 4. Concentrationꢀ
dependence for the selected compounds is shown in Figure 3, with IC50 values summarized in Table
4. Nonꢀtransfected FRT cells shows no signal upon ATP stimulation (Figure S1). By the shortꢀ
circuit current assay, 10aa showed an IC50 of 0.26 ± 0.10 ꢀM (n=3), similar to the
chlorodifluoroacetamide 10bk with IC50 of 0.23 ꢀM. Difluoroiodoacetamides 10bn and 10bt were
less potent with IC50 of 0.73 and 0.60 ꢀM, respectively. Notably, bromodifluoroacetamide 10bm had
IC50 of 0.030 ± 0.010 ꢀM (n=3). The IC50 of 4 using this assay was 0.11 ꢀM, close to 0.077 ꢀM
reported previously;32 compound 3 had IC50 of 1.6 ꢀM by the shortꢀcircuit current assay, significantly
less potent than the reported IC50 of 0.08 ꢀM using a Xenopus laevis oocyte assay.31 Inhibitor 2 was a
previously reported to have an IC50 of 1 ꢀM.29
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Figure 3. Shortꢀcircuit current measurement of TMEM16A inhibition by 10aa, 10bk and 10bm, and
previously reported compounds 3 and 4. Measurements were done in FRT cells expressing human
TMEM16A. A. Summary of doseꢀresponse data (mean ± SEM, n = 3). B. Examples of original data in
which inhibitors were added 5 min prior to TMEM16A activation by 100 ꢁM ATP.
Table 4. Characterization of AACT analogs. Concentrationꢀdependent inhibition of TMEM16A
measured by shortꢀcircuit current assay; TMEM16B and nonꢀTMEM16 anion conductance
measured using a fluorescence plate reader assay; cell viability measured in HTꢀ29 cells. (SEM in
parentheses; n=3).
Cellular
survival %
at 5 ꢀM
TMEM16A
IC50
TMEM16B
IC50
HTꢀ29
IC50
Inhibitor
R1
R2
R3
(µM)
(µM)
(µM)
–
–
–
–
~1ref 29
1.6
~4ref 32
13
5.0
>20
0.3
4.0
5.0
97
100
102
99
2
3
–
–
4
–
–
2ꢀ(CH3)
H
–
0.11
>20
10aa
10ae
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
CF3
CF3
0.26 (0.10)
0.13
1.4 (0.05)
4.6 (0.4)
97
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10bk
10bm
10bn
10bt
3
4
5
6
7
8
9
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
2ꢀ(CH3)
2ꢀ(CH3)
2ꢀ(CH3)
4ꢀF
CF2Cl
CF2Br
CF2I
0.23
0.030 (0.010)
0.73
0.4 (0.01)
0.4 (0.04)
1.4 (0.05)
1.1 (0.13)
9.5
5.4
3.5
5.0
98
97
96
97
CF2I
0.60
10
11
12
13
14
15
16
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Ion channel specificity and cytotoxicity were determined for the six most potent AACT
compounds as well as previously reported compounds 2, 3 and 4 (Table 4). Selectivity was studied
for TMEM16B, an isoform of TMEM16A that functions as a CaCC and regulates action potential
firing in olfaction.43 Inhibition of TMEM16B by the lowꢀpotency CaCC inhibitor anthraceneꢀ9ꢀ
carboxylic acid was recently reported.44 Compounds 3 and 4 (IC50 13 and >20 ꢀM, respectively) were
selective against TMEM16B, while 2 was less selective (IC50 4 ꢀM). The AACT compounds
displayed modest selectivity against TMEM16B, with an IC50 range of 0.4ꢀ1.4 ꢀM. Two of the more
potent AACT inhibitors of TMEM16A (10bk and 10bm) were also among the more potent against
TMEM16B with IC50 ~ 0.4 ꢀM. Compound 4 showed 181ꢀfold selectivity against TMEM16B, while
10bm achieved 13ꢀfold selectivity. We further assayed compound potency on endogenous nonꢀ
TMEM16A Ca2+ꢀactivated Clꢀ channel in HTꢀ29 cells.28 In general, the AACT compounds were
weak inhibitors of CaCCs in HTꢀ29 cells (IC50 3.5ꢀ9.5 ꢀM), contrasting with 2 (IC50 5 ꢀM), 3 (IC50 >
20 ꢀΜ), and 4 (IC50 0.3 ꢀM). None of the compounds examined showed significant toxicity using an
Alamar blue assay at concentrations up to 5 ꢀM. Additionally, none of the compounds inhibited the
cAMPꢀactivated Clꢀ channel cystic fibrosis transmembrane conductance regulator (CFTR) (data not
shown).
Inhibition of TMEM16A by 10bm was also investigated by the patchꢀclamp technique using the
insideꢀout configuration. Membrane patches were excised from TMEM16Aꢀexpressing FRT cells.
With the insideꢀout configuration, the cytosolic side of the membrane is exposed to the bath solution.
To activate TMEM16A, the bath solution contained a free Ca2+ concentration of 305 nM. Under
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these conditions, relatively large membrane currents were recorded due to the presence of multiple
TMEM16A channels (Figure 4). The currents had the typical characteristics of TMEM16A with
outward rectification and timeꢀdependent activation at positive membrane potentials. Addition to the
bath of 10bm (250 nM) by perfusion resulted in rapid inhibition of membrane currents, which fully
recovered following washout. To further examine the specificity of 10bm, shortꢀcircuit current
measurement showed that 10bm inhibited ionomycinꢀ and carbacholꢀactivated chloride conductance
(Figure S2A), indicating that 10bm did not inhibit chloride conductance through an ATPꢀstimulated
purinergic G proteinꢀcoupled receptor mechanism. Further, cytoplasmic calcium was not altered by 1
ꢁM 10bm as seen in Fluoꢀ4 fluorescence measurement of ATPꢀstimulated cytoplasmic calcium
elevation (Figure S2B). These results support reversible TMEM16A inhibition by 10bm by a direct
interaction mechanism.
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Figure 4. Patchꢀclamp studies of TMEM16A inhibition by 10bm. A. Representative currents recorded
from an insideꢀout membrane patch. Cytosolic (bath) free Ca2+ concentration was 305 nM. Each panel of
traces show superimposed membrane currents elicited at membrane potentials in the range ꢀ100 to +100
mV. Currents were recorded under control conditions, after application by perfusion of 10bm (250 nM),
and after washout. B. Currentꢀvoltage relationships from the experiment shown in A. Current values
were measured at the end of each voltage step. C. Summary of conductance values at +100 mV obtained
from five independent experiments (***, p < 0.001 by paired t test).
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6
7
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In vitro metabolic stability was determined using a hepatic microsome assay for the most potent
AACT inhibitor 10bm (Figure 5A) and previously reported compound 4. These compounds were
incubated with rat liver microsomes and NADPH, and nonꢀmetabolized compounds were quantified
by ESIꢀLCMS. Figure 5B shows near complete degradation of 4 at 180 min, whereas for the same
incubation time ~30% of 10bm remained. Figure 5C summarizes the time course of compound
degradation showing remarkably greater stability of 10bm compared to 4. Inhibitor 10bm could be
potentially metabolized by amideꢀbond hydrolysis or oxidation of the benzene or aryl methyl. We
speculate that 4 could be oxidized at the aryl methyl or NꢀN bond; or hydrolyzed at the amide or
hydrazone linkages.
We have found the bromodifluoroacetamide functional group present in 10bm to have longꢀterm
shelf stability (> 6 months) as a solid or in DMSO stock solution. In aqueous solution, there was
gradual degradation of 10bm, with 72% and 30% remaining at 1 and 2 days, respectively (Figure
S3A). ESIꢀLCMS analysis suggested that degradation of 10bm involves: solvolysis, elimination, and
hydrolysis of the resulting acid fluoride to a stable Nꢀsubstituted oxalamic acid (Figure S3B). This
solvolytic pathway has not been previously reported for the bromodifluoroacetamide functional
group. While we feel this degradation is slow enough to not effect potential pharmacological use of
10bm, these results discourage multiꢀday storage in aqueous solution. Kinetic aqueous solubility was
measured for 10bm to be 0.04 mg/mL by titration.
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Figure 5. Microsomal stability of 10bm and 4 in the presence of hepatic microsomes and NADPH, AND
inhibition of isometric smooth muscle contractions of ex vivo mouse ileum by 10bm. A. Structure of
10bm. B. LC/MS traces showing total ion counts as a function of incubation time. C. Summary of in
vitro metabolic stability shows percent of remaining compounds over time (mean ± S.E.M., n=3). D.
Isometric smooth muscle contraction in mouse ileum showing suppression by 10bm. Data representative
of three separate experiments.
To demonstrate one predicted biological action of TMEM16A inhibition, we measured intestinal
smooth muscle contraction. The effect of 10bm was determined when added to the bath in an ex vivo
preparation of mouse ileum. As shown in Figure 5D, 10bm strongly inhibited spontaneous isometric
contractions of ileum in a concentrationꢀdependent manner.
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CONCLUSION
5
6
7
8
In conclusion, we identified a new inhibitor of TMEM16A (10aa) by screening. The scaffold
(designated AACT) is modular, allowing the synthesis and characterization of 48 analogs (10abꢀ
10bw). The synthesis utilized substituted aryl cyanoacetamides (6aꢀ6k) which were converted to 2ꢀ
aminoꢀcycloalkylthiopheneꢀ3ꢀcarboxylic acid arylamides (9aꢀ9v) and acylated to afford the final
library. The most potent compound in the series (10bm) had an IC50 of 0.030 ꢀM, making it currently
the most potent reported TMEM16A inhibitor. Patchꢀclamp analysis supported reversible inhibition
of TMEM16A by 10bm by a mechanism involving direct interaction. 10bm had remarkably greater
metabolic stability than the previously reported compound 4. Finally, 10bm was shown to inhibit
smooth muscle contractions in mouse ileum in concentrationꢀdependent manner, illustrating one of its
potential biomedical applications. The high potency and microsomal stability of AACT 10bm
supports its potential as a pharmacological tool to study TMEM16A function and as a potential
candidate for further development.
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EXPERIMENTAL SECTION
Chemistry
The detailed synthesis of all newly reported synthetic precursor molecules and final inhibitor
candidates is presented in Supporting Information. Purity of assayed compounds was >95% based on
HPLCꢀLCMS analysis at 254 nm, and absence of impurities was additionally confirmed by inspection
of 1H NMR spectra, and examination of at least one other wavelength (typically 320 nm).
Biology
Cell lines and culture. Fischer Rat Thyroid (FRT) cells stably coꢀexpressing TMEM16A,
TMEM16B and human wildꢀtype CFTR and the halideꢀsensitive yellow fluorescent protein (YFP)ꢀ
H148Q were cultured as described.29 HTꢀ29 expressing YFP were cultured as described.28 FRT cells
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were cultured on plastic in Coon'sꢀmodified Ham's F12 medium supplemented with 10% fetal bovine
serum, 2 mM Lꢀglutamine, 100 units/mL penicillin, and 100 ꢁg/mL streptomycin. For plate reader
assays, cells were plated in black 96ꢀwell microplates (CorningꢀCostar Corp., New York, N.Y.) at a
density of 20,000 cells per well and assayed 24ꢀ48 hours after plating.
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TMEM16A functional assay. TMEM16A functional plateꢀreader assay was done as previously
described.29 Briefly, each well of 96ꢀwell plate containing the TMEM16Aꢀexpressing FRT cells was
washed twice with phosphate buffer saline (PBS) leaving 50 µl. Test compounds (0.5 µl in DMSO)
were added to each well at specified concentration. After 10 min each well was assayed individually
for TMEM16Aꢀmediated Iꢀ influx by recording fluorescence continuously (400 ms/point) for 2 s
(baseline), then 50 µl of 140 mM Iꢀ solution containing 300 ꢀM ATP was added at 2 s, and
fluorescence was further read for 12 s. The initial rate of Iꢀ influx following each of the solution
additions was computed from fluorescence data by nonlinear regression. TMEM16B activity was
assayed similarly as described29 using FRT cells coꢀexpressing YFP and TMEM16B.
In silico PAINS assay. A computational screen for panꢀassay interference compounds
(PAINS)42 was employed to identify substructures associated with promiscuous inhibition. The full
series of inhibitor candidates (10aaꢀ10bw) was converted to SMILES strings, and submitted to an
internetꢀbased implementation of the PAINS filter (http://cbligand.org/PAINS), maintained by Prof.
XiangꢀQun (Sean) Xie’s laboratory at University of Pittsburgh School of Pharmacy.
Shortꢀcircuit current measurement. Shortꢀcircuit current measurements were done as
described.30 Briefly, Snapwell inserts (Corning Costar, Corning, NY) containing TMEM16Aꢀ
expressing FRT cells were mounted in Ussing chambers (Physiological Instruments, San Diego, CA).
ꢀ
The hemichambers were filled with 5 ml of HCO3 buffered solution (basolateral) and halfꢀCl−
solution (apical), and the basolateral membrane was permeabilized with 250 µg/ml amphotericin B.
Solutions were bubbled with 95% O2/5% CO2 and maintained at 37°C, and shortꢀcircuit current was
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measured on a DVCꢀ1000 voltage clamp (World Precision Instruments Inc., Sarasota, FL) using
Ag/AgCl electrodes and 3 M KCl agar bridges.
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Patch clamp. Currents were recorded from insideꢀout membrane patches excised from FRT
cells stably expressing TMEM16A. The pipette (extracellular) solution contained (in mM): 150
NaCl, 1 CaCl2, 1 MgCl2, 10 glucose, 10 mannitol, 10 NaꢀHepes (pH 7.3). The bath (intracellular)
solution contained (in mM): 130 CsCl, 1 MgCl2, 10 EGTA, 10 NaꢀHepes (pH 7.3), and 8 CaCl2 to
obtain the desired free Ca2+ concentration of 305 nM. The electrical resistance of micropipettes was
3ꢀ7 Mꢂ. The protocol for stimulation consisted of 600 ms voltage steps in the range from ꢀ100 to
+100 mV (with 20 mV increments) starting from a holding potential of ꢀ60 mV. The interval between
steps was 4 s. Membrane currents were filtered at 1 kHz and digitized at 5 kHz. Data were analyzed
using the Igor software (Wavemetrics, Portland, OR) with custom software kindly provided by Dr.
Oscar Moran.
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In vitro metabolic stability. Compounds (each 10 uM) were incubated for specific time points
(2, 5, 15, 30, 60, 180 min) with shaking at 37 °C with rat liver microsomes (1 mg protein/mL, Sigmaꢀ
Aldrich, St. Louis, MO) in potassium phosphate buffer (100 mM) containing 1 mM NADPH. The
mixture was then chilled on ice, and 0.5 mL of iceꢀcold ethyl acetate was added. Samples were
centrifuged for 15 min at 3000 RPM. The supernatant was evaporated to dryness, and the residue was
dissolved in 80 ꢁL of mobile phase (acetonitrile:/water, 3:1, containing 0.1% formic acid) for LC/MS.
Reverseꢀphase HPLC separation was carried out using a Waters C18 column (2.1 mm × 100 mm, 3.5
mm particle size) equipped with a solvent delivery system (Waters model 2690, Milford, MA). The
solvent system consisted of a linear gradient from 5% to 95% acetonitrile run over 16 min (0.2
mL/min flow rate).
Plate reader assays of chloride channel function. CFTR inhibition was assayed as described.45
Briefly, FRT cells coꢀexpressing YFP and wildtype CFTR were washed with phosphateꢀbuffered
saline (PBS) and then incubated for 15 min with test compounds in PBS containing 20 ꢀM forskolin.
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Iꢀ influx was measured in a plate reader with initial baseline read for 2 s and then for 12 s after rapid
addition of an Iꢀ containing solution. Activity of nonꢀTMEM16A CaCC was assayed as described28 in
HTꢀ29 cells expressing YFP. In each assay initial rates of I− influx were computed as a linear
measure of channel function.
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Cytotoxicity. FRT cells were cultured overnight in black 96ꢀwell Costar microplates and
incubated with 5 ꢀM test compounds for 8 h. Cytotoxicity was measured by Alamar Blue assay
(Invitrogen, Carlsbad, CA) as per the manufacturer’s instructions.
Ex vivo intestinal contractility. Adult mice (CD1 genetic background) were euthanized by
avertin overdose (200 mg/kg, 2,2,2ꢀtribromethanol, SigmaꢀAldrich) and ileal segments of ~2 cm
length were isolated and washed with KrebsꢀHenseleit buffer (pH 7.4, in mM: 118 NaCl, 4.7 KCl, 1.2
MgSO4, 1.2 KH2PO4, 25 NaHCO3, 2.5 CaCl2, 11 Dꢀglucose). The ends of the ileal segments were
tied, connected to a force transducer (Biopac Systems, Goleta, CA) and tissues were transferred to an
organ chamber (Biopac Systems) containing KrebsꢀHenseleit buffer at 37°C aerated with 95% O2, 5%
CO2. Tissues were stabilized for 60 min with resting tension of 0.5 g and solutions were changed
every 15 min. Effects of 10bm on baseline isometric intestinal contractions were recorded. Animal
protocols were approved by the UCSF Institutional Animal Care and Use Committee (approval
number: AN108711ꢀ02A) and were conducted according with the NIH guidelines for the care and use
of animals.
Aqueous stability. Duplicate HPLC samples were prepared in H2O (20% DMSO) containing
10bm (100 ꢀM) and aqueous stable internal standard 5ꢀchloroꢀ3ꢀphenylꢀ2,1ꢀbenzisoxazole (100 ꢀM).
The samples were incubated at 25 oC for 3 days, and analyzed daily by ESIꢀLCMS showing gradual
decomposition to the corresponding oxalamic acid.
Aqueous kinetic solubility. To a series of 2 mL aliquots of PBS solution were added increasing
quantities of 10bm (30 mM DMSO stock solution). The titration point was determined at 6.5 ꢀL of
added stock solution, corresponding to 0.04 mg/ml kinetic solubility.
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AUTHOR INFORMATION
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Corresponding Author. *Phone: 415ꢀ338ꢀ6495. Fax: 415ꢀ405ꢀ0377. Eꢀmail: marc@sfsu.edu.
Notes. Drs. Anderson, Phuan and Verkman are named coꢀinventors on a provisional patent
filing with rights owned by UCSF and SFSU.
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ACKNOWLEDGEMENTS
This study was supported by NIH grants R15 GM102874, P30 DK072517, R01 DK099803, R01
DK075302, R01 DK101373, and R01 EY13574, and a grant from the Cystic Fibrosis Foundation.
Patchꢀclamp studies were supported by grants from Ministero della Salute (Ricerca Corrente: Cinque
per mille) and Telethon Foundation to LJVG (TMLGCBX16TT). The authors acknowledge Dr.
Robert Yen at the SFSU Mass Spectrometry Facility and Dr. Mark Swanson at SFSU NMR Facility.
ABBREVIATIONS USED
AACT, 2ꢀacylaminoꢀcycloalkylthiopheneꢀ3ꢀcarboxylic acid arylamide; ANO1, anoctamin 1;
CaCC, calciumꢀactivated chloride channel; CFTR, cystic fibrosis transmembrane regulator; DCM,
dichloromethane; 4ꢀDMAP, N,Nꢀdimethylaminopyridine; DMF, N,Nꢀdimethylformamide; DMSO,
dimethyl sulfoxide; EDCIꢀHCl, 1ꢀ(3ꢀdimethylaminopropyl)ꢀ3ꢀethylcarbodiimide hydrochloride; FPR,
fluorescent plate reader; FRT, Fischer Rat Thyroid; PAINS, pan assay interference compounds; PBS,
phosphateꢀbuffered saline; RT, room temperature; TLC, thin layer chromatography; TMEM16A,
transmembrane protein 16A; YFP, yellow fluorescent protein.
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ASSOCIATED CONTENT
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Supporting Information Available. Supporting Information is available free of charge on the
ACS Publications Website at DOI: xxxxxxxxx/yyyyyyyyy:
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Control measurement of shortꢀcircuit current in nonꢀtransfected cells; timeꢀcourse and proposed
mechanism of aqueous degradation of inhibitor 10bm; synthesis details and spectroscopic
characterization of new molecules (PDF).
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Molecular strings for all final inhibitor candidates (CSV).
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