Nanomolar-Potency Aminophenyl-1,3,5-triazine Activators of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Chloride Channel for Prosecretory Therapy of Dry Eye Diseases

Article abstract of DOI:10.1021/acs.jmedchem.6b01792

Dry eye disorders are a significant health problem for which limited therapeutic options are available. CFTR is a major prosecretory chloride channel at the ocular surface. We previously identified, by high-throughput screening, aminophenyl-1,3,5-triazine CFTR_act-K089 (1) that activated CFTR with EC₅₀ ≈ 250 nM, which when delivered topically increased tear fluid secretion in mice and showed efficacy in an experimental dry eye model. Here, functional analysis of aminophenyl-1,3,5-triazine analogs elucidated structure-activity relationships for CFTR activation and identified substantially more potent analogs than 1. The most potent compound, 12, fully activated CFTR chloride conductance with EC₅₀ ≈ 30 nM, without causing cAMP or calcium elevation. 12 was rapidly metabolized by hepatic microsomes, which supports its topical use. Single topical administration of 25 pmol of 12 increased tear volume in wild-type mice with sustained action for 8 h and was without effect in CFTR-deficient mice. Topically delivered 12 may be efficacious in human dry eye diseases.

Full text of DOI:10.1021/acs.jmedchem.6b01792

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Article
Nanomolar-potency aminophenyl-1,3,5-triazine activators of the
cystic fibrosis transmembrane conductance regulator (CFTR)
chloride channel for pro-secretory therapy of dry eye diseases
Sujin Lee, Puay-Wah Phuan, Christian M Felix, Joseph-Anthony Tan, Marc H Levin, and Alan S Verkman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01792 • Publication Date (Web): 18 Jan 2017
Downloaded from http://pubs.acs.org on January 19, 2017
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Journal of Medicinal Chemistry
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Nanomolar-potency aminophenyl-1,3,5-triazine activators of the cystic fibrosis trans-
membrane conductance regulator (CFTR) chloride channel for pro-secretory therapy of
dry eye diseases
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Sujin Lee, PuayꢀWah Phuan, Christian M. Felix, JosephꢀAnthony Tan, Marc H. Levin, and Alan
S. Verkman*
Departments of Medicine and Physiology, University of California, San Francisco, CA, 94143ꢀ
0521, USA
KEYWORDS Cystic fibrosis transmembrane conductance regulator, dry eye disease, chloride
channel, conjunctiva, aminophenyl-1,3,5-triazine
ABSTRACT: Dry eye disorders are a significant health problem for which limited therapeutic
options are available. CFTR is a major proꢀsecretory chloride channel at the ocular surꢀ
face. We previously identified, by highꢀthroughput screening, aminophenylꢀ1,3,5ꢀtriazine
CFTRact-K089 (1) that activated CFTR with EC50 ~250 nM, which when delivered topically inꢀ
creased tear fluid secretion in mice and showed efficacy in an experimental dry eye model.
Here, functional analysis of aminophenylꢀ1,3,5ꢀtriazine analogs elucidated structureꢀactivity
relationships for CFTR activation and identified substantially more potent analogs than 1. The
most potent compound, 12, fully activated CFTR chloride conductance with EC50 ~30 nM,
without causing cAMP or calcium elevation. 12 was rapidly metabolized by hepatic microꢀ
somes, which supports its topical use. Single topical administration of 25 pmol 12 increased
tear volume in wildtype mice with sustained action for 8 hours, and was without effect in
CFTRꢀdeficient mice. Topically delivered 12 may be efficacious in human dry eye diseases.
INTRODUCTION
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CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMPꢀgated chloride
channel with proꢀsecretory activity in the airways, gastrointestinal organs, testis and exocrine
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glands 1ꢀ4. Lossꢀofꢀfunction mutations in CFTR cause cystic fibrosis, and inappropriate activaꢀ
tion of CFTR causes certain secretory diarrheas such as cholera 5ꢀ7. CFTR is an important
drug target for which major advances have been made in the development of potentiators and
correctors of mutant CFTRs in cystic fibrosis, with two drugs having received FDA approval 8.
Activators of wildtype CFTR have potential clinical indications for proꢀsecretory therapy of conꢀ
stipation and dry eye disorders, and possibly for disorders of the liver, pancreas and airways 9ꢀ
12, and CFTR inhibitors are under development for certain secretory diarrheas and polycystic
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kidney disease 13, 14
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We previously identified, by highꢀthroughput screening, smallꢀmolecule activators of
wildtype CFTR that function by a direct activation mechanism 12, 15. One class of pheꢀ
nylquinoxalinone CFTR activators showed efficacy following oral administration in experiꢀ
mental mouse models of constipation 9, 10. A second class of CFTR activators, which includes
the aminophenylꢀ1,3,5ꢀtriazine CFTRact-K089 (compound 1, Fig. 1A), activated ocular surface
CFTR activity in mice and increased tear fluid secretion after topical delivery 12. In a mouse
model of aqueousꢀdeficient dry eye produced by lacrimal gland excision, topical delivery of 1
three times daily corrected defective tear fluid secretion and prevented ocular surface clinical
signs. An ideal dry eye therapeutic would produce sustained tear fluid secretion with once or
twice daily dosing.
Here, we report structureꢀactivity relationship studies of aminophenylꢀ1,3,5ꢀtriazine CFTR
activators with a focus on dry eye applications. Dry eye is a heterogeneous group of disorders
associated with reduced tear volume and tear fluid hyperosmolarity, resulting in ocular surface
inflammation, eye discomfort and visual disturbance. Dry eye is a major health problem in an
aging population, with five million patients in the U.S. age 50 and over 16, 17. The only approved
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dry eye drugs, topical cyclosporine and lifitegrast, act by an antiꢀinflammatory mechanism 18, 19
CFTR activators would be firstꢀinꢀclass dry eye therapeutics that function by a proꢀsecretory
mechanism. Synthesis and characterization of aminophenylꢀ1,3,5ꢀtriazines here produced poꢀ
tential lead candidates with substantially improved potency and in vivo proꢀsecretory efficacy
compared to 1, as well as compounds with greater metabolic stability for potential treatment of
nonꢀocular conditions.
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CHEMISTRY
General synthesis of aminophenylꢀ1,3,5ꢀtriazine analogs
The synthesis of aminophenylꢀ1,3,5ꢀtriazine analogs was accomplished by serial substituꢀ
tion of cyanuric chloride using fluorinated alkoxides, diethylamine, and anilines (Scheme 1).
Substitution of cyanuric chloride with 1,1,1,3,3,3ꢀhexafluoroisopropanol or 2,2,3,3ꢀtetrafluoroꢀ
1ꢀpropanol afforded 2 and 3, respectively. Amination of 2 and 3 using diethyl amine afforded
intermediates 4 and 5. Refluxing of 4 and 5 with aniline afforded 1 and 7a, respectively. Refluxꢀ
ing m-or p-F, Cl, NO2, CO2Hꢀsubstituted anilines with 4 and 5 afforded aminophenylꢀ1,3,5ꢀ
triazines 6a-g and 7b-g, respectively. 5 was refluxed with 5ꢀor 6ꢀaminoindazole, 6ꢀ
aminobenzoxazole, and 6ꢀaminobenzothiazole under basic condition to afford 6hꢀ6k.
Cl
Cl
NH
a or b
c
N
N
N
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Cl
N
Cl
RO
N
Cl
2 R = CH(CF3)2
3 R = CH2CF2CHF2
N
N
NH2
N
N
d
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N
RO
N
NH
X
RO
N
Cl
X
4 R = CH(CF3)2
5 R = CH2CF2CHF2
1, 6a-k R = CH(CF3)2
7a-g R = CH2CF2CHF2
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Scheme 1. Synthesis of N,N-diethylꢀ1,3,5ꢀtriazine amineanalogs. (a) (CF3)2CHOH, K2CO3,
THF, ꢀ80 oC, 62% for 2. (b) CHF2CF2CH2OH, K2CO3, THF, ꢀ80 oC, 68% for 3. (c) Et2NH,
DIPEA, THF, 0 oC, 55%. (d) DIPEA, THF, reflux, 25ꢀ81%.
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NꢀMethylꢀ1,3,5ꢀtriazine amine analogs 11 and 12 were synthesized as shown in Scheme 2.
Methylamine was substituted with cyanuric chloride to afford N-methylꢀ4,6ꢀdichloroꢀ1,3,5ꢀ
triazineꢀ2ꢀamine 8 in 72% yield. With 8 in hand, substitution with 1,1,1,3,3,3ꢀ
hexafluoroisopropanol or 2,2,3,3ꢀtetrafluoroꢀ1ꢀpropanol afforded 9 and 10, respectively. Aniline
substitution on 9 and 10 afforded N-methylꢀN’ꢀphenylꢀ1,3,5ꢀtriazine diamine analogs 11 and 12,
respectively.
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Scheme 2. Synthesis of N-methylꢀN’ꢀphenylꢀ1,3,5ꢀtriazine diamine analogs. (a) DIPEA, THF, 0
o
oC, 75%. (b) (CF3)2CHOH, K2CO3, THF, 0 C, 43% for 9. (c) CHF2CF2CH2OH, K2CO3, THF, 0
oC, 66% for 10. (d) DIPEA, THF, reflux, 74% for 12; 68% for 11.
RESULTS
Preliminary structureꢀactivity analysis of commercially available analogs
Initial evaluation of compound potency was done using a cellꢀbased plate reader assay of
CFTR function. Fig. 1B summarizes preliminary SAR analysis from functional assays on 91
commercially available analogs of 1 (data for all commercial compounds provided in Suppleꢀ
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mentary Table 1). The commercial 1,3,5ꢀtriazine analogs tested here were triꢀsubstituted at the
2ꢀ, 4ꢀ, 6ꢀpositions with various substituents, including alkoxide, aniline (aminoꢀphenyl), and
primary or secondary amines. SAR analysis revealed that trisubstituedꢀ1,3,5ꢀtriazines whose
three substituents contain a fluorinated alkoxide (R1), an aniline (R2) and an alkylamine (R3)
gave the best potency (compounds K032, K059 and K089, Supplementary Table 1). Analogs
with twoꢀalkoxide substituents or nonꢀfluorinated alkoxide greatly reduced potency (K010ꢀ018,
K023). Further, orthoꢀsubstituted aniline (R2) reduced potency as compared to metaꢀ and paraꢀ
substitution (K007, K032, K059). Electronꢀdonating groups including methoxy, acetyl, trifluoꢀ
romethoxy (K001, K024ꢀ028, K079) reduced activity as compared to electronꢀwithdrawing
group such as nitro as in K032. Though we focused on anilines here, benzylamine and substiꢀ
tuted benzylamines in place of anilines on R2 may be interesting to test. Finally, sterically bulky
alkyamines (R3) including cyclic, tert-butylꢀ and isobutylamine reduced potency (K029, K056,
K060ꢀ065). Short alkylamines such as methylamine and diethylamine gave greatest potency.
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Because the initial SAR data from commercial analogs was limited and confounded by
simultaneous variations in R1, R2 and R3, we synthesized targeted trisubstitutedꢀ1,3,5ꢀtriazine
analogs. EC50 values for CFTR activation for the synthesized compounds are summarized in
Tables 1ꢀ3. Active compounds produced full activation of CFTR, as seen by the similar efficacy
produced by maximal forskolin (20 ꢁM), but with different EC50 values.
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Figure 1. Structureꢀactivity analysis of aminophenylꢀ1,3,5ꢀtriazine CFTR activators. A. Chemꢀ
ical structure of 1. B. Preliminary SAR analysis based on commercial analogs (see Suppleꢀ
mentary Table 1 for data on all commercial analogs).
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Modifications of substituted anilines and fluorinated alkoxides
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Since both fluorinated alkoxide (R1) and short alkylamine (R3) appeared to be crucial for
activity, we first prepared Nꢀarylꢀ1,3,5ꢀtriazine amines with R3 as diethylamine and explored
the activity of two fluorinated alkoxides, 1,1,1,3,3,3ꢀhexafluoroꢀ2ꢀpropanol and 2,2,3,3ꢀ
tetrafluoroꢀ1ꢀpropanol. As shown in Table 1, hexafluoroisopropanol (1) was more potent than
tetrafluoropropanol (7a). With various aryl (R2) substituent, hexafluoroisopropanol analogs (6aꢀ
6f) were ~3 to 8ꢀfold more potent than corresponding tetrafluoropropanol analogs (7bꢀ7g). To
investigate aniline substituents (R2), meta- and paraꢀsubstituted aniline analogs with electronꢀ
neutral and electronꢀwithdrawing groups were prepared. In general, meta-substituted anilines
(6a and 6b) were more active than para-substituted aniline analogs (6e and 6f). Among meta-
substituted anilines, Fꢀ (6b), NO2ꢀ (6d), and CO2Hꢀaniline (6a) had comparable or slightly betꢀ
ter potency than nonꢀsubstituted aniline (1), but chloroaniline (6c) had significantly reduced
potency. From these findings we hypothesized that hydrogen bond donor or acceptor substituꢀ
ents are important for potency; we therefore synthesized more analogs with variations in R2.
Table 1. Structures of N, N-diethylꢀN’ꢀarylꢀ1,3,5ꢀtriazine diamine analogs and their EC50 (ꢁM).
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Modifications of bicyclic anilines
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To further investigate aniline substituents, compounds were synthesized with bicyclic aniꢀ
lines at the R2 position with diethylamine (R3) and hexafluoroisopropanol (R1), including 5ꢀ or
6ꢀaminoindazole (6h and 6i), 6ꢀaminobenzoxazole (6j) and 6ꢀaminobenzothiazole (6k). As
shown in Table 2, the indazole analog 6h was ~3ꢀfold more potent than its regioisomer 6i;
however, both were less potent than 1. Notably, replacement of indazole (6h) by benzoxazole
(6j) gave greater potency than 1; further, replacement of benzoxazole (6j) by benzothiazole
(6k) increased potency by ~3ꢀfold to EC50 ~ 50 nM. Analogs with electroneutral (6i) or hydroꢀ
gen bond donor (6h) substituents on the meta-position were less potent than the nonꢀ
substituted aniline analog (1), and analogs with hydrogen bond acceptor substituents, 6j and
6k, showed significantly greater potency than 1. These results support the importance of hyꢀ
drogen bond acceptor substituents at the aniline meta-position.
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Table 2. Substitution with bicyclic anilines and their EC50 (ꢁM).
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Modifications with Nꢀmethylamine
Finally, N,N-diethylamine at R3 was replaced by methylamine with two fluorinated alkoxides
(Table 3). Notably, N-methylꢀ2,2,3,3ꢀtetrafluoroꢀ1ꢀpropanoxyꢀ1,3,5ꢀtriazine amine 12 had EC50
~50ꢀfold better than diethylamine analog 6a. Interestingly, for diethylamine (R3) analogs, hexꢀ
afluoropropanol (1) is more potent than tetrafluoropropanol (7a), whereas for methylamine (R3)
analogs, tetrafluoropropanol analog 12 was ~3 fold more potent than hexafluoropropanol anaꢀ
log 11. Compounds that showed greater potency than 1 from the SAR analysis were chosen
for biological characterization and additional efficacy studies.
Table 3. N-methylꢀN’-phenylꢀ1,3,5ꢀtriazine diamine analogs and their EC50 (ꢁM).
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In vitro characterization of aminoꢀphenylꢀ1,3ꢀ5ꢀtriazines
Shortꢀcircuit current measurements were done on CFTRꢀexpressing FRT cells using a
transepithelial chloride gradient and following permeabilization of the cell basolateral memꢀ
brane, such that current provides a quantitative, linear measure of CFTR chloride conductance.
Representative data in Fig. 2A for 6k and 12 shows a small increase in current with addition of
a low concentration of forskolin, followed by concentrationꢀdependent increases in current folꢀ
lowing compound additions. The compounds produced full CFTR activation, as little further inꢀ
crease in current was seen following a maximal concentration of forskolin. EC50 values are 30
nM for 6k and 31 nM for 12 (Fig. 2B).
Figure 2. Shortꢀcircuit current measurement of CFTR activation by 6k and 12. A. Measureꢀ
ments done in FRT cells expressing human wildtype CFTR showing responses to indicated
concentrations of forskolin, 6k or 12, and 10 µM CFTR inhibitor CFTRinhꢀ172. B. Concentraꢀ
tionꢀdependent activation of CFTR (mean ± S.E.M., n=3).
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Possible offꢀtarget effects on cell signaling and relevant nonꢀCFTR ion channels were
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investigated. 6k and 12 at 10 µM did not elevate cellular cAMP (Fig. 3A), increase cytoplasmic
calcium, or inhibit the ATPꢀstimulated elevation in cytoplasmic calcium (Fig. 3B). The effects of
6k and 12 on TMEM16A and nonꢀTMEM16A calciumꢀactivated chloride channel (CaCCs) were
investigated in FRTꢀexpressing TMEM16A and in HTꢀ29 cells, respectively. Both cells types
were transfected with a cytoplasmic YFPꢀbased halide sensor. Upon CaCC activation, extraꢀ
cellular iodide addition results in CaCCꢀfacilitated iodide influx, as detected by fluorescence
quenching of the cytoplasmic YFP halide sensor. 6k and 12 at 10 µM neither inhibited nor actiꢀ
vated CaCCs in TMEM16Aꢀexpressing FRT cells (Fig. 3C) or HTꢀ29 cells (Fig. 3D). To further
investigate compound specificity, the major ion transport pathways in human bronchial epitheꢀ
lial (HBE) cells were studied by shortꢀcircuit current. HBE cells were sequentially treated with
amiloride to inhibit the epithelial sodium channel (ENaC), forskolin to activate CFTR, CFTRinhꢀ
172 to inhibit CFTR, and ATP to activate CaCC, which produced the anticipated changes in
shortꢀcircuit current in control cells (Fig. 3E). Pretreatment of cells for 10 min with 10 µM 6k or
12 did not alter shortꢀcircuit current responses to ENaC, maximal forskolin or ATP, indicating
that the compounds do not inhibitor ENaC, CaCC, or the supporting epithelial cell transporters
necessary to generate current (NKCC1, epithelial K+ channels, Na+/K+ pump). Finally, as found
in FRT cells expressing wildtype CFTR, 12 did not activate CFTR in HBE cells in the absence
of basal CFTR phosphorylation produced by forskolin (Fig. 3F, top), whereas 0.1 µM 12 fully
activated CFTR following basal CFTR phosphorylation by 100 nM forskolin (Fig 3F, bottom).
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Figure 3. Characterization of 6k and 12. A. Cellular cAMP in FRT cells following incubation for
10 min with 10 ꢁM 6k or 12, without or with 90 nM forskolin (fsk), as well as forskolin alone (90
nM and 20 ꢁM) and forskolin (20 µM) + IBMX (100 ꢁM) (mean ± S.E.M., n=4). B. Cytoplasmic
calcium measured by Fluoꢀ4 fluorescence. FRT cells were pretreated for 5 min with 10 ꢁM 6k
or 12 (or control), with 100 ꢁM ATP added as a calcium agonist as indicated. C. TMEM16A acꢀ
tivity measured in FRT cells expressing YFP showing no inhibition (left, iodide + ATP addition)
or activation (right, iodide addition) by 10 ꢁM 6k or 12. D. CaCC activity measured in HTꢀ29
cells expressing YFP showing no activation (iodide addition) or inhibition (iodide + ATP addiꢀ
tion) by 10 ꢁM 6k or 12. E. Short circuitꢀcurrent in primary cultures of human bronchial epitheꢀ
lial cells in response to agonists and inhibitors that target key ion transport processes: 20 ꢁM
amiloride (ami); 20 ꢁM forskolin (fsk); 10 ꢁM CFTRinhꢀ172; 100 ꢁM ATP. Experiments were
done following 10ꢀmin preincubation with 10 µM 6k or 12. F. CFTR activation by indicated conꢀ
centration of 12 without forskolin (top) and after 100 nM forskolin (bottom) addition. Studies in
BꢀF are representative of 2ꢀ4 separate sets of experiments.
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Pharmacological properties and in vivo efficacy
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Compounds were further studied for their solution and metabolic stability, and cellular toxꢀ
icity. Fig. 4A shows that 6k and 12 did not produce significant toxicity using an Alamar blue asꢀ
say, with 33% DMSO as positive control. When dissolved at 100 µM in an ophthalmic vehicle
containing 0.3% CMC in Ringer’s solution, 12 remained chemically stable for 24 h at 40 °C as
assayed by LC/MS (data not shown). To assess metabolic stability, compounds at 5 µM were
incubated with rat hepatic microsomes in the presence of NADPH. Fig. 4B shows the kinetics
of compound metabolism, and LC/MS profiles of 12. 1 and 12, which contain nonꢀsubstituted
anilines, were metabolized rapidly with < 40% of the original compounds remaining at 15 min,
whereas 6j and 6k, which contain bicyclic anilines, were metabolized slowly with ~60% of the
original compounds remaining at 60 min. The benzoxazoleꢀcontaining analog 6j showed
greater metabolic stability than benzothiazole analog 6k, which may be due to relative lability
of the sulfide in benzothiazole versus the oxygen in benzoxazole. Low metabolic stability is
desirable for a dry eye therapeutic to minimize systemic exposure following potential absorpꢀ
tion, whereas high metabolic stability is desirable for liver and lung applications of CFTR actiꢀ
vators where systemic exposure and organ accumulation are needed. Though possible offꢀ
target effect related to ion channels were investigated here, more extensive screening for offꢀ
target activity is indicated, particularly for liver and lung applications where systemic exposure
is required.
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Figure 4. Compound pharmacology. A. Cytotoxicity was measured by Alamar Blue assay in
FRT cells incubated for 8 h with 10 ꢁM 6k or 12, with 33% DMSO as positive control (mean ±
S.E.M., n = 8). B. In vitro metabolic stability. Compounds at 5 µM were incubated for indicated
times with 1 mg/ml hepatic microsomes in the presence of NADPH and parent compound asꢀ
sayed by LC/MS (mean ± S.E.M., n=3). LC/MS profile of 12 shown on the right with elution
time on the xꢀaxis for incubation times of 0, 15 and 60 min.
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Measurements of tear fluid volume were done following a single ophthalmic dosing of test
compound in 2.5 µL of an ophthalmic vehicle, or vehicle alone as control. Compound 12 was
selected following preliminary testing of compound efficacy at 6 hours. At 100 µM (250 pmol)
12 produced a sustained increase in tear volume for 8 hours that returned to baseline over 12ꢀ
24 h, with improved kinetics compared to 1 (Fig. 5A). As evidence for compound specificity, 12
did not increase tear volume in CF mice, which lack functional CFTR (Fig. 5B). A doseꢀ
response study was done to determine the minimal amount of 12 that produced sustained tear
fluid secretion. Fig. 5C shows similar activity of 12 at doses down to 25 pmol with maximal tear
volume at 1ꢀ3 hours and sustained effect to 8 hours.
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Figure 5. Tear fluid volume in mice following ocular delivery of 1 or 12. A. Tear volume was
measured just before and at the indicated times after single ocular delivery of vehicle, 1 (250
pmol) or 12 (250 pmol) in a 2.5 µL volume. B. Study as in A but in CF mice lacking functional
CFTR. C. Single dose study as in A with different amounts of 12. Data reported as mean ±
S.E.M, 5 mice, 10 eyes per condition, * p<0.05, ** p<0.01 compared to vehicle control.
DISCUSSION
The structureꢀactivity study here identified analogs of 1 with substantially greater CFTR acꢀ
tivation potency, with the most potent compound 12 having EC50 ~ 30 nM, CFTR selectivity,
and sustained in vivo efficacy in increasing tear volume for at least 8 hours in mice. CFTR seꢀ
lectivity was demonstrated in cellular studies and by the lack of increase in tear fluid volume in
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CFTRꢀdeficient mice. 12 was chemically stable, including when formulated in a standard ophꢀ
thalmic vehicle. Minimal systemic exposure of 12 is predicted based on the small amount
needed to stimulate tear secretion and its rapid predicted hepatic metabolism. Further studies
supporting its clinical development will be needed, such as ocular tissue pharmacokinetics,
longꢀterm toxicity and patient tolerability, which are beyond the scope of this medicinal chemisꢀ
try study. Of note, synthesized analogs 6j and 6k had substantially greater in vitro metabolic
stability than 1 or 12, and therefore have potential utility in therapy of liver and lung diseases
where systemic exposure is necessary.
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Various biological activities have been reported for the 1,3,5ꢀtriazines scaffold. Atrazine, a
chlorotriazine with two alkylamines substituent, is a widely used herbicide 20. Some N,Nꢀdiꢀ
aminobenzylꢀNꢀisopropylꢀ1,3,5ꢀtriazines were found to have antiꢀtubulin activities in vitro and
in vivo 21. In addition, triazines containing bulky piperazinylꢀ and cyclohexylaminoꢀ substitutions
were reported to have cathepsin inhibition activity 22. The substituents in these biologically acꢀ
tive triazines are very different from those that conferred CFTR activation in this study. Strucꢀ
tureꢀactivity analysis of commercial and synthesized aminophenylꢀ1,3,5ꢀtriazines determined
that three specific substituents – alkoxide, alkylamine, and phenylamine – on the triazine are
needed for CFTR activity. For each of the substituents, (i) fluorinated alkoxides showed supeꢀ
rior activity than nonꢀfluorinated alkoxides; (ii) short and linear alkylamines are crucial for activꢀ
ity while bulky amines greatly reduced activity; and (iii) phenylamines containing electronꢀ
withdrawing groups, including nitro, carboxylic acid or fluoride, in the meta-position increased
activity. Of note, compound 12 has drugꢀlike properties, including favorable molecular weight
(331 Da), topological polar surface area (70.3 Å2) and cLogP (4.51), that latter much improved
compared to cLogP of 5.99 for 1.
CFTR is a compelling target for dry eye therapy as it is expressed in corneal and conjunctiꢀ
val epithelia at the ocular surface 23ꢀ26 where it functions as a prosecretory chloride channel 27,
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28. While CFTR is largely inactive under basal conditions, when activated it has the capacity to
drive secretion of tear fluid onto the ocular surface. By analogy, CFTR is largely inactive in the
intestine under basal conditions, but when activated by bacterial enterotoxins as in cholera it
can promote massive secretion of fluid into the intestinal lumen. As topically delivered CFTR
activators act primary on ocular surface epithelia, they are predicted to be efficacious in dry
eye produced by multiple etiologies, including disorders targeting lacrimal glands such as
Sjogren’s syndrome. It is noted, however, that a pure prosecretory therapeutic replaces priꢀ
marily the aqueous compartment of the tear film, which also contains lipid and mucin compoꢀ
nents. This potential limitation also applies to antiꢀabsorptive therapeutics in development tarꢀ
geting the ENaC sodium channel 29. Notwithstanding this potential limitation, the demonstraꢀ
tion of efficacy in rodent models 30 and the known pathogenesis of dry eye in which tear film
hyperosmolality produces downstream pathology 31, 32 support the utility of CFTR activator
therapy in human dry eye.
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In conclusion, CFTR activator therapy is predicted to have broad clinical indications for
prosecretory therapy of dry eye caused by a variety of etiologies, including ‘idiopathic’ dry eye.
Because of their unique mechanism of action CFTR activators are combinable with approved
immunosuppressants and possible future therapeutics such as ENaC inhibitors. Compared
with immunosuppressant drugs, CFTR activator therapy rescues an early, initiating event in
dry eye pathogenesis in an etiologyꢀagnostic manner. The aminophenylꢀ1,3,5ꢀtriazines synꢀ
thesized and tested herein are candidate lead compounds for further preclinical development.
EXPERIMENTAL DETAILS AND DATA
Synthesis procedures
General Procedures. All solvents and chemicals were used as purchased without further puriꢀ
fication. Reaction progress was monitored on Merck preꢀcoated silica gel plates (with fluoresꢀ
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cence at 254 nm) using ethyl acetate/nꢀhexane as solvent system. Column chromatography
was done with Fluka silica gel 60 (230−400 mesh). Proton (1H) and carbon (13C) NMR spectra
were recorded on a Bruker Avance 300 or 500 (500.13 MHz for 1H; 125.76 MHz for 13C) using
CDCl3, CD3OD, acetoneꢀd6, or DMSOꢀd6 as solvents. Chemical shifts are given in parts per
million (ppm). HRMS was performed using a hybrid quadrupole Orbitrap mass analyzer,
QExactive (Thermo, Bremen, Germany), with an electrospray ionization source. The mass
resolution was set as 70,000 at m/z 200 and the mass accuracy was less than 3 ppm. Purity of
all final compounds was 95% or higher. Purities were determined by lowꢀresolution mass specꢀ
trometry using a LC/MS system (Waters 2695 HPLC with Micromass ZQ). LC was done on a
Xterra MS C18 column (2.1mm x 100 mm, 3.5 ꢁm) with 0.2 mL/min water/acetonitrile (containꢀ
ing 0.1 % formic acid), 16 min linear gradient, 5 to 95% acetonitrile. UV absorbance was deꢀ
tected at 254 nm. Below we report synthesis and analytic data for intermediates and repreꢀ
sentative final compounds 1 and 12. Analytic data for 6aꢀ6k, 7aꢀ7g, and 11 are reported in
Supporting Information.
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N,N-Diethyl-6-[(1,1,1,3,3,3-hexafluoropropan-2-yl)oxy]-N'-phenyl-1,3,5-triazine-2,4-
diamine (1). To a solution of 4 (500 mg, 1.42 mmol) in THF (30 ml) was added diisoproꢀ
pylethylamine (0.49 ml, 2.83 mmol) and aniline (0.15 ml, 1.7 mmol). The mixture was refluxed
for 16 h. After cooling to room temperature the reaction mixture was extracted with EtOAc,
washed with brine and dried over MgSO4. The solvent was removed under reduced pressure
and purified by flash chromatography (1:10 EtOAc/Hex) to afford 1 as a white powder (280 mg,
48%). 1H NMR (300 MHz, CDCl3): δ = 7.61 (d, 2H, J = 7.8 Hz), 7.55 (brs, 1H), 7.35 (t, 1H, J =
7.5 Hz), 7.11 (t, 1H, J = 7.4 Hz), 6.37 (sep, 1H, J = 6.2 Hz), 3.69ꢀ3.58 (m, 4H), 1.29ꢀ1.20 (m,
6H); 13C NMR (75 MHz, CDCl3): δ = 168.5, 165.1, 165.0, 138.4, 128.7, 123.4, 122.7, 120.3,
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119.0, 68.3 (t, 34 Hz), 42.4, 42.0, 12.9, 12.8; HRMS (ESI): m/z calculated for C16H18F6N5O [M
1
2
3
+ H+]: 410.1410. Found: 410.1411.
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8
Analogs 6a-6k and 7a-7g were prepared as described for 1, with various substituted anilines
and 4 or 5.
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2,4-Dichloro-6-[(1,1,1,3,3,3-hexafluoropropan-2-yl)oxy]-1,3,5-triazine (2). To a solution of
cyanuric chloride (2 g, 10.84 mmol) in THF (50 ml) at ꢀ80 oC was added potassium carbonate
(3.0 g, 21.74 mmol). A solution of 1,1,1,3,3,3ꢀhexafluoroꢀ2ꢀpropanol (1.36 ml, 10.30 mmol) in
THF (20 ml) was then added dropwise over 30 min. The mixture was stirred for additional 30
min at ꢀ80 oC and quenched with water. The reaction mixture was diluted with EtOAc and
washed with 1N HCl solution. The organic layer was washed with brine and dried over MgSO4.
The solvent was removed under reduced pressure to afford a white solid (1.5 g, 62%). The
residue was used for further reaction without purification. 1H NMR (300 MHz, CDCl3): δ = 6.25
(sep, 1H, J = 5.7 Hz).
2,4-Dichloro-6-[(2,2,3,3-tetrafluoropropan)oxy]-1,3,5-triazine (3). To a solution of cyanuric
chloride (2 g, 10.84 mmol) in THF (50 ml) at ꢀ80 oC was added potassium carbonate (3 g,
21.74 mmol). A solution of 2,2,3,3ꢀtetrafluoropropanol (1.2 ml, 10.3 mmol) in THF (20 ml) was
then added dropwise over 30 min. The mixture was stirred for additional 30 min at ꢀ80 oC and
quenched with water. The reaction mixture was diluted with EtOAc and the organic layer was
washed with brine and dried over MgSO4. The solvent was removed under reduced pressure
to afford a white solid (1.3 g, 68%). The residue was used for further reaction without purificaꢀ
tion. 1H NMR (300 MHz, CDCl3): δ = 6.01 (tt, 1H, J = 3.96, 52.9 Hz), 4.90ꢀ4.79 (m, 2H).
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4-Chloro-N,N-diethyl-6-[(1,1,1,3,3,3-hexafluoropropan-2-yl)oxy]-1,3,5-triazin-2-amine (4).
To a solution of 2 (1.6 g, 5.06 mmol) in THF (40 ml) was added diisopropylethylamine (1.06 ml,
6.08 mmol) and diethylamine (0.5 ml, 5.0 mmol) at 0 oC. The mixture was stirred for 1 h at 0 oC,
then quenched with water. The reaction mixture was extracted with EtOAc, washed with water
and brine, and dried over MgSO4. The solvent was removed under reduced pressure to afford
a white solid (950mg, 55%). 1H NMR (300 MHz, CDCl3): δ = 6.25 (sex, 1H, J = 6.0 Hz), 3.79ꢀ
3.53 (m, 4H), 1.25ꢀ1.16 (m, 6H).
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4-Chloro-N,N-diethyl-6-[(2,2,3,3,-tetrafluoropropan)oxy]-1,3,5-triazin-2-amine (5). To a soꢀ
lution of 3 (1.3 g, 5 mmol) in THF (30 ml) was added diisopropylethylamine (1 ml, 6 mmol) and
diethylamine (0.5 ml, 5.0 mmol) at 0 oC. The mixture was stirred for 1 h at 0 oC, then quenched
with water. The reaction mixture was extracted with EtOAc, washed with water and brine, and
dried over MgSO4. The solvent was removed under reduced pressure to afford yellow oil
(900mg, 57%). 1H NMR (300 MHz, CDCl3): δ = 6.02 (td, 1H, J = 1.60, 4.74 Hz), 4.70 (dt, 2H, J
= 1.5, 12.2 Hz), 3.65ꢀ3.55 (m, 4H), 1.19 (t, 6H, J = 7.08 Hz).
4,6-Dichloro-N-methyl-1,3,5-triazine-2-amine (8). To a solution of cyanuric chloride (5 g,
27.3 mmol) in acetone (30 ml) at 0 oC was added methylamine (13.7 ml, 2 M in THF) dropwise
over 30 min. The mixture was stirred for 1 h at 0 oC and then crushed ice was added. The reꢀ
sulting precipitate was filtered and washed with water to afford 8 as a white solid (3.7 g,
75.7%), which was used for further reaction without purification. 1H NMR (300 MHz, CDCl3) δ
= 7.02 (brs, 1H), 3.10 (dd, 3H, J = 1.2, 5.1 Hz); 13C NMR (75 MHz, CDCl3) δ = 171.0, 169.4,
166.2, 28.3.
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4-Chloro-6-((1,1,1,3,3,3-hexafluoropropan-2-yl)oxy)-N-methyl-1,3,5-triazin-2-amine (9). To
a solution of 8 (2 g, 11.17 mmol) in THF (40 ml) at 0 oC was added potassium carbonate
(11.56 g, 83.8 mmol) and 1,1,1,3,3,3ꢀhexafluoroꢀ2ꢀpropanol (1.4 ml, 13.4 mmol). The mixture
was stirred overnight at ambient temperature. The reaction mixture was diluted with EtOAc,
and then organic layer was washed with brine and dried over MgSO4. The solvent was reꢀ
moved under reduced pressure. A white solid was obtained (1.4 g, 43%). 1H NMR (300 MHz,
CDCl3) δ = 7.02 (brs, 1H), 6.27 (quint, 1H, J = 5.73 Hz), 3.11 (d, 3H, J = 5.1 Hz).
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4-Chloro-6-[(2,2,3,3-tetrafluoropropan)oxy]N-methyl-1,3,5-triazine-2-amine (10). To a soꢀ
lution of 8 (630 mg, 3.52 mmol) in THF (30 ml) at 0 oC was added potassium carbonate (970
mg, 7.04 mmol). A solution of 2,2,3,3ꢀtetrafluoropropanol (0.31 ml, 3.52 mmol) in THF (20 ml)
was added dropwise over 30 min. The mixture was stirred for additional 30 min at 0 oC, then
quenched with water. The reaction mixture was diluted with EtOAc, and the organic layer was
washed with brine and dried over MgSO4. Solvent was removed under reduced pressure to
afford a white solid (640 mg, 66%). The residue was used for further reaction without purificaꢀ
tion. 1H NMR (300 MHz, CDCl3): δ = 6.98ꢀ5.79 (m, 1H), 4.87ꢀ4.69 (m, 2H), 3.05 (m, 3H).
N-Methyl-N'-phenyl-6-(2,2,3,3-tetrafluoropropoxy)-1,3,5-triazine-2,4-diamine (12). To a soꢀ
lution of 10 (70 mg, 0.26 mmol) in THF (3 ml) was added diisopropylethylamine (0.09 ml, 0.51
mmol) and aniline (0.03 ml, 0.31 mmol). The mixture was refluxed for 16 h. After cooling to
room temperature the reaction mixture was extracted with EtOAc, washed with brine and dried
over MgSO4. Solvent was removed under reduced pressure and purified by flash chromatogꢀ
raphy (1:2 EtOAc/Hex) to afford 12 as a white powder (63 mg, 74%). 1H NMR (300 MHz,
CDCl3): δ = 7.61 (d, 2H, J = 7.8 Hz), 7.55 (brs, 1H), 7.35 (t, 1H, J = 7.5 Hz), 7.11 (t, 1H, J =
7.4 Hz), 6.37 (sep, 1H, J = 6.2 Hz), 3.69ꢀ3.58 (m, 4H), 1.29ꢀ1.20 (m, 6H); 13C NMR (75 MHz,
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CDCl3): δ = 168.5, 165.1, 165.0, 138.4, 128.7, 123.4, 122.7, 120.3, 119.0, 68.3 (t, 34 Hz), 42.4,
42.0, 12.9, 12.8; HRMS (ESI): m/z calculated for C13H14F4N5O [M + H+]: 332.1129. Found:
332.1127.
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Mice
Wildtype and CF (homozygous ∆F508ꢀCFTR mutant) mice in a CD1 genetic background were
bred at the University of California San Francisco (UCSF) Animal Facility. Mice aged 8 to 12
weeks (25 to 35 g) were used. Animal protocols were approved by the UCSF Institutional Aniꢀ
mal Care and Use Committee and were in compliance with the ARVO Statement for the Use of
Animals in Ophthalmic and Visual Research.
Cell culture
Fischer Rat Thyroid (FRT) cells stably coꢀexpressing human wildtype CFTR or TMEM16A and
the halideꢀsensitive yellow fluorescent protein (YFP)ꢀH148Q were cultured as described 15, 33
.
HTꢀ29 expressing YFP were cultured as described 34. Primary cultures of human bronchial epiꢀ
thelial (HBE) cells were maintained at an airꢀliquid interface as described previously 35.
Plate reader assays of chloride channel function
CFTR activity was assayed as described 15. FRT cells coꢀexpressing YFP and wildtype CFTR
were washed with phosphateꢀbuffered saline (PBS) and then incubated for 10 min with test
compounds in PBS containing 125 nM forskolin. 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.
TMEM16A activity was assayed similarly as described 33 using FRT cells coꢀexpressing YFP
and TMEM16A. Activity of nonꢀTMEM16A CaCC was assayed as described 34 in HTꢀ29 cells
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expressing YFP. In each assay initial rates of I− influx were computed as a linear measure of
channel function.
Page 22 of 30
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Shortꢀcircuit current measurement
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Shortꢀcircuit current measurements were done as described 9. Briefly, Snapwell (Corning Cosꢀ
tar, Corning, NY) inserts containing CFTRꢀexpressing FRT cells or human bronchial epithelial
cells were mounted in Ussing chambers (Physiological Instruments, San Diego, CA). For FRT
−
cells the hemichambers were filled with 5 ml of HCO3 ꢀbuffered solution (basolateral) and halfꢀ
Cl− solution (apical), and the basolateral membrane was permeabilized with 250 µg/ml amphoꢀ
−
tericin B. Symmetrical HCO3 ꢀbuffered solutions were used for human bronchial epithelial cells.
Solutions were bubbled with 95% O2/5% CO2 and maintained at 37°C, and shortꢀcircuit current
was measured on a DVCꢀ1000 voltage clamp (World Precision Instruments Inc., Sarasota, FL)
using Ag/AgCl electrodes and 3 M KCl agar bridges.
cAMP and Ca2+ assays
FRT cells expressing wildtype CFTR were cultured in 12ꢀwell plates. After washing with PBS,
cells were incubated for 10 min with compounds in the absence or presence of 90 nM forskolin
and then lysed. Lysates were assayed for cAMP content using a cAMP immunoassay kit (Paꢀ
rameter cAMP Immunoassay Kit, R&D Systems, Minneapolis, MN). For cytoplasmic calcium
measurements, FRT cells in 96ꢀwell blackꢀwalled microplates were loaded with Fluoꢀ4 NW
(Invitrogen, Carlsbad, CA) and Fluoꢀ4 fluorescence was measured with a plate reader
equipped with syringe pumps and monochromators.
Cytotoxicity
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Journal of Medicinal Chemistry
FRT cells were cultured overnight in black 96ꢀwell Costar microplates and incubated with test
compounds at up to 10 µM (the maximum solubility in PBS) for 8 h. Cytotoxicity was measured
by Alamar Blue assay (Invitrogen, Carlsbad, CA).
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Tear volume
Tear volume was measured using phenol red threads (ZoneꢀQuick, Oasis Medical, Glendora,
CA) as described 36. Threads were placed for 10 s in the lateral canthi of isofluoraneꢀ
anesthetized mice using jewelers’ forceps. Tear volume was determined from the length of
thread wetting as visualized under a dissecting microscope. Serial measurements were done
to evaluate compound pharmacodynamics after topical application of a single, 2.5ꢀ µL drop of
ophthalmic formulation containing 0ꢀ100 µM of 1 or 12 in Ringer’s solution with 0.3% carboxꢀ
ymethylcellulose (high viscosity, VWR, Radnor, PA), 0.015% benzalkonium chloride preservaꢀ
tive, and 1% DMSO. Both eyes in each mouse received the same treatment to control for poꢀ
tential contralateral effects of a given treatment.
In vitro metabolic stability
Compounds (each 5 µM) were incubated for specified times at 37 oC with rat liver microsomes
(1 mg/ml; SigmaꢀAldrich) in potassium phosphate buffer containing 1 mM NADPH, as deꢀ
scribed 14. Iceꢀcold EtOAc was then added and the mixture was centrifuged for 15 min at 3000
rpm. The supernatant was analyzed by lowꢀresolution mass spectrometry using a LC/MS sysꢀ
tem (Waters 2695 HPLC with Micromass ZQ). LC was done on a Xterra MS C18 column
(2.1mm x 100 mm, 3.5 ꢁm) with 0.2 mL/min water/acetonitrile (containing 0.1 % formic acid),
12 min linear gradient, 5 to 95% acetonitrile. UV absorbance was detected at 254 nm.
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Statistics
Statistical analysis was performed using Prism 5 GraphPad Software package (San Diego,
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CA). Serial tear volume measurements were analyzed by twoꢀway ANOVA with Dunnett postꢀ
hoc analysis. Data are presented as mean ± standard error of the mean (S.E.M.), and statistiꢀ
cal significance was set at p < 0.01 (*).
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ASSOCIATED CONTENT
Supporting Information.
Supporting Information Available:
Analytical data of synthetic compounds (6a-k, 7a-7g, and 11)
Supplementary Table 1
AUTHOR INFORMATION
Corresponding Author
* Alan S. Verkman, M.D., Ph.D.
1246 Health Sciences East Tower
University of California, San Francisco, CA 941430521 USA
Phone: +1ꢀ415ꢀ476ꢀ8530 — Fax: +1ꢀ415ꢀ665ꢀ3847
Eꢀmail: Alan.Verkman@ucsf.edu
ACKNOWLEDGMENTS
This work was supported by NIH Grants DK72517, EY13574, DK099803, EB00415, DK35124
and DK101373, and a NIH/NCATS UCSFꢀCTSI Grant, UL1 TR000004.
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Journal of Medicinal Chemistry
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ABBREVIATIONS
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CFTR, cystic fibrosis transmembrane conductance regulator; cAMP, cyclic adenosine monoꢀ
phosphate; YFP, yellow fluorescent protein; CF, cystic fibrosis; FRT cells, Fischer rat thyroid
cells; LC/MS, liquid chromatography / mass spectrometry; fsk, forskolin; SAR, structureꢀactivity
relationships.
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