Discovery of a novel chemotype of potent human ENaC blockers using a bioisostere approach. Part 1: Quaternary amines

Article abstract of DOI:10.1016/j.bmcl.2011.12.016

We report the identification of a novel series of human epithelial sodium channel (ENaC) blockers that are structurally distinct from the pyrazinoyl guanidine chemotype found in prototypical ENaC blockers such as amiloride. Following a rational design hypothesis a series of quaternary amines were prepared and evaluated for their ability to block ion transport via ENaC in human bronchial epithelial cells (HBECs). Compound 11 has an IC₅₀ of 200 nM and is efficacious in the Guinea-pig tracheal potential difference (TPD) model of ENaC blockade with an ED₅₀ of 44 μg kg^-1 at 1 h. As such, pyrazinoyl quaternary amines represent the first examples of a promising new class of human ENaC blockers.

Full text of DOI:10.1016/j.bmcl.2011.12.016

Bioorganic & Medicinal Chemistry Letters 22 (2012) 929–932
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a novel chemotype of potent human ENaC blockers using a
bioisostere approach. Part 1: Quaternary amines
Thomas Hunt a, , Hazel C. Atherton-Watson b, Jake Axford a, Stephen P. Collingwood a, Kevin J. Coote b,
⇑
Brian Cox a, Sarah Czarnecki b, Henry Danahay b, Nick Devereux a, Catherine Howsham a, Peter Hunt a,
Victoria Paddock a, Derek Paisley b, Alice Young b
a Novartis Institutes of Biomedical Research, Global Discovery Chemistry, Wimblehurst Road, Horsham, West Sussex RH12 5AB, United Kingdom
b Novartis Institutes of Biomedical Research, Respiratory Diseases Area, Wimblehurst Road, Horsham, West Sussex RH12 5AB, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the identification of a novel series of human epithelial sodium channel (ENaC) blockers that are
structurally distinct from the pyrazinoyl guanidine chemotype found in prototypical ENaC blockers such
as amiloride. Following a rational design hypothesis a series of quaternary amines were prepared and
evaluated for their ability to block ion transport via ENaC in human bronchial epithelial cells (HBECs).
Compound 11 has an IC50 of 200 nM and is efficacious in the Guinea-pig tracheal potential difference
(TPD) model of ENaC blockade with an ED50 of 44
represent the first examples of a promising new class of human ENaC blockers.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 20 October 2011
Revised 2 December 2011
Accepted 3 December 2011
Available online 8 December 2011
l
g kgꢀ1 at 1 h. As such, pyrazinoyl quaternary amines
Keywords:
ENaC blocker
Epithelial sodium channel
Quaternary amine
Cystic fibrosis
Amiloride
The epithelial sodium channel (ENaC) is a highly selective so-
dium ion channel that is involved in the reabsorption of sodium
ions across the apical membrane of a number of epithelia including
the lung, kidney and colon.1 ENaC is a heteromeric channel com-
O
NH
NH2
O
NH
Cl
N
N
Cl
N
N
N
H
N
H
N
H
H2N
NH2
Amiloride
1
NH2
Benzamil
H2N
prising a, b and c
subunits in a 1:1:1 or 2:1:1 stoichiometry.2 ENaC
2
and the cystic fibrosis transmembrane conductance regulator
(CFTR) are believed to be two of the key ion channels involved in
maintaining appropriate hydration of the airway surface liquid
(ASL) that lines the epithelia of the lungs. In cystic fibrosis (CF),
where CFTR function is impaired, the net action of ENaC is postu-
lated to lead to reduced mucosal hydration via absorption of so-
dium ions, creating an osmotic gradient that drives water out of
the ASL.3 This results in the formation of a thick, sticky layer of mu-
cus within the lungs that is difficult to remove via mucociliary
clearance (MCC) leading to chronic infection and inflammation.4
Blockade of ENaC is anticipated to increase hydration of the ASL,
enhance MCC and so help protect the lungs of CF patients from de-
cline in pulmonary function.5 Aerosolized amiloride (1, see Fig. 1),
a potassium-sparing diuretic that blocks ENaC has been shown to
improve MCC,6 but its effectiveness is limited by poor potency
and pharmacokinetic profile.7
Figure 1. Examples of pyrazinoyl guanidine ENaC blockers.
demonstrated that the 3,5-diamino-6-chloro-pyrazinoyl guanidine
is a privileged structure for ENaC blockade.8 Improvements in po-
tency have been obtained by derivatization of the guanidine with
lipophilic tail groups as in the case of benzamil (2, see Fig. 1).9
Although no crystal structure of ENaC exists modeling suggests that
the relative spatial arrangement of the pyrazine ring with respect to
the positively charged acylguanidinium functionalgroup is essential
for blockade of ENaC.10 There have been a number of isolated reports
where the acylguanidine group has been replaced with an appropri-
ately positioned amino group. For example, Li et al. demonstrated
that compound 3 (see Fig. 2) was up to 40-fold more potent than
amiloride against a range of amphibian epithelial sodium chan-
nels.11 A similar result was reported by Reid and co-workers who
demonstrated that tertiary amine 4 or alkyl guanidine 5 were effec-
tive diuretic, natiuretic and antikaliuretic agents when dosed iv to
rats, but both were less effective than amiloride.12
To date, the most potent ENaC blockers all contain the same pyr-
azinoyl guanidine chemotype based on amiloride and SAR has
In this Letter we wish to communicate our approach to identi-
fying novel and potent human ENaC blockers with a chemotype
⇑
Corresponding author. Tel.: +44 1403 323576; fax: +44 1403 323837.
E-mail address: tom.hunt@novartis.com (T. Hunt).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.016

930
T. Hunt et al. / Bioorg. Med. Chem. Lett. 22 (2012) 929–932
Table 1
O
O
Blockade of ENaC by amines 4, 8a and 8c–f in HBECs
Cl
N
Cl
N
N
N
H
NH2
N
H
NH2
NH2
N
3
H2N
N
4
O
H2N
Cl
N
N
R2
N
R1
O
H
N
NH2
H2N
Cl
H2N
N
NH2
N
H
R1
R2
C(N@H)NH2
C(N@H)NHCH2Ph
(CH2)2NMe2
(CH2)2NH2
(CH2)2NHMe
(CH2)3NMe2
(CH2)4NMe2
(CH2)2NMe2
HBEC IC50
(lM)
a,b
Compd
NH
N
NH2
Amilorideb
H
H
H
H
H
H
H
Me
0.22 (93)
0.020 (9)
4.78 (2)
3.97 (4)
4.57 (2)
17.35 (2)
Inactivec
>30 (2)
Benzamilb
5
4
Figure 2. Examples of non-pyrazinoyl guanidine ENaC blockers.
8a
8c
8d
8e
8f
distinct from the prototypical pyrazinoyl guanidines that will be
suitable for the treatment of CF via inhaled delivery (dry powder
or nebuliser).13 We speculated that a group that is significantly
ionized at physiological pH should function as a bioisostere for
the acylguanidinium cation. Our investigations began by exploring
amines as bioisosteres for the acyl guanidine present in amiloride;
although less basic than an acylguanidine an amine should be sig-
nificantly protonated at physiological pH.14 A small range of previ-
ously reported amines (4 and 8a–f),8b,12,15 were rapidly prepared
by either reaction of methyl ester 6 with neat amine under micro-
wave irradiation, or a O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetram-
ethyluronium hexafluorophosphate (HATU)-mediated amide
coupling reaction of carboxylic acid 7 (Scheme 1). Compound 8c
was synthesized via Boc-deprotection of compound 8b with triflu-
roacetic acid.
a
Mean IC50 data, number in parentheses refers to the number of repetitions.
A description of the assay conditions used and previously reported IC50 data for
b
amiloride and benzamil can be found in Ref. 16.
c
<60% inhibition at 30 lM.
O
O
I
Cl
N
N
Cl
N
N+
a
N
H
N
H
NH2
NH2
N
N
H2N
H2N
4
9
Scheme 2. Reagents and conditions: (a) MeI, CH2Cl2, rt, 97%.
Amine 4 demonstrates for the first time that blockade of human
ENaC is possible with a chemotype different to the prototypical
pyrazinoyl guanidines, albeit with a drop in potency (>10-fold)
compared to amiloride (Table 1). It was observed that a two-car-
bon linker between the amide and the amine was optimal (com-
pounds 4, 8a, 8c) with a large drop in potency observed as the
linker length increased to 3 or 4 carbons (compounds 8d,e).
Substitution of the distal amine with small alkyl groups was
well tolerated (compound 4) but a large drop in potency was ob-
served when the amide N–H was methylated (compound 8f) indi-
Quaternization of amine 4 with methyl iodide afforded quater-
nary amine 9 (Scheme 2) that showed comparable potency to
amine 4 illustrating that this strategy is tolerated but the antici-
pated increase in ENaC potency was not observed (Table 2). Previ-
ous work has demonstrated that the binding site in ENaC possesses
a hydrophobic region that is consistent with the increased potency
observed between amiloride and benzamil.9,16
To explore this hypothesis amines 4, 8a and 8c were quatern-
ized by mono-, di- or tri-alkylation with excess bromide 10 in
refluxing acetone for 1–3 days to generate quaternary amines
11–13, respectively (Scheme 3). Compound 13 could only be iso-
lated in low yield (0.3%); the major product isolated was the ter-
tiary amine resulting from dialkylation of amine 8a with bromide
10.
cating that the N–H is potentially providing
interaction with the ion channel.
a key binding
We speculated that generating a permanent cation via quatern-
ization of the amine would improve ENaC potency and increase
solubility.13,17 Increased solubility would enable either dry powder
or nebuliser formulations to be considered. Quaternization would
also reduce cellular permeability and this could potentially reduce
systemic absorption from the lung and provide a longer duration of
action for an inhaled ENaC blocker.
Pleasingly compound 11 showed improved potency with an IC50
of 0.27 lM (Table 2). Unlike the pyrazinoyl guanidine series, fur-
ther lipophilic groups can be incorporated around the quaternary
centre which dramatically improves ENaC blockade (compound
12 is approximately 100-fold more potent that amiloride).
The thermodynamic aqueous solubility of compounds 11 and
12 was assessed in a range of pH buffers and is summarized in
Table 3. Compared to amiloride quaternary amine 11 shows signif-
icantly improved solubility in a range of pH buffers. Although the
greater lipophilicity of compound 12 affords a 10-fold improve-
ment in potency versus compound 11, this is to the detriment of
solubility, which decreases 5- to 10-fold.
A parallel synthetic approach was adopted to generate a library
of 67 aryl and alkylaryl amides and sulfonamides to explore the
SAR around the hydrophobic pocket as outlined in Scheme 4. Reac-
tion of amine 4 with the corresponding N-phthalimido protected
alkyl bromides generated the series of quaternary ammonium bro-
mides 14 that were subsequently deprotected with hydrazine
monohydrate in ethanol to give amines 15.
O
O
Cl
N
Cl
N
OMe
NH2
OH
NH2
N
N
H2N
H2N
6
7
O
a
1
b
Cl
N
N
R
N
R
2
H2N
NH2
8d R1 = H, R2 = (CH2)3NMe2
8e R1 = H, R2 = (CH2)4NMe2
8f R1 = Me, R2 = (CH2)2NMe2
8a R1 = H, R2 = (CH2)2NH2
8b R1 = H, R2 = (CH2)2N(Me)Boc
8c R1 = H, R2 = (CH2)2NHMe
4 R1 = H, R2 = (CH2)2NMe2
c
The amines provided the necessary handle for the library and the
opportunity to introduce some polarity into the linker between the
quaternary amine and the terminal group. Guided by previously
Scheme 1. Reagents and conditions: (a) R1R2NH, neat,
HATU, 4-methylmorpholine, DMF, rt, 40%; (c) CF3CO2H, CH2Cl2, rt, 34%.
lwave, 130 °C, 31–58%; (b)

T. Hunt et al. / Bioorg. Med. Chem. Lett. 22 (2012) 929–932
931
Table 2
Blockade of ENaC by quaternary amines 9 and 11–13 in HBECs
1
O
R
Cl
N
N
N+
2
N
H
R
3
R
NH2
H2N
a,b
Compd
R1
R2
R3
HBEC IC50
5.81 (3)
(lM)
9
Me
Me
Me
OMe
OMe
OMe
11
12
13
Me
Me
Me
0.27 (11)
OMe
0.013 (24)
0.037 (4)
OMe
OMe
a
Mean IC50 data, number in parentheses refers to the number of repetitions.
A description of the assay conditions used to determine IC50’s can be found in Ref. 16.
b
OMe
1
1
R
N
O
Br
R
O
a
Cl
N
N
Cl
N
N
N+
R
2
N
H
R
N
H
2
OMe
NH2
H2N
Br
NH2
H2N
10
4; R1 = R2 = Me
11; R1 = R2 = Me
OMe
8c; R1 = Me, R2 = H
8a; R1 = R2 = H
12; R1 = Me, R2 =
13; R1 = R2 =
OMe
Scheme 3. Reagents and conditions: (a) Na2CO3, acetone, reflux, 0.3–90%.
Table 3
Thermodynamic solubility
Compd
Salt
Thermodynamic solubilitya (mg/mL)
pH 1
pH 4
pH 6.8
pH 9
Amiloride
11
12
HCl
Bromide
Bromide
0.4
4.8
0.6
1.0
4.9
0.5
<0.0005
5
0.9
0.8
4.7
1.0
a
Thermodynamic solubility was determined by the shake-flask methodology after 24 h equilibriation.
reported SAR,18 amines 15 were derivatized with a focused set of
sulfonyl chlorides and carboxylic acids to generate amides and sul-
fonamides of the general structure 16.
3.33 cm/s ꢁ 10ꢀ6, respectively). This was demonstrated further
by the activity of compound 16n on the hERG channel which
had high in vitro binding affinity (IC50 = 0.33
lM), but in patch
Selected results from this library are shown in Table 4. In the ser-
ies of amides where the terminal R group is 4-chlorophenyl (16a–d)
the optimal linker length, m, is 3 suggesting that there is a prefer-
ence for the position of the aromatic group. This trend was
confirmed with the analogous series of sulfonamides (16e–h) that
also demonstrated that a sulfonamide linker is equally well toler-
ated. Variation of the terminal aryl group indicated a preference
for 4-benzyloxy phenyl group (16n and 16r) suggesting that an
electron rich first aromatic ring is preferred with sufficient space
clamp studies inhibition was reduced (IC50 = 10.8
l
M), presum-
ably due to reduced cell permeation. As ENaC is expressed on
the surface of lung epithelia we anticipated that low cellular per-
meability would not affect the in vivo efficacy of an inhaled
therapeutic.
Cross-reactivity to Guinea-pig ENaC was assessed using Fischer
Rat Thyroid (FRT) cells transiently infected with Guinea-pig ENaC16
and in vivo efficacy was determined using the previously reported
Guinea-pig TPD model.16 Data for amiloride in these assays has
been previously reported and is shown in Table 5.16 Compound
11 shows excellent cross reactivity to Guinea-pig ENaC with an
IC50 of 180 nM. Intratracheal (i.t.) dosing of compound 11 in the
Guinea-pig TPD model showed compound 11 is efficacious with
for
a further large lipophilic group consistent with a large
hydrophobic pocket.
Compounds 11 and 16n were profiled further to establish if
quaternization led to reduced cellular permeability that we
anticipated. Pleasingly, both compounds do show poor cellular
permeability with Caco-2 ratios B–A/A–B of 1.67/0.93 and 2.55/
an ED50 at 1 h of 44
ED50 for amiloride at 1 h of 16
l
g kgꢀ1 which compares favorably with the
g kgꢀ1 (Table 5).
l

932
T. Hunt et al. / Bioorg. Med. Chem. Lett. 22 (2012) 929–932
O
O
Br-
m
N+
a
N
O
Cl
N
Cl
N
(
)
NPhth
N
H
N
H
Br
(
)
m
NPhth
NH2
H2N
N
NH2
N
14
NH2
H2N
4
O
S
X
Br-
)
O
Y
O
R
O
c
or
N+
N+
Cl
Cl
H2N
N
Cl
H2N
N
N
b
(
( )
m
N
H
R
N
H
N
H
O
N
NH2
NH2
d
HO
R
16
15
m = 1-4
R = Ph
CH2-(4-OBn-Ph)
CH2CH2-Ph
-
X = Br- or PF6
Y = C, S=O
4-Me-Ph
4-Cl-Ph
CH2-Ph
CH2CH2-(4-Me-Ph)
CH2CH2-(4-Cl-Ph)
CH2CH2-(4-OBn-Ph)
CH2-(4-Me-Ph)
CH2-(4-Cl-Ph)
Scheme 4. Reagents and conditions: (a) 2-butanone, reflux, 75–98%; (b) N2H4ꢂH2O, EtOH, reflux; (c) 4-methylmorpholine, CH2Cl2, rt, 14–54% (over two steps); (d) HATU, 4-
methylmorpholine, DMF, rt, 10–89% (over two steps).
that potently inhibit sodium ion transport via ENaC in HBECs and
Guinea-pig FRTs with activity comparable to amiloride resulting
Table 4
ENaC inhibition data for a series of quaternary amines 16a–r in HBECs
in in vivo efficacy in the Guinea-pig TPD model. In addition, quater-
nary amines provide a feasible route to enhancing the solubility of
O
Y
O
N+
ENaC blockers whilst reducing cellular permeability making them
suitable for nebuliser, as well as dry powder delivery. As such,
the discovery of the pyrazinoyl quaternary amines through a bio-
isostere approach represents a novel class of ENaC blockers that
are worthy of further investigation as potential inhaled therapeu-
tics for the improvement of mucociliary clearance in diseases such
as cystic fibrosis.
Cl
N
N
(
)
m
N
H
R
N
H
H2N
NH2
a
Compd
m
Y
R
HBEC IC50 (lM)
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
16l
16m
16n
16o
16p
16q
16r
1
2
3
4
1
2
3
4
3
3
3
3
3
3
3
3
3
3
C
C
C
C
S@O
S@O
S@O
S@O
C
C
C
C
C
4-Cl-Ph
1.66
0.74
0.51
0.75
1.12
0.29
0.25
0.96
1.23
0.88
0.95
0.75
0.72
0.06
0.91
0.59
0.16
0.08
Acknowledgment
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
Ph
4-Me-Ph
CH2-Ph
CH2-(4-Me-Ph)
CH2-(4-Cl-Ph)
CH2-(4-OBn-Ph)
CH2CH2-Ph
Colin Chinn and Ameet Ambarkhane for providing thermody-
namic solubility data.
References and notes
1. Garty, H.; Palmer, L. G. Physiol. Rev. 1997, 77, 359.
2. (a) Stockand, J. D.; Staruschenko, A.; Pochynyuk, O.; Booth, R. E.; Silverthorn, D.
U. IUBMB Life 2008, 60, 620; (b) Anantharam, A.; Palmer, L. G. J. Gen. Physiol.
2007, 130, 55.
3. Boucher, R. C. J. Intern. Med. 2007, 261, 5.
4. (a) Paisley, D.; Gosling, M.; Danahay, H. Expert Rev. Clin. Pharmacol. 2010, 3,
361; (b) Boucher, R. Trends Mol. Med. 2007, 13, 231.
C
C
C
C
CH2CH2-(4-Me-Ph)
CH2CH2-(4-Cl-Ph)
CH2CH2-(4-OBn-Ph)
5. (a) Ratjen, F. N. Eng. J. Med. 2006, 354, 291; (b) Clunes, M. T.; Boucher, R. Curr.
Opin. Pharmacol. 2008, 8, 292.
6. (a) Köhler, D.; App, E.; Schmitz-Schumann, M.; Würtemberger, G.; Matthys, H.
Eur. J. Respir. Dis. Suppl. 1986, 146, 319; (b) App, E. M.; King, M.; Helfesrieder, R.;
Köhler, D.; Matthys, H. Am. Rev. Resp. Dis. 1990, 141, 605.
7. Hoffmann, T.; Senier, I.; Bittner, P.; Hüls, G.; Schwandt, H. J.; Lindemann, H. J.
Aerosol. Med. 1997, 10, 147.
C
a
Mean IC50 data from at least two repetitions.
8. (a) Li, J. H.; Cragoe, E. J., Jr.; Lindemann, B. J. Membr. Biol. 1985, 83, 45; (b) Li, J.
H., ; Cragoe, E. J., Jr.; Lindemann, B. J. Membr. Biol. 1987, 95, 171.
9. (a) Cuthbert, A. W.; Fanelli, G. M. Br. J. Pharmacol. 1978, 63, 139; (b) Carr, M. J.;
Gover, T. D.; Weinreich, D.; Undem, B. J. Br. J. Pharmacol. 2001, 133, 1255.
10. Venanzi, C. A.; Plant, C.; Venanzi, T. J. J. Med. Chem. 1992, 35, 1643.
11. Li, J. H.; Shaw, A.; Kau, S. T. J. Pharmacol. Exp. Ther. 1993, 267, 1081.
12. Russ, T.; Reid, W.; Ullrich, F.; Mutschler, E. Arch. Pharm. 1992, 325, 761.
13. Collingwood, S. P.; Devereux, N. J.; Howsham, C.; Hunt, P.; Hunt, T. A.
International patent, WO 150 137 A2, 2009.
14. Collingwood, S. P.; Howsham, C.; Hunt, T. A. International patent, WO 138 378
A1, 2009.
15. Schwartz, J. A. European Patent 0293171, 1988.
16. Coote, K. J.; Atherton, H.; Sugar, R.; Burrows, R.; Smith, N. J.; Schlaeppi, J.-M.;
Groot-Kormelink, P. J.; Gosling, M.; Danahay, H. Br. J. Pharmacol. 2008, 155,
1025.
Table 5
ENaC Guinea-pig cross reactivity and in vivo Guinea-pig TPD efficacy for amiloride
and compound 11
a
Compd
HBEC IC50
M)
Guinea-pig FRT
Guinea-pig TPD 1 h ED50
g kgꢀ1
a
(
l
IC50
(lM)
(l
)
Amilorideb 0.22 (93)
11 0.27 (11)
0.54 (37)
0.18 (3)
16
44
a
Mean IC50 data, number in parentheses refers to the number of repetitions.
Data for amiloride has been previously reported using these assay conditions in
b
Ref. 16.
17. During the preparation of this manuscript a series of quaternary benzylamine
ENaC blockers were described: Laine, D. I.; Li, T.; Yan, H. International patent
WO 028 740 A1, 2011.
18. Hirsh, A. J.; Molino, B. F.; Zhang, J.; Astakhova, N.; Geiss, W. B.; Sargent, B. J.;
Swenson, B. D.; Usyatinsky, A.; Wyle, M. J.; Boucher, R. C.; Smith, R. T.; Zamurs,
A.; Johnson, M. R. J. Med. Chem. 2006, 49, 4098.
In summary, we have identified the first potent human ENaC
blockers which have an alternative core to the prototypical pyraz-
inoyl guanidine. Compound 11 represents a series of compounds