Discovery of benzylisothioureas as potent divalent metal transporter 1 (DMT1) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.05.129

Inhibition of intestinal brush border DMT1 offers a novel therapeutic approach to the prevention and treatment of disorders of iron overload. Several series of diaryl and tricyclic benzylisothiourea compounds as novel and potent DMT1 inhibitors were discovered from the original hit compound 1. These compounds demonstrated in vitro potency against DMT1, desirable cell permeability properties and a dose-dependent inhibition of iron uptake in an acute rat model of iron hyperabsorption. Tricyclic compounds increased the in vitro potency by up to 16-fold versus the original hit. Diaryl compounds 6b and 14a demonstrated significant iron absorption inhibition in vivo with both 25 and 50 mg/kg doses. The diaryl and tricyclic compounds described in this report represent promising structural templates for further optimization.

Full text of DOI:10.1016/j.bmcl.2012.05.129

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5108–5113
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of benzylisothioureas as potent divalent metal transporter 1
(DMT1) inhibitors
Zaihui Zhang a, Vishnumurthy Kodumuru a, Serguei Sviridov a, Shifeng Liu a, Mikhail Chafeev a,
Sultan Chowdhury a, Nagasree Chakka a, Jianyu Sun a, Simon J. Gauthier a, Maryanne Mattice b,
Laszlo G. Ratkay b, Rainbow Kwan b, Jay Thompson b, Alison Brownlie Cutts b, Jianmin Fu a,
Rajender Kamboj b, Y. Paul Goldberg c, Jay A. Cadieux a,
⇑
a Department of Medicinal Chemistry, Xenon Pharmaceuticals Inc., 3650 Gilmore Way, Burnaby, British Columbia, Canada V5G 4W8
b Department of Biological Sciences, Xenon Pharmaceuticals Inc., 3650 Gilmore Way, Burnaby, British Columbia, Canada V5G 4W8
c Department of Clinical Biology, Xenon Pharmaceuticals Inc., 3650 Gilmore Way, Burnaby, British Columbia, Canada V5G 4W8
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of intestinal brush border DMT1 offers a novel therapeutic approach to the prevention and
treatment of disorders of iron overload. Several series of diaryl and tricyclic benzylisothiourea com-
pounds as novel and potent DMT1 inhibitors were discovered from the original hit compound 1. These
compounds demonstrated in vitro potency against DMT1, desirable cell permeability properties and a
dose-dependent inhibition of iron uptake in an acute rat model of iron hyperabsorption. Tricyclic com-
pounds increased the in vitro potency by up to 16-fold versus the original hit. Diaryl compounds 6b
and 14a demonstrated significant iron absorption inhibition in vivo with both 25 and 50 mg/kg doses.
The diaryl and tricyclic compounds described in this report represent promising structural templates
for further optimization.
Received 5 March 2012
Revised 19 May 2012
Accepted 29 May 2012
Available online 15 June 2012
Keywords:
Hemochromatosis
DMT1
Iron overload
Diarylether
Ó 2012 Elsevier Ltd. All rights reserved.
Diarythioether
Dibenzofuran
Dibenzothiophene
Iron is an essential nutrient that is toxic in excess. Iron levels in
humans are primarily regulated at the level of intestinal iron
absorption. Divalent metal transporter 1 (DMT1) (also known as
natural resistance-associated macrophage protein-2 (NRAMP2),
divalent cation transporter-1 (DCT1) and solute carrier family 11,
member 2 (SLC11A2)) is responsible for the transport of several
divalent metal ions across the cell membrane.1–5 DMT1 is particu-
larly important for iron absorption in the duodenum, where it is
localized in the villus enterocytes and mediates the influx of die-
tary non-heme iron from the intestinal lumen.6 Once dietary iron
is absorbed across the intestinal wall, there is no physiologic mech-
anism for its excretion. Thus, excess absorbed iron is largely re-
tained in the body and accumulates in the heart, liver and other
vital organs.7,8 Excess tissue iron can lead to significant tissue dam-
age8 and has been associated with a number of disease states
including cardiomyopathy, cirrhosis, arthritis, hepatocellular carci-
noma and others.9–12 Hereditary hemochromatosis (HH) is charac-
terized by excessive intestinal iron absorption resulting in tissue
iron overload and damage. There is now compelling evidence
associating inappropriate excess DMT1 activity with HH13,14 and
related conditions. Currently, the standard treatment for HH is
repeat phlebotomy. However, poor patient compliance is a signifi-
cant issue with this invasive treatment.15 Furthermore, phlebot-
omy itself can exacerbate the underlying causal iron
hyperabsorption as the body responds to the loss of red blood cells
by increasing erythropoiesis and iron uptake via intestinal DMT1.16
Another potential indication for DMT1 inhibitor therapy is the
common hemoglobinopathy thalassemia major, in which patients
exhibit a severe anemia requiring chronic blood transfusions. The
iron overload (and ensuing complications) resulting from the
transfused erythrocytes is further compounded by the underlying
anemia, which in turn stimulates absorption of dietary iron
through DMT1. Patients with thalassemia intermedia typically do
not require significant blood transfusions, but nevertheless, due
to the anemia have DMT1 upregulation and intestinal iron hyper-
absorption resulting in iron overload. Phlebotomy is not an option
for patients with thalassemia (as they are anemic), and the present
standard of care for transfusional iron overload is chelation ther-
apy. However, these agents either have poor oral bioavailability
and/or suffer from serious dose-limiting side effects.17–19 Direct
blockade of DMT1-mediated iron influx by an orally-administered
⇑
Corresponding author. Tel.: +1 604 484 3331; fax: +1 604 484 3321.
E-mail address: jcadieux@xenon-pharma.com (J.A. Cadieux).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.129

Z. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5108–5113
5109
0.29
l
M
against DMT1 and modest Caco-2 permeability
NH
NH
NH2
(Papp = 0.9 ꢀ 10ꢁ6 cm/s (a?b), 1.2?10ꢁ6 cm/s (b?a)). Our initial
SAR effort was focused on the replacement of the isothiourea head
groups present in 1 with other moieties (e.g., amide, reverse amide,
amidine, guanidine, cyanoguanidine, sulfonamide and carbamate),
but this endeavor proved to be futile as all of these modifications
resulted in a dramatic decrease in inhibitory activity on DMT1.
Modifications to the chain length between the phenyl ring and
the terminal isothiourea groups of the two arms, the introduction
of substituents on the benzylic carbons and replacement of the
phenyl ring with pyridine or saturated hydrocarbon rings also
proved to be detrimental to target potency. Although we were
not able to generate improvements over the original hit compound
1 from these early investigations, we gained knowledge that the
terminal isothiourea groups and the distance between the two
isothiourea head groups were critical to retain the inhibitory activ-
ity against DMT1. Therefore, our efforts were then focused on ana-
logues containing the isothiourea head groups.
H2N
S
S
1
IC50 = 0.29 µM
Figure 1. Structure of the HTS hit 1.
HN
H2N
S
S
NH
NH2
L
L = O, S, C=O, NCH3
Figure 2. Structures of diaryl isothioureas.
When the phenyl anchor ring was replaced with a diaryl tem-
plate (Fig. 2), this transformation (which also afforded novelty)
led to the retention of inhibitory activity against DMT1. Based on
this development, optimization of the diaryl template was carried
out.
Syntheses of the diaryl compounds are outlined in Schemes 1
and 2. The commercially-available starting materials 2 (Scheme
1) were coupled with acids 3 under Ullmann conditions in the
presence of Cu powder and K2CO3 at 130–140 °C in a sealed tube
for 3 h. The di-acids 4 obtained were reduced to the di-alcohols 5
with borane/THF complex at room temperature overnight. The
di-alcohol 5 was treated with HBr and thiourea to afford the final
diaryl compounds 6a–s as their corresponding HBr salts.
small molecule inhibitor could be expected to lead to an improved
therapeutic approach for the treatment of iron overload. However,
DMT1 is also expressed in erythroid cells, macrophages and hepa-
tocytes and inhibitors potentially compromise iron metabolism.
Therefore, limiting the exposure of a compound to the gut (i.e.,
minimizing systemic bioavailability) could be advantageous from
a safety perspective.
To date, there have been limited reports of small-molecule com-
pounds that specifically modulate or inhibit DMT1.20,21 We previ-
ously reported a series of potent and efficacious pyrazole DMT1
blockers with significant Caco-2 permeability.22,23 In order to iden-
tify potent compounds with a higher potential for gut restriction, a
high throughput screen (HTS) using a calcein fluorescence assay
was carried out as previously described.22,24,25 This effort led to
the identification of the hit compound 1 (Fig. 1) with an IC50 of
Alternatively, the commercially-available phenols 7 (Scheme 2)
were coupled with halides 8 under Ullmann coupling conditions in
the presence of CuI and Cs2CO3 at 120 °C in a sealed tube overnight
HO
OH
H2N
S
S
NH2
NH
R5
HO
O
O
OH
HO
O
O
OH
L
NH
R1
L
RH
X
a
L
b
c
+
R1
R3
R5
R3
R1
R3
R5
R1
R3
R5
R2
R4
R2
R4
R2
R4
R2
R4
2
3
4
5
6a-s
L = S, NCH3
Scheme 1. Reagents and conditions: (a) Cu powder, K2CO3, H2O, 130–140 °C, 3 h; (b) BH3, THF, rt, 16 h; (c) thiourea, 48% aqueous HBr, 80 °C, 3 h.
OH
X
O
a
+
R1
R3
R5
R1
R3
R5
R2
R4
R2
R4
Br
Br
H2N
S
S
NH2
7
8
9
d
e
L
NH
R1
L
NH
R5
R1
R3
R5
R3
R'
R2
R4
R2
R4
MgBr
NC
R3
b
13
14a-m
+
R1
R5
R1
R3
R5
L = O, C=O
R2
10
R4
R2
R4
12a R' = NH
12b R' = O
11
c
Scheme 2. Reagents and conditions: (a) CuI, N,N-dimethylglycine hydrochloride, Cs2CO3, dioxane, 120 °C, 16 h; (b) 0–80 °C, under nitrogen atmosphere, 16 h; (c) 1-propanol,
6 N hydrochloric acid, rt, 16 h; (d) N-bromosuccinimide, benzoyl peroxide, CCl4, reflux, 12 h; (e) thiourea, ethanol, 80 °C, 16 h.

5110
Z. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5108–5113
to afford diarylethers 9. The diaryl ketone 12b was obtained via an
imine intermediate after the reaction between the Grignard re-
agent 10 and the nitrile 11, followed by treatment with acid. Both
methyl groups of compounds 9 and 12b were brominated under
free radical conditions using NBS and benzoyl peroxide under re-
flux for 12 h in CCl4 to generate the di-bromo compounds 13.
The bromo groups of 13 were replaced with thiourea to generate
diarylethers or diaryl ketones 14a–m as their corresponding HBr
salts.
The inhibitory activity of these analogues was assessed by cal-
cein fluorescence assay as previously reported22 and is summa-
rized in Table 1. The modification of the linker between the two
aryl groups seemed to have little effect on DMT1 inhibitory activity
as shown by compounds 6a (S), 6s (N-CH3), 14a (O) and 14i (C@O).
All of these linker-modified compounds had IC50 values in the cal-
strated by the results obtained using a Caco-2 cell permeability as-
say (Table 1), consistent with the desired gut-restricted profile.
A representative group of diaryl compounds (6a, 6b and 14a)
were then evaluated in an acute model of iron hyperabsorption
in anemic (hemoglobin <6 mg/dL, serum iron <50 lg/dL), conscious
rats.22,26 Serum iron was determined 1 h following an oral iron
challenge of 1 mg/kg Fe2+ po (as ferrous sulfate) which, in turn,
was administered 1 h post po dosing of the test article (formulated
in 5% DMSO, 10% EtOH, 30% PEG400, 35% propylene glycol, 20%
water). All compounds tested clearly demonstrated a dose-depen-
dent inhibition of iron absorption in vivo (Fig. 3) and the inhibitory
effects of all three compounds were statistically significant (p
<0.001) at the 50 mg/kg dose level. Diaryl compounds 6b and
14a also demonstrated significant inhibition of iron absorption in
this model at the 25 mg/kg dose level. Diaryl compound 14a was
the most efficacious among the compounds tested with an eight-
fold decrease in iron absorption after oral iron challenge at
50 mg/kg when compared to its vehicle control group. All com-
pounds were well-tolerated with no clinical observations noted
during these studies.
cein quench assay between 0.4 and 0.8 lM, about 2- to 3-fold less
potent than the original hit 1. The most potent compound was 6b
which was about threefold more potent than compound 1. Substit-
uents at the R2, R3 and R4 positions seemed to have more profound
effects on activity and resulted in dramatic decreases in potency as
demonstrated by compounds 6o, 6q, 6r, 14h and 14m. The simul-
taneous modification of substituents on both phenyl rings did not
afford a substantial increases in inhibitory activity. Most of the
compounds tested had very poor cell permeability as demon-
In an attempt to further improve in vitro activity against DMT1
by eliminating rotation about the aryl–aryl bond, the diaryl ether
compound 14a was rigidified, leading to the dibenzofuran 18a.
Interestingly, this transformation indeed generated a very potent
Table 1
SAR of diaryl compounds
H2N
S
S
NH2
NH
NH
R1
L
R3
R5
R2
R4
a
Compound
L
R1
R2
R3
R4
R5
Calcein IC50
(l
M)
Cell permeability (ꢀ 10ꢁ6 cm/s)b
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
H
F
F
F
H
H
H
H
H
H
F
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
Cl
F
H
H
Cl
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
NO2
H
F
F
OCH3
H
H
H
F
CF3
H
F
H
H
Cl
H
H
H
H
0.18
0.08
0.15
0.15
0.29
0.53
0.32
0.49
0.51
0.60
0.69
0.91
0.95
1.44
1.45
3.02
3.77
4.15
0.73
0.34
0.16
0.32
0.34
0.84
1.13
2.89
15.0
0.37
0.58
2.02
2.92
5.20
0.1/1.4
0.1/0.2
0.3/0.2
0.4/0.4
<0.1/0.1
2.4/2.4
1.3/1.4
1.5/1.4
0.3/0.2
<0.1/0.2
0.1/3.8
NDc
<0.1/0.1
ND
ND
ND
ND
SO2CH3
F
F
CH3
H
F
F
NH2
SO2N(CH3)2
OCH3
F
Cl
H
F
H
H
F
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
NO2
H
H
H
H
S
S
6r
6s
ND
ND
N-CH3
O
O
O
O
O
O
O
O
C@O
C@O
C@O
C@O
C@O
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
14l
14m
0.1/0.2
ND
<0.1/0.2
0.2/0.9
<0.1/0.4
ND
ND
ND
0.1/0.7
ND
ND
NO2
F
F
F
H
H
H
Cl
H
H
F
H
Cl
H
F
Cl
NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
ND
ND
a
b
c
IC50s are an average of at least two independent determinations.
Permeability determined using Caco-2 cells. Data expressed as Papp(a?b)/Papp(b?a).
ND: not determined.

Z. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5108–5113
5111
intramolecular cyclization of the diazonium salt in the presence
of a catalytic amount of palladium(II) acetate afforded the tricyclic
intermediates 16. Alternatively, intermediates 16 were obtained
via intramolecular cyclization of 2,20-diiodo-6,60-dimethyl-1,10-
biphenyl by heating at 250–270 °C in the presence of Cu for 2 h.
Bromination of intermediates 16 with N-bromosuccinimide gener-
ated di-bromo compounds 17 and subsequent displacement of the
bromo groups with thiourea afforded compounds 18a–d and 18g–
i as their corresponding HBr salts. Compounds 18e and 18f were
obtained similarly following the bromination of the corresponding
di-methyl intermediates, which were synthesized according to lit-
erature procedures.27,28 The di-acid 19 (Scheme 4) was reduced to
di-alcohol 20 with borane-THF complex. Bromination of 20 with
PBr3 followed by treatment with thiourea afforded compound 24.
Compound 25 was prepared by treatment of acridine 22 with
bromo(methoxy)methane in concentrated sulfuric acid followed
by replacement of the bromo groups of dibromide 23 with thio-
urea. Compound 26 (Scheme 5) was treated with bromine in acetic
acid at room temperature to generate either di- or mono-substi-
tuted bromo compounds 27 followed by treatment with n-butyl
lithium and subsequently either N-fluorobenzene-sulfonimide or
hexachloroethane to afford the fluoro or chloro compounds 28.
Bromination of the methyl groups with NBS generated compound
29. The displacement of the bromo groups with thiourea provided
compounds 30a–e as their corresponding HBr salts.
Figure 3. Effect of DMT1 inhibitors on iron absorption in an acute rat model of iron
hyperabsorption. Each bar represents the average of 8 animals and the error bars
represent standard errors of the mean. ⁄⁄⁄: one-way ANOVA analysis with Dunnett’s
multiple comparison tests when compared to its corresponding vehicle group (p
<0.001).
The inhibitory activities of these tricyclic compounds are sum-
marized in Tables 2 and 3. Dibenzofuran compound 18a was the
most potent compound with an IC50 of 51 nM. The biphenylene
compound 18b was also very potent (IC50 = 97 nM). Both dibenzo-
thiophene 18c and fluorenone 18d were less potent compared to
18a and 18b, but they were nevertheless comparable to compound
1. Interestingly, compound 18e was about 20-fold less potent than
compound 18c and compound 18f was not potent while compound
and novel compound. As a result, several tricyclic analogues were
then prepared (Schemes 3–5). The starting materials 15 (Scheme
3) were obtained following the procedures as outlined in Scheme
1 or Scheme 2. Compounds 15 were then treated with sodium
nitrite at 0 °C in the presence of tetrafluoroboric acid. The
J
a
R2
H2N
R5
H2N
S
S
NH2
NH
Br
Br
c
d
J
NH
R2
J
J
15
R2
R5
R5
R2
R5
16
17
18a-d, 18g-i
b
J = direct bond, O, S, C=O
I
I
Scheme 3. Reagents and conditions: (a) 48% aqueous tetrafluoroboric acid, NaNO2, THF/H2O, Pd(OAc)2, 0–60 °C to rt; (b) Cu, 250–270 °C, 2 h; (c) NBS, benzoyl peroxide, CCl4,
reflux, 12 h; (d) thiourea, ethanol, 80 °C, 16 h.
HO
O
O
OH
H2N
S
S
NH2
NH
HO
OH
Br
Br
O
NH
O
O
O
a
d
b
S
S
S
S
24
19
20
21
Br
Br
H2N
S
S
NH2
NH
N
d
c
N
NH
N
22
23
25
Scheme 4. Reagents and conditions: (a) BH3, THF, rt, 16 h; (b) PBr3, anhydrous diethyl ether, rt, 16 h; (c) bromo(methoxy)methane, concd H2SO4; (d) thiourea, ethanol, 80 °C,
16 h.

5112
Z. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5108–5113
R1
a
b
J
J
J
R6
R6
Br
26
27
28
H2N
NH
HN
NH2
Br
Br
S
S
d
c
R6
R1
J
R6
R1
J
29
3e-a0
J = O and S
Scheme 5. Reagents and conditions: (a) bromine, acetic acid, rt, 16 h; (b) n-butyllithium, N-fluorobenzenesulfonimide or hexachloroethane, THF, ꢁ78 °C, 5 h; (c) NBS, benzoyl
peroxide, CCl4, reflux, 12 h; (d) thiourea, ethanol, 80 °C, 16 h.
Table 2
SAR of tricyclic compounds
S
NH2
NH
R =
Cell permeability (ꢀ 10ꢁ6 cm/s)b
a
Compound
Structure
Calcein IC50
(lM)
R
R
R
O
18a
0.05
<0.1/0.2
R
R
18b
18c
18d
0.10
0.26
0.58
<0.1/0.4
<0.1/<0.1
0.4/0.3
R
S
O
R
R
R
R
18e
18f
5.04
>15
NDc
ND
S
R
R
O
R
R
R
N
24
25
0.15
0.29
ND
R
O
S
0.2/0.5
a
b
c
IC50s are an average of at least two independent determinations.
Permeability determined using Caco-2 cells. Data expressed as Papp(a?b)/Papp(b?a).
ND: not determined.
18d had sub-micromolar activity. The six-membered middle ring
analogues 24 and 25 retained activity against DMT1. Introduction
of electron-withdrawing groups to the meta-positions (R2 and R5)
of the phenyl rings increased the potency by about 2- to 3-fold
(compounds 18g, 18h, and 18i when compared to their un-substi-
tuted counterparts 18a and 18c, respectively). The introduction of

Z. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5108–5113
5113
Table 3
SAR of tricyclic compounds bearing further aryl ring substituents
NH2
H2N
HN
NH
S
S
J
R1
R6
R5
R2
R5
a
Compound
J
R1
R2
R6
Calcein IC50 (lM)
Cell permeability (ꢀ 10ꢁ6 cm/s)b
18g
18h
18i
30a
30b
30c
30d
30e
O
S
S
O
O
O
S
H
H
H
F
Br
Cl
Br
Br
F
F
F
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
F
Br
Cl
H
0.02
0.04
0.09
0.09
0.11
0.27
0.75
0.86
NDC
0.5/0.2
ND
5.0/1.0
0.2/0.1
<0.1/0.4
1.3/0.2
4.0/2.0
S
BR
a
b
c
IC50s are an average of at least two independent determinations.
Permeability determined using Caco-2 cells. Data expressed as Papp(a?b)/Papp(b?a).
ND: not determined.
fluoro groups to the ortho-positions (R1 and R6) of the phenyl rings
did not have much impact on activity (compound 30a). However,
the introduction of other halides at these same positions decreased
the activity by at least 10-fold (compounds 30b–e). None of these
tricyclic compounds demonstrated any significant cell permeabil-
ity. It should be noted that all compounds listed in Tables 1–3 were
found to be inactive (>10 lM) against the closely related transport
protein NRAMP1 (as determined in a variant of the calcein quench
assay using NRAMP1-transfected CHO cells).
References and notes
1. Garrick, M. D. Genes Nutr. 2011, 6, 45.
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G981.
4. Mims, M. P.; Prchal, J. T. Hematology 2005, 10, 339.
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F.; Nussberger, S.; Gollan, J. L.; Hediger, M. A. Nature 1997, 388, 482.
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7. Porter, J. Hematol. Oncol. Clin. North Am. 2005, 19, 7.
In summary, we discovered diaryl and tricyclic benzylisothiou-
rea compounds as novel and potent DMT1 inhibitors from the ori-
ginal hit compound 1. Both diaryl and tricyclic compounds
demonstrated in vitro potency against DMT1, desirable cell per-
meability properties and good selectivity against the closely re-
lated transporter protein NRAMP1. Several analogs reported
herein are the most potent DMT1 inhibitors reported to date
(e.g., 18g with threefold greater potency than compound 8a of
Ref. 22). The diaryl compounds also demonstrated a dose-depen-
dent inhibition of iron absorption in an acute rat model of iron
hyperabsorption. The tricyclic compounds showed increases in
in vitro potency of up to 16-fold with respect to 1. Diaryl com-
pounds 6b and 14a demonstrated significant iron absorption inhi-
bition in vivo with both 25 and 50 mg/kg doses. The diaryl and
tricyclic compounds described in this report represent promising
structural templates for further optimization, the results of which
will be reported in due course.
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Acknowledgments
The authors are grateful to Genome British Columbia and to the
Natural Sciences and Engineering Research Council of Canada for
financial support and to Monica Mork for technical assistance.
25. As previously reported (Ref. 22), a cell-free competition variant of the calcein
quench assay was developed in order to rule out chelation of ferrous iron as the
primary source of the observed activity. All compounds reported herein had
responses <20% of that of the known chelator desferoxamine in this assay.
26. All in vivo protocols described in this manuscript were reviewed and approved
by Xenon Pharmaceuticals Inc.’s Institutional Animal Care and Use Committee
(IACUC).
Supplementary data
Supplementary data (A detailed description of the conditions
employed for the calcein quench assay as well as representative
IC50 curves generated by this assay) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2012.05.129.
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