Synthesis and biological evaluation of substituted pyrazoles as blockers of divalent metal transporter 1 (DMT1)

Article abstract of DOI:10.1016/j.bmcl.2011.11.069

Three distinct series of substituted pyrazole blockers of divalent metal transporter 1 (DMT1) were elaborated from the high-throughput screening pyrazolone hit 1. Preliminary hit-to-lead efforts revealed a preference for electron-withdrawing substituents in the 4-amido-5-hydroxypyrazole series 6a-l. In turn, this preference was more pronounced in a series of 4-aryl-5- hydroxypyrazoles 8a-j. The representative analogs 6f and 12f were found to be efficacious in a rodent model of acute iron hyperabsorption. These three series represent promising starting points for lead optimization efforts aimed at the discovery of DMT1 blockers as iron overload therapeutics.

Full text of DOI:10.1016/j.bmcl.2011.11.069

Bioorganic & Medicinal Chemistry Letters 22 (2012) 90–95
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of substituted pyrazoles as blockers
of divalent metal transporter 1 (DMT1)
Jay A. Cadieux a, , Zaihui Zhang a, Maryanne Mattice b, Alison Brownlie-Cutts b, Jianmin Fu a,
⇑
Laszlo G. Ratkay b, Rainbow Kwan b, Jay Thompson b, Joseph Sanghara b, Jing Zhong b, Y. Paul Goldberg c
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:
Three distinct series of substituted pyrazole blockers of divalent metal transporter 1 (DMT1) were
elaborated from the high-throughput screening pyrazolone hit 1. Preliminary hit-to-lead efforts revealed
a preference for electron-withdrawing substituents in the 4-amido-5-hydroxypyrazole series 6a–l. In
turn, this preference was more pronounced in a series of 4-aryl-5-hydroxypyrazoles 8a–j. The represen-
tative analogs 6f and 12f were found to be efficacious in a rodent model of acute iron hyperabsorption.
These three series represent promising starting points for lead optimization efforts aimed at the discov-
ery of DMT1 blockers as iron overload therapeutics.
Received 12 October 2011
Revised 14 November 2011
Accepted 18 November 2011
Available online 25 November 2011
Keywords:
Pyrazole
Hemochromatosis
DMT1
Ó 2011 Elsevier Ltd. All rights reserved.
Iron overload
Thalassemia
The transport protein divalent metal transporter 1 (DMT1, also
known as NRAMP2, SLC11A2 and DCT1) is responsible for the
transport of several divalent metals across the cell membrane.1
This transporter, which is highly expressed in proximal duodenal
enterocytes, plays a pivotal role in iron homeostasis as it is respon-
sible for the uptake of dietary non-heme iron.2 Under normal phys-
iological conditions, DMT1 is active at a basal level whereas in
circumstances of iron depletion (e.g., iron-restricted anemia) it is
upregulated in enterocytes. Moreover, in certain conditions of iron
overload, regulation of DMT1 expression is impaired and the trans-
porter is inappropriately upregulated. This upregulation is ob-
served in hereditary hemochromatosis (HH), a common genetic
disorder in which dietary iron accumulates to toxic levels and
which is characterized by cardiomyopathy, diabetes, cirrhosis,
arthritis, hepatocellular carcinoma and ‘bronzing’ (tanning) of the
skin.3 The standard treatment for HH is life-long repeat phlebot-
omy. However, poor patient compliance is a significant issue with
this invasive treatment.4 Furthermore, phlebotomy itself can
exacerbate the underlying causal iron hyperabsorption as the body
responds to blood loss by increasing intestinal iron absorption via
upregulation of intestinal DMT1.5 Thus, a DMT1 inhibitor will serve
to prevent the iron hyperabsorption in HH.
patients exhibit a chronic anemia but often do not require blood
transfusions. Iron overload occurs due to intestinal hyperabsorp-
tion which is mediated through upregulated DMT1.
In light of the aforementioned unmet medical need in the treat-
ment of iron overload disorders, direct blockade of DMT1-medi-
ated iron influx by an orally-administered safe and well-tolerated
small molecule inhibitor could represent a novel therapeutic
approach.
Sporadic reports of small molecules capable of attenuating
DMT1-mediated Fe2+ uptake in vitro have appeared in the litera-
ture. However, these compounds were deemed unsuitable for an
early-stage hit-to-lead medicinal chemistry campaign on account
of modest potency,6 cytotoxicity6a or known off-target activities.6b
A high-throughput screening (HTS) campaign predicated on
fluorescence quenching of the intracellular dye acetomethoxycal-
cein (calcein-AM)7 was carried out on a library assembled in-house
from the diversity-oriented collections of five commercial vendors.
In this assay, Chinese hamster ovary (CHO) cells transformed with
human DMT1B + IRE are loaded with calcein-AM and incubated
with test compounds. Upon the subsequent addition of ferrous sul-
fate to the assay medium, DMT1-mediated uptake of ferrous ion
leads to the formation of a calcein–Fe2+ chelate, resulting in the
quenching of calcein’s inherent fluorescence. In the presence of
DMT1 blockers, ferrous ion uptake and the ensuing fluorescence
quenching are attenuated.8 From this screening campaign, the tri-
azinone pyrazolone 1 emerged as a candidate for hit-to-lead efforts
(Fig. 1). Preliminary studies aimed at simplifying the triazinone
A second potential indication for DMT1 inhibitor therapy is the
common hemoglobinopathy thalassemia intermedia, in which
⇑
Corresponding author. Tel.: +1 604 484 3300x157; fax: +1 604 484 3321.
E-mail address: jcadieux@xenon-pharma.com (J.A. Cadieux).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.069

J. A. Cadieux et al. / Bioorg. Med. Chem. Lett. 22 (2012) 90–95
91
moiety present in 1 led to the corresponding pyridyl analog 2 with
slightly improved potency against DMT1. The benzylidene function
present in 2 imparted several undesirable features, most notably
an extended conjugation motif affording an intense orange color
with 2-benzothiazole or 2-benzimidazole functions (9a–d) affor-
ded modest increases in potency, along with high microsomal
metabolic stability. On the other hand, comparable substitution
with a 2-thiazole ring led to a reduction in potency (9e). The
presence of a 2-pyridmidyl (9g) or 2-isoquinolyl (9i) substituent
was not tolerated, nor was the insertion of a methylene spacer
between the pyrazole and pyridine rings (9f).
as well as Michael acceptor properties at the benzylidene
a-car-
bon. The initial aim of the ensuing hit-to-lead efforts was the
replacement of the pyrazolone 4-benzylidene substituent with
benzamide or aryl functions in order to improve the potency and
ADME properties of 2.
The introduction of a cyclic constraint by means of tethering C-3
of the pyrazole ring and C-2 of the 4-aryl substituent with an ethyl-
ene linker (12a–o, Table 4) was then carried out. In addition to pro-
viding a series of novel analogs for further optimization, it was
hoped that the ensuing partial rigidification would in turn afford
an increase in target potency. In general, these analogs were equi-
potent to their corresponding ‘acyclic’ 4-aryl-5-hydroxypyrazoles
(Tables 2 and 3), less soluble and more metabolically labile (poten-
tially on account of benzylic oxidation at the ethylene linker). The
strong preference for electron-withdrawing substituents observed
for the 4-aryl-5-hydroxypyrazoles 8a–j did not extend to this rigid-
ified series (cf. 12j–o), as the presence of the ethylene linker pre-
vents optimal electronic interaction between the pyrazole and
aryl rings.15 Conversion of the active 2-pyridyl analog 12f into its
corresponding N-oxide 12h was not tolerated, nor was the intro-
duction of a methyl group (12i) to the 4-position of the benzothia-
zole ring in 12b. None of the analogs in Tables 1–4 were found to be
Treatment of the pyrazolones 5 obtained via condensation9 of
arylhydrazines 3 and ethyl acetoacetate 4 (Scheme 1) with aryl iso-
cyanates under basic conditions afforded the 4-amido-5-hydrox-
ypyrazoles 6a–l.10 Pd-catalyzed
a
-arylation of ethyl acetoacetate
4,11 followed by condensation of the resultant
a-aryl-b-ketoesters
7 with a variety of hydrazines led to the formation of 4-aryl-5-
hydroxypyrazoles 8a–i and 9a–j. The constrained analogs 12a–o
were in turn prepared by treating the sodium enolates of the 6-
substituted-2-tetralones 10 with diethyl carbonate12 followed by
suitable aryl or heteroaryl hydrazines.
The 4-amido derivatives 6a–l (for which some conformational
rigidity imparted by intramolecular hydrogen bonding was envis-
aged10b) generally exhibited acceptable solubility in phosphate-
buffered saline and were relatively resistant to metabolism in rat
liver microsome incubations (Table 1). This series was devoid of
appreciable CYP3A4 inhibition (in a recombinant human enzyme
assay), with the exception of the methylenedioxyphenyl analog
6k.13 However, permeability (Caco-2) data suggest that some of
these analogs (e.g., 6d, 6h and 6k) with Papp ratios in excess of 2
may be subject to efflux transport. In terms of potency against
DMT1, a variety of substituents on the 4-aryl ring were tolerated.
Electron-withdrawing substituents (6a, 6b, 6d) were slightly pre-
ferred over electron-donating ones (6g, 6k). Moreover, substitution
at the aryl ring’s 2-position was disfavored over substitution at the
corresponding 3- or 4-positions (cf. 6i vs 6b as well as 6j vs 6a and
6d). Replacement of the 2-pyridyl group at the pyrazole N-1 posi-
tion with a phenyl group (6l) was, however, not tolerated.
The 4-aryl-5-hydroxypyrazoles 8a–j were typically less soluble
and metabolically less stable than the 4-amido-5-hydroxypyraz-
oles 6a–l (Table 2). Analogs in this series were also free of signifi-
cant CYP3A4 inhibition liabilities and certain analogs here also
showed a tendency towards efflux transport. In this series, a
marked preference for electron-withdrawing substituents on the
aromatic ring was noted: the coefficient of determination R2 in a
cytotoxic to HepG2 cells after incubation at 10 l
M for 24 h.16
The calcein quench assay employed in this study and in earlier
investigations of DMT16b is a very sensitive measure of ferrous in-
flux, but inhibition of calcein quench can occur by mechanisms
other than direct effects on the DMT1 transporter. Since ferrous in-
flux involves electrogenic co-transport of protons and ferrous
ions,1e agents which chelate Fe2+, disturb the pH gradient, are re-
dox active or depolarize the cell membrane can all appear active.
One way to establish to establish direct interaction with DMT1 is
to measure the effect of test compounds on ionic current by
whole-cell voltage clamp. This technique allows control of the
intracellular solution and membrane voltage and can rule out all
indirect effects except Fe2+ chelation. In preliminary studies, com-
pounds 6a and 8b were evaluated with this methodology and were
found to be inactive at 10 lM, suggestive of an indirect effect. It
should, however, be noted that unlike some previously reported
antioxidant compounds6b hypothesized to act through modifica-
tion of cellular redox status, the pyrazole analogs in question are
not expected to possess redox activity under physiological
conditions.
In order to rule out the possibility that the majority of the ob-
served activity of these pyrazoles in the calcein quench assay
was attributable to chelation of Fe2+ (e.g., bidentate chelation
through the 5-hydroxy function and the pyridyl N in 8a–j) as op-
posed to intrinsic activity against DMT1, a cell-free competition
variant of this assay was carried out. Test compounds (final well
linear regression of potency (as pIC50
) and the substituent
Hammett constant
r
was 0.815.14 Potency in the 4-aryl series, in
which the 4-substituent is directly bonded to the pyrazole ring,
was far more sensitive to substituent effects than it was for the
4-amido analogs.
In order to probe the SAR at the pyrazole N-1 substituent, a
series of 1-substituted 3-methyl-4-phenyl-5-hydroxypyrazoles
9a–i was then prepared (Table 3). Several heterocycles possessing
concentration 10
were mixed, freshly-prepared Fe2+ (as ferrous ammonium sulfate,
2 lM) was added and the calcein fluorescence was measured after
lM in 12.6% aqueous DMSO) and calcein (1 lM)
heteroatoms
a to the site of linkage to the pyrazole ring were
found to be potent. Replacement of the 2-pyridyl moiety in 8g
incubation for 20 min. As Fe2+ quenches the fluorescence of calcein,
the residual fluorescence persisting after the addition of Fe2+ is a
direct measure of the ability of a compound to chelate Fe2+ away
from calcein. Desferoxamine17 was employed as a positive control.
The pyrazole compounds described herein had responses in this
assay <20% of that of desferoxamine. These data suggest that the
contribution of direct Fe2+ chelation to the observed activity, if
any, was minimal. Moreover, the preference for electron-with-
drawing substituents observed in the 4-aryl-5-hydroxypyrazoles
8a–j is inconsistent with a chelation mechanism; under such a par-
adigm, one would anticipate a preference for electron-releasing
substituents as the latter would increase the Lewis basicity of the
putative chelation sites.18
O
N
N
N
NH
O
N
N
O
N
N
N
N
1
2
IC50 = 2.57 µM
IC50 = 1.53 µM
Figure 1. Structures of the HTS hit 1 and its simplified analog 2.

92
J. A. Cadieux et al. / Bioorg. Med. Chem. Lett. 22 (2012) 90–95
X
N
O
O
X
OH
O
N
a
b
+
OEt
N
X
NHNH2
O
HN
H3C
H3C
6a-l
4
3
HN
5
R
O
O
R1
O
O
N
OEt
OH
N
c
d
OEt
H3C
4
R2
R2
8a-j, 9a-i
7
R1
N
CO2Et
OH
OH
O
N
e
d
R2
R2
10
11
12a-o
R2
Scheme 1. Reagents and conditions: (a) EtOH or HOAc, reflux, 1 h, 32–92%; (b) ArNCO, NEt3, PhH, rt, 16 h, 6–63%; (c) ArBr, K3PO4, Pd(OAc)2 (cat.), 2-di-tert-butylphosphino-
20-methylbiphenyl (cat.), PhMe, 90 °C, 16 h, 32–79%; (d) R1NHNH2, HOAc, reflux, 1–2 h; (e) NaH, (EtO)2CO, reflux, 2 h, 40–74%.
Table 1
SAR of 1-aryl-3-methyl-4-amido-5-hydroxypyrazoles
X
N
OH
N
O
H3C
HN
R
6a-l
Entry
X
R
Calcein assay
Solubilitya
Rat microsome
Permeability (Caco-2)c
CYP3A4 (% inhib. at
10 lM)
IC50
(lM)
(l
g/mL)
metabolic stabilityb
(ꢀ10ꢁ6 cm/s)
2
N
N
N
N
N
N
N
N
N
N
N
N
Not applicable
3-CF3
4-NO2
3-F-4-OCH3
4-CF3
3-CH3
H
3-OCH3
4-CH3
2-NO2
2-CF3
1.53
0.17
0.20
0.26
0.29
0.41
0.64
0.66
0.69
0.71
1.00
1.22
15
>73
34
>73
>73
54
9
0.3/0.6
4.5/8.9
74
12
13
6
18
19
8
14
13
18
18
41
6a
6b
6c
6d
6e
6f
6g
6h
6i
67
82
94
98
71
94
85
74
82
96
91
7.6/11.5
6.6/13.2
0.6/3.9
9.9/15.6
10.4/19.6
9.0/15.4
6.1/15.3
11.2/15.5
7.8/14.2
9.3/20.8
48
>73
>73
45
>73
50
6j
6k
3,4-(–OCH2O–
)
H
6l
CH
>5
>73
NDd
ND
ND
a
b
c
Determined in phosphate-buffered saline, pH 7.4.
Percentage of parent compound remaining following a 30 min incubation at 5 lM.
Transport in a ? b/b ? a directions.
Direct inhibitors of DMT1 are expected to block iron uptake by
and iron-hyperabsorptive through DMT1 upregulation.20 After
confirmation of the rats’ anemic status (hemoglobin <6 mg/dL, ser-
um iron <50 lg/dL) was obtained, 6f (25 or 50 mg/kg po), 12f
(50 mg/kg po) or vehicle (EtOH/PEG400/propylene glycol/DMSO/
H2O 10:30:35:5:20 v/v) was administered by oral gavage followed
enterocytes in vivo and this was evaluated by testing two repre-
sentative analogs (6f and 12f) in an acute rat model of iron hyper-
absorption.19 Briefly, weanling (3–4 weeks of age) rats were placed
on a low (2 ppm) iron diet for four weeks, rendering them anemic

J. A. Cadieux et al. / Bioorg. Med. Chem. Lett. 22 (2012) 90–95
93
Table 2
SAR of 1-(2-pyridyl)-3-methyl-4-aryl-5-hydroxypyrazoles
N
N
OH
N
H3C
R
8a-j
Entry
R
ra
Calcein assay
Solubilityb
g/mL)
Rat microsome
Permeability (Caco-2)d
CYP3A4 (% inhib. at
10 lM)
IC50
(l
M)
(
l
metabolic stabilityc
(ꢀ10ꢁ6 cm/s)
2
Not applicable
4-NO2
4-CF3
3-CF3
4-CN
4-CO2Et
4-F
H
4-OCH3
4-NH2
4-NEt2
Not applicable
0.78
0.54
0.43
0.66
0.45
0.06
0.00
ꢁ0.27
ꢁ0.66
ꢁ0.83
1.53
0.06
0.15
0.32
0.36
0.77
0.91
1.70
2.82
>5
15
14
6
9
0.3/0.6
3.5/5.6
3.1/7.9
7.0/5.8
21.4/27.8
ND
11.2/15.9
8.2/16.7
12.7/16.3
ND
74
8
15
45
20
ND
38
26
8a
8b
8c
8d
8e
8f
8g
8h
8i
84
16
41
32
NDe
16
61
12
ND
ND
18
<5
51
32
37
>73
>73
29
35
ND
ND
8j
>5
ND
a
b
c
Hammett constant (rm or
Determined in phosphate-buffered saline, pH 7.4.
Percentage of parent compound remaining following a 30 min. incubation at 5
Transport in a ? b/b ? a directions.
ND = not determined.
r
p, as appropriate) for R.
lM.
d
e
Table 3
SAR of 1-(2-pyridyl)-3-methyl-4-aryl-5-hydroxypyrazoles
R
N
OH
Ph
N
H3C
9a-i
Entry
R
Calcein assay
Solubilitya
g/mL)
Rat microsome
Permeability (Caco-2)c
CYP3A4 (% inhib. at
10 lM)
IC50
(lM)
(
l
metabolic stabilityb
(ꢀ10ꢁ6 cm/s)
2
Not applicable
6-Fluoro-2-benzothiazyl
2-Benzothiazyl
2-Benzimidazyl
6-Methoxy-2-
benzothiazyl
1.53
0.39
0.43
0.52
1.04
15
16
>73
51
12
9
0.3/0.6
16.8/20.1
6.7/6.6
10.1/13.5
ND
74
6
24
93
9a
9b
9c
9d
98
93
95
NDd
ND
8g
9e
9f
9g
9h
9i
2-Pyridyl
2-Thiazoyl
(2-Pyridyl)methyl
2-Pyrimidyl
4-Hydroxy-6-pyridazyl
2-Isoquinolyl
1.70
2.19
>5
>5
>5
37
>73
47
>73
>73
>73
61
91
ND
ND
ND
ND
8.2/16.7
23.9/23.8
ND
ND
ND
26
21
ND
ND
ND
ND
>5
ND
a
b
c
Determined in phosphate-buffered saline, pH 7.4.
Percentage of parent compound remaining following a 30 min incubation at 5 lM.
Transport in a ? b/b ? a directions.
1 h later by an iron challenge of 1 mg/kg po Fe2+ (as ferrous sul-
fate). In the vehicle-treated hyperabsorptive animals, the iron chal-
lenge resulted in a robust increase in serum iron levels as
measured 1 h post oral iron challenge (Fig. 2). A statistically-signif-
icant attenuation in the post-challenge serum iron increase was
observed in the groups treated with 25 or 50 mg/kg 6f or with
50 mg/kg 12f. The hyperabsorptive state of the rats in this model
was confirmed by comparison with a further vehicle-treated con-
trol group (‘diet control’, Fig. 2) which had been raised on a stan-
dard (230 ppm iron) diet; this group exhibited a ꢂ4-fold lower
iron uptake following the ferrous sulfate challenge relative to the
low-iron diet vehicle control group.
The substituted pyrazoles described in this communication are
among the most potent inhibitors of DMT1-mediated iron influx
reported to date and are devoid of HepG2 cytotoxicity. Direct inhi-
bition is supported by in vivo activity consistent with calcein
quench assay activity as well as the lack of either redox or chela-
tion effects. The three series elaborated from the original screening
hit 1 through the simplified analog 2 represent promising avenues
for subsequent lead optimization activities.

94
J. A. Cadieux et al. / Bioorg. Med. Chem. Lett. 22 (2012) 90–95
Table 4
SAR of tricyclic pyrazole analogs
R1
N
OH
N
R2
12a-o
Entry
R1
R2
Calcein assay
Solubilitya
g/mL)
Rat microsome
Permeability (Caco-2)c
CYP3A4 (% inhib. at
10 lM)
IC50
(lM)
(
l
metabolic stabilityb
(ꢀ10ꢁ6 cm/s)
2
12a
12b
Not applicable
6-Methyl-2-benzothiazyl
6-Methoxy-2-
benzothiazyl
Not applicable
H
H
1.53
0.15
0.33
15
10
32
9
16
74
0.3/0.6
5.2/3.4
12.7/16.7
74
8
9
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
6-Fluoro-2-benzothiazyl
6-Carboxy-2-benzothiazyl
2-Benzothiazyl
2-Pyridyl
2-Benzimidazyl
2-Pyridyl-N-oxide
4-Methyl-2-benzothiazyl
2-Pyridyl
2-Pyridyl
2-Pyridyl
2-Pyridyl
2-Pyridyl
H
H
H
H
H
H
H
0.40
0.56
0.72
0.83
1.76
>5
11
>73
62
>73
17
>73
5
<5
8
13
5
69
4.3/1.1
ND
20.8/24.0
8.9/11.7
5.1/6.3
ND
11
ND
1
NDd
75
63
86
ND
ND
ND
40
52
30
33
ND
44
56
ND
ND
ND
27
ND
21
28
ND
>5
ND
ND
4-(OCH3)Ph
Ph
4-MePh
Br
CN
OCH3
0.21
0.26
0.27
0.31
0.52
2.77
0.1/0.9
ND
3.2/7.6
2.3/13.1
ND
12m
12n
12o
44
52
2-Pyridyl
a
b
c
Determined in phosphate-buffered saline, pH 7.4.
Percentage of parent compound remaining following a 30 min incubation at 5 lM.
Transport in a ? b/b ? a directions.
Gastrointest. Liver Physiol. 2005, 289, G981; (d) Mims, M. P.; Prchal, J. T.
Hematology 2005, 10, 339; (e) Gunshin, H.; Mackenzie, B.; Berger, U. V.;
Gunshin, Y.; Romero, M. F.; Boron, W. F.; Nussberger, S.; Gollan, J. L.; Hediger,
M. A. Nature 1997, 388, 482.
2. (a) Theil, E. C. J. Nutr. 2011, 141, 724S; (b) Sargent, P. J.; Farnaud, S.; Evans, R. W.
Curr. Med. Chem. 2005, 12, 2683; (c) Anderson, G. J.; Frazer, D. M.; McKie, A. T.;
Vulpe, C. D.; Smith, A. Biometals 2005, 18, 339; (d) Aisen, P.; Enns, C.; Wessling-
Resnick, M. Int. J. Biochem. Cell Biol. 2001, 33, 940.
3. (a) van Bokhoven, M. A.; van Deursen, C. T.; Swinkels, D. W. Br. Med. J. 2011,
342, C7251; (b) Pietrangelo, A. Gastroenterology 2010, 139, 393; (c) Hazin, R.;
Abu-Rajab, T. T. I.; Abuzetun, J. Y.; Zein, N. N. Cleve. Clin. J. Med. 2009, 76, 599;
(d) Brissot, P.; Troadec, M. B.; Bardou-Jacquet, E.; Le Lan, C.; Jouanolle, A. M.;
Deugnier, Y.; Loréal, O. Blood Rev. 2008, 22, 195.
4. Hicken, B. L.; Tucker, D. C.; Barton, J. C. Am. J. Gastroenterol. 2003, 98, 2072.
5. Kelleher, T.; Ryan, E.; Barrett, S.; Sweeney, M.; Byrnes, V.; O’Keane, C.; Crowe, J.
Gut 2004, 54, 1174.
6. (a) Buckett, P. D.; Wessling-Resnick, M. Am. J. Physiol. Gastrointest. Liver Physiol.
2009, 296, G798; (b) Wetli, H. A.; Buckett, P. D.; Wessling-Resnick, M. Chem.
Biol. 2006, 13, 965.
7. Hollo, Z.; Homolya, L.; Davis, C. W.; Sarkadi, B. Biochim. Biophys. Acta 1994,
1191, 384.
8. All IC50 data reported herein are means of no fewer than two replicate
experiments.
9. (a) Makhija, M. T.; Kasliwal, R. T.; Kulkarni, V. M.; Neamati, N. Bioorg. Med.
Chem. 2004, 12, 2317; (b) Pal, S.; Mareddy, J.; Devi, N. S. J. Braz. Chem. Soc. 2008,
19, 1207.
Figure 2. Inhibition of intestinal iron uptake by 6f and 12f in an acute rat model of
iron hyperabsorption. Data represent mean SEM (n = 8/group); p values were
determined using a one-way ANOVA with Dunnett’s post-hoc test (for 6f) or an
unpaired t-test (for 12f).
10. (a) Huppatz, J. L. Aust. J. Chem. 1983, 36, 135; for
a discussion on
regioisomerism and tautomerism in this ring system, see: (b) Holzer, W.;
Hallak, L. Heterocycles 2004, 63, 1311. and references cited therein.
11. Zeevaart, J. G.; Parkinson, C. J.; de Koning, C. B. Tetrahedron Lett. 2004, 45, 4261.
12. Li, C. J.; Chen, D. L.; Lu, Y.-Q.; Haberman, J. X.; Mague, J. T. Tetrahedron 1998, 54,
2347.
Acknowledgments
13. The methylenedioxyphenyl moiety is a known structural alert for CYP3A4
inhibition; see: Staack, R. F.; Maurer, H. H. Curr. Drug Metab. 2005, 6, 259.
14. Hansch, C.; Leo, A. J. Substituent Constants for Correlation Analysis in Chemistry
and Biology; Wiley-Interscience: New York, 1979.
15. Lowry, T. H.; Richardson, K. S. Mechanism and Theory in Organic Chemistry, 3rd
ed.; Harper & Row: New York, 1987.
The authors are grateful to Genome British Columbia and to the
Natural Sciences and Engineering Research Council of Canada for
financial support. Charles Cohen and Clint Young are thanked for
helpful discussions during the preparation of this manuscript.
16. Nociari, M. M.; Shalev, A.; Benias, P.; Russo, C. J. Immunol. Methods 1998, 213,
157.
References and notes
17. Hann, H. W.; Stahlhut, M. W.; Hann, C. L. Hepatology 1990, 11, 566.
18. Laurence, C.; Gal, J.-F. Lewis Basicity and Affinity Scales: Data and Measurement;
Wiley: New York, 2010. Chapter 1.
1. (a) Garrick, M. D. Genes Nutr. 2011, 6, 45; (b) Thwaites, D. T.; Anderson, C. M.
Exp. Physiol. 2007, 92, 603; (c) Mackenzie, B.; Garrick, M. D. Am. J. Physiol.

J. A. Cadieux et al. / Bioorg. Med. Chem. Lett. 22 (2012) 90–95
95
19. Brownlie-Cutts, A; Ratkay, L.; Waldbrook, M.; Cadieux, J. A.; Chahal, N.;
Chowdhury, S.; Cohen, C.J.; Cosic, M.; Fu, J.; Grimwood, M. E.; Huang, J.;
Namdari, R.; Samra, P.; Sojo, L. E.; Webb, M.; Winters, C.; Zhang, Z.; Pimstone,
S.; Hayden, M. R.; Goldberg, Y. P. Presented at the 4th Meeting of the
International BioIron Society, Vancouver, BC, May 2011; paper #95. Meeting
abstracts published as Am. J. Hematol. 2011, 86, E1.
20. Ferguson, C. J.; Wareing, M.; Delannoy, M.; Fenton, R.; McLarnon, S. J.; Ashton,
N.; Cox, A. G.; McMahon, R. F.; Garrick, L. M.; Green, R.; Smith, C. P.; Riccardi, D.
Kidney Int. 2003, 64, 1755.