4-(((4-Iodophenyl)methyl)-4H-1,2,4-triazol-4-ylamino)-benzonitrile: A Potential Imaging Agent for Aromatase

Article abstract of DOI:10.1021/acs.jmedchem.6b00849

Aromatase (CYP19) is a rate-limiting enzyme that catalyzes the biosynthesis of estrogens. Imaging agents based on aromatase inhibitors (AIs) have been developed for a PET/SPECT study. A series of compounds was synthesized based on YM511, which has previou

Full text of DOI:10.1021/acs.jmedchem.6b00849

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Article
4-(((4-Iodophenyl)methyl)-4H-1,2,4-triazol-4-ylamino)-
benzonitrile: A potential imaging agent for aromatase
Jin Song, Zehui Wu, Phumvadee Wangtrakuldee, Seok Rye Choi,
Zhihao Zha, Karl Ploessl, Robert H. Mach, and Hank F. Kung
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00849 • Publication Date (Web): 02 Oct 2016
Downloaded from http://pubs.acs.org on October 3, 2016
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4-(((4-Iodophenyl)methyl)-4H-1,2,4-triazol-4-ylamino)-benzo
nitrile: A potential imaging agent for aromatase
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Jin Song1,2, Zehui Wu2, Beau Wangtrakuldee3, Seok Rye Choi2,4, Zhihao
Zha2, Karl Ploessl2,4, Robert H Mach2, Hank Kung*1,2,4
1Beijing Institute for Brain Disorders, Capital Medical University, Beijing, 100069,
China
2Department of Radiology, University of Pennsylvania, Philadelphia, 3700 Market Street,
Suite 305, Pennsylvania 19104
3Department of Systems Pharmacology and Translational Therapeutics and the Center for
Excellence in Environmental Toxicology, Perelman School of Medicine, University of
Pennsylvania, 1315 BRBII/III, 421 Curie Blvd, Philadelphia, PA 19104ꢀ6160
4Five Eleven Pharma Inc., Philadelphia, PA 19104
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Abstract
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Aromatase (CYP19) is a rateꢀlimiting enzyme that catalyzes the biosynthesis of
estrogens. Imaging agents based on aromatase inhibitors (AIs) has been developed for a
PET/SPECT study. A series of compounds was synthesized based on YM511, which has
previously been used for breast cancer treatment. Two examples of these derivatives,
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4ꢀ(((4ꢀiodophenyl)methyl)ꢀ4Hꢀ1,2,4ꢀtriazolꢀ4ꢀylꢀamino)ꢀbenzonitrile
(5)
and
4ꢀ((1Hꢀimidazolꢀ1ꢀyl)(4ꢀiodobenzyl)amino)benzonitrile (11), displayed potent binding
affinities to human aromatase (IC50 = 0.17 and 0.04 nM, respectively). Biodistribution
and autoradiographic studies revealed that [125I]5 and [125I]11 were highly accumulated in
the stomach (16.21 and 10.88% dose/g, respectively) and ovaries (8.56 and 3.32% dose/g,
respectively) of female rats. Log P of [125I]5 was 2.49, meaning good brain penetration.
Autoradiograms of brain sections showed a high uptake in the bed nucleus of the stria
terminalis and amygdala. These results suggest that [125I]5 and [125I]11 are potent probes
for aromatase imaging in both the brain and peripheral organs.
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Introduction
Aromatase, the product of the CYP19 gene (also known as estrogen synthetase or
estrogen synthase), is the obligatory enzyme catalyzing the biosynthesis of estrogens
from androgen precursors. It is a cytochrome P450 protein of the 19A family, also known
as P450AROM estrogen synthase.1 Aromatase is widely distributed in peripheral organs
such as the ovaries,2 skin,3 adipose tissue4 as well as the brain.5 Aromatase has been
reported overexpressed in women diagnosed with breast cancer,6 endometriosis,7 and
liver cancer.8 Brain aromatase is involved in regulating sexual behavior, aggression,
cognition, memory, and neuroprotection.9 It has been reported that the aromatase
immunoreactivity changed during the pathophysiology of Alzheimer’s disease.10
Aromatase inhibitors (AIs) have been increasingly used in the hormonal treatment
of breast cancer.11, 12 The aromatase inhibitors fall into two categories: A) steroidal AIs
such as formestane and exemestane, which bind irreversibly to the active site in
aromatase, and B) nonꢀsteroidal AIs such as aminoglutethimide, fadrozole, anastrozole,
letrozole, and vorozole, whose binding is competitive and reversible to the active site in
aromatase.11 Several AIs, including vorozole, letrozole, and cetrozole have been labeled
with 11C.13ꢀ16 [11C]vorozole was the first aromatase radiotracer used in human brain
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studies and has been wellꢀstudied in rodents, primates, and humans. It accumulated
highly in the amygdala and hypothalamic areas of the brain14, 17, 18 as well as in rat
stomach.15 Because of the absence of regional specificity, [11C]letrozole was deemed
unsuitable as a PET radiotracer for brain aromatase.13 Recently, [11C]cetrozole has been
reported to show high specific binding in the amygdala, hypothalamus, and nucleus
accumbens of rhesus monkeys with a higher signalꢀtoꢀnoise ratio than [11C]vorozole.19
However, the existing 11C radiotracers for aromatase imaging showed several
disadvantages. It is difficult to synthesize the precursor of [11C]vorozole, and the
nonspecific signal in PET imaging is high.19 Although [11C]cetrozole showed a higher
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signal to noise ratio, its radiosynthesis is difficult. The PET imaging study of Fꢀlabeled
vorozole,
6ꢀ[(S)ꢀ(4ꢀchlorophenyl)(1Hꢀ1,2,4ꢀtriazolꢀ1ꢀyl)methyl]ꢀ1ꢀ(2ꢀ[18F]fluoroethyl)ꢀ1Hꢀbenzotri
azole, in monkey brains also showed high nonꢀspecific binding.20
YM511, an aminotriazole derivative, and a nonꢀsteroidal aromatase inhibitor, has
been used for the treatment of breast cancer in postmenopausal women. The early phase
II study of YM511 appeared to be effective and safe in postmenopausal patients with
breast cancer.21 However YM511 was abandoned due to competitive market reasons.22
YM511 has a high binding affinity to human aromatase and a simple structure that is easy
to synthesize. It has potential to be a good PET/SPECT imaging agent of aromatase.
Moreover, the binding patterns of aromatase tracers in peripheral organs need further
study. In order to explore new tracers with potentially better in vivo characteristics for
aromatase imaging, we synthesized a series of compounds, radiolabeled them with Iꢀ125,
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and investigated regional patterns of their specific binding through in vivo and ex vivo
experiments.
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Results
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Chemical synthesis. In order to develop [123I] SPECT imaging agents, 5, 8 and
11 were synthesized according to schemes 1, 2, and 3. Ligands 5, 8, and 11 were derived
from YM511 (1a),23 6a,24 and 925 respectively, ligands which have been reported to show
high binding affinities to aromatase. The synthesis of 1a-c was accomplished by the
reactions described in Scheme 1. These reactions followed methods reported in the
literature. Compound 4 was prepared by reacting 1a with bis(tributyltin). Ligand 4 was
treated with iodine to give 5. Ligands 6a-b and 9 were prepared by a method similar to
the reported procedure.25 Ligands 8 and 11 were prepared in similar fashion as ligand 5.
In order to develop [18F]ꢀlabeled PET imaging agents, 3a and b were synthesized
according to Scheme 1. Ligand 1c was treated with BBr3 to give 2. Ligand 2 was then
treated with either 1ꢀbromoꢀ2ꢀfluoroꢀethane or 3ꢀfluoropropyl 4ꢀmethylbenzenesulfonate
to give 3a or 3b.
Two high performance liquid chromatography (HPLC) conditions were
performed to test the purity of the final compounds, 3a, 3b, 5, 8, and 11. An Agilent
HPLC 1100 series with A) a 5 ꢁm C18 250 mm × 10 mm column, eluted with ACN/10
mM ammonium formate buffer, 6/4, 3 mL/min or B) a 5 ꢁm C18 250 mm × 10 mm
column, eluted with MeOH/10 mM ammonium formate buffer, 7/3, 3 mL/min. All of the
final tested samples showed a purity > 95%. The HPLC profiles are included in the
supplementary information.
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Scheme 1: Synthesis of (4ꢀbenzyl(4Hꢀ1,2,4ꢀtriazolꢀ4ꢀyl)amino)benzonitrile derivatives
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Scheme 2. Synthesis of 4ꢀ((4ꢀiodobenzyl)(pyrimidinꢀ5ꢀyl)amino)benzonitrile
N
N
N
N
N
N
N
N
Pd(PPh3)4 toluene
(SnBu3)2
K2CO3
DMF
N
N
X
N
Br
I2, CH2Cl2
CN
HN
CN
CN
RT, 0.5h
yield 94%
Reflux, 2h
yield 32%
RT, 2h
Br
CN
I
yield 33% Br
SnBu3
8
6a
: X =C, yield 41 %
6b
7
: X =N, yield 81 %
Scheme 3. Synthesis of 4ꢀ((1Hꢀimidazolꢀ1ꢀyl)(4ꢀiodobenzyl)amino)benzonitrile
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In vitro binding assays to human CYP19 (aromatase). An in vitro binding
assay was employed to measure the inhibition of the new compounds to [125I]5 binding to
human CYP19. The half maximal inhibitory concentrations (IC50, nM) of the new
compounds against the binding to human CYP19 are shown in Table 1. High binding
affinities
were
obtained
for
several
compounds.
benzonitrile
(8)
The
ligands
(5),
4ꢀ((4ꢀiodobenzyl)(4Hꢀ1,2,4ꢀtriazolꢀ4ꢀyl)amino)
4ꢀ((4ꢀiodobenzyl)(pyrimidinꢀ5ꢀyl)amino)benzonitrile
and
4ꢀ((1Hꢀimidazolꢀ1ꢀyl)(4ꢀiodobenzyl)amino)benzonitrile (11), showed excellent binding
affinities (IC50 = 0.17, 0.04, and 0.04 nM, respectively), compared to YM511 (IC50 =
0.32 nM). However, ligands 3a and 6b showed a significantly lower affinity (IC50 = 40.2
and 11.6 nM, respectively), and the ligand 3b also showed dramatically low binding
affinity (IC50 = 273 nM).
Table 1. Comparison of the half maximal inhibitory concentration (IC50) of the
human CYP19 (aromatase) targeting ligands against [125I]5 binding to human
CYP19.
compd
YM511
IC50 (nM)
0.32 ± 0.07
7.56 ± 1.79
compd
1c
IC50 (nM)
5.06 ± 0.42
2.88 ± 0.27
40.2 ± 12.3
273 ± 23
compd
IC50 (nM)
0.06 ± 0.02
11.6 ± 3.50
0.04 ± 0.03
0.93 ± 0.48
6a
6b
8
Letrozole
1d
Anastrozole 7.98 ± 1.93
Fadrozole 1.14 ± 0.09
3a
3b
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Exemestane
318 ±133
0.17 ± 0.05
0.04 ± 0.02
5
11
5
6
n = 4. Values were obtained from the mean ± SD of four independent experiments, each in duplicates.
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Radiolabeling of 4-((4-iodobenzyl)(4H-1,2,4-triazol-4-yl)amino)benzonitrile,
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4-((4-iodobenzyl)(pyrimidin-5-yl)amino)benzonitrile,
and
4-((1H-imidazol-1-yl)(4-iodobenzyl)amino)benzonitrile. The radioiodinated ligand,
[125I]5, was prepared from the corresponding tributyltin precursor, 4, by an
iododestannylation reaction (see Scheme 4). After the reaction was terminated, HPLC
purification was performed on a 5 ꢁm C18 250 mm × 4.6 mm column. Radiochemical
yield was 57% (decay corrected (dc)) with a radiochemical purity of >99% after HPLC
purification. The radiochemical identity of the radioiodinated ligand was verified by a
coꢀinjection of the “cold compound”, 5 (see Figure 1). The radiolabeling of [125I]8 (RCY
63% (dc); RCP >99%) and [125I]11 (RCY 61% (dc); RCP >99%) were successfully
performed using the same protocol as [125I]5. The same procedure will be suitable for
preparation of 123I labeled agents, which are suitable for SPECT imaging.
Scheme 4. Radiosynthesis of [125I]5, [125I]8 and [125I]11
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UV @ 280 nm of cold standard 5
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Radio trace of [125I]5
Figure 1. HPLC profiles of cold standard 5 and [125I]5 on a 5 ꢁm C18 250 mm × 4.6 mm
column with the following gradient and a flow rate: 1 mL/min, 0ꢀ15 min ammonium
formate buffer (10 mM) 90 to 0%, ACN 10ꢀ100%; 15ꢀ20 min ACN 100%.
Partition coefficient (Log P). Partition coefficients between 1ꢀoctanol/buffer of
[125I]5, [125I]8, and [125I]11 were determined to be 2.49, 2.16, and 1.59, respectively.
According to Hansch and coꢀworkers’ study26, 27, optimum CNS penetration
approximately Log P = 2 ± 0.7, meaning the lipophilicity of the three compounds is
suitable for imaging aromatase activity in the CNS.
In vivo biodistribution. As shown in Table 2, [125I]5, [125I]8, and [125I]11 showed
high uptakes in the stomach at 60 min postꢀinjection, with [125I]5 showing the highest
uptake among the three ligands (16.21 ± 0.62% dose/g). The female rats displayed
significantly higher [125I]5 uptake in the stomach than male rats (16.21 vs. 10.12%
dose/g, Ρ = 0.002). [125I]8 and [125I]11 also showed higher stomach uptake, 6.29 vs.
4.86% dose/g for [125I]8 and 10.8 vs. 4.90% dose/g for [125I]11, respectively. The uptake
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in ovaries at 60 min postꢀinjection was also high, 8.56 ± 3.79% dose/g for [125I]5, 2.25 ±
0.49% dose/g for [125I]8 and 3.32 ± 0.11% dose/g for [125I]11, respectively. There was no
uptake in the testes for all three ligands. The liver and adrenal glands also showed uptake
to varying degrees. However, radioactivity levels in liver and adrenal glands were not
changed by preꢀtreatment of anastrozole (2 mg/kg, iv, data not shown), suggesting that
the binding was nonꢀspecific.
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Table 2.1. Comparison of biodistribution of [125I]5, [125I]8, and [125I]11 in normal
rats (% dose/g)
[125I]5
[125I]8
Female
[125I]11
Female Male
Organ
Blood
Female
Male
Male
0.32 ± 0.03
0.62 ± 0.01
0.30 ± 0.02
0.52 ± 0.02
0.77 ± 0.02
0.42 ± 0.02
0.82 ± 0.02
0.43 ± 0.07 0.54 ± 0.10 0.43 ± 0.06 0.21 ± 0.03 0.24 ± 0.03
0.57 ± 0.01 0.28 ± 0.03 0.21 ± 0.01 0.37 ± 0.01 0.23 ± 0.04
0.29 ± 0.01 0.13 ± 0.02 0.14 ± 0.00 0.17 ± 0.01 0.12 ± 0.01
0.49 ± 0.01 0.34 ± 0.05 0.26 ± 0.02 0.38 ± 0.03 0.28 ± 0.03
0.81 ± 0.01 0.75 ± 0.02 0.60 ± 0.03 1.10 ± 0.09 0.59 ± 0.11
0.38 ± 0.01 0.17 ± 0.02 0.15 ± 0.03 0.32 ± 0.04 0.18 ± 0.03
0.67 ± 0.02 0.68 ± 0.04 0.52 ± 0.23 0.81 ± 0.07 0.37 ± 0.04
Heart
Muscle
Lung
Kidneys
Spleen
Pancreas
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3.37 ± 0.15
0.21 ± 0.02
0.31 ± 0.01
0.51 ± 0.02
2.77 ± 0.04 1.32 ± 0.05 0.99 ± 0.09 3.93 ± 0.16 7.47 ± 1.67
0.26 ± 0.18 0.26 ± 0.03 0.31 ± 0.04 0.28 ± 0.05 0.19 ± 0.03
Liver
Skin
0.26 ± 0.01 0.12 ± 0.01 0.08 ± 0.01 0.27 ± 0.02
0.14 ±0.02
Brain
Thyroid
0.54 ± 0.01 0.69 ± 0.04 0.48 ± 0.26 0.58 ± 0.06 0.33 ± 0.09
0.88 ± 0.06 1.83 ± 0.35 1.13 ± 0.15 2.11 ± 0.16 1.20 ± 0.14
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Adrenal
glands
1.06 ± 0.09
16.21 ± 0.62 10.12 ± 0.04 6.29 ± 0.59 4.86 ± 0.22 10.88 ± 1.97 4.90 ± 0.91
Stomach
Intestine
0.65 ± 0.04
8.56 ± 3.79
0.49 ± 0.46 0.92 ± 0.10 0.86 ± 0.22 0.85 ± 0.07 0.57 ± 0.16
0.35 ± 0.04 2.25 ± 0.49 0.41 ± 0.08 3.32 ± 0.11 0.34 ± 0.06
Ovaries/
Testes
Table 2.2. In vivo blocking study (% dose/g)
[125I]5
[125I]8
Female
[125I]11
Female Male
Organ
Blood
Female
Male
Male
0.36 ± 0.01
0.81 ± 0.02
0.38 ± 0.01
0.64 ± 0.03
0.95 ± 0.03
0.53 ± 0.03
0.97 ± 0.06
0.39 ± 0.02 0.54 ± 0.03 0.43 ± 0.06 0.24 ± 0.01 0.26 ± 0.03
0.71 ± 0.03 0.34 ± 0.02 0.26 ± 0.02 0.48 ± 0.04 0.29 ± 0.00
0.36 ± 0.02 0.16 ± 0.01 0.17 ± 0.01 0.23 ± 0.01 0.14 ± 0.01
0.58 ± 0.02 0.39 ± 0.03 0.28 ± 0.03 0.49 ± 0.04 0.32 ± 0.02
1.11 ± 0.14 0.96 ± 0.08 0.94 ± 0.03 1.40 ± 0.17 0.89 ± 0.04
0.47 ± 0.05 0.27 ± 0.06 0.15 ± 0.01 0.38 ± 0.03 0.23 ± 0.03
0.85 ± 0.08 0.95 ± 0.08 0.59 ± 0.14 0.96 ± 0.06 0.55 ± 0.02
Heart
Muscle
Lung
Kidneys
Spleen
Pancreas
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3.66 ± 0.68
0.26 ± 0.02
0.37 ± 0.01
0.63 ± 0.03
3.49 ± 0.58 1.60 ± 0.35 1.18 ± 0.19 4.72 ± 0.69 8.48 ± 1.20
0.30 ± 0.01 0.29 ± 0.03 0.36 ± 0.01 0.27 ± 0.07 0.22 ± 0.02
Liver
Skin
0.32 ± 0.01 0.16 ± 0.02 0.10 ± 0.01 0.38 ± 0.03
0.18 ±0.02
Brain
Thyroid
0.66 ± 0.08 0.61 ± 0.08 0.50 ± 0.04 0.58 ± 0.03 0.39 ± 0.05
1.12 ± 0.09 1.90 ± 0.21 1.60 ± 0.19 2.63 ± 0.17 1.55 ± 0.09
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Adrenal
glands
1.23 ± 0.12
0.69 ± 0.03
0.74 ± 0.04
0.63 ± 0.05 0.45 ± 0.04 0.35 ± 0.01 1.57 ± 0.11 0.67 ± 0.55
0.63 ± 0.06 1.11 ± 0.27 0.87 ± 0.24 1.15 ± 0.03 0.84 ± 0.03
Stomach
Intestine
Ovaries/
Testes
0.70 ± 0.03
0.37 ± 0.04 0.97 ± 0.14 0.40 ± 0.10 1.20 ± 0.21 0.41 ± 0.01
About 10 µCi of each ligand in a saline solution was injected. For in vivo blocking study, anastrozole (2
mg/kg) was injected at 2 min prior to the injection of radiotracers. Rats were sacrificed at 60 min
postꢀinjection, organs of interest were removed and weighed, and the radioactivity was counted. Data
shown are the mean ± SD of three rats..
Pharmacological specificity of [125I]5 binding in vivo. Effects of the preꢀtreatment
with various compounds on stomach and ovary distribution of [125I]5 were examined to
assess the in vivo pharmacological specificity. Specific binding of [125I]5 in the stomach
and ovaries were significantly reduced by preꢀtreatment with selective aromatase
inhibitors, anastrozole, letrozole, and YM511 (Figure 2A, P < 0.05), suggesting a potent
in vivo competitive binding of these compounds with [125I]5 for aromatase. However,
exemestane which is an steroidal aromatase inhibitor showed no effect on the specific
binding of [125I]5 (Figure 2A). Furthermore, an in vivo competition of [125I]5 binding
with anastrozole (dose range 0.0001 mg/kg ꢀ 2 mg/kg) was conducted. Anastrozole
inhibited the specific bindings of [125I]5 doseꢀdependently, in both the stomach and
ovaries (Figure 2B), thus indicating the binding of [125I]5 to aromatase was saturable.
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Control
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Letrozole
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YM511
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*
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0
Stomach
Ovaries
Anastrozole (mg/kg)
Figure 2. In vivo blockade on the uptake of [125I]5 in stomach and ovaries of female rats.
A. Various drugs with a dose of 2 mg/kg was injected at 2 min prior to the injection of 10
ꢁCi [125I]5. Rats were sacrificed at 60 min after the injection of [125I]5. The stomach and
ovaries were removed and counted. The % dose/g uptakes in the stomach and ovaries
were compared between salineꢀpretreated (control) and drugꢀpretreated rats. Data shown
are the mean ± SD of three rats.
B. Anastrozole (dose range 0.0001 mg/kg – 2 mg/kg) was injected at 2 min prior to the
injection of [125I]5 (10 ꢁCi). Rats were sacrificed at 60 min after the injection of [125I]5.
The stomach and ovaries were removed and counted. The % dose/g uptakes in the
stomach and ovaries of rats under treatment of the highest dose of anastrozole (2 mg/kg)
were used as the background and the specific binding was calculated as the ratios of
uptake value/background on a % dose/g basis. Data shown are the mean ± SEM of three
rats.
Ex vivo autoradiography of [125I]5 and [125I]11. At 60 min postꢀinjection of [125I]5
and [125I]11, autoradiograms of sections from rat stomach and ovaries showed strong
binding which was completely blocked in the presence of anastrozole (2 mg/kg, injected
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at 2 min prior to the injection of [125I]5 and [125I]11, Figure 3A, B). However, the addition
of anastrozole did not show any effect on the binding of [125I]5 and [125I]11 in the adrenal
glands (Figure 3C). The autoradiograms of brain sections showed intense labeling by
[125I]5 in the amygdala and the bed nucleus of the stria terminalis (BNST), areas known
to have high densities of aromatase. In rats pretreated with anastrozole (2 mg/kg, injected
at 2 min prior to the injection of [125I]5), the binding signals were significantly reduced
compared to the matched sections of the control rats (Figure 3D). The regional
distribution observed with [125I]5 was consistent with those reported for other aromatase
ligands.15, 17 [125I]11 was also detected in the BNST and the amygdala of male rats, while
[125I]11 displayed a weak signal, compared with [125I]5 (Figure 3E). Similar as [125I]5 and
[125I]11, autoradiograms of sections from rat stomach and ovaries also showed strong
binding at 60 min postꢀinjection of [125I]8. The binding signal was completely blocked in
the presence of anastrozole. However, the autoradiograms of brain sections failed to show
any labeling by [125I]8 (data not shown).
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Stomach
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[125I]5
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[125I]11
Blocking with
anastrozole
[125I]5
Male
[125I]11
E
D
Blocking with
anastrozole
Male
Female
BNST
Amygdala
Figure 3. Ex vivo autoradiograms of [125I]5 and [125I]11 in rats at 60 min post iv
injection. High levels of radioactivity were observed in stomach (A) and ovaries (B)
sections. The high regional uptakes in BNST and amygdala were also observed in brain
sections (D, E). The bindings in stomach, ovaries and brain were blocked in the presence
of anastrozole (2 mg/kg, injected at 2 min prior to the injection of [125I]5 and [125I]11).
High nonꢀspecific binding was observed in adrenal glands (C). BNST: bed nucleus of
stria terminalis.
X-ray crystal structures of 5 and 11. Xꢀray crystal structures of 5 and 11 were
obtained in order to gain reliable structural information which is needed for molecular
docking calculations. A crystal of 5 was grown slowly from a mixture of methanol/THF
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and ethyl acetate (EtOAc). The size of the crystal used for structure determination was
0.25 × 0.22 × 0.2 mm, whereas the size of the crystal for 11 was 0.28 × 0.21 × 0.1 mm
(grown from methanol/THF/EtOAc). The crystal for 5 showed the space group P 212121
(orthorhombic) whereas 11 crystallyzed in P21 (monoclinic) (Figure 4). Both compounds
5 and 11 have two crystallographicallyꢀindependent structures with 50% probability each.
ORTEP drawings of one of the two independent structures for 5 and 11 are shown in
Figure 4.
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A
B
Figure 4: ORTEP drawing of 5 (A) and 11 (B) of the asymmetric unit with 50% thermal
ellipsoids.
Molecular docking study of interactions between 5, 11, and human aromatase.
Docking of compounds 5 and 11 to the aromatase enzyme was performed to investigate
their binding modes. The binding of both compounds displayed similar πꢀπ stacking and
interactions of their benzyl rings with residues of aromatase; Ile 133, Phe 134, Phe 221,
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Thr 224, Ala 306, Thr 310, Val 370, Leu 372, Leu 477 (Figure 5). Compound 5 formed a
hydrogen bond between the second nitrogen of its triazole ring and Ser 478, the latter of
which plays an important role in antiꢀaromatase activity when androstenedione is a
natural substrate.28 The amino acid forms hydrogen bonds through a waterꢀmediated
network, initiating enolization of the aromatase and the C3ꢀketo oxygen of
androstenedione.28 Additional studies identified that Ser 478 is an important target to
achieve selectivity of aromatase inhibition.29ꢀ32 Interactions between compound 5 and Ser
478 may partially contribute to the observed aromatase inhibition. The atomic structures
of compound 5 and 11 share some similarities, including the presence of a heterocyclic
triazole and imidazole ring system, which differ in the presence of either a carbon or
nitrogen in the ring. Both compounds may bind similarly to aromatase via hydrophobic
interactions with Ser 478, and through hydrogenꢀbond interactions between the shared
nitrogen facing the residue of the triazole/imidazole moieties with a water molecule. This
water molecule also forms a hydrogenꢀbond bridge with both the hydroxyl of Ser 478 and
another water molecule that coordinates to the catalytic site Fe2+ on the heme group of
aromatase, similar to the proposed binding modes of Verdenafil and Sildenafil to PDE5.33
This proposed interaction fits well with the comparable IC50 values of two compounds
observed in a binding assay with human aromatase (CYP19) since these compounds bind
in a similar fashion even in the absence of a direct hydrogenꢀbond interaction with Ser
478. Other binding modes were observed outside the active site of aromatase (data not
shown). Previous studies mention possible steric interactions when docking azole
fungicides into the active site of aromatase.34 It is possible that both compounds 5 and 11
bind at an away site, inducing allosteric changes at the active site and inhibiting
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aromatase from binding to androstenedione as observed in higher energy binding modes.
More details are provided in the supplementary information.
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Figure 5. Molecular docking poses of compound 5 represented in cyan (A) and 11
represented in white (B) in aromatase’s active site (PDB ID: 4KQ8)51; yellow dash line
indicates a possible hydrogen bond formed between Ser 478 and triazole nitrogen of
compound 5.
Discussion and conclusions
The present study showed that [125I]5, [125I]8, and [125I]11 had an abundant
accumulation in rat stomach, which was completely blocked in the presence of aromatase
inhibitors. Our result is consistent with a study by Ozawa et al. in which they showed the
presence of gastric aromatase in parietal cells using [11C]vorozole.15 In addition, for the
first time, we found that the uptake of [125I]5 in females was significantly higher than in
males. The underlying mechanisms responsible for high accumulation of aromatase
tracers in stomach as well as gender difference still unclear. It probably related to
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ghrelin’s function and energy metabolism. As is known to all, ghrelin is an important
hormone regulating energy metabolism what shows significant gender difference. It has
been reported that the aromatase mRNAꢀexpressing cells and ghrelin immunoꢀpositive
cells are localized close to each other in stomach of female and male rats, and that
estrogen treatment can induce ghrelin mRNA expression and peptide production in
isolated stomach cells.35 Another human study showed estrogen replacement therapy
increased human plasma ghrelin levels, and according to clinical statistics, women have
higher plasma ghrelin levels than men.36, 37 Since evidence showed estrogen treatment
induced ghrelin releasing in isolated stomach cells, gastric aromatase, which also showed
a significant gender difference, may play an important role in regulating ghrelin, further
affected energy metabolism.
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Our present study also showed a high accumulation of [125I]5 in rat ovaries, which
is expected as the ovaries are the main site of peripheral estrogen production. The
standard deviation was large because the female rats used in the present study are in
random stages of the estrus cycle. Ovarian aromatase expression, [11C]vorozole binding,
and enzymatic activity are reported to be strongly dependent on the stage of the estrus
cycle.38, 39 According to another study by Kobayashi et al., the protein and mRNA levels
of ovary aromatase significantly decrease during estrus and diestrus of female rats while
stomach aromatase remained unchanged.40 These studies suggest that gastric aromatase is
not significantly related to the estrus cycle.
The autoradiograms of the brain showed a high specific binding of [125I]5 in the
BNST and amygdala. The present data agree with an earlier report by Takahashi et al.
using [11C]vorozole.17 These regions are also reported by other researchers to show high
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aromatase activity,5 high levels of aromatase mRNA,41, 42 and immunoactivity43, 44 in rat
brain. Aromatase immunoreactive neurons have also been detected in the hypothalamic
area of rat brain,45 however, we failed to observe any specific binding of our compounds
in the hypothalamus in the present study. This may be due to the varying sensitivities
between the different techniques. Moreover, the aromataseꢀrich areas detected by
[11C]vorozole varies slightly among rodents, nonꢀhuman primates and humans.14, 17, 18
While there were no significant differences in aromatase expression between gender or
gonadal/hormonal conditions in rat brain,42 the sex differences of specific binding
patterns in rat brain requires further study.
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Aromatase has been reported as overexpressed and implicated in women
diagnosed with breast cancer,6 endometriosis,46 brain, lung, and hepatic cancer.8, 47, 48
Moreover, aromatase is also reported to be linked to brain disorders including
Alzheimer’s disease10 and autism.49 Although current studies on aromatase imaging are
still restricted to expression and physiological function of aromatase, the radiotracers
described here might be developed as potential radiopharmaceuticals for the diagnosis
and clinical assessment of aromataseꢀrelated diseases.
In summary, several radioiodinated agents targeting aromatase have been
prepared and evaluated. Of these, [125I]5 and [125I]11 are useful tools for aromatase
studies in peripheral organs and [125I]5 is also a good tool for brain aromatase study.
When labeled with 123I, [123I]5 may be a good SPECT agent for brain aromatase imaging
studies. Further characterization of these new ligands for human aromatase imaging
studies is currently ongoing in our laboratory.
Experimental section
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1 General
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1.1 All reagents and solvents were purchased from commercial sources (Aldrich, Acros,
or Alfa Inc.) and used without further purification, unless otherwise indicated. Solvents
were dried through a molecular sieve system (Pure Solve Solvent Purification System;
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1
Innovative Technology, Inc.). H spectra and 13C NMR were recorded on an Avance
spectrometer at 400 MHz and 100 MHz, respectively, and referenced to NMR solvents as
indicated. Chemical shifts are reported in units of ppm (δ), with a coupling constant, J, in
Hz. Multiplicity is defined by singlet (s), doublet (d), triplet (t), broad (br), and multiplet
(m). Highꢀresolution mass spectrometry (HRMS) data was obtained with an Agilent
(Santa Clara, CA) G3250AA LC/MSD TOF system. Thinꢀlayer chromatography (TLC)
analyses were performed using Merck (Darmstadt, Germany) silica gel 60 F254 plates.
Generally, crude compounds were purified by flash column chromatography (FC) packed
with silica gel (Aldrich). HPLC was performed on an Agilent 1100 series system. Two
HPLC conditions were used to test the purity of the final compounds. All compounds
showed a purity > 95%. A Cobra II autoꢀgamma counter (PerkinꢀElmer) measured 125I
radioactivity. Reactions of nonꢀradioactive chemical compounds were monitored by TLC
analysis with preꢀcoated plates of silica gel 60 F254. Solidꢀphase extraction cartridges
(SEP Pak® Light QMA, Oasis® HLB 3cc) were obtained from Waters (Milford, MA,
USA).
1.2 Animals. Male and female SpagueꢀDawley rats weighing 220ꢀ250 g were used in
biodistribution and ex vivo autoradiography studies. The rats were housed in an animal
facility with 12 hr light and dark cycles with access to food and water freely. All animal
experiments were carried out according to the ethics and animal welfare regulation
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requirements approved by the Institutional Animal Care and Use Committee (University
of Pennsylvania).
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2 Synthesis of 4-Benzyl(4H-1,2,4-triazol-4-yl)amino)benzonitrile derivatives
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2.1
4-((4H-1,2,4-triazol-4-yl)amino)benzonitrile
and
(1a).
and
4-((4-bromobenzyl)(4H-1,2,4-triazol-4-yl)amino)benzonitrile
4ꢀ((4Hꢀ1,2,4ꢀtriazolꢀ4ꢀyl)amino)benzonitrile
4ꢀ((4ꢀbromobenzyl)(4Hꢀ1,2,4ꢀtriazolꢀ4ꢀyl)amino)benzonitrile (1a) were synthesized
according to the published report by Wanatanabe et al.23 as summarized in Scheme 1.
2.2 4-((4-Fluorobenzyl)(4H-1,2,4-triazol-4-yl)amino)benzonitrile (1b). To a stirred
solution of 4ꢀ((4Hꢀ1,2,4ꢀtriazolꢀ4ꢀyl)amino)benzonitrile (100 mg, 0.54 mmol) in DMF
(10 mL) at room temperature, K2CO3 (82 mg, 0.59 mmol) and
1ꢀ(bromomethyl)ꢀ4ꢀfluorobenzene (111 mg, 0.59 mmol) were added. After maintaining
the mixture at room temperature for 2 h, it was then extracted with EtOAc. The combined
organic layers were dried over MgSO4, filtered, and concentrated in vacuo to afford a
crude product. Purification by FC (EtOAc/Hexanes, 50/50, V/V) afforded 1b (134 mg,
85.4%) as white solid. 1H NMR (400 MHz, CDCl3) δ: 8.11(s, 2H), 7.52 (dd, J = 2.0, 2.4
Hz, 2H), 7.16 (dd, J = 5.2, 5.2 Hz, 2H), 6.97 (t, J = 8.4 Hz, 2H), 6.64 (dd, J = 2.0, 2.0 Hz,
1H), 4.85 (s, 2H). 13C NMR (100 MHz, CDCl3) δ: 164.08, 162.42, 150.49, 142.64,
133.98, 130.12, 130.04, 129.43, 129.40, 118.45, 116.37, 116.16, 113.60, 105.11, 57.42.
HRMS calcd for; C16H12FN5 293.1077 found, 294.1117 [M+H]+.
2.3 4-((4-Methoxybenzyl)(4H-1,2,4-triazol-4-yl)amino)benzonitrile (1b). To a stirred
solution of 4ꢀ((4Hꢀ1,2,4ꢀtriazolꢀ4ꢀyl)amino)benzonitrile (100 mg, 0.54 mmol) in
dimethylformamide (DMF) (10 mL) at room temperature, K2CO3 (82 mg, 0.59 mmol)
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and 1ꢀ(bromomethyl)ꢀ4ꢀmethoxybenzene (117 mg, 0.59 mmol) were added. After
maintaining the mixture at room temperature for 2 h, it was then extracted with EtOAc.
The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo
to afford a crude product. Purification by FC (EtOAc/Hexanes, 50/50, V/V) afforded 1c
(142 mg, 86.6%) as a white solid. 1H NMR (400 MHz, CDCl3) δ: 8.05 (s, 2H), 7.98 (s,
2H), 7.55 (d, J = 9.2Hz, 2H), 7.07 (d, J = 8.8 Hz, 2H), 6.82 (d, J = 8.8 Hz, 2H), 6.98 (d, J
= 8.8 Hz, 2H), 4.81 (s, 2H), 3.76 (s, 3H). 13C NMR (100 MHz, CDCl3) δ: 150.56, 142.70,
134.01, 129.74, 125.20, 118.55, 114.65, 113.52, 104.92, 57.51, 55.27. HRMS calcd for:
C17H15N5O 305.1277; found, 306.1282 [M+H]+.
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2.4 4-(((6-Bromopyridin-3-yl)methyl)(4H-1,2,4-triazol-4-yl)amino)benzonitrile (1d). To a
stirred solution of 4ꢀ((4Hꢀ1,2,4ꢀtriazolꢀ4ꢀyl)amino)benzonitrile (100 mg, 0.54 mmol) in
DMF (10 mL) at room temperature, K2CO3 (82 mg, 0.59 mmol) and
2ꢀbromoꢀ5ꢀ(bromomethyl)pyridine (146 mg, 0.59 mmol) were added. After maintaining
at room temperature for 2 h, the mixture was extracted with EtOAc. The combined
organic layers were dried over MgSO4, filtered, and concentrated in vacuo to afford a
crude product. Purification by FC (EtOAc/Hexanes, 50/50, V/V) afforded 1d (160 mg,
84.1%) as a white solid. 1H NMR (400 MHz, CDCl3) δ: 8.35 (d, J = 2.4 Hz, 1H), 8.21 (s,
2H), 7.64 (dd, J = 2.0, 2.0 Hz, 2H), 7.52 (d, J = 8.0 Hz, 1H), 7.40 (dd, J = 2.0, 2.0 Hz,
13
1H), 6.70 (d, J = 9.2 Hz, 1H), 4.91 (s, 2H). C NMR (100 MHz, CDCl3) δ: 149.94,
149.65, 143.26, 142.27, 137.85, 134.27, 128.78, 128.60, 118.10, 113.84, 106.40, 55.59.
HRMS calcd for C15H11BrN6 354.0229; found, 355.0231 [M+H]+.
2.5
4-((4H-1,2,4-triazol-4-yl)(4-(tributylstannyl)benzyl)amino)benzonitrile
(4).
Compound 1a (100 mg, 0.28 mmol) and bis(tributyltin) (820 mg, 1.42 mmol) were
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dissolved in toluene (10 mL), and tetrakis(triphenylphosphine)palladium(0) (60 mg, 0.05
mmol) was added. The mixture was refluxed under argon for 2 h and condensed under
reduced pressure. The residue was then applied to FC (hexanes/EtOAc 3:1), which
afforded 4 (59 mg, 37.6%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ: 8.13 (s, 2H),
7.56 (d, J = 9.2 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.15 (d, J = 8.0 Hz, 2H), 6.66 (d, J =
9.2 Hz, 2H), 4.95 (s, 2H), 1.56ꢀ1.52 (m, 6H), 1.51ꢀ1.48 (m, 7H), 1.36ꢀ1.27 (m, 5H),
0.91ꢀ0.87 (m, 9H). 13C NMR (100 MHz, CDCl3) δ: 150.55, 143.72, 142.70, 137.31,
134.05, 133.13, 127.54, 127.34, 118.59, 113.27, 109.83, 58.28, 29.01, 27.28, 13.64, 9.63.
HRMS calcd for C28H39N5Sn 565.2227; found, 566.2382 [M+H]+.
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2.6 4-((4-Iodobenzyl)(4H-1,2,4-triazol-4-yl)amino)benzonitrile (5). Compound 4 (30 mg,
0.053 mmol) and I2 (28 mg, 0.11 mmol) were dissolved in dichloromethane (DCM) (15
mL), and the reaction mixture was stirred at room temperature for a half hour. Then, 2M
Na2S2O3 was added, DCM was extracted, the reaction mixture was condensed, and the
crude product was purified by flash column to afford 5 as a white solid (20 mg, 95.6%).
1H NMR (400 MHz, CDCl3) δ: 8.43 (s, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 9.2 Hz,
2H), 7.05 (d, J = 8.1 Hz, 2H), 6.75 (d, J = 9.2 Hz, 2H), 4.95 (s, 2H). 13C NMR (100 MHz,
CDCl3) δ: 150.80, 143.17, 138.21, 133.95, 133.68, 130.13, 118.47, 113.91, 104.88, 94.28,
57.60. HRMS calcd for C16H12IN5; found, 402.0209 [M+H]+.
2.7 4-((4-Hydroxybenzyl)(4H-1,2,4-triazol-4-yl)amino)benzonitrile (2). A solution of 1c
(100 mg, 0.33 mmol) and 1M BBr3 (1 mL) was dissolved in DCM (15 mL), and the
°
reaction was stirred at ꢀ78 C for 15 min. The solution was then stirred at room
temperature for 1 h, and CH3OH (10 mL) was added. The reaction was purified by FC,
which afforded 2 as a white solid (57 mg, 60.1%). 1H NMR (400 MHz, CDCl3) δ: 8.47 (s,
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2H), 7.70 (d, J = 9.2 Hz, 2H), 7.18 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 8.8 Hz, 2H), 6.80 (d,
5
6
13
J = 8.4 Hz, 2H), 5.02 (s, 2H). C NMR (100 MHz, CDCl3) δ: 151.72, 142.86, 133.70,
7
8
9
130.19, 125.25, 118.53, 115.52, 113.94, 113.82, 103.86, 56.98, 56.27. HRMS calcd for
C16H13N5O 291.1120; found, 292.1122 [M+H]+.
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2.8 4-((4-(2-Fluoroethoxy)benzyl)(4H-1,2,4-triazol-4-yl)amino)benzonitrile (3a). To a
stirred solution of 2 (100 mg, 0.34 mmol) in DMF (10 mL) at room temperature, K2CO3
(95 mg, 0.68 mmol) and 1ꢀbromoꢀ2ꢀfluoroꢀethane (48 mg, 0.38 mmol) were added. After
maintaining at room temperature for 2 h, the mixture was then extracted with EtOAc. The
combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo to
afford a crude product. Purification by FC (EtOAc/hexanes, 50/50, V/V) yielded 3a (99
mg, 86.3%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ: 8.07 (d, J = 2.8Hz, 2H),
8.02 (s, 1H), 7.59 (dd, J = 2.4, 2.0 Hz, 2H), 7.11 (dd, J = 2.0, 2.0 Hz, 2H), 6.70 (dd, J =
2.0, 2.4 Hz, 2H), 4.83ꢀ4.80 (m, 3H), 4.70 (t, J = 4.0 Hz, 1H), 4.23 (t, J = 4.0 Hz, 1H),
4.16 (t, J = 4.0 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ: 150.48, 142.65, 134.07, 129.81,
125.85, 118.50, 115.38, 113.52, 105.15, 82.57, 80.87, 67.27, 67.06, 57.55. HRMS calcd.
for C18H16FN5O 337.1339; found, 338.1419 [M+H]+.
2.9 4-((4-(3-Fluoropropoxy)benzyl)(4H-1,2,4-triazol-4-yl)amino)benzonitrile (3b). To a
stirred solution of 2 (100 mg, 0.34 mmol) in DMF (10 mL) at room temperature, K2CO3
(95 mg, 0.68 mmol) and 3ꢀfluoropropyl 4ꢀmethylbenzenesulfonate (88 mg, 0.38 mmol)
were added. After maintaining at room temperature for 2 h, the mixture was extracted
with EtOAc. The combined organic layers were then dried over MgSO4, filtered, and
concentrated in vacuo to give a crude product. Purification by FC (EtOAc/hexanes,
50/50, V/V) yielded 3b (102 mg, 85.9%) as a yellow solid. 1H NMR (400 MHz, CDCl3)
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δ: 8.07 (s, 2H), 7.61 (d, J = 8.8, 2H), 7.10 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.8Hz, 2H),
6.71 (d, J = 8.8Hz, 2H), 4.83 (s, 2H), 4.72 (t, J = 4.0 Hz, 1H), 4.60 (t, J = 4.0 Hz, 1H),
7
8
9
13
4.15ꢀ4.08 (m, 2H), 2.25ꢀ2.14 (m, 2H). C NMR (100 MHz, CDCl3) δ: 159.42, 150.47,
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142.66, 134.09, 129.76, 125.30, 118.49, 115.26, 113.50, 105.18, 81.36, 79.72, 63.66,
63.61, 57.59, 30.42, 30.23. HRMS calcd for; C19H18FN5O 351.1495 found, 352.1509
[M+H]+.
3 Synthesis of 4-((4-iodobenzyl)(pyrimidin-5-yl)amino)benzonitrile
3.1 4-(Pyrimidin-5-ylamino)benzonitrile and 4-((4-bromobenzyl)(pyrimidin-5-yl)
amino)benzonitrile
(6a).
4ꢀ(pyrimidinꢀ5ꢀylamino)benzonitrile
and
4ꢀ((4ꢀbromobenzyl)(pyrimidinꢀ5ꢀyl)amino) benzonitrile (6a) were prepared according to
the published reported by Okada et al.50 as summarized in Scheme 2.
3.2 4-(((6-Bromopyridin-3-yl)methyl)(pyrimidin-5-yl)amino)benzonitrile (6b). To a
stirred solution of 4ꢀ(pyrimidinꢀ5ꢀylamino)benzonitrile (100 mg, 0.51 mmol) in DMF (10
mL)
at
room
temperature,
K2CO3
(141
mg,
1
mmol)
and
2ꢀbromoꢀ5ꢀ(bromomethyl)pyridine (139 mg, 0.56 mmol) were added. After maintaining
at room temperature for 2 h, the mixture was then extracted with EtOAc. The combined
organic layers were dried over MgSO4, filtered, and concentrated in vacuo to afford a
crude product. Purification by FC (EtOAc/hexanes, 50/50, V/V) yielded 6b (151 mg,
81.3%) as a white solid. 1H NMR (400 MHz, CDCl3) δ: 9.03 (s, 1H), 8.67 (s, 2H), 8.63
(s, 2H), 8.33 (t, J = 0.8 Hz, 1H), 7.56 (d, J = 8.8 Hz, 2H), 7.48 (t, J = 0.8 Hz, 2H), 6.96
(d, J = 8.8 Hz, 2H), 5.02 (s, 2H). 13C NMR (100 MHz, CDCl3) δ: 154.74, 152.24, 149.32,
148.58, 148.23, 141.91, 140.61, 136.66, 134.10, 131.12, 128.54, 118.66, 117.63, 115.93,
104.91, 53.12. HRMS calcd for C17H12BrN5 365.0276; found, 366.0420 [M+H]+.
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3.3 4-(Pyrimidin-5-yl(4-(tributylstannyl)benzyl)amino)benzonitrile (7). Compound 6a
(100 mg, 0.27 mmol) and bis(tributyltin) (820 mg, 1.42 mmol) were dissolved in toluene
(10 mL), and tetrakis(triphenylphosphine)palladium(0) (60 mg, 0.05 mmol) was
subsequently added. The mixture was refluxed under argon for 2 h and condensed under
reduced pressure. The resulting residue was applied to FC (hexanes/EtOAc 3:1) which
afforded 7 (65 mg, 41.3%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ: 8.99 (s, 1H),
8.67 (s, 2H), 7.73 (d, J = 6.0 Hz, 2H), 7.53 (d, J = 8.8 Hz, 2H), 7.45 (d, J = 8.0 Hz, 2H),
7.40 (d, J = 7.6 Hz, 2H), 7.22 (d, J = 7.6 Hz, 2H), 7.16 (d, J = 8.8 Hz, 2H), 5.04 (s, 2H),
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13
1.58ꢀ1.52 (m, 5H), 1.36ꢀ1.28 (m, 7H), 1.08ꢀ1.04 (m, 5H), 0.91ꢀ0.87 (m, 9H). C NMR
(100 MHz, CDCl3) δ: 154.17, 152.22, 150.04, 142.03, 137.17, 133.87, 130.36, 127.92,
127.87, 125.81, 119.09, 117.37, 103.88, 55.93, 29.04, 27.33, 13.64, 9.61. HRMS calcd
for; C30H40N4Sn 576.2275 found, 577.2451 [M+H]+.
3.4 4-((4-Iodobenzyl)(pyrimidin-5-yl)amino)benzonitrile (8). A solution of 7 (30 mg,
0.052 mmol) and I2 (28 mg, 0.11 mmol) dissolved in DCM (15 mL) was stirred at room
temperature for a half hour before 20 mL 2M Na2S2O3 was added. DCM was then
extracted, the reaction mixture was condensed, and the crude product was purified by FC
to afford 8 as a white solid (20 mg, 94.2%). 1H NMR (400 MHz, CDCl3) δ: 9.01 (s, 1H),
8.65 (s, 2H), 7.70 (dd, J = 2.0, 0.8 Hz, 2H), 7.55 (dd, J = 2.0, 2.0 Hz, 2H), 7.02 (d, J = 8.8
13
Hz, 2H), 7.55 (dd, J = 2.4, 2.0 Hz, 2H), 4.99 (s, 2H). C NMR (100 MHz, CDCl3) δ:
154.42, 152.17, 149.68, 140.82, 138.31, 135.69, 133.96, 128.88, 117.45, 105.11, 93.30,
55.53. HRMS calcd for; C18H13IN4 412.0185 found, 413.0212 [M+H]+.
4 Synthesis of 4-((1H-imidazol-1-yl)(4-iodobenzyl)amino)benzonitrile
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4.1
4-((4-Bromobenzyl)(1H-imidazol-1-yl)amino)benzonitrile
(9).
5
6
4ꢀ((4ꢀbromobenzyl)(1Hꢀimidazolꢀ1ꢀyl)amino)benzonitrile (9) was prepared according to
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9
the published report by Adje et al.25
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4.2 4-((1H-imidazol-1-yl)(4-(tributylstannyl)benzyl)amino)benzonitrile (10). To a
solution of 9 (100 mg, 0.28 mmol) and bis(tributyltin) (820 mg, 1.42 mmol) dissolved in
toluene (10 mL), tetrakis(triphenylphosphine)palladium(0) (60 mg, 0.05 mmol) was
added. The mixture was refluxed under argon for 2 h and condensed under reduced
pressure. The residue was applied to FC (hexanes/EtOAc 3:1) which afforded 10 (53 mg,
1
32.9%) as a colorless oil. H NMR (400 MHz, CDCl3) δ: 7.54ꢀ7.46 (m, 5H), 7.21 (d, J =
8.0 Hz, 2H), 7.14 (t, J = 1.2 Hz, 1H), 6.93 (t, J = 1.2 Hz, 1H), 6.58 (d, J = 9.2 Hz, 2H),
4.89 (s, 2H), 1.56ꢀ1.52 (m, 5H), 1.36ꢀ1.25 (m, 7H), 0.95ꢀ0.92 (m, 5H), 0.91ꢀ0.87 (m,
13
9H). C NMR (100 MHz, CDCl3) δ: 151.46, 142.79, 137.08, 136.92, 134.28, 133.80,
129.08, 126.99, 119.04, 118.57, 112.81, 103,57, 60.37, 58.26, 29.04, 27.31, 14.19, 9.61.
HRMS calcd for C29H40N4Sn 564.2275; found, 565.3219 [M+H]+.
4.3 4-((1H-imidazol-1-yl)(4-iodobenzyl)amino)benzonitrile (11). Compound 10 (30 mg,
0.053 mmol) and I2 (28 mg, 0.11mmol) were dissolved in DCM (15 mL). The reaction
mixture was stirred at room temperature for a half hour, 2M Na2S2O3 was added, DCM
was extracted, the mixture was condensed, and the crude product was purified by FC to
afford 11 as a white solid (20 mg, 92.9%). 1H NMR (400 MHz, CDCl3) δ: 7.70 (d, J = 8.4
Hz, 2H), 7.54 (d, J = 8.8 Hz, 2H), 7.46 (s, 1H), 7.15 (d, J = 1.2 Hz, 1H), 7.02 (d, J = 8.4
13
Hz, 2H), 6.93 (t, J = 1.2 Hz, 2H), 6.58 (d, J = 9.2 Hz, 2H), 4.84 (s, 2H). C NMR (100
MHz, CDCl3) δ: 151.24, 138.24, 136.85, 134.31, 133.88, 129.53, 129.38, 118.86, 118.32,
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