Synthesis of 5- and 6-substituted 2-(4-dimethylaminophenyl)-1,3-benzoxazoles and their in vitro and in vivo evaluation as imaging agents for amyloid plaque

Article abstract of DOI:10.1016/j.bmcl.2008.05.033

A series of novel 5- and 6-substituted 2-(4-dimethylaminophenyl)-1,3-benzoxazoles was synthesized and their potential as imaging probes for Alzheimer's Disease (AD)-related amyloid plaque was evaluated in vitro and in vivo. In vitro binding affinities for

Full text of DOI:10.1016/j.bmcl.2008.05.033

Bioorganic & Medicinal Chemistry Letters 19 (2009) 543–545
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 5- and 6-substituted 2-(4-dimethylaminophenyl)-1,
3-benzoxazoles and their in vitro and in vivo evaluation
as imaging agents for amyloid plaque
Sven H. Hausner a, , David Alagille a, Andrei O. Koren b, Louis Amici a, Julie K. Staley a,
Kelly P. Cosgrove a, Ronald M. Baldwin a,à, Gilles D. Tamagnan a,b,
*
a School of Medicine, Department of Psychiatry and Diagnostic Radiology, Yale University, VACHS, West Haven, CT 06516, USA
b Institute for Neurodegenerative Disorders, New Haven, CT 06510, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 5- and 6-substituted 2-(4-dimethylaminophenyl)-1,3-benzoxazoles was synthesized
and their potential as imaging probes for Alzheimer’s Disease (AD)-related amyloid plaque was evaluated
in vitro and in vivo. In vitro binding affinities for Ab1–40 peptide of several of these compounds were in
the low-nanomolar range . The lowest Ki of 9.3 nM was found for N-(2-(4-(dimethylamino)phenyl)-1,3-
benzoxazol-5-yl)-4-iodobenzamide (1e). Its 123I-radiolabeled form ([123I]1e) was subsequently prepared
by iododestannylation of the corresponding tributylstannyl precursor and evaluated in vivo in a baboon
model using SPECT imaging. Contrary to our expectations, 1e did not cross the blood–brain barrier (BBB)
to any significant extent.
Received 13 March 2008
Revised 7 May 2008
Accepted 8 May 2008
Available online 15 May 2008
Keywords:
Amyloid plaques
Imaging
Ó 2008 Elsevier Ltd. All rights reserved.
SPECT
Benzoxazole
Labeling
Alzheimer’s Disease (AD) is the most common form of dementia
in people aged 65 and older. Currently, several million patients suf-
fer from AD in the US alone, and with the aging of the baby-boom
generation, this number is expected to increase. AD is a neurode-
generative disease that gradually leads to severe memory loss.
Abundant insoluble neurotoxic deposits of Ab1–40 and Ab1–42
peptides on neurons (‘Ab-plaque’ or ‘amyloid plaque’), resulting
from cleavage of the amyloid precursor protein (APP) by specific
proteases, are one of the characteristic pathological changes occur-
ring in AD. Currently, a definite confirmation of AD is attained only
by post-mortem histopathology of the brain. The ability to image
amyloid plaque noninvasively in vivo would allow for early diag-
nosis of AD and evaluation of medication treatment progress in
AD patients. Recently, it was reported that non-ionic analogues
of Thioflavin T, an ionic imaging dye, penetrate the blood–brain
barrier (BBB) and show high affinity for Ab-plaque.1 Radioiodin-
ated varieties of these compounds have been used for single pho-
ton emission computed tomography (SPECT) imaging of amyloid
plaque.2–4
N
N
S
Thioflavin T
N
N
N
X
I
I
IBOX (X= O); TZDM (X= S)
I-Stilbene
A number of these ligands share a 4-(dimethylamino)phenyl
fragment linked to another aromatic moiety, such as 5- or 6-substi-
tuted benzothiazole or benzoxazole.5–8 Given the favorable Ab-pla-
que binding properties shown in initial studies, we decided to
further explore IBOX-related structures.2 We reasoned that replac-
ing the original iodo substituent with an amine functionality
would allow for easy attachment of different groups via an amide
bond. Here we report the synthesis and in vitro and in vivo evalu-
ation of 5- and 6-substituted 2-(4-dimethylaminophenyl)-1,3-
benzoxazoles (1) as potential imaging agents for amyloid plaque.
* Corresponding author.
O
N
O
E-mail address: gtamagnan@indd.org (G.D. Tamagnan).
Present address: Department of Biomedical Engineering, UC Davis, Davis, CA
95616, USA.
H
N
N
Ar
à
Present address: Medical School, Radiology, Vanderbilt University, Nashville, TN
1
37235, USA.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.033

544
S. H. Hausner et al. / Bioorg. Med. Chem. Lett. 19 (2009) 543–545
NH2
OH
B(OH)3 (1 eq)
N
O
O2N
N
O2N
N
Xylenes, Δ, 1 d
O
HO
H2, Pd/C
N
Ar-COCl (1.0-1.1 eq)
1
H2N
N
2,6-Lutidine,THF, -5 ºC to r.t.
EtOAc/EtOH
O
Scheme 1. Synthesis of the target substituted benzoxazoles.
had the least effect on binding (cf. 1a, 1e, 1g, and 1i). Fortunately,
1e, the compound that exhibited the lowest Ki value in the series
studied, was also the ideal candidate for developing a 123I-labeled
SPECT imaging probe.
Compounds of the general structure 1 were synthesized accord-
ing to Scheme 1. The 1,3-benzoxazole nucleus was formed via a
boronic acid-catalyzed condensation of the corresponding aminon-
itrophenols with 4-(dimethylamino)benzoic acid. Reduction of the
nitro group to amino followed by condensation with the appropri-
ate aromatic acid chloride gave the intended structures. Purity of
the target compounds was confirmed by 1H and 13C NMR, high-res-
olution mass spectrometry, and elemental analysis (C, H, N) (see
Table 1).
The radioiodinated [123I]1e was prepared by reaction of the cor-
responding tributylstannyl precursor with electrophilic [123I]io-
dine species generated in situ from sodium [123I]iodide (Scheme
2). In a typical procedure, to a vial containing Na[123I]I and NaOH
were added: reaction solvent (50% THF); 0.8 M H3PO4 to adjust
pH to 3; 50–100 lg of the radiolabeling precursor in 50 lL of
THF; and peracetic acid so that its final concentration was at least
1%. The reaction mixture was heated at 80 °C for 30 min, quenched
with 100 lL (100 lg) of Na2S2O5/NaHCO3 solution, and injected
Compounds were then screened for binding to Ab-plaque by
ELISA.6 Ki values in the low-nanomolar range were found for sev-
eral compounds. The in vitro data showed that a benzamide substi-
tuent in position 5 of the benzoxazole nucleus resulted in higher
Ab-plaque binding affinity compared to the same substituent in
position 6. Further substitution of the benzamide moiety with a
small, nonpolar group resulted in only minor to moderate effects
on Ki: substitution at positions 3 and 4 of the benzamide phenyl
onto a C18 reverse-phase HPLC column eluted with a mixture of
MeCN–H2O–Et3N (70:30:0.2, v/v/v). The fraction containing
[
123I]1e was collected, diluted with water, and the resulting solu-
tion was passed through a C18 solid-phase extraction cartridge.
Table 1
Properties of the novel 5- and 6-substituted 2-(4-dimethylaminophenyl)-1,3-benzoxazoles
5
O
N
O
H
N
N
Ar
6
Compound
Ar
Position
Yielda (%)
Mp (°C)
Kib (nM)
ClogPc
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
PIBd
Phenyl
Phenyl
5
6
5
6
5
6
5
6
5
6
5
6
5
6
58
41
47
64
34
59
42
45
24
44
38
50
30
52
251–254
259–263
224–226
215–217
274–279
273–278
255–258
270–273
193–195
192–196
176–178
195–198
262–266
244–246
12.0
26.0
109
628
9.3
60.1
13.2
86.0
13.4
31.5
18.9
112
17.2
19.7
4.3
4.92
4.92
4.28
4.28
6.11
6.11
5.42
5.42
5.42
5.42
5.08
5.08
4.60
4.60
3.99
3,4,5-Trimethoxyphenyl
3,4,5-Trimethoxyphenyl
4-Iodophenyl
4-Iodophenyl
p-Tolyl
p-Tolyl
m-Tolyl
m-Tolyl
o-Tolyl
o-Tolyl
3,4-(Methylenedioxy)phenyl
3,4-(Methylenedioxy)phenyl
a
After purification, last step, not optimized.
Against [3H]BTA-1 as obtained in ELISA.6
b
c
As calculated in ChemDraw Ultra 8.0 (CambridgeSoft, Cambridge, MA).
d
Reference compound.6
H
H
N
Na123I, AcOOH, H3PO4
THF/H2O, 80 ºC, 30 min
O
N
O
N
O
N
O
N
N
1e*
SnBu3
123I
Scheme 2. Synthesis of the putative [123I]-labeled SPECT imaging probe [123I]1e.

S. H. Hausner et al. / Bioorg. Med. Chem. Lett. 19 (2009) 543–545
545
The product retained on the cartridge was eluted with 0.9 mL of
100% ethanol and filtered through a 0.22 membrane filter, then
ered generally acceptable10). Modifications of the benzamide moi-
ety of the general structure 1 aimed at lowering the lipophilicity
might allow for penetrating the brain while maintaining favorable
Ab-plaque binding characteristics. Recent studies with modified
amyloid ligands11–13 provide encouragement to further explore
this route.
l
diluted with 9 mL of sterile normal saline. Radiolabeling yield
was in the range of 60–70%, and the radiochemical purity of the fi-
nal product exceeded 99%.
In vivo studies with [123I]1e were carried out in ovariectomized
female baboons (Papio Anubis). SPECT imaging was carried out as
previously described by Staley et al.9 under institutional animal
care protocols complying with Federal regulations. In brief, a fasted
(18–24 h) animal was immobilized using ketamine (10 mg/kg im),
Acknowledgments
We thank Drs. Chester Mathis and William Klunk from the Uni-
versity of Pittsburg for helpful comments. Supported in part by Na-
tional Institutes of Health (Neuroimaging Sciences Training
Program/NISTP and 1R43AG024717).
combined with glycopyrrolate (10 lg/kg im) 2 h prior to study. The
animal was intubated and maintained on 2.5% isoflurane. Vital
signs, including heart rate, respiration rate, oxygen saturation,
and body temperature, were monitored every 15 min throughout
the study. SPECT data were acquired with the nonhuman primate
brain-dedicated multislice CERASPECT camera (Digital Scinti-
graphics, Waltham, MA, USA). Brain images (128 Â 128 Â 64 ma-
trix; pixel size = 1.67 Â 1.67 mm, slice, thickness = 1.67 mm, voxel
volume = 4.66 mm3) were acquired continuously at 159 keV for
1 h after injection of radiotracer (3.2 mCi in 8 mL). SPECT images
were reconstructed using a ramp and a Butterworth filter (cut-
off = 0.65 cmÀ1, power factor = 10). Images showed low uptake in
the brain and no distinct localization within brain regions.
These animal studies showed that, despite the observed in vitro
binding of 1e, radiolabeled [123I]1e would not be a suitable radio-
ligand for in vivo imaging of amyloid plaque. The inability of this
compound to cross the BBB may be attributable to its excessively
high lipophilicity (CLogP 6.11 compared to a range of 1.5–4 consid-
References and notes
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