Fully automated synthesis of [18F]T807, a PET tau tracer for Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2015.05.035

The authentic standard T807 and its nitro-precursor T807P as well as t-Boc-protected T807P precursor for radiolabeling were synthesized from (4-bromophenyl)boronic acid, 3-bromo-4-nitropyridine and 3-bromo-6-nitropyridine with overall chemical yield 27% i

Full text of DOI:10.1016/j.bmcl.2015.05.035

Bioorganic & Medicinal Chemistry Letters 25 (2015) 2953–2957
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fully automated synthesis of [18F]T807, a PET tau tracer
for Alzheimer’s disease
⇑
Mingzhang Gao, Min Wang, Qi-Huang Zheng
Department of Radiology and Imaging Sciences, Indiana University School of Medicine, 1345 West 16th Street, Room 202, Indianapolis, IN 46202, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The authentic standard T807 and its nitro-precursor T807P as well as t-Boc-protected T807P precursor for
radiolabeling were synthesized from (4-bromophenyl)boronic acid, 3-bromo-4-nitropyridine and
3-bromo-6-nitropyridine with overall chemical yield 27% in three steps, 4–7% in three to five steps,
and 3–8% in four to five steps, respectively. [18F]T807 was synthesized from T807P by the nucleophilic
Received 24 April 2015
Revised 12 May 2015
Accepted 13 May 2015
Available online 21 May 2015
[
18F]fluorination with K[18F]F/Kryptofix 2.2.2 in DMSO at 140 °C followed by reduction with Fe
powder/HCOOH through manual synthesis with 5–10% decay corrected radiochemical yield in two steps.
Keywords:
[
Tau tracer
Automation
Positron emission tomography (PET)
Alzheimer’s disease (AD)
[
18F]T807 was also synthesized from t-Boc-protected T807P by a concurrent [18F]fluorination and depro-
18F]T807
tection with K[18F]F/Kryptofix 2.2.2 in DMSO at 140 °C and purified by HPLC-SPE method in a home-built
automated [18F]radiosynthesis module with 20–30% decay corrected radiochemical yield in one step. The
specific activity of [18F]T807 at end of bombardment (EOB) was 37–370 GBq/
lmol.
Ó 2015 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is an irreversible, progressive brain
disease that slowly loses memory and other important mental
functions, and nearly 35 million people suffer this disease world-
wide.1–3 The exact causes of AD are not fully understood.4
Currently there is no prevention or cure, and clinical drug trials
have greater than 90% failure rate.5 The lack of a reliable early diag-
nosis for AD results in the difficulty to find an efficient treatment
for AD, and the lack of effective prognostic biomarkers also
hampers the development of therapeutics for AD.6 Therefore, the
development of pathology-specific diagnostic tools has been
strongly desired, and advanced molecular imaging technique posi-
tron emission tomography (PET) is a promising modality for early
detection, disease staging, drug discovery and progression of AD
patients.7 Amyloid and tau are two major interesting targets for
the development of therapeutic solutions and diagnostic biomark-
ers such as in vivo PET probes for AD.8 Amyloid cascade hypothesis
has generated a number of Ab PET tracers, and the representative
radiopharmaceuticals are [11C]PIB9 and [18F]Amyvid (formerly
known as [18F]AV-45),10 as indicated in Figure 1. Since more and
more evidences demonstrate that increased tau levels appear to
correlate with clinical AD disease severity, recently the researchers
have switched their focus to tau hypothesis, several highly
selective and specific PET tau tracers like [18F]T808 ([18F]AV-680),
developed, and promising clinical PET imaging results with these
tracers have been reported.11–15
The importance of PET tau tracers is well recognized, and
broader research investigation to fully explore and validate the
utility of tau tracer-PET is important. However, the limited com-
mercial availability, complicated and/or patented synthetic proce-
dure, and high costs of starting materials and precursor can present
an obstacle to more widespread evaluation of these intriguing
agents. In an attempt to study these compounds in our PET center,
we decided to make our own material by following the literature
methods. In our previous work, we have developed a concise and
high-yield synthesis of T808 and T808P, and a fully automated
radiosynthesis of [18F]T808.16 In this ongoing study, we select
[
18F]T807 as another target PET tau tracer. The published and
patented synthesis of the authentic standard T807 and target tra-
cer [18F]T807 lacks synthetic detail, and the key steps gave poor
yield and was difficult to reproduce in our hands. Therefore, we
revisited the reported literature methods12,13,17,18 and investigated
alternate approaches and modifications that eventually resulted in
a high-yield synthesis of T807 and [18F]T807 starting from very
beginning materials (4-bromophenyl)boronic acid, 3-bromo-4-
nitropyridine and 3-bromo-6-nitropyridine that was superior to
previous works or addressed more synthetic details to reveal and
explain technical artifices. In this letter we report the first synthe-
sis of the nitro-precursor T807P as well as t-Boc-protected T807P
precursor, provide complete experiment procedures, yields,
analytical details and new findings for the synthesis of T807 and
[
18F]T807 ([18F]AV-1451) and [11C]PBB3 (Figure 1) have been
⇑
Corresponding author. Tel.: +1 317 278 4671; fax: +1 317 278 9711.
E-mail address: qzheng@iupui.edu (Q.-H. Zheng).
http://dx.doi.org/10.1016/j.bmcl.2015.05.035
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

2954
M. Gao et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2953–2957
H3C
HN
i
O2N
SnMe3
Me
Me
Me
Me
H311C
HN
O2N
Br
18F
3
N
N
N
O
4
N
S
OH
[
18F]Amyvid ([18F]AV-45)
[
11C]PIB
O
O2N
B
N
N
N
O
N
S
N
or
ii
Br
O2N
N
H
11CH3
N
HO
N
H
2
N
H
O2N
SnMe3
T807P (5)
[
11C]PBB3
N
4
N
N
N
N
iii
N
N
O2N
N
Boc
N
H
N
18F
18F
N
t-Boc-protected T807P (6)
18F]T808 ([18F]AV-680)
[
18F]T807 ([18F]AV-1451)
Kd (tau affinity) 14.6 nM
[
Scheme 2. Synthesis of T807P (5) and t-Boc-protected T807P (6). Reagents,
conditions and yields: (i) (Me3Sn)2, Pd(PPh3)4, 1,4-dioxane, reflux; 55%. (ii)
Pd(PPh3)4, Cs2CO3, 1,4-dioxane, reflux; 7% (from 2-nitro-5-pyridineboronic acid
pinacol ester) and 26% (from 4). (iii) DMAP, (Boc)2O, CH2Cl2; 85%.
Figure 1. Chemical structure of
[
11C]PIB,
[
18F]Amyvid ([18F]AV-45),
[
11C]PBB3,
[
18F]T808 ([18F]AV-680), and [18F]T807 ([18F]AV-1451).
reactivity of the boronic acid ester. Thus alternate reagent and
more reactive method need to be developed. To improve the yield
of T807P, a Stille cross-coupling reaction was employed to prepare
T807P in 26% yield, using 2-nitro-5-(trimethylstannyl)pyridine (4)
as the replacement of the boronic acid ester to react with the inter-
mediate 2. The halogen-tin exchange reaction of 5-bromo-2-
nitropyridine with hexamethylditin in refluxing dioxane in the
presence of the catalyst Pd(PPh3)4 gave 4 in 55% yield. Another
precursor t-Boc-protected T807P was prepared from T807P with
di-tert-butyl-dicarbonate {(Boc)2O} using base catalyst 4-N,N-
dimethylaminopyridine (DMAP) in 85% yield.19
Further studies showed the ÀNH in compound 2 may result in
by-product and lower yield in Suzuki coupling and Stille cross-
coupling reactions (Scheme 2). Consequently, alternate synthesis
of T807P and t-Boc-protected T807P was designed and conducted
as shown in Scheme 3. Boc-protected intermediate 7 was synthe-
sized from compound 2 with (Boc)2O and DMAP in 86% yield.
Compound 7 was reacted with 2-nitro-5-pyridineboronic acid
pinacol ester or 4 using the same procedure for the preparation
of T807P (Scheme 2) to afford two precursors 5 (10–16% yield)
and 6 (15–32% yield) at the same time, and the yields of 5 and 6
was changed while reaction time was different. If the reaction time
is longer, the yield of 6 will decrease, since the t-Boc protecting
group is not very stable under high reaction temperature, and
can be deprotected under mildly basic conditions.18,19
[
18F]T807, and present
a fully automated one-step-one-pot
radiosynthesis of [18F]T807 with relatively high radiochemical
yields and shortened radiosynthesis time.
The synthesis of reference standard T807 (7-(6-fluoropyridin-3-
yl)-5H-pyrido[4,3-b]indole, 3) was shown in Scheme 1, according
to the literature method17 with modifications. (4-Bromophenyl)-
boronic acid and 3-bromo-4-nitropyridine were undergone
Suzuki coupling reaction in the presence of the catalyst
tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) to produce
3-(4-bromophenyl)-4-nitropyridine (1) in 72% yield, which was
cyclized using triethyl phosphite (4–8 equiv) at 110 °C to provide
7-bromo-5H-pyrido[4,3-b]indole (2) in 70% yield. The literature
yield was 40%.17 Careful control of reaction temperature and time
(3 h) in the cyclization step improved the yield of 2. Compound 2
was used for further Suzuki coupling reaction with (6-fluoropy-
ridin-3-yl)boronic acid to give T807 in 53% yield.
The synthesis of nitro-precursor T807P (7-(6-nitropyridin-3-yl)-
5H-pyrido[4,3-b]indole, 5) and t-Boc-protected T807P (tert-butyl
7-(6-nitropyridin-3-yl)-5H-pyrido[4,3-b]indole-5-carboxylate, 6)
was outlined in Scheme 2. The Suzuki coupling reaction of the
intermediate 2 with 2-nitro-5-pyridineboronic acid pinacol ester
gave T807P in only 7% yield, although the reaction conditions
including catalyst ligands, solvents and bases have been optimized.
The low yield is attributed to the low reactivity of boronic acid
ester, since this boronic acid ester contains: (1) strong electron-
withdrawing nitro group in pyridinyl ring at its para-position;
and (2) pyridinyl nitrogen at its meta-position resulting in the
electron deficiency of pyridinyl ring, and both all decrease the
Me
O
Me
Me
O2N
O2N
B
N
N
O
Me
or
N
OH
B
SnMe3
N
N
N
i
ii
ii
OH
i
4
Br
Br
Br
iii
N
N
H
2
NO2
Br
Br
NO2
Br
Boc
7
1
N
N
N
N
F
N
H
N
N
O2N
N
Boc
H
O2N
N
H
N
N
2
T807 (3)
t-Boc-protected T807P (6)
T807P (5)
Scheme 1. Synthesis of T807 (3). Reagents, conditions and yields: (i) Pd(PPh3)4,
Na2CO3, 1,4-dioxane, H2O, reflux; 72%. (ii) P(OEt)3, 110 °C; 70%. (iii) (6-fluoropy-
ridin-3-yl)boronic acid, Pd(OAc)2, 2-dicyclohexylphosphinobiphenyl, K2CO3,
dimethoxyethane (DME), H2O, reflux; 53%.
Scheme 3. Alternate synthesis of T807P (5) and t-Boc-protected T807P (6).
Reagents, conditions and yields: (i) DMAP, (Boc)2O, CH2Cl2; 86%. (ii) Pd(PPh3)4,
Cs2CO3, 1,4-dioxane, reflux; 5 (10–16%) and 6 (15–32%).

M. Gao et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2953–2957
2955
Synthesis of the target tracer [18F]T807 (7-(6-[18F]fluoropy-
ridin-3-yl)-5H-pyrido[4,3-b]indole, [18F]3) from T807P is shown
in Scheme 4. The nitro-precursor T807P was labeled with
K[18F]F/Kryptofix 2.2.2 through nucleophilic substitution at
140 °C to form [18F]T807.12,13,16,18 It is difficult to directly purify
labeled product [18F]T807 from unlabeled precursor T807P by a
semi-preparative reverse-phase (RP) high performance liquid chro-
matography (HPLC) due to their close polarity. Therefore, the
reduction of –NO2 to –NH2 is needed after F-18 labeling step. The
reaction mixture was treated with Fe powder with formic acid,
quenched with 0.1 M NaHCO3, filtered, and purified by HPLC com-
bined with solid-phase extraction (SPE) using a C18 Plus Sep-Pak
cartridge to give pure tracer [18F]T807, which was formulated in
ethanol/saline. This is a two-step-two-pot radiosynthesis. DMSO
is a good reaction solvent for [18F]fluorination at high reaction tem-
perature, since it resulted in higher radiochemical yield.20–24
However, the solubility of T807P in DMSO is not very high, and
thus F-18 labeling yield was low.18 To reduce excess amount of
the unreacted precursor T807P (1 mg) to amino-T807, huge
amount of Fe powder (90–100 mg) need to be used, thus it is not
easy to perform routine automation, and instead manual synthesis
is required. The overall synthesis, purification and formulation
time was ꢀ90 min from end of bombardment (EOB), and decay
corrected radiochemical yield at EOB based on H[18F]F was 5–
10%. Other group18 has proved it was unable to reach the reported
high radiochemical yield (29–47%),12,13 and our manual procedure
reported here was not able to give high radiochemical yield as well.
Synthesis of the target tracer [18F]T807 from t-Boc-protected
T807P is shown in Scheme 5. t-Boc-protected T807P was labeled
with K[18F]F/Kryptofix 2.2.2 in DMSO at 140 °C via a concurrent
separation of labeled product [18F]T807 from its corresponding
precursor t-Boc-protected T807P and unreacted [18F]fluoride. The
amounts of the precursor t-Boc-protected T807P used were
ꢀ1 mg. Small amount of the precursor was used for radiolabeling
instead of large amount of the precursor (2.5 mg) reported in the
literature,11 which improved the chemical purity of the final tracer
solution. A large amount of precursor would increase the radio-
chemical yield, but decrease the chemical purity of [18F]T807 tracer
solution due to precursor contamination. In addition, a large
amount of precursor would also decrease the SA of final labeled
product due to potential F-18/F-19 exchange during the radiolabel-
ing.16 The SA was 37–370 GBq/
that the maximum in-target SA for our
ꢀ370 GBq/ mol at EOB produced in our cyclotron (Siemens RDS-
111 Eclipse) and home-built automated [18F]-radiosynthesis unit.
The SA for our
18F]-tracers usually ranges from 37 to
370 GBq/ mol at EOB according to our previous works for the
18F]-tracers produced in this facility, including [18F]Fallypride,
l
mol at EOB. Our study has proved
[
18F]-tracers is
l
[
l
[
[
18F]PBR06, [18F]FEDAA1106, etc.20–23 Theoretically, all [18F]-trac-
ers have same SA, and actual SA of [18F]-tracers is mainly related
to the type of cyclotron and synthetic module.27 The SA of the title
tracer [18F]T807 was 37–370 GBq/
l
mol at EOB, which was similar
to the values previously reported by our lab.20–23 No-carrier-added
18F]fluoride ion in [18O]water was trapped without a QMA car-
[
tridge. This way20–23,28 significantly increased the SA of the pre-
pared F-18 labeled product. As indicated in the literature,28 when
the cyclotron-produced [18F]fluoride ion was dried without the
use of a cartridge, but through cycles of evaporation with added
acetonitrile, the SA of the prepared [18F]T807 was substantially
higher, and was similar to that we achieved in the radiosynthesis
of other F-18 tracers such as [18F]fallypride and [18F]PBR06
previously reported.20–23 The reason was that there was a low-
level contamination of QMA anionic resins with fluoride ion.28
Furthermore, in order to make more product radioactivity, we
also modified the published semi-preparative HPLC condi-
tions12,13,18 including column, mobile phase and flow rate to
shorten the retention time of [18F]T807. Although this modification
helped to shorten the overall production time, it potentially could
bring the organic solvent residue issue (CH3CN from mobile phase)
since CH3CN residue has limit restriction for clinical usage. In addi-
tion, to our F-18 labeling experiences on F-18 tracers,16,20–23
although the HPLC systems we employed have shown good separa-
tion of products from precursors, there always was a co-elution of
the F-18 labeled product with its corresponding precursor from the
[
18F]fluorination and deprotection and purified by RP-HPLC/SPE
method to give [18F]T807 in 20–30% decay corrected radiochemical
yield. This is a one-step-one-pot radiosynthesis, which was per-
formed in a self-designed and fully automated multi-purpose
[
18F]-radiosynthesis module.23,25,26 The solubility of t-Boc-
protected T807P in DMSO is increased compared to T807P, and
thus it improved F-18 labeling yield. t-Boc-protected T807P is con-
currently deprotected during nucleophilic F-18 substitution with
K[18F]F/Kryptofix 2.2.2 at high reaction temperature (140 °C).18
The overall synthesis, purification and formulation time was
60–70 min from EOB. t-Boc-protected T807P has made more easily
amenable for routine automation with relatively short radiosyn-
thesis time and much higher radiochemical yield.
Several modifications have been made to improve the chemical
purity and specific activity (SA) of the target tracer [18F]T807.
Addition of NaHCO3 to quench the radiolabeling reaction and to
dilute the radiolabeling mixture prior to the injection onto the
semi-preparative HPLC column for purification20–23 gave better
HPLC column, very tiny amount of the precursor (0.1–0.3 lg/mL)
contaminating the tracer solution. [18F]T807 was also in the same
case. Therefore, we used a C-18 Plus Sep-Pak cartridge for the pur-
poses to further remove the organic solvent residue from HPLC
mobile phase, the precursor and most of possible non-radiolabeled
undesired side-products. The product fraction (5–10 mL) was
diluted in 30 mL water, passed through the C18 cartridge, and
washed with 4 Â 5 mL water. Thus, the CH3CN residue in the final
product (EtOH/saline solution) is lower than limit, which can be
detected by gas chromatography (GC) in quality control (QC) anal-
ysis. A C-18 Plus Sep-Pak cartridge instead of rotatory evaporation
was used to significantly improve the chemical purity of the tracer
solution.16,20–23 In this study, the Sep-Pak purification can further
increase the chemical purity more than 10%.
N
N
N
H
i
N
H
18F
N
O2N
N
3
)
T807P (5)
[
18F]T807 ([18F]
N
ii
Chemical purity and radiochemical purity were determined by
analytical HPLC.29 The chemical purity of T807, T807P and t-Boc-
protected T807P was >93% determined by HPLC through UV flow
detector. The radiochemical purity of the target tracer [18F]T807
N
H
H2N
N
Amino-T807
was >98% determined by radio-HPLC through
flow detector.
c-ray (PIN diode)
Scheme 4. Synthesis of [
18F]T807 ([18F]3) from T807P (5). Reagents, reaction
conditions and yields: (i) K[18F]/K2.2.2, DMSO, 140 °C, 15 min; (ii) Fe powder/
HCOOH, 110 °C, 10 min; HPLC-SPE; 5–10%.
The experimental details and characterization data for com-
pounds 1–7 and for the tracer [18F]3 are given.30

2956
M. Gao et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2953–2957
N
N
N
i
N
i
Boc
O2N
N
Boc
N
H
18F
t-Boc-protected [18F]T807
N
N
18F
N
[
t-Boc-protected T807P (6)
18F]T807 ([18F]
)
3
Scheme 5. Synthesis of [18F]T807 ([18F]3) from t-Boc-protected T807P (6). Reagents, reaction conditions and yields: (i) K[18F]/K2.2.2, DMSO, 140 °C, 15 min; HPLC-SPE;
20–30%.
12. Xia, C. F.; Arteaga, J.; Chen, G.; Gangadharmath, U.; Gomez, L. F.; Kasi, D.; Lam,
In summary, T807 was synthesized by the patent method with
modifications and improved yields. Synthesis of the precursors
C.; Liang, Q.; Liu, C.; Mocharla, V. P.; Mu, F.; Sinha, A.; Su, H.; Szardenings, A. K.;
Walsh, J. C.; Wang, E.; Yu, C.; Zhang, W.; Zhao, T.; Kolb, H. C. Alzheimer’s Dement
T807P and t-Boc-protected T807P is first reported. A two-step-
two-pot manual radiosynthesis of [18F]T807 from T807P has
described for comparison. An improved, concise and automated
one-step-one-pot radiosynthesis of [18F]T807 from t-Boc-protected
T807P has been developed, which were superior to previous works.
The radiosynthesis employed a concurrent [18F]fluorination and
deprotection. The target tracer was isolated and purified by a
semi-preparative HPLC combined with SPE procedure in relatively
high radiochemical yields, shortened overall synthesis time, and
high specific activity, radiochemical purity and chemical purity.
New and improved results in the synthetic methodology, radiola-
beling, preparative separation and analytical details for T807,
T807P, t-Boc-protected T807P and [18F]T807 have been presented.
These methods are efficient and convenient. It is anticipated that
the approaches for the design, synthesis and automation of new
tracer and radiolabeling precursor, and improvements to increase
radiochemical yield, chemical purity and specific activity of the
tracer described here can be applied with advantage to the synthe-
sis of other 18F-radiotracers for PET imaging. These results facilitate
the potential preclinical and clinical PET studies of [18F]T807 as a
PET AD tau tracer in animals and humans.
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30. (a). General: All commercial reagents and solvents were purchased from
Sigma–Aldrich and Fisher Scientific, and used without further purification.
Melting points were determined on a MEL-TEMP II capillary tube apparatus
and were uncorrected. 1H NMR spectra were recorded on a Bruker Avance II
500 MHz NMR spectrometer using tetramethylsilane (TMS) as an internal
standard. Chemical shift data for the proton resonances were reported in parts
per million (ppm, d scale) relative to internal standard TMS (d 0.0), and
coupling constants (J) were reported in hertz (Hz). Liquid chromatography-
mass spectra (LC–MS) analysis was performed on an Agilent system, consisting
of an 1100 series HPLC connected to a diode array detector and a 1946D mass
spectrometer configured for positive-ion/negative-ion electrospray ionization.
Chromatographic solvent proportions are indicated as volume: volume ratio.
Thin-layer chromatography (TLC) was run using Analtech silica gel GF
uniplates (5 Â 10 cm2). Plates were visualized under UV light. Normal phase
flash column chromatography was carried out on EM Science silica gel 60
(230–400 mesh) with a forced flow of the indicated solvent system in the
proportions described below. All moisture- and air-sensitive reactions were
performed under a positive pressure of nitrogen maintained by a direct line
from a nitrogen source. Analytical RP HPLC was performed using a Prodigy
This work was partially supported by the United States Indiana
State Department of Health (ISDH) Indiana Spinal Cord & Brain
Injury Fund (ISDH EDS-A70-2-079612). 1H NMR spectra were
recorded on a Bruker Avance II 500 MHz NMR spectrometer in
the Department of Chemistry and Chemical Biology at Indiana
University Purdue University Indianapolis (IUPUI), which is
supported by the United States National Science Foundation
(NSF) Major Research Instrumentation Program (MRI) grant
CHE-0619254.
References and notes
1. James, O. G.; Doraiswamy, P. M.; Borges-Neto, S. Front. Neurol. 2015, 6, 38.
2. Ariza, M.; Kolb, H. C.; Moechars, D.; Rombouts, F.; Andrés, J. I. J. Med. Chem.
2015. http://dx.doi.org/10.1021/jm5017544.
3. Adlard, P. A.; Tran, B. A.; Finkelstein, D. I.; Desmond, P. M.; Johnston, L. A.; Bush,
A. I.; Egan, G. F. Front. Neurosci. 2014, 8, 327.
4. Zimmer, E. R.; Leuzy, A.; Benedet, A. L.; Breitner, J.; Gauthier, S.; Rosa-Neto, P. J.
Neuroinflammation 2014, 11, 120.
5. Catafau, A. M.; Bullich, S. Clin. Transl. Imaging 2015, 3, 39.
6. Villemagne, V. L.; Fodero-Tavoletti, M. T.; Masters, C. L.; Rowe, C. C. Lancet
Neurol. 2015, 14, 114.
(Phenomenex) 5
77% 20 mM H3PO4; flow rate 1.0 mL/min; and UV (254 nm) and
diode) flow detectors. Semi-preparative RP HPLC was performed using
Prodigy (Phenomenex) 5
m C-18 column, 12 nm, 10 Â 250 mm; mobile phase
25% CH3CN/75% 20 mM H3PO4; 5.0 mL/min flow rate; UV (254 nm) and -ray
(PIN diode) flow detectors. C18 Plus Sep-Pak cartridges were obtained from
l
m C-18 column, 4.6 Â 250 mm; mobile phase 23% CH3CN/
c
-ray (PIN
a
l
c
7. Sabri, O.; Seibyl, J.; Rowe, C.; Barthel, H. Clin. Transl. Imaging 2015, 3, 13.
8. Wurtman, R. Metabolism 2015, 64, S47.
Waters Corporation (Milford, MA). Sterile Millex-FG 0.2
obtained from Millipore Corporation (Bedford, MA).
lm filter units were
9. Klunk, W. E.; Engler, H.; Nordberg, A.; Bacskai, B. J.; Wang, Y.; Price, J. C.;
Bergström, M.; Hyman, B. T.; Långström, B.; Mathis, C. A. Neuroimaging Clin. N.
Am. 2003, 13, 781.
10. Carpenter, A. P., Jr.; Pontecorvo, M. J.; Hefti, F. F.; Skovronsky, D. M. Q. J. Nucl.
Med. Mol. Imaging 2009, 53, 387.
11. Zhang, W.; Arteaga, J.; Cashion, D. K.; Chen, G.; Gangadharmath, U.; Gomez, L.
F.; Kasi, D.; Lam, C.; Liang, Q.; Liu, C.; Mocharla, V. P.; Mu, F.; Sinha, A.;
Szardenings, A. K.; Wang, E.; Walsh, J. C.; Xia, C.; Yu, C.; Zhao, T.; Kolb, H. C. J.
Alzheimer’s Dis. 2012, 31, 601.
(b). 3-(4-Bromophenyl)-4-nitropyridine (1): To a solution of 1,4-dioxane (60 mL)
and water (10 mL) was added 3-bromo-4-nitropyridine (1.02 g, 5.0 mmol), (4-
bromophenyl)boronic acid (1.10 g, 5.5 mmol), Pd(PPh3)4 (0.23 g, 0.2 mmol),
and Na2CO3 (1.33 g, 12.5 mmol). Then the reaction was allowed to stir at reflux
temperature (110 °C) for 18 h. The reaction was cooled down, poured into
water (40 mL) and extracted with EtOAc (80 mL Â 3). The combined organic
extracts were washed with brine, dried over MgSO4 and concentrated in
reduced pressure. The residue was purified by silica gel column
chromatography using an eluent (10:90 EtOAc/hexanes) to afford a yellow

M. Gao et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2953–2957
solid product 1 (1.0 g, 72%). Rf = 0.50 (1:2 EtOAc/hexanes), mp 135–137 °C. 1H
yellow solid product
NMR (CDCl3): d 7.21 (d, J = 8.0 Hz, 2H, Ph-H), 7.61 (d, J = 8.0 Hz, 2H, Ph-H), 7.68
(d, J = 5.2 Hz, 1H, Py-H), 8.78 (s, 1H, Py-H), 8.84 (d, J = 5.2 Hz, 1H, Py-H). 1H
NMR (DMSO-d6): d 7.41 (dt, J = 2.2, 9.0 Hz, 2H, Ph-H), 7.71 (dt, J = 2.2, 9.0 Hz,
2H, Ph-H), 8.03 (d, J = 5.5 Hz, 1H, Py-H), 8.87 (s, 1H, Py-H), 8.93 (d, J = 5.5 Hz,
1H, Py-H). MS (ESI): 279 ([M+H]+, 100%).
2957
6
(100 mg, 85%). Rf = 0.77 (1:12 MeOH/CH2Cl2), mp
>300 °C. 1H NMR (CDCl3): d 1.80 (s, 9H, 3 Â CH3), 7.70 (dd, J = 1.5, 8.0 Hz, 1H,
Ar-H), 8.14 (d, J = 5.5 Hz, 1H, Ar-H), 8.22 (d, J = 8.5 Hz, 1H, Ar-H), 8.32 (dd,
J = 2.5, 8.5 Hz, 1H, Ar-H), 8.39 (dd, J = 0.5, 8.5 Hz, 1H, Ar-H), 8.69 (d, J = 6.0 Hz,
1H, Ar-H), 8.70 (s, 1H, Ar-H), 8.97 (dd, J = 0.5, 2.0 Hz, 1H, Ar-H), 9.34 (s, 1H, Ar-
H). MS (ESI): 391 ([M+H]+, 100%). MS (ESI): 413 ([M+Na]+, 20%).
(c). 7-Bromo-5H-pyrido[4,3-b]indole (2): A solution of compound 1 (0.84 g,
3.0 mmol) in 30 mL of triethyl phosphate was heated at 110 °C for 3 h under
N2. The reaction was monitored with TLC for every 1 h. After the starting
material was disappeared, the reaction mixture was cooled down, and volatiles
were removed in vacuo. The residue was purified by silica gel chromatography
using an eluent (2:98 MeOH/CH2Cl2) to give a light brown color solid product 2
(0.52 g, 70%). Rf = 0.48 (1:12 MeOH/CH2Cl2), mp 258–260 °C (dec.). 1H NMR
(DMSO-d6): d 7.42 (dd, J = 2.0, 8.5 Hz, 1H, Ar-H), 7.49 (dd, J = 1.0, 5.5 Hz, 1H, Ar-
H), 7.76 (d, J = 2.0 Hz, 1H, Ar-H), 8.1 (d, J = 8.5 Hz, 1H, Ar-H), 8.4 (d, J = 5.5 Hz,
1H, Ar-H), 9.36 (s, 1H, Ar-H), 11.82 (s, 1H, NH). MS (ESI): 247 ([M+H]+, 100%).
(d). 7-(6-Fluoropyridin-3-yl)-5H-pyrido[4,3-b]indole (3, T807): Palladium(II)
acetate (18 mg, 0.08 mmol) was added to a solution containing compound 2
(198 mg, 0.8 mmol), (6-fluoropyridin-3-yl)boronic acid (158 mg, 1.12 mmol),
dicyclohexylphosphinobiphenyl (30.8 mg, 0.088 mmol) and K2CO3 (332 mg,
2.4 mmol) in DME (50 mL) and water (6 mL). The reaction mixture was
refluxed for 48 h, cooled down to room temperature (rt), diluted with water
and extracted with ethyl acetate (50 mL Â 3). The combined organic extracts
were washed with brine, dried over MgSO4 and concentrated in reduced
pressure. The residue was purified by silica gel chromatography using an
eluent (2:98 MeOH/CH2Cl2) to obtain a white solid product 3 (112 mg, 53%).
Rf = 0.33 (1:12 MeOH/CH2Cl2), mp >300 °C. 1H NMR (acetone-d6): d 7.20 (ddd,
J = 0.5, 3.5, 8.5 Hz, 1H, Ar-H), 7.50 (dd, J = 0.8, 6.0 Hz, 1H, Ar-H), 7.61 (dd, J = 1.5,
8.0 Hz, 1H, Ar-H), 7.89 (d, J = 1.0 Hz, 1H, Ar-H), 8.30–8.33 (m, 1H, Ar-H), 8.35 (d,
J = 8.0 Hz, 1H, Ar-H), 8.47 (d, J = 5.0 Hz, 1H, Ar-H), 8.58 (dd, J = 0.5, 2.0 Hz, 1H,
Ar-H), 9.39 (s, 1H, Ar-H), 10.95 (br s, 1H, NH). MS (ESI): 264 ([M+H]+, 100%). MS
(ESI): 262 ([MÀH]À, 20%).
(h). tert-Butyl 7-bromo-5H-pyrido[4,3-b]indole-5-carboxylate (7): The mixture of
compound 2 (495 mg, 2.0 mmol), DMAP (293 mg, 2.4 mmol), and (Boc)2O
(698 mg, 3.2 mmol) in CH2Cl2 (70 mL) was stirred at rt for 6 h, and then was
concentrated to dryness. The solid residue was purified by silica gel
chromatography using an eluent (2:98 MeOH/CH2Cl2) to give a white solid
product 7 (597 mg, 86%). Rf = 0.79 (1:12 MeOH/CH2Cl2), mp 156–158 °C. 1H
NMR (CDCl3): d 1.77 (s, 9H, 3 Â CH3), 7.53 (dd, J = 1.5, 8.0 Hz, 1H, Ar-H), 7.89 (d,
J = 8.0 Hz, 1H, Ar-H), 8.08 (d, J = 6.0 Hz, 1H, Ar-H), 8.54 (d, J = 0.5 Hz, 1H, Ar-H),
8.64 (d, J = 6.0 Hz, 1H, Ar-H), 9.23 (d, J = 1.0 Hz, 1H, Ar-H). MS (ESI): 347
([M+H]+, 100%).
(i). General procedure for preparation of compound
compounds were synthesized from compound
pyridineboronic acid pinacol ester or compound
5 and 6: These two
7
and 2-nitro-5-
4
according to the
procedure previously described for the synthesis of compound 5. From 2-
nitro-5-pyridineboronic acid pinacol ester, yield (5, 10% and 6, 15%); from 4,
yield (5, 16% and 6, 32%). Analytical data was identical with that previously
described.
(j). 7-(6-[18F]Fluoropyridin-3-yl)-5H-pyrido[4,3-b]indole ([18F]3,
[
18F]T807):
Method A (from 5): No-carrier-added (NCA) aqueous H[18F]F was produced
by 18O(p,n)18F nuclear reaction using a Siemens Eclipse RDS-111 cyclotron by
irradiation of H218O (2.5 mL). H[18F]F (7.4–18.5 GBq) in [18O]water plus 0.1 mL
K2CO3 solution (1.7 mg) and Kryptofix 2.2.2 (10 mg) in 1.0 mL CH3CN with
additional 1 mL CH3CN were placed in the fluorination reaction vial (10-mL V-
vial) and repeated azeotropic distillation (17 min) was performed at 110 °C to
remove water and to form the anhydrous K[18F]F-Kryptofix 2.2.2 complex. The
precursor T807P (5, 1 mg) dissolved in DMSO (1.0 mL) was introduced to the
reaction vessel and heated at 140 °C for 15 min to affect radiofluorination.
After cooling to 60 °C, the reaction mixture was transferred to another 10-mL
V-vial containing 90–100 mg Fe powder with 1.0 mL formic acid. The mixture
was heated to 110 °C for 10 min, cooled to 60 °C, and then quenched with
0.1 M NaHCO3 (3.0 mL). The mixture was filtered, and injected onto the semi-
preparative HPLC column with 3 mL injection loop for purification. The product
fraction was collected in a recovery vial containing 30 mL water. The diluted
tracer solution was then passed through a C-18 Sep-Pak Plus cartridge, and
washed with water (5 mL Â 4). The cartridge was eluted with EtOH (1 mL Â 2)
to release [18F]3, followed by saline (10 mL). The eluted product was then
(e). 2-Nitro-5-(trimethylstannyl)pyridine (4):
A
mixture of 5-bromo-2-
nitropyridine (0.81 g, 4.0 mmol), hexamethylditin (1.84 g, 5.6 mmol),
Pd(PPh3)4 (169 mg, 0.16 mmol) in 1,4-dioxane (50 mL) was heated to reflux
for 6 h. Then the reaction mixture was diluted with water, and extracted with
ethyl acetate (60 mL Â 3). The organic layer was dried over Na2SO4 and
concentrated in vacuo. The crude product was purified by silica gel column
chromatography using an eluent (10:90 EtOAc/hexanes) to give a colorless
solid product 4 (0.63 g, 55%). Rf = 0.52 (1:5 EtOAc/hexanes), mp 109–110 °C. 1
H
NMR (CDCl3): d 0.42 (s, 9H, 3 Â CH3), 8.12–8.18 (m, 2H, Ar-H), 8.65–8.66 (m,
1H, Ar-H). MS (ESI): 289 ([M+H]+, 100%).
(f). 7-(6-Nitropyridin-3-yl)-5H-pyrido[4,3-b]indole (5, T807P): Method A:
A
sterile-filtered through a Millex-FG 0.2 lm membrane into a sterile vial. Total
mixture of compound 2 (99 mg, 0.4 mmol), 2-nitro-5-pyridineboronic acid
pinacol ester (160 mg, 0.64 mmol), Pd(PPh3)4 (46 mg, 0.04 mmol), and Cs2CO3
(391 mg, 1.2 mmol) in 1,4-dioxane (100 mL) was stirred at 100 °C under N2
atmosphere for 20 h. After cooling to RT, the volatiles were removed under
reduced pressure. The residue was purified by silica gel chromatography using
an eluent (3:97 MeOH/CH2Cl2) to afford a yellow solid product 5 (8 mg, 7%).
Rf = 0.32 (1:12 MeOH/CH2Cl2), mp >300 °C. 1H NMR (DMSO-d6): d 7.52 (d,
J = 5.5 Hz, 1H, Ar-H), 7.76 (dd, J = 1.5, 8.0 Hz, 1H, Ar-H), 7.98–8.04 (m, 1H, Ar-
H), 8.42–8.47 (m, 3H, Ar-H), 8.61 (dd, J = 2.5, 8.5 Hz, 1H, Ar-H), 9.12 (d,
J = 2.0 Hz, 1H, Ar-H), 9.42 (s, 1H, Ar-H), 11.96 (s, 1H, NH). MS (ESI): 291
([M+H]+, 100%). MS (ESI): 289 ([MÀH]À, 40%). Method B (from 4): A mixture of
compound 2 (99 mg, 0.4 mmol), 4 (184 mg, 0.64 mmol), Pd(PPh3)4 (46 mg,
0.04 mmol), and Cs2CO3 (391 mg, 1.2 mmol) in 1,4-dioxane (100 mL) was
stirred at 100 °C under N2 atmosphere for 20 h. After cooling to RT, the volatiles
were removed under reduced pressure. The residue was purified by silica gel
chromatography using an eluent (3:97 MeOH/CH2Cl2) to obtain a yellow solid
product 5 (30 mg, 26%). Analytical data was identical with Method A.
radioactivity was assayed and total volume was noted for tracer dose
dispensing. Retention times in the semi-preparative HPLC system were: tR
5 = 7.85 min, tR 3 = 9.88 min, tR
analytical HPLC system were: tR 5 = 6.56 min, tR 3 = 7.53 min, tR
18F]3 = 7.65 min. The decay corrected radiochemical yields of [18F]3 were 5–
10%. Method (from 6): The precursor t-Boc-protected T807P (6, 1 mg)
[
18F]3 = 9.91 min. Retention times in the
[
B
dissolved in DMSO (1.0 mL) was introduced to the reaction vial containing dry
K[18F]F/Kryptofix 2.2.2 complex as previously described and heated at 140 °C
for 15 min for [18F]fluorination to afford t-Boc-protected [18F]T807, which was
concurrently deprotected during
[ [
18F]fluorination to form 18F]T807. The
contents of the reaction vial were cooled down to ꢀ90 °C and diluted with
0.1 M NaHCO3 (1 mL), and injected onto the semi-preparative HPLC column
with 3 mL injection loop for purification. The product fraction was collected in
a recovery vial containing 30 mL water. The diluted tracer solution was then
passed through
a C-18 Sep-Pak Plus cartridge, and washed with water
(5 mL Â 4). The cartridge was eluted with EtOH (1 mL Â 2) to release [18F]3,
followed by saline (10 mL). The eluted product was then sterile-filtered
(g). tert-Butyl 7-(6-nitropyridin-3-yl)-5H-pyrido[4,3-b]indole-5-carboxylate (6, t-
Boc-protected T807P): Di-tert-butyl-dicarbonate ((Boc)2O, 105 mg, 0.48 mmol)
was added to a mixture of DMAP (44 mg, 0.36 mmol) and compound 5 (87 mg,
0.3 mmol) in dichloromethane (50 mL) at rt. The resulting reaction mixture
was stirred for 8 h and then was concentrated. The residue was purified by
silica gel chromatography using an eluent (2:98 MeOH/CH2Cl2) to afford a
through a Millex-FG 0.2 lm membrane into a sterile vial. Total radioactivity
was assayed and total volume was noted for tracer dose dispensing. Retention
times in the semi-preparative HPLC system were: tR 6 = 13.45 min, tR
3 = 9.88 min, tR
system were: tR 6 = 9.04 min, tR 3 = 7.53 min, tR
corrected radiochemical yields of [18F]3 were 20–30%.
[
18F]3 = 9.91 min. Retention times in the analytical HPLC
[
18F]3 = 7.65 min. The decay