Radiosynthesis of a carbon-11-labeled AMPAR allosteric modulator as a new PET radioligand candidate for imaging of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2019.03.027

To develop PET tracers for imaging of Alzheimer's disease, a new carbon-11-labeled AMPAR allosteric modulator 4-cyclopropyl-7-(3-[¹¹C]methoxyphenoxy)-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide ([¹¹C]8) has been synthesized

Full text of DOI:10.1016/j.bmcl.2019.03.027

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Radiosynthesis of a carbon-11-labeled AMPAR allosteric modulator as a new
PET radioligand candidate for imaging of Alzheimer’s disease
⁎
Caihong Miaoa, Fugui Donga, Limeng Jiaa, Wei Lia, Min Wangb, Qi-Huang Zhengb,
,
⁎
Zhidong Xua,c,d,
a Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, College of Chemistry and Environmental Science, Hebei University, Baoding,
Hebei 071002, China
b Department of Radiology and Imaging Sciences, Indiana University School of Medicine, 1345 West 16th Street, Room 202, Indianapolis, IN 46202, USA
c College of Chemical & Pharmaceutical Engineering, Key Laboratory of Molecular Chemistry for Medicine of Hebei Province, Hebei University of Science & Technology,
Shijiazhuang, Hebei 050018, China
d Shijiazhuang Vince Pharmatech Co., Ltd., Shijiazhuang, Hebei 050030, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
To develop PET tracers for imaging of Alzheimer’s disease, a new carbon-11-labeled AMPAR allosteric modulator
4-cyclopropyl-7-(3-[11C]methoxyphenoxy)-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide ([11C]8) has
been synthesized. The reference standard 4-cyclopropyl-7-(3-methoxyphenoxy)-3,4-dihydro-2H-benzo[e][1,2,4]
thiadiazine 1,1-dioxide (8) and its corresponding desmethylated precursor 4-cyclopropyl-7-(3-hydroxyphenoxy)-
3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (9) were synthesized from 4-methoxyabiline and chlor-
osulfonyl isocyanate in eight and nine steps with 3% and 1% overall chemical yield, respectively. The target
tracer [11C]8 was prepared from the precursor 9 with [11C]CH3OTf through O-[11C]methylation and isolated by
HPLC combined with SPE in 10–15% radiochemical yield, based on [11C]CO2 and decay corrected to end of
α-Amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid receptor (AMPAR)
Carbon-11-labeled AMPAR allosteric
modulator
Radiosynthesis
Positron emission tomography (PET)
Alzheimer’s disease (AD)
bombardment (EOB). The radiochemical purity was > 99%, and the molar activity (AM
) at EOB was
370–740 GBq/μmol with a total synthesis time of 35–40-minutes from EOB.
Imaging has played a variety of important roles in the study of
Alzheimer’s disease (AD).1 Neuroimaging of AD becomes one of the
particularly active as well as most challenging areas in neuroscience.2
Advanced biomedical imaging technique positron emission tomography
(PET) is a promising modality for AD, and significant advances have
occurred in this field of molecular imaging.3 The development of PET
imaging probes for in vivo detection of Alzheimer’s brains is critical for
early and accurate diagnosis and for the successful discovery of AD
therapies.4 Previous PET AD imaging agent development is based on
cholinergic hypothesis, amyloid hypothesis, and tau hypothesis. The
representative PET tracers in clinical evaluations such as carbon-11-
labeled AChEIs [11C]AMP ([11C]MP4A), [11C]PMP ([11C]MP4P), and
toward the development of PET agents for AD diagnosis have been
ongoing quite some time, and a series of enzyme- or receptor-based PET
agents has been developed in this laboratory. For example, we have
targeted the enzyme glycogen synthase kinase-3 (GSK-3) and developed
carbon-11-labeled GSK-3 inhibitors12,13 as PET AD imaging agents
(Fig. 1).
In this continued effort, we revisit α-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid receptor (AMPAR), which is a novel and at-
tractive molecular target for treatment and PET imaging of AD.14,15 We
and other groups have developed several AMPAR PET tracers,15–21 and
representative radioligands are shown in Fig. 2. However, preclinical
and clinical evaluation indicated these radioligands have significant
drawbacks like not potent enough in vitro IC50 and/or Ki values, low
specific binding, high non-specific binding, poor brain entry, incon-
sistent brain uptake compared to known AMPAR distribution, and small
dynamic range and metabolite in the brain.15–21 Thus an ideal AMPAR
radioligand that can be used in the clinical setting to study AMPAR
[
[
11C]Donepezil;5–7 β-amyloid plaques (Aβ) tracers
[
11C]PIB and
18F]Amyvid ([18F]AV-45);8,9 and tau tracers [11C]PBB3 and [18F]T807
([18F]AV-1451)10,11 are listed in Fig. 1. The success and limitations of
Aβ imaging and tau imaging have spurred efforts worldwide to develop
new selective PET tracers for different imaging targets. Our efforts
⁎ Corresponding authors at: Department of Radiology and Imaging Sciences, Indiana University School of Medicine, 1345 West 16th Street, Room 202,
Indianapolis, IN 46202, USA (Q-H. Zheng); Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, College of Chemistry and
Environmental Science, Hebei University, Baoding, Hebei 071002, China (Z. Xu).
E-mail addresses: qzheng@iupui.edu (Q.-H. Zheng), zhidongxu@hbu.edu.cn (Z. Xu).
https://doi.org/10.1016/j.bmcl.2019.03.027
Received 11 February 2019; Received in revised form 13 March 2019; Accepted 20 March 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:CaihongMiao,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2019.03.027

C. Miao, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Fig. 1. PET radiotracers for imaging of AD.
Scheme 1. Synthesis of reference standard (8) and precursor (9). Conditions:
(i) (a) ClSO2NCO, 1-nitro-propane, (b) AlCl3, 100 °C; (ii) H2SO4 (50% wt),
130 °C; (iii) 1-ethoxycyclopropyloxy trimethylsilane, AcOH, CH3OH, room
temperature (RT); (iv) NaBH4, boron trifluoride diethyl etherate, THF, reflux;
(v) triethyl orthoformate, 150 °C; (vi) NaBH4, isopropanol, 50 °C; (vii) BBr3,
CH2Cl2, 0 °C; (viii) 3-methoxybenzeneboronic acid, copper (II) acetate, mole-
cular sieves, pyridine, CH2Cl2, 40 °C; (ix) BBr3, CH2Cl2, 0 °C.
Synthesis of the reference standard 8 and its desmethylated pre-
cursor 4-cyclopropyl-7-(3-hydroxyphenoxy)-3,4-dihydro-2H-benzo[e]
[1,2,4]thiadiazine 1,1-dioxide (9) is outlined in Scheme 1 according to
the reported procedures.14,22–27 The commercially available starting
material 4-methoxyaniline was reacted with sulfurisocyanatidic
chloride following a modified procedure22 by using 1-nitropropane as
solvent to give 7-methoxy-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-
dioxide (1), which resulted in a better yield (82%) and safer operation.
An easy and efficient preparation for hydrolysis of compound 1 in 50%
aqueous H2SO4 under heating afforded 2-amino-5-methox-
ybenzenesulfonamide (2) in 46% yield. Compound 2 was reacted with
1-ethoxycyclopropyloxy trimethylsilane in methanol through a transa-
cetalization reaction in the presence of glacial acetic acid (AcOH) to
generate 5-methoxy-2-((1-methoxycyclopropyl)amino)benzenesulfona-
mide (3) in 61% yield. After the reduction of NaBH4 with boron tri-
fluoride diethyl etherate, methoxyl group of compound 3 was removed
Fig. 2. AMPAR PET radioligands.
and
further
converted
to
2-(cyclopropylamino)-5-methox-
ybenzenesulfonamide (4) in 63% yield. 4-Cyclopropyl-7-methoxy-4H-
benzo[e][1,2,4]thiadiazine 1,1-dioxide (5) was formed by a cyclization
of compound 4 in triethyl orthoformate at 150 °C in 75% yield. Re-
duction of compound 5 with NaBH4 in isopropanol provided 4-cyclo-
propyl-7-methoxy-3, 4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-di-
oxide (6) in 76% yield. Desmethylation of compound 6 with boron
tribromide in dichloromethane gave 4-cyclopropyl-7-hydroxy-3, 4-di-
hydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (7) in 73% yield.
Compound 7 was reacted with 3-methoxybenzeneboronic acid in the
presence of copper (II) acetate in dichloromethane through a Chan-Lam
coupling reaction27 to produce the reference standard compound 8 in
47% yield. The desmethylated precursor 9 was obtained by des-
methylation of compound 8 with BBr3 in dichloromethane in 50%
yield. The overall chemical yield for the standard 8 and precursor 9 in
eight and nine steps was 3% and 1%, respectively. There was no much
expression levels in AD remains to be discovered. Recently a novel
series of 7-phenoxy-substituted 3,4-dihydro-2H-1,2,4-benzothiadiazine
1,1-dioxides have been developed as positive allosteric modulators of
AMPARs with nanomolar potency for potential treatment of AD, and
the lead compound, 4-cyclopropyl-7-(3-methoxyphenoxy)-3,4-dihydro-
2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (8), exhibited high potency
with EC50 = 2.0 nM.14 This compound has the combination of favorable
in vitro activity to AMPAR, and O- or N-methyl positions amenable to
labeling with carbon-11, therefore, its carbon-11-labeled radioligand is
expected to have high specific binding. Here, we report the design,
synthesis and labeling of a carbon-11-labeled AMPAR allosteric mod-
ulator,
4-cyclopropyl-7-(3-[11C]methoxyphenoxy)-3,4-dihydro-2H-
benzo[e][1,2,4]thiadiazine 1,1-dioxide ([11C]8) (Fig. 2), as a new
candidate PET radioligand for imaging of AD.
2

C. Miao, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
(CH3CN, 2 N NaOH, 80 °C, 3 min) are listed and the results are sum-
marized in Scheme 2. [11C]8 N2-isomer was not detected, [11C]8 N4-
isomer was a major radiolabeled product, and [11C]8 was a minor
radiolabeled product. These results suggested that 4-thiadiazine ni-
trogen position of 9 is more easily methylated than phenol hydroxyl
oxygen position to yield undesired radiolabeled by-product. The
radiochemical yield of [11C]8 was optimized to 10–15%. The radio-
chemical yield of [11C]8 N4-isomer was 45–50%. Likewise, there was no
much room to improve the labeling reaction in one-step radiosynthesis
of the desired product [11C]8 using non-protected desmethylated pre-
cursor due to its chemistry nature. To make the target compound [11C]8
as a major product from radiochemistry and synthetic chemistry per-
spectives, the potential strategy will be redesign of the synthetic route
to prepare N-protected desmethylated precursor for two-step radio-
synthesis of [11C]8 in the future work.
The radiosynthesis was performed in a home-built automated multi-
purpose [11C]-radiosynthesis module.30–32 Our radiosynthesis module
facilitated the overall design of the reaction, purification and re-
formulation capabilities in a fashion suitable for adaptation to pre-
paration of human doses. The radiosynthesis includes three stages: 1)
labeling reaction; 2) purification; and 3) formulation. The overall
synthesis time was 35–40 min from EOB. Our module is also designed to
allow in-process measurement of [11C]-tracer molar activity (AM, GBq/
μmol at EOB) using a radiation detector with a UV detector at the outlet
of the HPLC-portion of the system. At the end of synthesis (EOS), the AM
of [11C]-tracer was determined again by analytical RP-HPLC, calcu-
lated, decay corrected to EOB, and based on [11C]CO2, which was in
agreement with the ‘on line’ determined value. The AM of [11C]8 at EOB
was 370–740 GBq/μmol.
Scheme 2. [11C]Methylation reaction of the precursor 9 for the synthesis of
target tracer [11C]8.
room to significantly improve the overall yield in reported multiple step
synthetic approach. The potential strategy will be redesign of the syn-
thetic route to decrease the reaction steps in the future work.
Synthesis of the target tracer [11C]8 is presented in Scheme 2. The
desmethylated precursor 9 underwent O-[11C]methylation15 using the
reactive
[
11C]methylating
agent
[
11C]methyl
triflate
([11C]CH3OTf)28,29 in acetonitrile at 80 °C under basic conditions (2 N
NaOH). The product was isolated by semi-preparative reverse-phase
(RP) high performance liquid chromatography (HPLC) with a C-18
column, and then concentrated by solid-phase extraction (SPE) with a
disposable C-18 Plus Sep-Pak cartridge to produce the corresponding
pure radiolabeled compound [11C]8 in 10–15% radiochemical yield,
decay corrected to end of bombardment (EOB), based on [11C]CO2.
The precursor 9 contains phenol hydroxyl, 4- and 2- thiadiazine
nitrogen positions that can be O- and N-[11C]methylated to form [11C]
8, [11C]8 N4-isomer, and [11C]8 N2-isomer, respectively, as indicated in
Scheme 2. Based on the Log P and calculated Log P (CLog P) values of
Chemical purity and radiochemical purity were determined by
analytical HPLC.33 The chemical purity of the precursor and reference
standard was > 93% determined by RP-HPLC through UV flow de-
tector. The radiochemical purity of the target tracer was > 99% de-
termined by radio-HPLC through γ-ray (PIN diode) flow detector.
The octanol-water partition coefficient (commonly expressed as Log
P) is an important physical parameter directly correlated with the
biological activities of a wide variety of organic compounds.34 Log P
provides an assessment of lipophilicity that often correlates with a
compound’s ability to penetrate the blood brain barrier (BBB). Table 1
gives Log P and CLog P values of [11C]8 and its isomers in comparison
with [11C]PIB, [18F]Amyvid, [11C]PBB3 and [18F]T807 (Fig. 1), which
are obtained from ChemDraw Professional 18.0 (ChemOffice). Log P
data of [11C]8 (3.04) is in the range of those of [11C]PIB, [18F]Amyvid,
[
11C]8 and its isomers obtained from ChemDraw Professional 18.0
(ChemOffice) as listed in Table 1, we can predict their retention time
(tR) sequence in semi-preparative RP-HPLC system would be tR
[
11C]8
N2-isomer > tR
[
11C]8 > tR
[
11C]8 N4-isomer. Base effect on the
radiosynthesis of [11C]8 was investigated to maximize the radio-
chemical yield of desired radiolabeled product. The different bases in-
cluding solid base sodium hydride (NaH) powder, sodium hydroxide
(NaOH), potassium hydroxide (KOH), sodium carbonate (Na2CO3),
potassium carbonate (K2CO3), NaOH-Na2CO3 (mixed); and liquid base
aqueous NaOH, KOH, Na2CO3, K2CO3, sodium bicarbonate (NaHCO3)
were tested for the radiosynthesis, and 2 N NaOH was identified as the
best base. The reaction conditions including the reaction solvent,
temperature and time were optimized as well. The optimized conditions
[
11C]PBB3 and [18F]T807 (2.25–4.09), which are PET AD imaging
agents in clinical evaluation. These data suggest [11C]8 has an appro-
priate lipophilicity for brain uptake.
The stability of the labeled tracer [11C]8 was evaluated by analytical
HPLC from EOS up to 3 h, one injection of the tracer solution in EtOH/
saline onto HPLC column per hour. The HPLC chromatograms showed
[
11C]8 was stable without decomposition.
The experimental details and characterization data for compounds
1–9 and for the tracer [11C]8 are given.35
In summary, facile synthetic routes with moderate to excellent
yields have been developed to produce the precursor 9, the reference
standard 8, and the target PET radiotracer [11C]8. The radiosynthesis
employed [11C]CH3OTf for O-[11C]methylation at the phenyl hydroxyl
position of the precursor, followed by product purification and isolation
by a semi-preparative RP-HPLC combined with SPE. The base effect on
the radiotracer production of [11C]8 has been investigated. The desired
Table 1
Log P and CLog P values of [11C]8 and its isomers in comparison with [11C]PIB,
[
18F]Amyvid, [11C]PBB3 and [18F]T807 obtained from ChemDraw Professional
18.0 (ChemOffice).
Compound
Log P
CLog P
[
11C]8 was obtained in reasonable radiochemical yield, and high
[
[
[
[
[
[
[
11C]8
3.04
N/A
3.07
3.41
3.16
4.09
2.25
3.12
0.023
3.27
3.99
3.91
4.05
3.18
11C]8 N4-isomer
11C]8 N2-isomer
11C]PIB
radiochemical purity, with a reasonably short overall synthesis time,
and high molar activity. A new AMPAR radioligand has been success-
fully radiosynthesized. This will facilitate studies to evaluate the
carbon-11-labeled AMPAR allosteric modulator as a new candidate PET
radioligand for imaging of AD.
18F]Amyvid
11C]PBB3
18F]T807
3

C. Miao, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
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16. Arstad E, Gitto R, Chimirri A, et al. Closing in on the AMPA receptor: synthesis and
evaluation of 2-acetyl-1-(4'-chlorophenyl)-6-methoxy-7-[11C]methoxy-1,2,3,4-tetra-
hydroisoquinoline as a potential PET tracer. Bioorg Med Chem. 2006;14:4712–4717.
17. Oi N, Tokunaga M, Suzuki M, et al. Development of novel PET probes for central 2-
amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid receptors. J Med Chem.
2015;58:8444–8462.
18. Takahata K, Kimura Y, Seki C, et al. A human PET study of [11C]HMS011, a potential
radioligand for AMPA receptors. EJNMMI Res. 2017;7:63.
J = 8.8 Hz, 1H), 3.75 (s, 3H). LC-MS (ESI, m/z): Calcd for C8H9N2O4S ([M+H]+
)
19. Yuan G, Jones GB, Vasdev N, Liang SH. Radiosynthesis and preliminary PET eva-
luation of 18F-labeled 2-(1-(3-fluorophenyl)-2-oxo-5-(pyrimidin-2-yl)-1,2-dihy-
dropyridin-3-yl)benzonitrile for imaging AMPA receptors. Bioorg Med Chem Lett.
2016;26:4857–4860.
229.03, found: 229.03. (c). 2-Amino-5-methoxybenzenesulfonamide (2): Compound
1 (0.51 g, 2.2 mmol) was added to a stirred solution of H2SO4 (15 mL, 50% wt) at RT,
the resulting reaction mixture was heated to 130 - 140 °C, and the reaction was
continued for 3 h. The reaction mixture was cooled to 25 °C, and then poured into ice
water (100 mL). The solution was neutralized with NaOH (40%), and extracted with
EtOAc (3 × 60 mL). The combined organic layer was washed with water, brine,
dried over anhydrous Na2SO4 and filtered. The solvent was evaporated under va-
cuum. The crude product was purified by silica gel column chromatography with
EtOAc/petroleum ether (PE) (1:10 to 1:4) as eluent to afford 2 as a pale yellow solid
(0.21 g, 46%), mp 118.3-119.7 °C. 1H NMR (600 MHz, DMSO-d6): δ 7.23 (s, 2H), 7.
11 (d, J = 3.0 Hz, 1H), 6.93 (dd, J = 9.0, 3.0 Hz, 1H), 6.77 (d, J = 9.0 Hz, 1H), 5.43
(s, 2H), 3.67 (s, 3H). LC-MS (ESI, m/z): Calcd for C7H11N2O3S ([M+H]+) 203.05,
found: 203.01. (d). 5-Methoxy-2-((1-methoxycyclopropyl)amino)benzenesulfona-
mide (3): To a stirred solution of 2 (1.00 g; 4.9 mmol) in methanol (20 mL), (1-
ethoxycyclopropyloxy) trimethylsilane (4 mL) and glacial acetic acid (4 mL) were
added at RT. After refluxed for 18 h, the reaction mixture was evaporated to dry
under reduced pressure. The resulted oily residue was treated with water (30 mL),
and extracted with CHCl3 (3 × 30 mL). The combined organic layer was washed
20. Chen Z, Mori W, Zhang X, et al. Synthesis, pharmacology and preclinical evaluation
of 11C-labeled 1,3-dihydro-2H-benzo[d]imidazole-2-ones for imaging γ8-dependent
transmembrane AMPA receptor regulatory protein. Eur J Med Chem.
2018;157:898–908.
21. Fu H, Chen Z, Josephson L, Li Z, Liang SH. Positron emission tomography (PET)
ligand development for ionotropic glutamate receptors: challenges and opportunities
for radiotracer targeting N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-me-
thyl-4-isoxazolepropionic acid (AMPA), and kainate receptors. J Med Chem.
2019;62:403–419.
22. Girard Y, Atkinson JG, Rokach J. A new synthesis of 1,2,4-benzothiadiazines and a
selective preparation of o-aminobenzenesulfonamides. J Chem Soc Perkin Trans.
1979;1:1043–1047.
23. Adams JL, Ator LE, Duffy KJ, Graybill TL, Kiesow TJ, Lian Y, Moore ML, Palph JM,
Ridgers LH. Benzothiadiazine compounds. WO 2017098421A1.
4

C. Miao, et al.
with water, brine, dried over anhydrous Na2SO4 and filtered. The solvent was re-
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Calcd for C10H13N2O3S ([M+H]+) 241.06, found: 241.06. (i). 4-Cyclopropyl-7-(3-
methoxyphenoxy)-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (8): To a
stirred solution of 7 (0.32 g, 1.3 mmol) in CH2Cl2 (40 mL), pyridine (8 drops),
molecular sieves (4 g), 3-methoxybenzeneboronic acid (0.30 g, 2.0 mmol), and
copper (II) acetate (0.63 g, 2.0 mmol) were added at RT. The reaction mixture was
stirred at 40 °C for 5 h, and then CH2Cl2 (15 mL) was added to the reaction mixture.
The solid was removed from the reaction mixture by filtration, and the resulted
organic solution was evaporated under vacuum. The resulted residue was suspended
with aqueous HCl (pH = 5-6) solution (50 mL), and then the resulted aqueous so-
lution was extracted with EtOAc (3 × 50 mL). The combined organic layer was
washed with water, brine, dried over anhydrous Na2SO4 and filtered. The solvent
was evaporated under vacuum. The crude product was purified by silica gel column
chromatography with EtOAc/PE (1:10 to 1:5) as eluent to afford 8 as a white solid (0.
22 g, 47%), mp 151.2-152.7 °C. 1H NMR (600 MHz,CDCl3): δ 7.26 (d, J = 2.4 Hz,
1H), 7.32 (d, J = 9.0 Hz, 1H), 7.21 (t, J = 8.4 Hz, 1H), 7.14 (dd, J = 9.0, 2.4 Hz,
1H), 6.65 - 6.64 (m, 1H), 6.54 - 6.52 (m, 2H), 4.76 - 4.70 (m, 3H), 3.78 (s, 3H), 2.42 -
2.38 (m, 1H), 0.98 - 0.95 (m, 2H), 0.73 - 0.70 (m, 2H). LC-MS (ESI, m/z): Calcd for
moved under vacuum. The crude product was purified by silica gel column chro-
matography with EtOAc/PE (1:20 to 1:5) as eluent to afford 3 as a white solid (0.80
g, 61%), mp 147.5-149.4 °C. 1H NMR (600 MHz, CDCl3): δ 7.41 (d, J = 9.0 Hz, 1H),
7.34 (d, J = 3.0 Hz, 1H), 7.07 (dd, J = 9.0, 3.0 Hz, 1H), 6.48 (s, 1H), 4.86 (s, 2H), 3.
78 (s, 3H), 3.28 (s, 3H), 1.15 (t, J = 5.4 Hz, 2H), 0.90 (t, J = 4.8 Hz, 2H). LC-MS
(ESI, m/z): Calcd for C11H17N2O4S ([M+H]+) 273.09, found: 273.10. (e). 2-(Cy-
clopropylamino)-5-methoxybenzenesulfonamide (4): To a stirred solution of 3 (0.80
g, 2.94 mmol) in THF (50 mL), NaBH4 (2.00 g, 52.9 mmol), and boron trifluoride
diethyl etherate (2 mL) were added at RT. The reaction mixture was then refluxed for
16 h. The solvent was evaporated under vacuum. The resulted oily residue was
treated with water (30 mL) and the pH was adjusted to 4 with aqueous 6 N HCl
solution. The mixture was extracted with CHCl3 (3 × 30 mL), and the combined
organic layer was washed with water, brine, dried over anhydrous Na2SO4 and fil-
tered. The organic mixture was evaporated under vacuum, and the crude product
was purified by silica gel column chromatography with EtOAc/PE (1:10 to 1:4) as
eluent to afford 4 as a white solid (0.46 g, 63%), mp 116.5-118.7 °C. 1H NMR (600
MHz, CDCl3): δ 7.35 (d, J = 3.0 Hz, 1H), 7.23 (d, J = 9.0 Hz, 1H), 7.07 (dd, J = 9.0,
3.0 Hz, 1H), 5.65 (s, 1H), 4.84 (s, 2H), 3.78 (s, 3H), 2.47 - 2.45 (m, 1H), 0.81 - 0.78
C
17H19N2O4S ([M+H]+) 347.11, found: 347.10. (j). 4-Cyclopropyl-7-(3-hydro-
xyphenoxy)-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (9): Compound
9 was prepared by referring to the synthesis procedure of compound 7 as a white
solid (0.13 g, 50%), mp 166.4-168.4 °C. 1H NMR (400 MHz, CD3OD): δ 7.43 (d, J =
9.2 Hz, 1H), 7.23 - 7.14 (m, 3H), 6.56 (dd, J = 8.4, 2.4 Hz, 1H), 6.45 - 6.41 (m, 2H),
4.75 (s, 2H), 2.52 - 2.45 (m, 1H), 1.01 - 0.97 (m, 2H), 0.79 - 0.76 (m, 2H). 13C NMR
(100 MHz, CD3OD): δ 158.88, 158.84, 148.23, 140.98, 129.94, 124.83, 123.77, 116.
48, 114.54, 110.05, 108.44, 104.90, 61.31, 29.48, 7.86 (overlap, 2C). HRMS (ESI, m/
z): Calcd for C16H17N2O4S ([M+H]+) 333.0904, found: 333.0904. (k). 4-Cyclo-
propyl-7-(3-[11C]methoxyphenoxy)-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-
dioxide ([11C]8): [11C]CO2 was produced by the 14N(p,α)11C nuclear reaction in the
small volume (9.5 cm3) aluminum gas target provided with the Siemens RDS-111
Eclipse cyclotron. The target gas consisted of 1% oxygen in nitrogen purchased as a
specialty gas from Praxair, Indianapolis, IN. Typical irradiations used for the de-
velopment were 58 μA beam current and 20 min on target. The production run
produced approximately 37.0 GBq of [11C]CO2 at EOB. The precursor 9 (0.1-0.3 mg)
was dissolved in CH3CN (500 μL). To this solution was added aqueous NaOH (2 N, 2
μL). The mixture was transferred to a small reaction vial. No-carrier-added (high
molar activity) [11C]CH3OTf that was produced by the gas-phase production
method29 within 12 min from [11C]CO2 through [11C]CH4 and [11C]CH3Br with
AgOTf column was passed into the reaction vial at RT until radioactivity reached a
maximum (2 min), and then the reaction vial was isolated and heated at 80 °C for 3
min. The contents of the reaction vial were diluted with aqueous NaHCO3 (0.1 M, 1
mL). The reaction vial was connected to a 3-mL HPLC injection loop. The labeled
product mixture solution was injected onto the semi-preparative HPLC column 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 Plus Sep-Pak
cartridge, and washed with water (3 × 10 mL). The cartridge was eluted with EtOH
(3 × 0.4 mL) to release the labeled product, followed by saline (10-11 mL). The
eluted product was then sterile-filtered through a Millex-FG 0.2 μm membrane into a
sterile vial. Total radioactivity was assayed and total volume (10-11 mL) was noted
for tracer dose dispensing. The overall synthesis time including HPLC-SPE purifica-
tion and reformulation was 35-40 min from EOB. The decay corrected radiochemical
yield of [11C]8 was 10-15%. Retention times in the analytical RP-HPLC system were:
(m, 2H), 0.53 - 0.51 (m, 2H). LC-MS (ESI, m/z): Calcd for C10H15N2O3S ([M+H]+
)
243.08, found: 243.20. (f). 4-Cyclopropyl-7-methoxy-4H-benzo[e][1,2,4]thiadiazine
1,1-dioxide (5): A stirred solution of 4 (0.45 g, 1.86 mmol) in triethyl orthoformate
(10 mL) was heated to 150 °C, and the reaction mixture was refluxed for 6 h. After
cooled on an ice-bath, the solid product was collected by filtration, and washed with
diethyl ether. The product was purified by recrystallization in methanol to afford 5 as
a white solid (0.35 g, 75%), mp 229.8-230.2 °C. 1H NMR (600 MHz, DMSO-d6): δ 8.
07 (s, 1H), 7.80 (d, J = 9.0 Hz, 1H), 7.41 (dd, J = 9.6, 3.0 Hz, 1H), 7.31 (d, J = 3.0
Hz, 1H), 3.87 (s, 3H), 3.39 - 3.36 (m, 1H), 1.17 - 1.14 (m, 2H), 1.10 - 0.98 (m, 2H).
LC-MS (ESI, m/z): Calcd for C11H13N2O3S ([M+H]+) 253.06, found: 253.07. (g). 4-
Cyclopropyl-7-methoxy-3, 4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (6):
To a stirred solution of 5 (0.28 g, 1.11 mmol) in isopropyl alcohol (7 mL), NaBH4 (0.
14 g, 3.71 mmol) was added at RT, and the reaction mixture was heated to 50 °C for
10 min. The reaction mixture was evaporated to dry under vacuum. The resulted oily
residue was treated with water (10 mL) and the pH was adjusted to 4 with aqueous 6
N HCl solution. The resulted solution was extracted with CH2Cl2 (3 × 20 mL). The
combined organic layer was washed with water, brine, dried over anhydrous Na2SO4
and filtered. The organic mixture was evaporated under vacuum. The resulted crude
product was purified by silica gel column chromatography with EtOAc/PE (1:10 to
1:4) as eluent to afford 6 as a white solid (0.21 g, 76%), mp 160.8-161.2 °C. 1H NMR
(600 MHz,CDCl3): δ 7.27 (s, 1H), 7.15 (d, J = 3.0 Hz, 1H), 7.02 (dd, J = 9.6, 3.0 Hz,
1H), 4.82 (t, J = 8.4 Hz, 1H), 4.69 (d, J = 8.4 Hz, 2H), 3.78 (s, 3H), 2.36 - 2.35 (m,
1H), 0.94 - 0.91 (m, 2H), 0.70 - 0.68 (m, 2H). LC-MS (ESI, m/z): Calcd for
C
11H15N2O3S ([M+H]+) 255.08, found: 255.08. (h). 4-Cyclopropyl-7-hydroxy-3,4-
dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (7): To a stirred solution of 6 (0.
2 g, 0.79 mmol) in CH2Cl2 (12 mL), BBr3 (0.3 mL) was added slowly at 0 °C, and the
reaction continued at 0 °C for 20 h. The reaction mixture was poured into ice water
(10 mL), and then the dichloromethane was removed under reduced pressure. The
resulted aqueous solution was extracted with EtOAc (3 × 40 mL), and the combined
organic layer was washed with water, brine, dried over anhydrous Na2SO4 and fil-
tered. The organic solution was evaporated under vacuum, and the resulted crude
product was purified by silica gel column chromatography with EtOAc/PE (1:10 to
1:2) as eluent to afford 7 as a white solid (0.14 g, 73%), mp 175.5-177.9 °C. 1H NMR
(600 MHz, DMSO-d6): δ 9.32 (s, 1H), 7.82 (t, J = 7.8 Hz, 1H), 0.19 (d, J = 9.0 Hz,
1H), 6.94 (dd, J = 9.0, 3.0 Hz, 1H), 6.92 (d, J = 3.0 Hz, 1H), 4.55 (d, J = 7.8 Hz,
2H), 2.39 - 2.36 (m, 1H), 0.86 - 0.83 (m, 2H), 0.62 - 0.60 (m, 2H). LC-MS (ESI, m/z):
t
R 9 = 4.55 min, tR 8 = 7.02 min, and tR
[
11C]8 = 7.10 min. Retention times in the
preparative RP-HPLC system were: tR 9 = 4.87 min, tR 8 = 8.10 min, and tR
[
11C]8
= 8.21 min.
5