Synthesis and preliminary biological evaluation of radiolabeled 5-BDBD analogs as new candidate PET radioligands for P2X4 receptor

Article abstract of DOI:10.1016/j.bmc.2017.05.031

P2X4 receptor has become an interesting molecular target for treatment and PET imaging of neuroinflammation and associated brain diseases such as Alzheimer's disease. This study reports the first design, synthesis, radiolabeling and biological evaluation

Full text of DOI:10.1016/j.bmc.2017.05.031

Accepted Manuscript
Synthesis and preliminary biological evaluation of radiolabeled 5-BDBD ana-
logs as new candidate PET radioligands for P2X4 receptor
Min Wang, Mingzhang Gao, Jill A. Meyer, Jonathan S. Peters, Hamideh
Zarrinmayeh, Paul R. Territo, Gary D. Hutchins, Qi-Huang Zheng
PII:
S0968-0896(17)30824-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.031
BMC 13749
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 April 2017
10 May 2017
15 May 2017
Please cite this article as: Wang, M., Gao, M., Meyer, J.A., Peters, J.S., Zarrinmayeh, H., Territo, P.R., Hutchins,
G.D., Zheng, Q-H., Synthesis and preliminary biological evaluation of radiolabeled 5-BDBD analogs as new
candidate PET radioligands for P2X4 receptor, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/
10.1016/j.bmc.2017.05.031
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Synthesis and preliminary biological evaluation of radiolabeled 5-BDBD analogs as new
candidate PET radioligands for P2X4 receptor
Min Wang, Mingzhang Gao, Jill A. Meyer, Jonathan S. Peters, Hamideh Zarrinmayeh,
Paul R. Territo, Gary D. Hutchins, Qi-Huang Zheng*
Department of Radiology and Imaging Sciences, Indiana University School of Medicine, 1345
West 16th Street, L3-208, Indianapolis, IN 46202, USA
*Corresponding author:
Qi-Huang Zheng, Ph.D.
Department of Radiology
Indiana University School of Medicine
1345 West 16th Street, L3-208
Indianapolis, IN 46202-2111
USA
Tel: 317-278-4671
Fax: 317-278-9711
E-mail: qzheng@iupui.edu
1

Abstract:
P2X4 receptor has become an interesting molecular target for treatment and PET imaging of
neuroinflammation and associated brain diseases such as Alzheimer’s disease. This study reports
the first design, synthesis, radiolabeling and biological evaluation of new candidate PET P2X4
receptor radioligands using 5-BDBD, a specific P2X4 receptor antagonist, as a scaffold. 5-(3-
Hydroxyphenyl)-1-[11C]methyl-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one
(N-
[11C]Me-5-BDBD analog, [11C]9) and 5-(3-Bromophenyl)-1-[11C]methyl-1,3-dihydro-2H-
benzofuro[3,2-e][1,4]diazepin-2-one (N-[11C]Me-5-BDBD, [11C]8c) were prepared from their
corresponding desmethylated precursors with [11C]CH3OTf through N-[11C]methylation and
isolated by HPLC combined with SPE in 30-50% decay corrected radiochemical yields with
370-1110 GBq/µmol specific activity at EOB. 5-(3-[18F]Fluorophenyl)-1,3-dihydro-2H-
benzofuro[3,2-e][1,4]diazepin-2-one
([18F]F-5-BDBD,
[18F]5a)
and
5-(3-(2-
[18F]fluoroethoxy)phenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one
([18F]FE-5-
BDBD, [18F]11) were prepared from their corresponding nitro- and tosylated precursors by
nucleophilic substitution with K[18F]F/Kryptofix 2.2.2 and isolated by HPLC-SPE in 5-25%
decay corrected radiochemical yields with 111-740 GBq/µmol specific activity at EOB. The
preliminary biological evaluation of radiolabeled 5-BDBD analogs indicated these new
radioligands have similar biological activity with their parent compound 5-BDBD.
Keywords: Radiolabeled 5-BDBD analogs; P2X4 receptor; Radiosynthesis; Radioligand
depletion experiment; Competitive binding assay; Positron emission tomography (PET).
2

1. Introduction
The purinergic P2X receptors are a family of cation-permeable ligand gated ion channels that
open in response to the binding of extracellular adenosine 5’-triphosphate (ATP).1 This
ionotropic receptor family contains seven subunits P2X1-7. P2X receptors are involved in
various physiological processes and associated with a variety of diseases including cancer,
cardiovascular and neurological diseases.2 P2X7 and P2X4 receptors are two predominant
subtypes expressed on immune and neural cells. The over expression of P2X7 and P2X4
receptors is linked to neuroinflammation, which is an essential step in the progression of brain
diseases.3 Both P2X7 and P2X4 receptors have become a novel molecular target for treatment
and PET (positron emission tomography) imaging of neuroinflammation and associated brain
diseases such as Alzheimer’s disease.4 In our previous work, we have developed
[11C]GSK14821605,6 as a new PET probe for imaging of P2X7 receptor as indicated in Figure 1.
In this ongoing study, we first target P2X4 receptor and develop PET P2X4 receptor
radioligands. Despite the therapeutic potential of P2X4 receptor antagonists, only a very limited
number of inhibitors are known so far. Subsequently, the structure-activity relationship (SAR)
information of P2X4 receptor antagonists is limited as well. However, potent and selective P2X4
receptor antagonists are required as research tools for investigating the physiological functions of
P2X4 receptor. 5-BDBD (5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-
one) is a specific P2X4 receptor antagonist with an IC50 of 500 nM in Chinese hamster ovary
cells (CHO).7 Here we report the design, synthesis, radiolabeling and preliminary biological
evaluation of a series of radiolabeled 5-BDBD analogs as new candidate P2X4 receptor
radioligands as shown in Figure 2.
3

Figure 1. P2X7 receptor ligand GSK1482160 and radioligand [11C]GSK1482160.
Figure 2. P2X4 receptor antagonist 5-BDBD and radiolabeled 5-BDBD analogs.
4

2. Results and discussion
2.1. Chemistry
5-BDBD can be directly radiolabeled with bromine-76 as [76Br]5-BDBD ([76Br]5c) using either
chloro-precursor or tributylstannyl precursor.8,9 However, our cyclotron does not produce
bromine-76, thus we focus on the synthesis of carbon-11 and fluorine-18 labeled 5-BDBD
analogs. The novel P2X4 receptor radioligands proposed for development are based on direct N-
[11C]methylation at amide position of 5-BDBD and replacement of the bromo group of 5-BDBD
with an O-[11C]methoxy, [18F]fluoro or [18F]fluoroethoxy group. Our initial design is to
synthesize O-[11C]Me-5-BDBD ([11C]5f), N-[11C]Me-5-BDBD ([11C]8c), [18F]F-5-BDBD
([18F]5a) and [18F]FE-5-BDBD ([18F]11) (Figure 2).
5-(3-Substituted phenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one analogs 5a-5f
were synthesized as outlined in Scheme 1. Starting from commercially available 2-
hydroxybenzonitrile, it underwent alkylation with 3-substituted phenacyl bromides 1a-1f in the
presence of Na2CO3 in N,N-dimethylformamide (DMF) to obtain intermediate 2-(2-(3-
substituted phenyl)-2-oxoethoxy)benzonotriles 2a-2f, which were cyclized with sodium
methoxide in MeOH to provide (3-aminobenzofuran-2-yl)-(3-substituted phenyl)methanones 3a-
3f in yields 82-98%.10 As shown in Scheme 1, compound 1d was achieved by α-bromination of
3-iodoacetophenone with copper (II) bromide in EtOAc/CHCl3 in 67% yield.11,12 Acylation of
5

amines 3a-3f with bromoacetyl bromide in the presence of NaHCO3 in CHCl3 afforded 2-bromo-
N-(2-(3-substituted benzoyl)benzofuran-3-yl)acetamide 4a-4f in yields 75-89%. Treatment of
4a-4f with ammonia in dioxane for cyclization to give 5a-5f in yields 58-92%.13
Scheme 1. Synthesis of 5-BDBD analogs 5a-5f.
6

Scheme 2. Synthesis of 5-BDBD analogs 6, 8c, 8f, 10 and 11.
As depicted in Scheme 2, desmethylation14,15 of 5f with BBr3 in CH2Cl2 afforded the precursor 5-
(3-hydroxyphenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (6) in 81% yield.
Methylation of 5c, 5f with CH3I in the presence of NaH in DMF gave N-
methylbenzodiazepinone 8c, 8f in 65% and 90% yield, respectively. O-alkylation of 6 with 1,2-
7

bis(tosyloxy)ethane or 2-fluoroethyl tosylate in the presence of NaH in DMF,16 gave the
tosylated precursor 2-(3-(2-oxo-2,3-dihydro-1H-benzofuro[3,2-e][1,4]diazepin-5-
yl)phenoxy)ethyl 4-methylbenzenesulfonate (10) and reference standard 5-(3-(2-
fluoroethoxy)phenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (11) in 65% and 43%
yield, respectively.
Scheme 3. Radiosynthesis of N-[11C]Me-5-BDBD analog ([11C]9).
The precursor 6 underwent [11C]methylation17,18 using a reactive [11C]methylating agent
ο
[11C]methyl triflate ([11C]CH3OTf)19,20 in acetonitrile at 80 C under basic condition (NaH) to
obtain an unexpected radiolabeled product 5-(3-hydroxyphenyl)-1-[11C]methyl-1,3-dihydro-2H-
benzofuro[3,2-e][1,4]diazepin-2-one (N-[11C]Me-5-BDBD analog, [11C]9). The expected
radiolabeled product O-[11C]Me-5-BDBD ([11C]5f) was not formed. For compound 6, its amide
N-H is more reactive than the phenol O-H. [11C]Methylation of the precursor 6 is outlined in
8

Scheme 3. 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)21,22 with a disposable C-18 Light Sep-Pak cartridge to produce the
corresponding pure radiolabeled compound [11C]9 in 30-50% radiochemical yield, decay
corrected to end of bombardment (EOB), based on [11C]CO2. [11C]CH3OTf is more reactive than
commonly used [11C]methyl iodide ([11C]CH3I), and we use [11C]CH3OTf instead of [11C]CH3I
as the radiolabeled precursor to improve the radiochemical yield.17,18 Acetonitrile (CH3CN) was
found to be an appropriate reaction solvent for [11C]CH3OTf, and N,N-dimethylformamide
(DMF) was found to be an inappropriate reaction solvent for [11C]CH3OTf since it reacted with
and consumed a large portion of the radiolabel, especially under basic condition.
[11C]CH3OTf/CH3CN for [11C]methylation would improve the radiochemical yield.
H
O
N
Boc
N
1) 11CH3OTf, CH3CN
O
NaH
N
O
2) HCl
N
O
H
N
O
(Boc)2O, DMAP
O11CH3
CH3CN
X
O-[11C]Me-5-BDBD ([11C]5f)
OH
O
N
O
(Boc)2O, DMAP
CH3CN
H
N
OH
6
N
O
OBoc
7
Scheme 4. The protecting reaction of 5-BDBD analog 6 with (Boc)2O.
9

As indicated in Scheme 4, in order to synthesize O-[11C]Me-5-BDBD, we attempted to protect
the amide N-H of 6 with tert-butyloxycarbonyl (Boc) protecting group using 4-
dimethylaminopyridine (DMAP) as a catalyst,5,23 but we only obtained a phenol O-H protected
product 7 in 44% yield. The bulky Boc-group would react with the phenol O-H of 6, instead of
the amide N-H, and the steric effect was involved.
Scheme 5. Synthesis of N-Me-5-BDBD analog (9).
10

Thus, we have no way to synthesize O-[11C]Me-5-BDBD. Then, we have to synthesize the
reference standard 5-(3-hydroxyphenyl)-1-methyl-1,3-dihydro-2H-benzofuro[3,2-
e][1,4]diazepin-2-one (9) for N-[11C]Me-5-BDBD analog. As shown in Scheme 5, the direct
methylation of 6 with CH3I in DMF provided a mixture containing 9, 5f, and 8f. The reaction
was monitored by thin layer chromatography (TLC). From TLC, only very little 9 was observed.
In addition, it was difficult to purify the methylation mixture. Therefore, we conducted O-
desmethylation14,15 of 8f with BBr3 in dichloromethane to afford 9 in 72% yield. CH3I/DMF for
methylation would improve the chemical yield.
The
synthesis
of
5-(3-bromophenyl)-1-[11C]methyl-1,3-dihydro-2H-benzofuro[3,2-
e][1,4]diazepin-2-one (N-[11C]Me-5-BDBD, [11C]8c) is depicted in Scheme 6. 5-BDBD was
labeled with [11C]CH3OTf through N-[11C]Methylation24 under basic condition (NaH) and
isolated by HPLC-SPE method to give the target tracer N-[11C]Me-5-BDBD in 30-50% decay
corrected radiochemical yield.
Scheme 6. Radiosynthesis of N-[11C]Me-5-BDBD ([11C]8c).
11

The 11C-radiosynthesis was performed in a home-built automated multipurpose [11C]-
radiosynthesis module.25,26 It included three stages: 1) labeling reaction; 2) purification; and 3)
formulation. We used a vial-method for reaction; semi-preparative HPLC for purification; and
Light Sep-Pak trap/release for formulation. Overall, it took ~40 min for synthesis, purification
and dose formulation. Our [11C]-radiosynthesis module is designed to allow in-process
measurement of [11C]-tracer specific activity (SA, GBq/µmol at EOB) using a radiation detector
at the outlet of the HPLC-portion of the system. For the reported syntheses, product SA was in a
range of 370-1110 GBq/µmol at EOB. The major factors including [11C]-target and [11C]-
radiosynthesis unit that affect the EOB SA significantly to lead to such a wide range from 370 to
1110 GBq/µmol have been discussed in our previous works.18 The general methods to increase
SA have been described as well, and the SA of our [11C]-tracers is significantly improved.18 The
‘wide range’ of SA we reported is for the same [11C]-tracer produced in different days, because
very different [11C]-target and [11C]-radiosynthesis unit situations would make SA in a wide
range. For a [11C]-tracer produced in the same day, the SA of the same tracer in different
production runs will be in a small range, because [11C]-target and [11C]-radiosynthesis unit would
not be much different in the same day. Likewise, the methods to minimize such wide range of
SA from practice perspective have been provided in our previous works.18 At the end of
synthesis (EOS), the SA of [11C]-tracer was actually determined again by analytical HPLC,27
calculated, decay corrected to EOB, and based on [11C]CO2, which was in agreement with the
‘on line’ determined value. In each our [11C]-tracer production, if semi-preparative HPLC was
used for purification, then the SA of [11C]-tracer was assessed by both semi-preparative HPLC
(during synthesis) and analytical HPLC (EOS).
12

Scheme 7. Radiosynthesis of [18F]F-5-BDBD ([18F]5a) and [18F]FE-5-BDBD ([18F]11).
As shown in Scheme 7, the nitro-precursor 5e was labeled with K[18F]F/Kryptofix 2.2.2 through
nucleophilic substitution28 and isolated by the HPLC-SPE method to produce the corresponding
radiolabeled product 5-(3-[18F]fluorophenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-
one ([18F]F-5-BDBD, [18F]5a) in 5-15% decay corrected radiochemical yield from K[18F]F at
EOB. Likewise, the tosylated precursor 10 was labeled with K[18F]F/Kryptofix 2.2.229 and
isolated by HPLC-SPE to produce the corresponding radiolabeled product 5-(3-(2-
[18F]fluoroethoxy)phenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one
([18F]FE-5-
BDBD, [18F]11) in 15-25% decay corrected radiochemical yield. Using dimethyl sulfoxide
(DMSO) as the reaction solvent and running radiolabelling reaction at 140 οC would increase the
radiochemical yield.30 The effect of the reaction solvent and temperature on the radiochemical
13

ο
yield has been studied in our previous work by direct comparison of DMSO/140 C with
CH3CN/120 οC.29
The 18F-radiosynthesis was performed in an automated multipurpose [18F]-radiosynthesis module
of our own design.16 The radiosynthesis process included labeling reaction, purification and
formulation. The overall synthesis, purification and formulation time was ~70 min from EOB.
The SA of [18F]-tracer can be assessed by both semi-preparative HPLC (during synthesis) and
analytical HPLC (EOS).27 18F-product SA was in a range of 111-740 GBq/µmol at EOB.
Chemical purity and radiochemical purity were determined by analytical HPLC.27 The chemical
purity of the precursors (6, 5c, 5e, and 10) and reference standards (9, 8c, 5a, and 11) was >90%.
The radiochemical purity of the target tracers ([11C]9, [11C]8c, [18F]5a, and [18F]11) was >99%
determined by radio-HPLC through γ-ray (PIN diode) flow detector, and the chemical purity of
the target tracers was >90% determined by reversed-phase HPLC through UV flow detector.
2.2. In vitro binding studies
The preliminary biological evaluation of the carbon-11 radioligands [11C]9 and [11C]8c was
performed by a radioligand depletion experiment.6,31 In this experiment, a wide range of protein
was used for the optimization of the assay conditions. For the law of mass action to be valid, the
protein and radioligand concentration need to be at levels where no more than 10% of the total
radioligand added is bound to the protein. Otherwise the radioligand is considered depleted and
specialized formulas for radioligand depletion must be used for analysis of the data in saturation
14

or competitive binding assays. An optimized binding assay uses protein levels where the
radioligand is depleted no more than 10%, and where sufficient signal to background binding
levels, at least 5 fold total binding/non-specific binding ratio, are obtained. To achieve both of
these ends, a wide range of membrane protein concentrations was tested to get the optimal level
for the assay. The results indicated low affinity of [11C]9 and [11C]8c to P2X4 receptor. We
suspected the radioligands might be too sticky. Then the method was further optimized by
improving the solubility of the tracers [11C]9 and [11C]8c, and the results remained same. Thus
the competitive binding assay of ligands 5-BDBD (5c), N-Me-5-BDBD (8c), N-Me-5-BDBD
analog (9), F-5-BDBD (5a) and FE-5-BDBD (11) was conducted following the literature
method31,32 with modifications using [35S]ATPγS as the radioligand, ATP as a positive control,
and buffer as a negative control, respectively. The assays were incubated at 4 °C for 2 h. The
results are shown in Figure 3, which showed new ligands we synthesized have similar biological
activity in comparison with the parent compound 5-BDBD, because their binding curves are
similar. These results suggest more potent PET P2X4 receptor radioligands remain to be
discovered and developed.
Figure 3. The results of the ligand competitive binding assay of 5-BDBD analogs.
15

3. Materials and methods
3.1. General
All commercial reagents and solvents were purchased from Sigma-Aldrich and Fisher Scientific,
and used without further purification. [11C]CH3OTf was prepared according to a literature
procedure.20 Melting points were determined on a MEL-TEMP II capillary tube apparatus and
1
were uncorrected. H and 13C NMR spectra were recorded on a Bruker Avance II 500 MHz
NMR Fourier transform spectrometer at 500 and 125 MHz, respectively. Chemical shifts (δ) are
reported in parts per million (ppm) relative to an internal standard tetramethylsilane (TMS, δ 0.0)
(1H NMR) and to the solvent signal (13C NMR), and coupling constants (J) are 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. The high
resolution mass spectra (HRMS) were obtained using a Waters/Micromass LCT Classic
spectrometer. Chromatographic solvent proportions are indicated as volume: volume ratio. TLC
was run using Analtech silica gel GF uniplates (5 × 10 cm2). Plates were visualized under UV
light. Preparative TLC was run using Analtech silica gel UV254 plates (20 × 20 cm2). 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
16

maintained by a direct line from a nitrogen source. C18 Sep-Pak Light and Plus cartridges were
obtained from Waters Corporation (Milford, MA). Sterile Millex-FG 0.2 µm filter units were
obtained from Millipore Corporation (Bedford, MA).
3.2. 2-Bromo-1-(3-iodophenyl)ethanone (1d)
To a stirred solution of 3-iodoacetophenone (5.0 g, 20.3 mmol) in EtOAc/CHCl3 (100 mL/100
mL) was added CuBr2 (9.1 g, 40.6 mmol). After the reaction mixture was heated under reflux for
3.5 h, it was cooled and filtered through Celite. The solvent was evaporated in vacuo to afford 1d
as a white solid (4.4 g, 67%), which was used for next step without further purification. 1H NMR
(DMSO-d6): δ 8.30 (t, J = 2.0 Hz, 1H), 8.04 (dt, J = 1.0, 8.0 Hz, 1H), 8.00 (dt, J = 1.0, 8.0 Hz,
1H), 7.37 (t, J = 8.0 Hz, 1H), 4.96 (s, 2H).
3.3. General procedure for preparation of compound 3
(3-Aminobenzofuran-2-yl)-(3-fluorophenyl)methanone (3a). A mixture of 2-hydroxybenzonitrile
(1.0 g, 8.4 mmol), 2-bromo-1-(3-fluorophenyl)ethanone (1a, 2.0 g, 9.2 mmol) and Na2CO3
(1.33g, 12.6 mmol) in anhydrous DMF (15 mL) was stirred at room temperature (RT) overnight.
The reaction mixture was poured into water. The precipitate was collected by filtration and
washed with water. The solid was dissolved in EtOAc, washed with brine, dried over anhydrous
MgSO4, and filtered. After the solvent was evaporated in vacuo, MeOH (25 mL) was added to
the intermediate solid, followed by addition of sodium methoxide (463 mg, 8.6 mmol). The
reaction mixture was stirred and heated under reflux for 30 min. The mixture was cooled and
17

pour into ice-water. The precipitate was collected by filtration and washed with water. The solid
ο
1
was dried in vacuo to afford 3a as a yellow solid (2.0 g, 91%); mp 126-128 C. H NMR
(DMSO-d6): δ 8.08 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 7.5 Hz, 1H), 7.86-7.84 (m, 1H), 7.66-7.57
(m, 5H), 7.48-7.45 (m, 1H), 7.34-7.30 (m, 1H).
(3-Aminobenzofuran-2-yl)-(3-chlorophenyl)methanone (3b). Compound 3b was prepared with
similar procedure as compound 3a from 1b. Yield: 82%, yellow solid; mp 122-124 οC. 1H NMR
(DMSO-d6): δ 8.09-8.06 (m, 3H), 7.69-7.67 (m, 1H), 7.62-7.56 (m, 5H), 7.33-7.30 (m, 1H).
(3-Aminobenzofuran-2-yl)-(3-bromophenyl)methanone (3c). Compound 3c was prepared with
ο
similar procedure as compound 3a from 1c. Yield: 87%, yellow solid; mp 102-104 C. 1H NMR
(DMSO-d6): δ 8.19 (t, J = 2.0 Hz, 1H), 8.13 (dt, J = 1.0, 8.0 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H),
7.82-7.80 (m, 1H), 7.61-7.53 (m, 5H), 7.33-7.30 (m, 1H).
(3-Aminobenzofuran-2-yl)-(3-iodophenyl)methanone (3d). Compound 3d was prepared with
similar procedure as compound 3a from 1d. Yield: 88%, yellow solid; mp 138-140 οC. 1H NMR
(DMSO-d6): δ 8.35 (t, J = 1.5 Hz, 1H), 8.14 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.97-
7.96 (m, 1H), 7.60-7.54 (m, 4H), 7.39 (t, J = 8.0 Hz, 1H), 7.33-7.30 (m, 1H).
(3-Aminobenzofuran-2-yl)-(3-nitrophenyl)methanone (3e). Compound 3e was prepared with
ο
similar procedure as compound 3a from 1e. Yield: 84%, yellow solid; mp 166-168 C. 1H NMR
(DMSO-d6): δ 8.85 (t, J = 2.0 Hz, 1H), 8.56 (d, J = 7.5 Hz, 1H), 8.46-8.44 (m, 1H), 8.11 (d, J =
8.0 Hz, 1H), 7.88 (t, J = 8.0 Hz, 1H), 7.72 (br s, 2H), 7.63-7.60 (m, 1H), 7.56 (d, J = 8.0 Hz, 1H),
7.35-7.32 (m, 1H).
(3-Aminobenzofuran-2-yl)-(3-methoxyphenyl)methanone (3f). Compound 3f was prepared with
ο
similar procedure as compound 3a from 1f. Yield: 98%, yellow solid; mp 126-128 C. H NMR
1
18

(DMSO-d6): δ 8.07 (d, J = 7.5 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.61 (br s, 1H), 7.58-7.48 (m,
5H), 7.33-7.30 (m, 1H), 7.18 (dd, J = 2.0, 8.0 Hz, 1H).
3.4. General procedure for preparation of compound 4
2-Bromo-N-(2-(3-fluorobenzoyl)benzofuran-3-yl)acetamide (4a). To a stirred mixture of
compound 3a (1.4 g, 5.3 mmol) and NaHCO3 (1.8 g, 51.3 mmol) in CHCl3 (25 mL) was added
bromoacetyl bromide (0.6 mL, 6.9 mmol) dropwise at 0 οC. The reaction mixture was stirred at 0
οC for 1.5 h. After the solvent was evaporated in vacuo, EtOAc was added to dissolve the
residue. The organic solution was washed with saturated aqueous NaHCO3 and brine, dried over
anhydrous Na2SO4, and filtered. The solvent was evaporated in vacuo. The crude product was
ο
1
washed with hexanes and Et2O to afford 4a as a yellow solid (1.8 g, 89%); mp 106-108 C. H
NMR (DMSO-d6): δ 10.90 (s, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.88-7.86 (m, 1H), 7.79-7.75 (m,
2H), 7.68-7.61 (m, 2H), 7.57-7.54 (m, 1H), 7.43-7.40 (m, 1H), 4.23 (s, 2H).
2-Bromo-N-(2-(3-chlorobenzoyl)benzofuran-3-yl)acetamide (4b). Compound 4b was prepared
with similar procedure as compound 4a from 3b. Yield: 75%, pale yellow solid; mp 112-114 οC.
1H NMR (DMSO-d6): δ 10.90 (s, 1H), 7.98-7.95 (m, 3H), 7.77-7.74 (m, 2H), 7.64-7.61 (m, 2H),
7.43-7.40 (m, 1H), 4.21 (s, 2H).
2-Bromo-N-(2-(3-bromobenzoyl)benzofuran-3-yl)acetamide (4c). Compound 4c was prepared
ο
1
with similar procedure as compound 4a from 3c. Yield: 78%, yellow solid; mp 118-120 C. H
NMR (DMSO-d6): δ 10.89 (s, 1H), 8.08 (t, J = 2.0 Hz, 1H), 8.00 (dt, J = 1.0, 7.5 Hz, 1H), 7.97
(d, J = 8.0 Hz, 1H), 7.90-7.88 (m, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.64-7.60 (m, 1H), 7.56 (t, J =
8.0 Hz, 1H), 7.43-7.40 (m, 1H), 4.21 (s, 2H).
19

2-Bromo-N-(2-(3-iodobenzoyl)benzofuran-3-yl)acetamide (4d). Compound 4d was prepared with
similar procedure as compound 4a from 3d. Yield: 88%, yellow solid; mp 140-142 οC. 1H NMR
(DMSO-d6): δ 10.88 (s, 1H), 8.23 (t, J = 1.5 Hz, 1H), 8.06-8.04 (m, 1H), 8.01 (dt, J = 1.5, 7.5
Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.64-7.60 (m, 1H), 7.43-7.38 (m,
2H), 4.21 (s, 2H).
2-Bromo-N-(2-(3-nitrobenzoyl)benzofuran-3-yl)acetamide (4e). Compound 4e was prepared with
ο
similar procedure as compound 4a from 3e. Yield: 87%, yellow solid; mp 150-152 C. 1H NMR
(DMSO-d6): δ 10.96 (s, 1H), 8.66 (t, J = 2.0 Hz, 1H), 8.52-8.50 (m, 1H), 8.41 (dt, J = 1.0, 8.0
Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.89 (t, J = 8.0 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.66-7.63
(m, 1H), 7.46-7.42 (m, 1H), 4.16 (s, 2H).
2-Bromo-N-(2-(3-methoxybenzoyl)benzofuran-3-yl)acetamide (4f). Compound 4f was prepared
ο
1
with similar procedure as compound 4a from 3f. Yield: 87%, yellow solid; mp 122-124 C. H
NMR (DMSO-d6): δ 10.88 (s, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 7.5 Hz, 1H), 7.64-7.60
(m, 2H), 7.54-7.51 (m, 2H), 7.41 (t, J = 7.5 Hz, 1H), 7.28 (dd, J = 2.5, 8.5 Hz, 1H), 4.24 (s, 2H),
3.85 (s, 3H).
3.5. General procedure for preparation of compound 5
5-(3-Fluorophenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (F-5-BDBD, 5a). To a
stirred compound 4a (1.0 g, 2.7 mmol) in dioxane (5 mL) was added ammonia (0.5 M solution in
dioxane, 60 mL, 30.0 mmol). After the reaction mixture was stirred at RT for 2 days, EtOAc was
added. The mixture was washed with water, brine, dried over anhydrous Na2SO4 and filtered.
The solvent was evaporated in vacuo. The crude product was washed with MeOH to afford 5a as
20

ο
1
a pale yellow solid (724 mg, 92%); mp 256-258 C. H NMR (DMSO-d6): δ 11.60 (s, 1H), 8.00
(d, J = 7.5 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.61-7.53 (m, 4H), 7.44-7.39 (m, 2H), 4.46 (s, 2H).
13C NMR (DMSO-d6): δ 165.7, 162.9, 161.0 (d, J = 122 Hz), 153.6, 138.3, 130.4, 129.0, 128.6,
125.2, 123.5, 120.8, 120.7, 117.5, 115.4, 112.4, 59.2. HRMS (ESI, m/z): Calcd for C17H12FN2O2
([M+H]+) 295.0883, found: 295.0883.
5-(3-Chlorophenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (5b). Compound 5b
was prepared with similar procedure as compound 5a from 4b. Yield: 62%, pale yellow solid;
1
mp 263-264 οC. H NMR (DMSO-d6): δ 11.59 (s, 1H), 8.00 (d, J = 7.5 Hz, 1H), 7.77 (t, J = 2.0
Hz, 1H), 7.72-7.70 (m, 2H), 7.63-7.61 (m, 1H), 7.60-7.56 (m, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.44-
7.41 (m, 1H), 4.46 (s, 2H). 13C NMR (DMSO-d6): δ 165.7, 160.0, 153.6, 138.2, 138.0, 133.2,
130.4, 130.3, 129.0, 128.6, 128.4, 127.7, 123.5, 120.8, 120.7, 112.4, 59.2. HRMS (ESI, m/z):
Calcd for C17H12ClN2O2 ([M+H]+) 311.0587, found: 311.0573.
5-(3-Bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one
(5-BDBD,
5c).
Compound 5c was prepared with similar procedure as compound 5a from 4c. Yield: 84%, pale
ο
1
yellow solid; mp 244-246 C. H NMR (DMSO-d6): δ 11.59 (s, 1H), 8.00 (d, J = 7.5 Hz, 1H),
7.91 (s, 1H), 7.76-7.73 (m, 2H), 7.70 (d, J = 8.0 Hz, 1H), 7.59-7.56 (m, 1H), 7.47 (t, J = 8.0 Hz,
1H), 7.43 (t, J = 7.5 Hz, 1H), 4.46 (s, 2H).
5-(3-Iodophenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (5d). Compound 5d was
prepared with similar procedure as compound 5a from 4d. Yield: 58%, pale yellow solid; mp
245-246 οC. 1H NMR (DMSO-d6): δ 11.58 (s, 1H), 8.08 (t, J = 1.5 Hz, 1H), 8.00 (d, J = 8.0 Hz,
1H), 7.91 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.59-7.56 (m,
1H), 7.43 (t, J = 7.5 Hz, 1H), 7.30 (t, J = 8.0 Hz, 1H), 4.45 (s, 2H). 13C NMR (DMSO-d6): δ
165.7, 159.9, 153.6, 139.1, 138.2, 138.0, 137.1, 130.5, 129.0, 128.6, 128.4, 123.5, 120.8, 120.7,
21

112.4, 94.6, 59.2. HRMS (ESI, m/z): Calcd for C17H12IN2O2 ([M+H]+) 402.9944, found:
402.9930.
5-(3-Nitrophenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (5e). Compound 5e was
prepared with similar procedure as compound 5a from 4e. Yield: 84%, pale yellow solid; mp 273
1
οC (dec.). H NMR (DMSO-d6): δ 11.68 (s, 1H), 8.57 (t, J = 2.0 Hz, 1H), 8.41-8.39 (m, 1H),
8.22 (dt, J = 1.0, 8.0 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.70 (J = 8.5 Hz,
1H), 7.62-7.58 (m, 1H), 7.46-7.43 (m, 1H), 4.52 (s, 2H).
5-(3-Methoxyphenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (O-Me-5-BDBD, 5f).
Compound 5f was prepared with similar procedure as compound 5a from 4f. Yield: 75%, off
white solid; mp 224-225 οC. 1H NMR (DMSO-d6): δ 11.53 (s, 1H), 7.99 (d, J = 7.5 Hz, 1H), 7.69
(d, J = 8.5 Hz, 1H), 7.59-7.55 (m, 1H), 7.44-7.28 (m, 2H), 7.30-7.28 (m, 2H), 7.13-7.11 (m, 1H),
13
4.44 (s, 2H), 3.80 (s, 3H). C NMR (DMSO-d6): δ 165.9, 161.0, 159.1, 153.5, 138.8, 137.4,
129.4, 128.6, 128.4, 123.4, 121.4, 120.8, 120.8, 116.4, 113.8, 112.3, 59.1, 55.2. HRMS (ESI,
m/z): Calcd for C18H15N2O3 ([M+H]+) 307.1083, found: 307.1077.
3.6. 5-(3-Hydroxyphenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (6)
To a stirred suspension of compound 5f (1.1 g, 3.4 mmol) in anhydrous CH2Cl2 (30 mL) was
ο
added BBr3 (1.0 M solution in CH2Cl2, 30 mL, 30.0 mmol) dropwise at -78 C under N2
atmosphere. The reaction mixture was stirred overnight after warming up to RT slowly. The
reaction was quenched with water in an ice-cooled bath, it was neutralized with aqueous 1 N
NaHCO3 solution. The precipitate was collected by filtration and washed with water. The solid
ο
was dried in vacuo to afford 6 as a yellow solid (846 mg, 81%); mp 234 C (dec.). H NMR
1
22

(DMSO-d6): δ 11.50 (s, 1H), 9.62 (s, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.57
(t, J = 7.5 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.15 (s, 2H), 6.93 (d, J = 7.5
13
Hz, 1H), 4.41 (s, 2H). C NMR (DMSO-d6): δ 164.7, 162.5, 157.5, 156.2, 135.1, 131.7, 130.1,
124.4, 122.4, 121.9, 120.8, 120.1, 117.5, 112.8, 54.0. LC-MS (ESI, m/z): Calcd for C17H13N2O3
([M+H]+) 293.1, found: 293.0. HRMS (ESI, m/z): Calcd for C18H15N2O3 ([M+H]+) 293.0926,
found: 293.0916.
3.7. tert-Butyl (3-(2-oxo-2,3-dihydro-1H-benzofuro[3,2-e][1,4]diazepin-5-yl)phenyl)carbonate
(7)
A suspension of compound 6 (50 mg, 0.17 mmol), (Boc)2O (250 mg, 1.14 mmol) and DMAP (9
mg, 0.07 mmol) in CH3CN (2 mL) was stirred at RT for 2.5 h. The solvent was evaporated in
vacuo. The crude product was purified by preparative TLC plate using CH2Cl2/MeOH (100:4) as
eluent to afford 7 as a pale yellow solid (29 mg, 44%); mp 182-183 οC. 1H NMR (CDCl3): δ 11.2
(s, 1H), 7.92 (d, J = 7.5 Hz, 1H), 7.69-7.67 (m, 2H), 7.53-7.52 (m, 2H), 7.45 (t, J = 8.0 Hz, 1H),
7.42-7.38 (m, 1H), 7.33-7.30 (m, 1H), 4.63 (s, 2H), 1.55 (s, 9H). 13C NMR (DMSO-d6): δ 167.9,
161.8, 154.2, 151.8, 151.2, 139.6, 137.6, 129.4, 128.8, 128.4, 126.8, 124.1, 123.8, 122.2, 121.0,
120.2, 112.6, 83.9, 58.8, 27.8. HRMS (ESI, m/z): Calcd for C22H21N2O5 ([M+H]+) 393.1450,
found: 393.1434.
3.8. General procedure for preparation of compound 8
23

5-(3-Bromophenyl)-1-methyl-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one
(N-Me-5-
BDBD, 8c). To a stirred solution of compound 5c (200 mg, 0.56 mmol) in anhydrous DMF (2
mL) was added NaH (60% dispersion in mineral oil, 16 mg, 0.67 mmol). The reaction mixture
was stirred at RT for 15 min, and methyl iodide (70 µL, 1.12 mmol) was then added. After the
mixture was stirred at RT for 30 min, it was poured into ice-water and extracted with EtOAc.
The organic layer was washed with water, brine, dried over anhydrous Na2SO4 and filtered. The
solvent was evaporated in vacuo. The crude product was purified by preparative TLC plate using
CH2Cl2/MeOH (100:4) as eluent to afford 8c as a pale yellow solid (136 mg, 65%); mp 158-160
οC. 1H NMR (DMSO-d6): δ 8.05 (d, J = 8.0 Hz, 1H), 7.94 (s, 1H), 7.80-7.75 (m, 3H), 7.60 (t, J =
13
8.0 Hz, 1H), 7.47 (q, J = 8.0 Hz, 2H), 4.51 (br s, 2H), 3.57 (s, 3H). C NMR (CDCl3): δ 164.8,
159.9, 153.4, 139.6, 137.7, 133.5, 131.9, 131.2, 130.6, 128.3, 128.0, 123.9, 121.8, 121.7, 121.5,
112.8, 58.4, 33.9. HRMS (ESI, m/z): Calcd for C18H14BrN2O2 ([M+H]+) 369.0239, found:
369.0254.
5-(3-Methoxyphenyl)-1-methyl-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one
(8f).
Compound 8f was prepared with similar procedure as compound 8c from 5f. Yield: 90%, pale
ο
1
yellow solid; mp 62-64 C. H NMR (CDCl3): δ 7.81 (d, J = 8.5 Hz, 1H), 7.57 (d, J = 8.5 Hz,
1H), 7.53 (dt, J =1.5, 7.0 Hz, 1H), 7.43-7.35 (m, 4H), 7.08-7.05(m, 1H), 4.57 (br s, 2H), 3.86 (s,
13
3H), 3.64 (s, 3H). C NMR (CDCl3): δ 166.1, 162.1, 159.8, 153.9, 141.1, 137.2, 131.5, 129.5,
128.1, 123.9, 122.0, 121.9, 121.1, 117.4, 113.8, 113.1, 58.7, 55.6, 34.4. HRMS (ESI, m/z): Calcd
for C19H17N2O3 ([M+H]+) 321.1239, found: 321.1234.
3.9. 5-(3-Hydroxyphenyl)-1-methyl-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (N-Me-
5-BDBD analog, 9)
24

To a stirred solution of compound 8f (60 mg, 0.18 mmol) in anhydrous CH2Cl2 (1 mL) was
ο
added BBr3 (1.0 M solution in CH2Cl2, 1.4 mL, 1.4 mmol) dropwise at -78 C under N2
atmosphere. The reaction mixture was stirred overnight after warming up to RT slowly. The
reaction was quenched with water in an ice-cooled bath, it was neutralized with aqueous 1 N
NaHCO3 solution and extracted with CH2Cl2. The solvent was evaporated in vacuo. The crude
product was purified by preparative TLC plate using CH2Cl2/MeOH (100:4) as eluent to afford 9
ο
as a pale yellow solid (41 mg, 72%); mp 227-228 C. 1H NMR (DMSO-d6): δ 9.65 (s, 1H), 8.04
(d, J = 8.0 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.61-7.58 (m, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.29 (t,
13
J = 8.0 Hz, 1H), 7.20-7.19 (m, 2H), 6.95-6.93 (m, 1H), 4.46 (br s, 2H), 3.56 (s, 3H). C NMR
(DMSO-d6): δ 165.3, 161.1, 157.3, 153.2, 140.4, 136.8, 131.3, 129.4, 128.1, 123.8, 121.7, 121.5,
119.8, 117.9, 115.4, 112.7, 58.2, 33.8. LC-MS (ESI, m/z): Calcd for C18H15N2O3 ([M+H]+)
307.1, found: 307.1. HRMS (ESI, m/z): Calcd for C18H15N2O3 ([M+H]+) 307.1083, found:
307.1070.
3.10.
2-(3-(2-Oxo-2,3-dihydro-1H-benzofuro[3,2-e][1,4]diazepin-5-yl)phenoxy)ethyl
4-
methylbenzenesulfonate (10)
To a stirred solution of compound 6 (200 mg, 0.7 mmol) in anhydrous DMF (4 mL) was added
ο
NaH (60% dispersion in mineral oil, 82 mg, 2.0 mmol) at 0 C under N2 atmosphere and the
mixture was stirred at 0 οC for 45 min, followed by addition of 1,2-bis(tosyloxy)ethane (504 mg,
1.4 mmol). After the reaction mixture was stirred at 0 οC for 6 h, it was poured into ice-water and
extracted with EtOAc. The organic layer was washed with water, brine, dried over anhydrous
25

Na2SO4 and filtered. The solvent was evaporated in vacuo. The crude product was purified by
preparative TLC plate using CH2Cl2/MeOH (100:4) as eluent to afford 10 as a yellow solid (144
mg, 43%); mp 188-189 οC. 1H NMR (DMSO-d6): δ 11.55 (s, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.79
(d, J = 8.5 Hz, 2H), 7.69 (d, J = 8.5 Hz, 1H), 7.60-7.56 (m, 1H), 7.44-7.36 (m, 4H), 7.30 (d, J =
7.5 Hz, 1H), 7.13 (t, J = 2.0 Hz, 1H), 7.03-7.01(m, 1H), 4.44 (s, 2H), 4.37-4.35 (m, 2H), 4.21-
13
4.19 (m, 2H), 2.36 (s, 3H). C NMR (DMSO-d6): δ 165.9, 160.9, 157.6, 153.5, 144.9, 138.7,
137.4, 132.3, 130.1, 129.5, 128.7, 128.5, 127.7, 123.5, 121.9, 120.8, 116.8, 114.6, 112.4, 69.1,
65.4, 59.1, 21.0. LC-MS (ESI, m/z): Calcd for C26H23N2O6S ([M+H]+) 491.1, found: 491.1.
HRMS (ESI, m/z): Calcd for C26H23N2O6S ([M+H]+) 491.1277, found: 491.1265.
3.11. 5-(3-(2-Fluoroethoxy)phenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (FE-5-
BDBD, 11)
To a stirred solution of compound 6 (100 mg, 0.3 mmol) in anhydrous DMF (2 mL) was added
ο
NaH (60% dispersion in mineral oil, 41 mg, 1.0 mmol) at 0 C under N2 atmosphere and the
mixture was stirred 0 οC for 45 min, followed by addition of a solution of 2-fluoroethyl tosylate
(111 mg, 0.5 mmol) in anhydrous DMF (1 mL). After the reaction mixture was stirred at 0 οC for
6 h, it was poured into ice-water and extracted with EtOAc. The organic layer was washed with
water, brine, dried over anhydrous Na2SO4 and filtered. The solvent was evaporated in vacuo.
The crude product was purified by preparative TLC plate using CH2Cl2/MeOH (100:4) as eluent
ο
1
to afford 11 as a yellow solid (75 mg, 65%); mp 215-216 C. H NMR (DMSO-d6): δ 11.55 (s,
1H), 8.00 (d, J = 8.0 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.59-7.56 (m, 1H), 7.44-7.41 (m, 2H),
7.34-7.32 (m, 2H), 7.17-7.15 (m, 1H), 4.80 (t, J = 4.0 Hz, 1H), 4.71 (t, J = 4.0 Hz, 1H), 4.44 (s,
26

13
2H), 4.32 (t, J = 4.0 Hz, 1H), 4.26 (t, J = 4.0 Hz, 1H). C NMR (DMSO-d6): δ 165.9, 161.0,
158.1, 153.5, 138.7, 137.4, 129.6, 128.7, 128.5, 123.5, 121.9, 120.8, 120.8, 117.1, 114.5, 112.4,
82.7 (d, J = 165 Hz), 67.2, 59.1. LC-MS (ESI, m/z): Calcd for C19H16FN2O3 ([M+H]+) 339.1,
found: 339.0. HRMS (ESI, m/z): Calcd for C19H16FN2O3 ([M+H]+) 339.1145, found: 339.1140.
3.12. General procedure for preparation of carbon-11-labeled 5-BDBD analogs
5-(3-Hydroxyphenyl)-1-[11C]methyl-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one
(N-
[11C]Me-5-BDBD analog, [11C]9) and 5-(3-bromophenyl)-1-[11C]methyl-1,3-dihydro-2H-
benzofuro[3,2-e][1,4]diazepin-2-one (N-[11C]Me-5-BDBD, [11C]8c). [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
development were 58 µA beam current and 15 min on target. The production run produced
approximately 25.9 GBq of [11C]CO2 at EOB. The precursor (6 or 5c, 0.1-0.3 mg) was dissolved
in CH3CN (300 µL). To this solution was added NaH (95% dispersion in mineral oil, 1 mg). The
mixture was transferred to a small reaction vial. No-carrier-added (high specific activity)
[11C]CH3OTf that was produced by the gas-phase production method20 within 12 min from
[11C]CO2 through [11C]CH4 and [11C]CH3Br with silver triflate (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
27

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 Light 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 of [11C]9 or [11C]8c was
assayed and total volume (10-11 mL) was noted for tracer dose dispensing. The overall synthesis
time including HPLC-SPE purification and reformulation was ~40 min from EOB. The decay
corrected radiochemical yield for [11C]9 and [11C]8c was 30-50%. Retention times in the
analytical HPLC system performed using a Prodigy (Phenomenex) 5 µm C-18 column, 4.6 × 250
mm; mobile phase 30% CH3CN/70% 20 mM H3PO4; flow rate 1.0 mL/min; and UV (254 nm)
and γ-ray (PIN diode) flow detectors were: tR 6 = 3.68 min, tR 9 = 6.61 min, tR [11C]9 = 6.66 min.
Retention times in the semi-preparative HPLC system performed using a Prodigy (Phenomenex)
5 µm C-18 column, 10 × 250 mm; mobile phase 30% CH3CN/70% 20 mM H3PO4; flow rate 5.0
mL/min; and UV (254 nm) and γ-ray (PIN diode) flow detectors were: tR 6 = 4.88 min, tR 9 =
7.65 min, tR [11C]9 = 7.77 min. Retention times in the analytical HPLC system performed using a
Prodigy (Phenomenex) 5 µm C-18 column, 4.6 × 250 mm; mobile phase 70% CH3CN/30% H2O;
flow rate 1.3 mL/min; and UV (254 nm) and γ-ray (PIN diode) flow detectors were: tR 5c = 4.11
min, tR 8c = 6.12 min, tR [11C]8c = 6.19 min. Retention times in the semi-preparative HPLC
system performed using a Prodigy (Phenomenex) 5 µm C-18 column, 10 × 250 mm; mobile
phase 70% CH3CN/30% H2O; flow rate 5.0 mL/min; and UV (254 nm) and γ-ray (PIN diode)
flow detectors were: tR 5c = 3.56 min, tR 8c = 7.91 min, tR [11C]8c = 8.00 min.
3.13. General procedure for preparation of fluorine-18-labeled 5-BDBD analogs
28

5-(3-[18F]Fluorophenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one ([18F]F-5-BDBD,
[18F]5a) and 5-(3-(2-[18F]fluoroethoxy)phenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-
2-one ([18F]FE-5-BDBD, [18F]11). 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 H2 O
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(2.5 mL). [18F]Fluoride (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 were placed in the fluorination reaction vial (5-mL
V-vial) and repeated azeotropic distillation was performed at 110 °C to remove water and to
form the anhydrous K[18F]F-Kryptofix 2.2.2 complex. Nitro- precursor 5e or tosylated precursor
10 (1 mg) dissolved in DMSO (1.0 mL) was introduced to the reaction vessel and heated at 140
°C for 20 min to affect radiofluorination. After cooling to ~90 °C, aqueous NaHCO3 (0.1 M, 1
mL) was added to the reaction vessel for dilution, at which time the reaction mixture was loaded
into a 3-mL injection loop for introduction to the semi-preparative reverse-phase (C18) HPLC
column for initial purification of the labeled product. The HPLC fraction containing [18F]5a or
[18F]11 eluted from the column was automatically 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 × 5 mL). The cartridge was eluted with EtOH (2 × 1 mL) to release [18F]5a
or [18F]11. The eluted product was then sterile-filtered through a Millex-FG 0.2 µm membrane
into a sterile vial and formulated with 10 mL saline. Total radioactivity was assayed and total
volume was noted for tracer dose dispensing. The overall decay-corrected radiochemical yield
from K[18F]F for [18F]5a and [18F]11 was 5-15% and 15-25%, respectively. Retention times in
the semi-preparative HPLC system performed using a YMC-Pack ODS-A (Phenomenex), S-5
µm, 12 nm, 10×250 mm C-18 column; 30% CH3CN/70% 20 mM H3PO4 (pH 2.5) mobile phase;
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