Selective targeting of melanoma using N-(2-diethylaminoethyl) 4-[18F]fluoroethoxy benzamide (4-[18F]FEBZA): a novel PET imaging probe

Article abstract of DOI:10.1186/s13550-017-0311-2

Background: The purpose of this study was to develop a positron emission tomography (PET) imaging probe that is easy to synthesize and selectively targets melanoma in vivo. Herein, we report the synthesis and preclinical evaluation of N-(2-diethylaminoeth

Full text of DOI:10.1186/s13550-017-0311-2

Garg et al. EJNMMI Research (2017) 7:61
DOI 10.1186/s13550-017-0311-2
ORIGINAL RESEARCH
Open Access
Selective targeting of melanoma using N-
1
8
(
2-diethylaminoethyl) 4-[ F]fluoroethoxy
18
benzamide (4-[ F]FEBZA): a novel PET
imaging probe
1,2
1
,2*
1,2
2
3
Pradeep K. Garg
, Rachid Nazih , Yanjun Wu , Vladimir P. Grinevich and Sudha Garg
Abstract
Background: The purpose of this study was to develop a positron emission tomography (PET) imaging probe
that is easy to synthesize and selectively targets melanoma in vivo. Herein, we report the synthesis and
1
8
18
preclinical evaluation of N-(2-diethylaminoethyl) 4-[ F]Fluoroethoxy benzamide (4-[ F]FEBZA).
1
8
A one-step synthesis was developed to prepare 4-[ F]FEBZA in high radiochemical yields and specific activity. The
binding affinity, the in vitro binding, and internalization studies were performed using B16F1 melanoma cell line. The
biodistribution studies were performed in C57BL/6 normal mice, C57BL/6 mice bearing B16F1 melanoma tumor
xenografts, and nu/nu athymic mice bearing HT-29 human adenocarcinoma tumor and C-32 amelanotic
melanoma tumor xenografts. MicroPET studies were performed in mice bearing B16F1 and HT-29 tumor xenografts.
1
8
Results: 4-[ F]FEBZA was prepared in 53 ± 14% radiochemical yields and a specific activity of 8.7 ± 1.1 Ci/μmol. The
18
overall synthesis time for 4-[ F]FEBZA was 54 ± 7 min. The in vitro binding to B16F1 cells was 60.03 ± 0.48% after 1 h
incubation at 37 °C. The in vivo biodistribution studies show a rapid and high uptake of F-18 in B16F1 tumor with 8.
66 ± 1.02%IA/g in this tumor at 1 h. In contrast, the uptake at 1 h in HT-29 colorectal adenocarcinoma and C-32
amelanotic melanoma tumors was significantly lower with 3.68 ± 0.47%IA/g and 3.91 ± 0.23%IA/g in HT-29 and C-32
tumors, respectively. On microPET images, the melanoma tumor was clearly visible by 10 min post-injection and
the intensity in the tumor continued to increase with time. In contrast, the HT-29 tumor was not visible on the
microPET scans.
1
8
Conclusions: A rapid and facile synthesis of 4-[ F]FEBZA is developed. This method offers a reliable production
1
8
of 4-[ F]FEBZA in high radiochemical yields and specific activity. A high binding affinity to melanoma cells and
high uptake in tumor was noted. The microPET scan clearly delineates the melanoma tumor by 10 min post-
1
8
injection. The results from these preclinical studies support the potential of 4-[ F]FEBZA as an effective probe
to image melanoma.
1
8
Keywords: Melanoma, 4-[ F]FEBZA, Radiochemical synthesis, Radiofluorination, Binding affinity, Cell binding,
Biodistribution, MicroPET imaging
*
Correspondence: pgarg@biomed.org
1
Wake Forest University Health Sciences, Winston Salem, NC, USA
Center for Molecular Imaging and Therapy, Biomedical Research
2
Foundation, 1505 Kings Highway, Shreveport, LA 71133, USA
Full list of author information is available at the end of the article
©
The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made.

Garg et al. EJNMMI Research (2017) 7:61
Page 2 of 11
Background
experimental animals [24]. With the wider acceptance of
An estimated 87,000 new cases of melanoma of the skin PET as a clinical diagnostic tool for accurate diagnosis as
will be diagnosed in the USA in 2017, and approximately well as encouraging findings on the ability of radioio-
9
730 individuals are anticipated to die of melanoma this dobenzamide to target melanoma, there has been a
year [1]. Among many factors, lacking monthly self- significant impetus to develop PET probes which in-
inspection of the skin by patients, routine visits to the corporate benzamide moiety [17, 20, 25–30]. One such
1
8
dermatologist combined with lack of effective diagnostic compound, F-DAFBA, which was initially reported
tools training for the accurate detection of melanoma at by our group [25] and subsequently explored by other in-
an early stage, malignant melanoma remains a signi- vestigators, showed promising results [26]. Nonetheless,
ficant health problem and remains as the sixth most the synthesis of DAFBA requires ~3 h and includes two or
prevalent type of cancer [1]. While melanoma accounts more high-performance liquid chromatography (HPLC)
for a small percentage of all skin cancer cases, 75% of purifications [25]. To overcome that hurdle, we subse-
11
deaths from skin cancer are associated with melanoma. quently developed C-MBZA with a shorter and easier
11
Among many factors, late diagnosis and poor localization synthesis [31]. Preclinical evaluations of this C-probe
of metastatic lesions in patients with melanoma results in showed superior in vitro and in vivo properties compared
18
11
high mortality from this cancer [2]. If diagnosed at stage 1 to that for F-DAFBA. While C-MBZA has many desi-
of this disease, the overall 10-year survival for melanoma rable features and properties, it incorporates a short half-
patients is ~95%. By contrast, if this disease is diagnosed life radionuclide (T1/2 = 20 min). Therefore, we focused
at stage IV, the overall survival drops to 10–15% [2]. our efforts towards developing an F-18 benzamide with a
11
Therefore, early diagnosis and accurate assessment of short and facile radiochemical synthesis akin to C-
metastatic lesions is crucial for appropriate treatment MBZA while bearing similar or better biological proper-
planning, improved outcome, and disease-free survival [3]. ties. Herein, we report the synthesis, radiochemistry, and
18
Positron Emission Tomography (PET) is a powerful tool preclinical evaluation of 4-[ F]FEBZA as a PET probe to
to non-invasively study organ function and related diso- selectively and non-invasively target melanoma.
rders such as tissue perfusion and metabolism in vivo [4].
PET imaging with F-18 fluorodeoxy glucose (FDG) has
shown high sensitivity and specificity in the diagnosis and Methods
staging of various cancers [5] primarily due to its signifi- All chemicals were purchased from Sigma (Sigma-Aldrich,
cantly higher uptake in malignant cells with elevated Milwaukee, WI) and were used without further purifica-
metabolic activity [6]. While quite effective in many types tion. C-18 plus Sep-Pak cartridges were obtained from the
of cancers, FDG-PET has shown a low mean sensitivity of Waters Corporation (Waters Corporation, Milford, MA).
17.3% for detecting small lymph node metastases in pa- Flash column chromatography was carried out over silica
tients with early-stage melanoma cancer [6–8], thus offe- gel (60 Å, Mallinckrodt Baker). HPLC was performed in
ring limited value in the initial staging of AJCC (American the isocratic mode using an HPLC system (Varian Corp,
Joint Commission on Cancer) stage I, or stage II melan- Palo Alto, CA) equipped with an LC pump, a variable
oma [6, 9, 10]. In another study, FDG-PET failed to detect wavelength UV/VIS detector set at 254 nm, and an in-line
melanoma in all 14 patients showing a positive sentinel radioisotope detector (Bioscan Inc., Washington, DC).
18
lymph node biopsy for cancer [11]. Additionally, the high The HPLC column for the purification of 4-[ F]FEBZA
uptake of FDG in brain tissues further limits its utility in was a C-18 reverse-phase, 10 mm × 250 mm, 5-μ Luna
detecting brain metastases [12]. Therefore, developing a column (Phenomenex, Torrence, CA), and elution was
PET probe with high specificity and selectivity for mela- performed at a flow rate of 2 mL/min using a solution of
noma and a low uptake in normal tissues has been the methanol:0.1 M ammonium acetate (50:50) containing
goal of many investigators [13–15].
trimethylamine (40 μL/100 mL). The quality of the
To selectively target melanoma, the role of F-18- and purified product was checked using a C-18 column
Cu-64 labeled α–melanocyte-stimulating hormone ana- (4.6 mm × 250 mm, 5 μ), eluted at a flow rate of 1 mL/
log to image melanoma by targeting melanocortin-1 re- min using methanol:0.1 M ammonium acetate (50:50)
18
ceptors was evaluated [16–19] but showed limited containing trimethylamine (40 μL/100 mL). F-Fluoride
18
18
success. In addition, potential of various fluorinated was produced by the O(p,n) F nuclear reaction using
iodoquinoxaline carboxamide was also explored as imaging O-18 enriched water (95% enrichment) on a GE PET-
and therapeutic probe to target melanoma [20]. Addition- Trace 800 series cyclotron (GE Healthcare, Milwaukee,
ally, the efforts were made to develop radioiodinated benza- WI). Kryptofix solution was prepared by dissolving
mides as potential melanoma-targeting agents [21–24], 120 mg of K2.2.2. and 720 mg of K CO in 720 μL of water
2
3
partially inspired from the incidental finding of a high up- followed by the addition of 12 mL of acetonitrile. The
take of iodo-benzamides in uveal tissues of pigmented accell plus QMA-cartridge was activated by successively

Garg et al. EJNMMI Research (2017) 7:61
Page 3 of 11
flushing it with 5 mL of 1 N sodium bicarbonate solution mixture for 24 h at room temperature. After evaporating
and 10 mL of sterile water, followed by air flushing. The the solvent, the residue was dissolved in 200 mL of dichlo-
C-18 plus cartridge was activated by washing with 5 mL romethane and was washed successively with water
of ethanol followed by 10 mL of sterile water.
(2 × 50 mL), 1 M sodium bicarbonate (2 × 20 mL), and
B16F1 melanoma cells, HT-29 human colorectal water (2 × 100 mL). The resulting crude product was puri-
adenocarcinoma cells, and C-32 amelanotic melanoma fied using silica gel flash column chromatography (5%
cells were purchased from the American Type Culture methanol in dichloromethane).
Collection (ATCC, Rockville, MD). B16F1 murine mel-
anotic melanoma cells were maintained in Dulbecco’s
modified Eagles medium supplemented with 10% fetal Preparation of N-(2-diethylaminoethyl) 4-(2-
calf-serum, 2 mM L-glutamine, and 1 mM pyruvate. HT- tolunesulphonyloxyethoxy) benzamide (3)
2
9 and C-32 cells were maintained in RPMI supplemented A suspension of NaH (120 mg, 5 mmol) in tetrahydro-
with 10% fetal calf serum and 2 mM L-glutamine. C57BL/ furan (THF) (50 mL) was cooled to 0 °C, and a solution
and athymic nu/nu mice were purchased from Charles of 4-HBZA (1) (590 mg, 2.5 mmol) in 20 mL of THF
6
River Laboratories (Charles River Laboratories, Boston, was added. After stirring the reaction mixture for
MA). All animal studies were performed using an IACUC- 10 min, a solution of 1,2-di-toluenesulphonyloxyethane
approved protocol, and the work was performed under the (1.85 g, 5 mmol) in 10 mL of THF was added, and the
guidelines established by Wake Forest University. The reaction mixture was refluxed overnight. The contents
cells were grown to confluency, harvested, washed with were extracted in 100 mL of dichloromethane, and the
phosphate-buffered saline and then were suspended in organic layer was washed with 1 M HCl followed by
a 1:1 mixture of phosphate-buffered saline and Matrigel 2 × 50 mL of water. Dichloromethane was removed, and
(
BD Life Sciences, San Jose, CA) at a concentration of the residue was purified using flash column chromatog-
6
5
× 10 cells/mL. For subcutaneous tumor xenografts, 6 raphy (5% methanol in CH
mice were inoculated/6 mice were inoculated in the was as follows: C22
right flank with 100 μL of a suspension of B16F1 cells, NMR (CDCl , δ): 1.33 (t, 6H, 2 × N–(CH
and female athymic nu/nu mice were injected in the (s, 3H, CH –Ar–SO ), 3.17 (q, 4H, 2 × N–(CH
), 3.33 (t, 2H, −CH –N–(CH –CH ), 3.82 (m,
H, −CONH–CH –), 4.12 (m, 2H, Ar–O–CH –CH –),
2 2
Cl ). The molecular formula
1
1
H
N
O
S. H NMR (CDCl3, δ). H
–CH ), 2.36
30
2
5
)
3 2
3
2
–
2
3
2
right flank with 100 μL of HT-29 cells or C-32 cells.
CH
3
)
2
2
2
)
3 2
2
4
2
2
2
.36 (m, 2H, Ar–O–CH –CH –), 7.17 (m, 2H, Ar–H–
2
2
Preparation of 1-fluoro 2-tolunesulphonyloxyethane
To a solution of 2-fluoroethanol (480 mg, 7.52 mmol) in
dry dichloromethane (100 mL) were successively added p-
toluenesulfonyl chloride (2.16 g, 11.28 mmol), triethyla-
mine (1.6 mL, 11.28 mmol), and 4-dimethylaminopyridine
C3 and C5–), 7.27 (m, 2H, Ar–H–C2 and C6). ESI-MS
m/z [MH]+ calcd 434, found 435.19.
Preparation of N-(2-diethylaminoethyl)
(
108 mg, 0.88 mmol). After stirring the reaction mixture
4
-fluoroethoxybenzamide (4-FEBZA; 2)
for 8 h at room temperature, the contents were washed
with 1 M HCl (50 mL) and water (2 × 50 mL). The
organic layer was separated, dried over anhydrous magne-
sium sulfate, filtered and evaporated to dryness. The
residue was purified by silica gel flash column chromatog-
raphy (10% ethyl acetate in hexane).
A suspension of NaH (120 mg, 5 mmol) in tetrahydro-
furan (50 mL) was cooled to 0 °C, and a solution of 4-
HBZA (590 mg, 2.5 mmol) in tetrahydrofuran (20 mL)
was added. After stirring the reaction mixture for 10 min,
a solution of 1-fluoro-2-tosyloxyethane (1.09 g, 5 mmol)
in 10 mL of tetrahydrofuran was added, and the reaction
mixture was refluxed overnight. After adding 100 mL of
dichloromethane, the contents were washed with 1 M
HCl followed with 2 × 50 mL of water. After drying the
Preparation of N-(2-diethylaminoethyl) 4-
hydroxybenzamide (4-HBZA; 1)
This intermediate was synthesized as described previously organic layer over anhydrous magnesium sulfate, the solv-
31]. Briefly, a mixture of thionyl chloride (30 mL) and 4- ent was removed, and the residue was purified using flash
acetoxy benzoic acid (7.4 g, 43 mmol) in dimethylforma- column chromatography (5% methanol in CH Cl ) to af-
[
2
2
mide (5 mL) was refluxed for 4 h. After removal of thionyl ford the desired product. The molecular formula was as
1
chloride on a rotary evaporator, triethylamine (6 mL, follows: C H N O F. H NMR (CDCl , δ): 1.07 (t, 6H,
15
23
2
2
3
4
3 mmol) and N,N-diethylethylenediamine (5 g, 43 mmol) 2 × −CH –CH ), 2.61 (q, 4H, 2 × −CH CH ), 2.70 (t, 2H,
2 3 2 3
were added slowly at 0 °C, and the contents were stirred −CH –N–(CH –CH ) ), 3.50 (q, 2H, −CONH–CH –),
2
2
3 2
2
overnight at room temperature. After the usual work-up 4.25 (m, 2H, F–CH –CH ), 4.78 (m 2H F–CH –CH ),
2
2
2
2
of the reaction, 50 mL of sodium methoxide in methanol 6.95 (m, 2H, Ar–H–C3 and C5–), 7.78 (m, 2H, Ar–H–C2
0.5 M) was added followed by stirring the reaction and C6). ESI-MS m/z [MH]+ calcd 282; found 283.29.
(

Garg et al. EJNMMI Research (2017) 7:61
Page 4 of 11
1
8
Radiochemical synthesis of N-(2-diethylaminoethyl)
were incubated at 0, 22, and 37 °C with 4-[ F]FEBZA in
quadruplicate sets for 15, 30, 60, 120, and 180 min. After
1
8
18
4
-[ F]fluoroethoxybenzamide (4-[ F]FEBZA; 4)
1
8
After irradiation, the O–H O bolus containing 450– the incubation, the media were removed, and each well
00 mCi of [ F]fluoride was transferred from the cyclo- was washed with fresh ice-cold media (2 × 2 mL). The
2
1
8
8
tron target to a pre-activated QMA cartridge in the hot adherent cells were removed by lysing cells in 500 μL of
1
8
cell. The trapped [ F]fluoride was eluted from the cart- PBS containing 10% Triton X-100 (Sigma-Aldrich, St.
ridge with 1.5 mL of kryptofix (K2.2.2.) solution prepared Louis, MO). The combined washes and lysate were
as described previously [32]. The remaining water from analyzed for radioactivity content using a γ-counter.
this mixture was removed azeotropically using aceto-
nitrile. The dried [ F]fluoride thus obtained was reacted [ F]FEBZA to B16F1 cells through internalization ex-
Next, we evaluated the binding characteristics of 4-
1
8
18
with a freshly prepared solution of 1–5 mg of precursor periments [33, 34]. The cells were treated with a low pH
3
in 200 μL of anhydrous DMSO or acetonitrile at 115 ° buffer to sequester membrane-bound radioactivity (acid
C for 5–20 min. Subsequently, the contents were diluted wash-releasable) from the internalized1 (acid wash-
8
with 1 mL of water and loaded on to a reverse phase resistant) fraction [34]. For this assay, 4-[ F]FEBZA was
5
semi-preparatory HPLC column. The product was eluted added to 12-well plates with each well containing 4 × 10
from the column into a flask containing 70 mL of water. B16F1cells/well (in quadruplicate), and the plates were in-
Subsequently, the contents were transferred and trapped cubated at 37 °C for 15, 30, 60, 120, and 180 min. After
onto a C-18 Sep-Pak, and the cartridge was washed with the incubation, the media were removed, each well was
1
0 mL of water to remove solvent residue. The desired washed with ice-cold PBS (2 × 1 mL), and the washes
product was eluted from the Sep-Pak with 1 mL of etha- were collected in the tubes. Subsequently, each well was
nol followed by 9 mL of saline and collection in a sterile gently treated with 1 mL of an ice-cold mildly acidic
product vial.
medium (sodium acetate buffer, pH 4.5) to remove
surface-bound radioactivity, and the rinses were collected
in tubes (acidic wash). The adherent cells were then lysed
In vitro binding assay
1
8
The binding affinity of 4-[ F]FEBZA was measured using 500 μL of 10% Triton X-100 in PBS, and the lysate
using the homologous completion assay in 12-well plates was transferred into tubes. The total radioactivity in each
containing a uniform number of fixed B16F1 cells/well. of the lysate, acidic wash, and the PBS wash was measured
After adding 0.1 nmol of 4-[ F]FEBZA/well as the radi- using a γ-counter.
oligand, unlabeled 4-FEBZA (2) was added to the quad-
1
8
ruplicate sets of wells in concentrations ranging from 10 Tissue distribution studies
−
11
−4
to 10 M and the plates were incubated for an hour The biodistribution studies were performed in mice bear-
at 37 °C under 5% CO . At the end of the incubation, ing B16F1 (1 h n = 5; 2 h n = 6), C-32 (1 h n = 3; 2 h
2
the media were removed, and the cells were washed with n = 3), and HT-29 (1 h n = 3; 2 h n = 3) tumor xenografts.
5
1
00 μL of ice-cold media. The cells were lysed using The tumor-bearing mice were injected with ~40 μCi of 4-
mL of phosphate-buffered saline containing 10% [ F]FEBZA. A separate group of normal C57BL/6 mice
18
Triton X-100, and the lysates were collected in culture with no tumor implants (1 h n = 3; 2 h n = 3) was also
18
tubes to measure the radioactivity contents using a γ- injected with ~40 μCi of 4-[ F]FEBZA to study the distri-
18
counter. We performed a non-linear regression analysis bution characteristics of 4-[ F]FEBZA in the absence of
of the decay-corrected CPM values to calculate the in- tumors. At 1 h and 2 h post-injection, the mice were eu-
hibition constant value (IC ), the concentration of thanized, and the tissues of interest were removed, rinsed
5
0
competitor required to inhibit 50% of radiotracer bind- with PBS, weighed, and counted for radioactivity contents
ing, utilizing the GraphPad Prism software program using a γ-counter.
(
GraphPad Software, Inc., CA). The binding curve was
generated by fitting the data simultaneously to one-site MicroPET imaging studies
18
and two-site competitive binding models, and the F test In vivo imaging of 4-[ F]FEBZA in mice bearing B16F1
provided a preferred model for the mean IC50 values (n = 2) and HT-29 (n = 1) tumor was performed using a
(confidence interval 95%).
P4 microPET™ scanner (Concorde Microsystems, Inc.,
The in vitro cell binding was assessed by growing B16F1 Knoxville, TN). After administering ~100 μCi of 4-
18
cells in culture flasks to 80–90% confluence, trypsinizing, [ F]FEBZA via tail-vein injection, whole body PET scans
washing in PBS (pH 7.4), aliquoting into 12-well plates were acquired in list mode. The images were recon-
and allowing adherence to the wells overnight. Initially, structed using filtered back projection with attenuation
we studied the influence of the incubation time and correction. Regions Of Interest (ROIs) were drawn on
temperature on the binding to B16F1 melanoma cells. For select organs using the summed image from the entire
5
this study, 12-well plates containing ~2.5 × 10 cells/well scan acquisition session, and the data were analyzed

Garg et al. EJNMMI Research (2017) 7:61
Page 5 of 11
1
8
using ASIPro software (Siemens Preclinical Solutions, 4-[ F]FEBZA including the HPLC purification and re-
Knoxville, TN). The time activity curves (TACs) were formulation was 54 ± 7 min.
1
8
generated from the list mode data reconstructed into
multiple 5-min frames.
The binding affinity of 4-[ F]FEBZA was assessed using
a homologous competitive binding assay. The results from
this assay are shown in Fig. 3. In this assay, two distinct
18
binding sites were noted for the 4-[ F]FEBZA, with IC
50
Results
values of 1.8 nM and 1.3 μM. A nanomolar binding is an
The synthetic procedure to prepare various intermediates attractive property of this compound, perhaps indicating a
and the non-radioactive final product is shown in Fig. 1. high targeting ability of this compound to melanoma.
The compound 1-fluoro 2-toluenesuIfonyloxyethane was
obtained as a colorless oil in 88% yields. The intermediate total binding of 4-[ F]FEBZA to melanoma cell was
The impact of incubation time and temperature on
18
5
4-HBZA (1) was prepared in 28% yields as a light-yellow- assessed using 2.5 × 10 cells/well using 12-well plates.
colored oil following a previously published procedure. The total binding to B16F1 cells at 37, 22, and 0 °C was
The reference standard for the title compound N-(2- 60.03 ± 0.48%, 44.45 ± 0.61%, and 4.17 ± 0.30%, respec-
diethylaminoethyl) 4-fluorothoxybenzamide (2) was pre- tively, and is shown in Fig. 4.
pared in 40% yields as an amorphous white compound.
The tosylate-precursor (3) was prepared as an amorphous C57BL/6 mice bearing B16F1 and athymic nu/nu mice
white compound in 32% chemical yields. bearing HT-29 and C-32 tumor xenografts at 1 and 2 h
Results from the biodistribution studies performed in
1
8
The radiochemical synthesis of 4-[ F]FEBZA (4) was are presented in Table 1.
accomplished through a simple one-step fluoro-for-tosyl
exchange reaction and is shown in Fig. 2. The total time Discussion
required for the synthesis, HPLC purification, and refor- Towards an ongoing effort to develop a PET imaging
mulation of the final product in buffer required less than probe to target melanoma with high specificity and select-
1
h. On a semi-prep column, the final product was ivity, we recently reported the synthesis and preliminary
11
eluted at ~16–17 min; on an analytical-HPLC column, it preclinical evaluations of a novel PET imaging probe C-
eluted at ~8–9 min. The radiochemical yields ranged MBZA. Despite its attractive in vitro binding and in vivo
from 11 to 20% (16 ± 4%) using 1 mg of precursor. The biodistribution characteristics, the short half-life of C-11
radiochemical yields increased to 53 ± 14% with doub- radionuclide (t1/2 = 20 min) remained a small set-back for
ling the precursor amount. No significant change in the the wide use of this very promising probe. Our next goal
radiochemical yields was noted using 5 mg of precursor. was to develop an imaging probe incorporating a facile
1
8
The radiochemical purity of 4-[ F]FEBZA was >99%, and rapid radiochemical synthesis along with biological
1
1
and the specific activity was 8.7 ± 1.1 Ci/μmol. The traits similar C-MBZA. To that end, we have now de-
1
8
specific activity of 4-[ F]FEBZA was significantly veloped an F-18-labeled fluoroethoxy analog, i.e., 4-
1
8
18
higher than the specific activity reported for F- [ F]FEBZA, and herein, we present those findings.
1
8
FPBZA (30–40 GBq/μMol) and comparable to specific
The radiosynthesis procedure for 4-[ F]FEBZA is quite
1
8
11
activity reported for MEL050, F-DAFBA and C- simple and straight forward. This procedure provides the
MBZA [25, 28, 35]. The overall synthesis time for the desired compound in high radiochemical yields and
Fig. 1 Preparation of the title compound and various other intermediates is shown. Compound (1) was prepared following previously published
method and is shown above. The synthesis of non-radioactive title compound (2) was prepared as the reference standard and was accomplished
by reacting compound (1) with 1-fluoro 2-toluenesulphonyloxyethane. Compound (3) was prepared as precursor for the radiochemical synthesis.
The preparation of compound (3) was accomplished by reacting compound (1) with commercially available ditosylethane

Garg et al. EJNMMI Research (2017) 7:61
Page 6 of 11
1
8
Fig. 2 The radiochemical synthesis of 4-[ F]FEBZA (4) was accomplished using a single-step method and is shown above. The precursor (3)
was reacted with F-18 fluoride in kryptofix/K CO and the crude reaction mixture was subsequently purified using HPLC
2
3
6
purity. The synthesis time and the radiochemical yields despite a higher number of cells (1 × 10 cells) used in
1
8
for the 4-[ F]FEBZA are quite comparable to those re- the assay [28].
ported for many of the other melanoma-targeting agents In addition to rapid and high binding of 4-[ F]FEBZA
reported earlier such as F-DAFBA [25], F-18-labeled to B16F1 melanoma cells, the majority of the cell-
18
1
8
1
8
iodoquinoxaline carboxamide [20],
F-FPBZA [28], associated radioactivity was internalized and the results
MEL050, and other F-18-labeled fluoronicotinamides [27]. from this assay are shown in Fig. 5. A combination of
1
8
Additionally, the synthesis of 4-[ F]FEBZA is quite adapt- attributes such as the high IC50 value and strong inter-
able to automation using many of the commercially avail- nalization could be beneficial and further allude to the
1
8
able synthesis modules.
4
potential of 4-[ F]FEBZA as an effective melanoma
-[ F]FEBZA showed a rapid and high binding to targeting probe.
B16F1 melanoma cells at 37 and 22 °C. In contrast, it A two- to threefold higher uptake of radioactivity was
showed insignificant binding to B16F1 cells at 0 °C; noted in B16F1 melanoma tumors as compared to that
1
8
18
perhaps indicating the involvement of an active uptake in the HT-29 or the amelanotic C-32 tumors. The F-
pathway towards its binding to melanoma cells. In FEBZA uptake in B16F1 tumor is quite similar to tumor
addition to temperature dependency, 4-[ F]FEBZA uptake reported for various other fluorinated benza-
also showed a time-dependent increase in binding. A mides. For example, the F-18 uptake in B16F1 tumor at
threefold higher binding was noted when the cells 1 h was 8.66 ± 1.02%IA/g, 8.4 ± 1.5%IA/g, and
were incubated for 60 min as compared to a 15 min 8.31 ± 1.00%IA/g for the F-FEBZA, MEL050, and F-
incubation. While the cell binding continued to fur- FPBZA, respectively [25, 26, 28, 35]. Similarly, the up-
ther increase with time, the increase was modest be- take of these three compounds in non-melanoma tumor
yond 120 min incubation. The increase in binding for was low.
1
8
18
18
cells incubated at 22 °C was slightly lower than that at
The biodistribution studies show a low uptake of 4-
1
8
18
3
7 °C. For comparison, the overall binding of F- [ F]FEBZA in normal tissue and the uptake levels
FPBZA to B16F0 melanoma cells was 4.12 ± 0.20% continued to decrease further with time, leading to sig-
after 2 h incubation at 37 °C, a 20-fold lower binding nificantly reduced radioactivity levels in normal tissues.
For example, the uptake in the liver and the lungs from
B16F1 tumor-bearing mice at 1 h was 3.25 ± 0.34%IA/g
100
1
00
0
2
3
°C
2°C
7°C
8
6
4
0
0
0
50
0
20
0
-11
-10
-9
-8
-7
-6
-5
-4
log [FEBENZ] concentration (M)
15
30
60
120
180
1
8
Incubation time (min)
Fig. 3 Results from homologs competition of 4-[ F]FEBZA for the
5
varying concentration of FEBZA binding to B16F1 cells. The B16F1
cells were incubated with non-radioactive FEBZA (10
M) at room temp for 1 h followed by adding a fixed amount of
-[ F]FEBZA to each well. Each data point (mean ± SD; n = 4 wells)
represents the percent of added 4-[ F]FEBZA bound to the cells in
presence of varying concentration of non-radioactive FEBZA
Fig. 4 The B16F1 cells (~2.5 × 10 cells/well) were incubated
−
11
18
M–10
with 4-[ F]FEBZA for 15, 30, 60, 120, and 180 min at 0, 22, and
−
4
37 °C. Significantly higher binding was noted at 22 and 37 °C as
compared to that at 0 °C. The data is expressed as percent of total
radioactivity added to each well and the values are the mean ± SD
(n = 4 wells). Data for 0 °C was determined only at 30, 60, and 180 min
18
4
18

Garg et al. EJNMMI Research (2017) 7:61
Page 7 of 11
18
Table 1 Biodistribution of 4-[ F]FEBZA in mice bearing tumor xenografts
%IA/g tissue (mean ± SD)
Organ
B16F1 tumor
HT-29 tumor
1 h
C-32 tumor
1 h
1
h
2 h
2 h
2 h
(
n = 6)
(n = 5)
(n = 3)
(n = 3)
(n = 3)
(n = 3)
Liver
3.25 ± 0.34
2.24 ± 0.36
2.12 ± 0.17
2.82 ± 0.25
3.10 ± 0.28
3.78 ± 0.45
2.49 ± 0.25
2.48 ± 0.35
1.51 ± 0.21
2.55 ± 0.61
1.10 ± 0.36
3.06 ± 0.52
8.66 ± 1.02
2.28 ± 0.49
1.58 ± 0.18
1.49 ± 0.49
2.12 ± 0.58
2.20 ± 0.38
5.45 ± 0.98
1.60 ± 0.31
1.73 ± 0.14
1.14 ± 0.21
2.25 ± 0.50
0.88 ± 0.06
2.51 ± 0.93
7.47 ± 1.34
4.84 ± 0.95
2.11 ± 0.25
2.19 ± 0.33
2.52 ± 0.12
2.91 ± 0.24
3.83 ± 0.39
1.98 ± 0.62
2.41 ± 0.26
1.61 ± 0.28
1.68 ± 0.18
1.07 ± 0.08
2.80 ± 0.26
3.68 ± 0.47
2.50 ± 0.29
1.51 ± 0.12
1.63 ± 0.16
2.19 ± 0.37
2.08 ± 0.29
5.71 ± 1.11
1.42 ± 0.10
1.56 ± 0.18
1.40 ± 0.24
1.52 ± 0.28
0.91 ± 0.19
2.29 ± 0.23
3.07 ± 0.36
3.11 ± 0.49
1.96 ± 0.38
1.94 ± 0.67
2.52 ± 0.15
2.85 ± 0.37
3.47 ± 0.78
2.23 ± 0.21
2.49 ± 0.42
2.35 ± 1.06
1.87 ± 0.21
1.26 ± 0.39
3.22 ± 0.64
3.91 ± 0.23
1.99 ± 0.12
1.22 ± 0.08
1.33 ± 0.35
1.75 ± 0.35
1.84 ± 0.12
5.09 ± 0.72
1.33 ± 0.06
1.40 ± 0.10
1.05 ± 0.14
1.36 ± 0.17
0.77 ± 0.18
2.40 ± 0.30
2.53 ± 0.19
Spleen
Lung
Heart
Kidneys
Bone
Sm. Intestine
Pancreas
Muscles
Brain
Adrenals
Blood
Tumor
and 2.12 ± 0.17%IA/g, respectively, and these levels
Additionally, the uptake levels in the liver, lungs, intes-
decreased to 2.28 ± 0.49%IA/g and 1.49 ± 0.49%IAD/g, tine, and several other organs were comparable between
respectively, by 2 h. The radioactivity contents in the the three types of tumor-bearing mice used in this study.
small intestine of mice bearing B16F1, HT-29, and C- For a radiotracer to qualify as good imaging agent, it
3
2 tumors was 2.48 ± 0.35%IA/g, 1.98 ± 0.62%IA/g, should exhibit low uptake in normal tissues along with a
and 2.23 ± 0.21%IA/g, respectively, at 1 h. Uptake in rapid and high uptake in the tumor. A high lipophilicity
intestine seems high but radioactivity levels in intestine of the compound has been identified as one of the many
was comparable to uptake levels seen in other organs. The factors responsible for higher uptake in normal tissues.
1
8
18
intestinal uptake for F-DAFBA, MEL050, and F- Therefore, a radiotracer with lower lipophilicity would
FPBZA at 1 h in mice bearing B16F melanoma tumor was be desirable to minimize accumulation in the normal tis-
3.04 ± 0.24%IA/g, 2.1 ± 0.3%IA/g, and 2.45 ± 0.38%IA/g, sues, but could alongside, has the potential to reduce its
respectively.
uptake in the tumor. Therefore, a good balance between
lipophilicity and functional groups within the molecular
moiety is a key when designing biologically active im-
aging agents.
The radioactivity levels decreased in normal tissues
with time. The uptake in B16F1 tumor was rapid, high
and the tumor uptake levels plateaued by 1 h. A combi-
nation of these two factors resulted in favorable tumor
to tissue ratios for the melanoma group as compared to
those for the HT-29 tumor group. The tumor-to-tissue
ratio at 1 h post-injection for various groups is presented
in Fig. 6. The tumor-to-muscle ratio at 2 h for the
B16F1, C-32, and HT-29 mice groups was 6.64 ± 1.11,
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
Cell Surface Bound
Internalized
1
5
30
60
120
180
Incubation time (min)
2
.43 ± 0.14, and 2.21 ± 0.17, respectively. The tumor-to-
18
5
Fig. 5 Internalization of 4-[ F]FEBZA into B16F1 cells (~5 × 10 cells/
well) at 37 °C. The data represents the percent of total cell-associated
radioactivity and is the mean ± SD (n = 4). Majority of the cell-
associated radioactivity is internalized and the cell surface bound
fraction amount is negligible (<1%) at all time points and the
remainder of the radioactivity was present in the media. The
internalized fraction increased with time and reached plateau
by about 120 min
18
tissue ratio for the 4-[ F]FEBZA is quite comparable to
those reported for F-DAFBA and other C-labeled
and F-labeled benzamides [20, 25–27].
18
11
18
A low uptake of radioactivity was noted in most
normal tissues except for the bones. A high uptake in
the bones is generally an indirect measure of in vivo
defluorination [25, 36, 37], thus indicating metabolic

Garg et al. EJNMMI Research (2017) 7:61
Page 8 of 11
8
Tumor to Tissue Ratio
B16F1
7
6
5
4
3
2
1
0
HT-29
C-32
Liver
Spleen
Lung
Heart
Kidneys Pancrease Muscle
Blood
Brain
Fig. 6 Tumor-to-normal tissue ratio at 1 h is presented for the mice bearing B16F1, C-32, and HT-29 tumors. The tumor-to-tissue ratio for
the amelanotic C-32 and colorectal adenocarcinoma HT-29 group of mice were quite similar and these ratios were appreciably lower than
that for the mice from B16F1 group. A twofold lower tumor-to-muscle ratio was noted for the C-32 and HT-29 as compared to that from
B16F1 group
1
8
dehalogenation of 4-[ F]FEBZA. Although high bone feature of probes to target metastatic lesions to the
uptake has been reported for MEL050 (0.58 ± 0.05%IA/g brain. The tissue distribution studies showed a modest
1
8
at 2 h) and F-FPBZA (3.21 ± 0.16%IA/g at 2 h), high uptake of radioactivity in the mouse brain
1
8
bone uptake observed for the F-FEBZA is somewhat (2.55 ± 0.61%IA/g at 1 h) demonstrating the ability of
1
8
unexpected since the structurally close analog F- this compound to cross the blood-brain barrier. In com-
DAFBA showed low bone uptake (0.20 ± 0.06%IA/g parison, the uptake of MEL050 in mouse brain as deter-
at 2 h) [26]. Placement of fluorine group on the alkyl chain mined from microPET imaging studies was
1
8
of 4-[ F]FEBZA may be a likely contributor towards en- 1.38 ± 0.40%IA/g at 60–70 min [38]. More importantly,
hanced defluorination. The covalently bound fluorine to the levels in the brain were significantly lower than those
1
8
aryl group in F-DAFBA and MEL050 may have provided in the tumor. The tumor-to-brain tissue ratios for the
extra stability to Aryl–F bond towards catabolic defluori- B16F1 and HT-29 groups were 3.55 ± 0.80 and
nation in vivo. To further investigate whether the en- 2.19 ± 0.04, respectively, at 1 h, and 3.45 ± 0.81 and
1
8
hanced defluorination of F-FEBZA and subsequent 2.04 ± 0.19, respectively, at 2 h. A high tumor-to-brain
1
8
accumulation of F-fluoride in the bone was tumor me- ratio (Fig. 6) further supports the ability of 4-
18
diated phenomenon, we repeated the biodistribution study [ F]FEBZA to delineate metastatic lesions present in
in C57BL/6 mice with no implanted tumor. The bone up- the brain.
take at 1 h in mice with no tumor, and the mice with
B16F1, HT-29, and C-32 tumors was 3.23 ± 0.24%IA/g, the imaging characteristics of 4-[ F]FEBZA through
.78 ± 0.45%IA/g, 3.83 ± 0.39%IA/g, and 3.47 ± 0.78%IA/ microPET studies in mice bearing B16F1 and HT-29
g, respectively (p > 0.1). In addition, the uptake of 4- tumor xenografts. The maximum intensity projection
Based on these encouraging results, we further explored
18
3
18
[
F]FEBZA in various organs was similar in mice with no (MIP) images and the axial view from microPET images
tumors and the tumor bearing mice. For example, at 1 h acquired at 30 min for mice bearing B16F1 and HT-29
IA/g in the liver, lungs, muscle, blood, and brain was tumor xenografts is shown in Fig. 7. In order to provide
%
3
2
.25 ± 0.34, 2.12 ± 0.17, 1.51 ± 0.21, 3.06 ± 0.52, and appropriate comparison between the two independently
.55 ± 0.61, respectively, in mice bearing B16F1 tumor acquired microPET imaging studies, each image set was
(Table 1) and 4.11 ± 0.63, 2.52 ± 0.47, 1.10 ± 0.13, normalized to injected dose levels (Fig. 7). The normalized
2.81 ± 0.31, and 2.46 ± 0.41, respectively, in mice without MIP projection over coronal display is that MIP images
the implanted tumor. The differences in tissue uptake for minimize the frame selection bias and enables direct com-
the two groups are statistically insignificant (p > 0.1). Simi- parison between the two subjects. As anticipated, the MIP
18
larity in tissue distribution data and bone uptake levels for images show distinct uptake of 4-[ F]FEBZA in the eyes
the two groups of mice indicate that the defluorination and tumor of mouse bearing B16F1 tumor xenograft. In
might be resulting from weaker C–F alkyl bond and per- addition, the black skin coat of the C57BL/6 mouse is vis-
haps not due to tumor mediated catabolic defluorination ible due to binding of this radiotracer to skin containing
of the parent molecule.
melanin (Fig. 7, right panel). In contrast, the skin of
The ability of a melanoma imaging agent to cross the athymic mice does not contain melanin and thus show
blood brain barrier is considered another important lack of F-18 accumulation in the skin (Fig. 7, left panel).

Garg et al. EJNMMI Research (2017) 7:61
Page 9 of 11
Eyes
Tumor
Tumor
Stomach
Bladder
Stomach
Bladder
Fig. 7 Maximum intensity projection (MIP) image from MicroPET scans of mice bearing B16F1 melanoma (right panel) and HT-29 human colorectal
left panel) tumor xenograft. An intense uptake of radioactivity is seen in B16F1 tumor. In contrast, HT 29 tumor shows a significantly low uptake and it
(
is quite difficult to visualize this tumor on microPET images (left panel). Eyes show a distinct uptake pattern in B16F1 tumor bearing mice (right panel).
Additionally, the uptake of radiotracer in skin of black mice (C57/BL6) was evident from darker background (right panel) in comparison to lack of such
uptake in the skin for HT-29 tumor bearing athymic mice (left panel)
Most other melanoma-targeting agents display similar
The radioactivity levels were measured from microPET
characteristic uptake in the eyes and the skin of C57BL/6 images by drawing ROIs on major organs visible on the
mice, a unique feature of this family of compounds [27, scans. The standardized uptake values (SUVs) derived
2
8, 39]. On microPET images, B16F1 tumor was clearly from these microPET images for the B16F1 and HT-29
visible on the summed image. In contrast, the HT-29 tumors were 1.53 ± 1.01 and 0.25 ± 0.04, respectively. The
tumor was not discernable. MIP image from microPET study for the mouse bearing
To further understand the uptake characteristics, B16F1 tumor at various time points is shown in Fig. 9.
time activity curves were generated from the ROIs
drawn over the organs visible on microPET images.
4
3
3
.0
.5
.0
1
8
HT29
The time activity curves for 4-[ F]FEBZA uptake in
B16F1 melanoma tumor and HT-29 (colorectal car-
cinoma tumor) are shown in Fig. 8. After an initial
uptake, the radioactivity levels in B16F1 tumor in-
creased with time reaching over 3.5%IA/g tumor by
B16F1
2.5
2
1
1
.0
.5
.0
4
7 min, whereas, the levels in HT-29 tumor decreased
significantly with time; resulting in threefold higher
uptake in B16F1 tumor (Fig. 8). It is noteworthy that
the tumor uptake values reported as %IA/g derived
from microPET images are 2- to 3-times lower than
the values obtained from the biodistribution studies.
While the uptake data derived from microPET images
is lower than that obtained from biodistribution stu-
dies, the tumor-to-tissue ratios and the melanoma to
non-melanoma tumor uptake ratios were comparable
between the microPET images and biodistribution studies.
One of the likely explanations for this discrepancy could
be due to a mismatch of radioactivity level calibration be-
tween the gamma counter and the scanner.
0.5
.0
0
0
5
10
15
20
25
30
35
40
Time (min)
Fig. 8 Time activity curves showing tumor uptake in mice bearing
B16F1 and HT-29 tumors. The curves were generated from microPET
images. Each data point represents the percent of injected Activity
per gram (%IA/g) of B16F1 tumor (red) and HT-29 (Blue) at various
time points after injecting 4-[ F]FEBZA. After an initial accumulation
in both the tumor, the uptake levels in melanoma tumor increased
with time (red), whereas the levels in HT-29 tumor decreased and
remained low (blue)
18

Garg et al. EJNMMI Research (2017) 7:61
Page 10 of 11
Eyes
Eyes
Kidneys
Tumor
Tumor
Stomach
Bladder
Bladder
2.5 min
10 min
20 min
30 min
40 min
Fig. 9 Maximum Intensity projection (MIP) images from MicroPET scans of a mouse bearing B16F1 melanoma tumors xenograft that was injected
18
with ~284 μCi of 4-[ F]FEBZA. These MIP images show radioactivity accumulation at 2.5, 10, 20, 30, and 40 min post-injection. Uptake in eyes was
quite prominent as early as 2.5 min post-injection. The kidneys show an intense accumulation of radioactivity within 2.5 min post-injection
followed by a rapid clearance thereafter. The tumor is clearly visible by 10 min post-injection and the uptake intensity in tumor continued
to increase with time
On these reconstructed images, the B16F1 tumor is
clearly visible as early as 10 min post-injection and the
uptake intensity continued to increase with time. In
addition, a characteristic uptake in the eyes is also
distinctly visible in images at all time points.
In summary, 4-[ F]FEBZA shows a selective and rapid
uptake by melanoma cells in vitro. Preferential uptake in
melanoma tumor xenograft along with a favorable whole
body distribution pattern in vivo suggests a significant
potential of this radiotracer in delineating and localizing
melanoma.
experiments were executed under PG guidance. All authors read and
approved the final manuscript.
Ethics approval
This manuscript does not include human studies. All studies that involved the
use of animals were performed under IACUC-approved protocol (Institutional
Animal Care and Use Committee) and the work was performed following the
guidelines established by the Wake Forest University.
1
8
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published
maps and institutional affiliations.
Conclusions
Herein, we have developed a rapid and reliable one-step
Author details
1
8
1
2
synthesis of 4-[ F]FEBZA that provides the desired pro-
duct in high radiochemical yields and high specific acti-
vity. The in vitro studies show a high binding affinity of
this compound to melanoma cells. The biodistribution
and microPET imaging studies show a rapid and prefe-
Wake Forest University Health Sciences, Winston Salem, NC, USA. Center for
Molecular Imaging and Therapy, Biomedical Research Foundation, 1505
Kings Highway, Shreveport, LA 71133, USA. Current Address: Asinex
Corporation, 10 N. Chestnut Street, St 104, Winston Salem, NC 27101, USA.
3
Received: 19 April 2017 Accepted: 25 July 2017
1
8
rential accumulation of 4-[ F]FEBZA in melanoma
tumors. Results from these preclinical studies suggests
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