Facile synthesis of para-[18F]fluorohippurate via iodonium ylide-mediated radiofluorination for PET renography

Article abstract of DOI:10.1016/j.bmcl.2015.11.091

para-[¹⁸F]fluorohippurate ([¹⁸F]PFH) is a renal tubular agent suitable for conducting positron emission tomography (PET) renography. [¹⁸F]PFH is currently synthesized by a four-step two-pot procedure utilizing a classical

Full text of DOI:10.1016/j.bmcl.2015.11.091

Bioorganic & Medicinal Chemistry Letters 26 (2016) 479–483
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Facile synthesis of para-[18F]fluorohippurate via iodonium
ylide-mediated radiofluorination for PET renography
⇑
Gregory N. Nkepang, Andria F. Hedrick, Vibhudutta Awasthi, Hariprasad Gali
Department of Pharmaceutical Sciences, College of Pharmacy, The University of Oklahoma Health Sciences Center, 1110 N. Stonewall Avenue, Room 301, Oklahoma City, OK
73117, USA
a r t i c l e i n f o
a b s t r a c t
para-[18F]fluorohippurate ([18F]PFH) is a renal tubular agent suitable for conducting positron emission
tomography (PET) renography. [18F]PFH is currently synthesized by a four-step two-pot procedure utiliz-
ing a classical prosthetic group, N-succinimidyl-4-[18F]fluorobenzoate, followed by glycine conjugation.
Considering the short half-life of fluorine-18 (110 min), it is important to reduce the number of synthetic
steps and overall production time for successful translation of any fluorine-18 radiopharmaceutical in to
clinical practice. Here, we report a new two-step one-pot procedure using a novel spirocyclic iodonium
ylide precursor for producing a dose of [18F]PFH suitable for human use in 45 min including HPLC
purification with an overall decay-corrected radiochemical yield of 46.4 2.9% (n = 3) and radiochemical
purity of >99%.
Article history:
Received 6 October 2015
Revised 20 November 2015
Accepted 24 November 2015
Available online 25 November 2015
Keywords:
[
18F]PFH
PET
Renography
Iodonium
Ylide
Ó 2015 Elsevier Ltd. All rights reserved.
Currently there is a need to develop suitable radiotracers to uti-
In this regard, we and others have reported a few potential PET
renal agents in recent years.4–13 Renal agents used for renography
generally fall under two categories: filtration agents and tubular
secretion agents. Renal filtration agents are only filtered by the
glomerulus, whereas tubular secretion agents undergo both
glomerular filtration (depending on their molecular weight and
plasma protein binding affinity) and tubular secretion. Renal tubu-
lar secretion agents are extracted from the plasma present in the
peritubular capillaries into the tubular cells via the basolateral
membrane governed by the organic anion transporter (OAT) sys-
tem expressed in the proximal convoluted tubules, for example,
OAT1 and OAT3.14,15 The higher clearance of renal tubular secre-
tion agents due to both glomerular filtration and tubular secretion
enables them to provide better quality images and, thus, are pre-
ferred radiopharmaceuticals for renography, especially in patients
with poor renal function.16
lize positron emission tomography (PET) in clinical evaluation of
renal function. The main advantage of PET is that it enables the
use of dynamic tomographic imaging and provides accurate cam-
era-based quantification.1 The PET quantification is superior to pla-
nar gamma imaging, which is predominantly used in current
clinical renal imaging studies such as renography. In addition, pla-
nar imaging provides limited structural information and lower-
quality images. Although drawbacks of planar imaging could be
overcome by using single photon emission computed tomography
(SPECT), conducting a clinical dynamic SPECT study is technically
challenging. The clinical SPECT camera depends on rotational
movement (step-and-shoot) of detector heads, which cannot be
used for renography due to fast pharmacokinetics exhibited by
the renal agents. Whereas PET enables list-mode acquisition of
imaging data using a series of detector blocks arranged in a cylin-
drical geometry around the subject. List-mode provides extremely
high temporal resolution with full spatial resolution and allows
frame durations to be set after the data acquisition.2 In addition,
clinical PET cameras offer higher sensitivity, spatial resolution,
and signal-to-noise ratios than clinical gamma/SPECT cameras.3
For example, spatial resolution of a clinical PET camera and a clin-
ical gamma/SPECT camera is 4–6 mm and 10–20 mm, respectively.
Of all the PET renal agents reported so far, only para-[18F]fluoro-
hippurate ([18F]PFH) and meta-cyano-para-[18F]fluorohippurate
([18F]CNPFH) reported by us are tubular secretion agents.4–6 Both
of them are structural analogs of ortho-[131I]iodohippurate (Hippu-
ran, a gold standard tubular secretion agent).17 Hippuran labeled
with iodine-131 (a beta emitter, half-life—8 d) and iodine-123 (a
gamma emitter, half-life—13.2 h) were approved by FDA for clini-
cal use and later discontinued after the introduction of [99mTc]
MAG3 (a renal tubular secretion agent widely used in current clin-
ical renography studies) because of higher costs of iodine-123 and
suboptimal nuclear properties of iodine-131 for imaging. Among
⇑
Corresponding author. Tel.: +1 (405) 271 6593x47877; fax: +1 (405) 271 7505.
E-mail address: hgali@ouhsc.edu (H. Gali).
http://dx.doi.org/10.1016/j.bmcl.2015.11.091
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

480
G. N. Nkepang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 479–483
[
18F]PFH and [18F]CNPFH, [18F]PFH was found to be rapidly and
handle, and suffer from suboptimal radiochemical yields or poor
regiospecificity.22 In this regard, iodonium(III) ylide based nucle-
ophilic radiofluorination methodologies are very promising for
the preparation of non-activated [18F]fluoroarenes and thus are
gaining popularity.19–21,23 In our case, the aromatic ring of [18F]
PFH is deactivated for a nucleophilic substitution reaction for
radiofluorination using the common leaving groups such as nitro
and trimethylammonium salts due to presence of an amide group.
Thus, in our previous work, we have started the radiosynthesis
with the ethyl 4-(trimethylammonium triflate)benzoate precursor
in which the aromatic ring is activated due to presence of an ester
group.4
exclusively cleared by the kidneys and provided much higher qual-
ity renogram and images obtained by dynamic PET studies com-
pared to those obtained with
[
99mTc]MAG3 dynamic planar
imaging studies.6 However, [18F]PFH radiosynthesis was long
(ꢀ95 min including HPLC purification) due to its four-step two-
pot procedure utilizing a classical prosthetic group, N-succin-
imidyl-4-[18F]fluorobenzoate ([18F]SFB),18 followed by glycine con-
jugation.4 Considering the short half-life of fluorine-18 (110 min),
it is important to reduce the number of synthetic steps and overall
production time for successful translation of any fluorine-18 radio-
pharmaceutical in to clinical practice. Thus, [18F]CNPFH was devel-
oped utilizing
a
simpler process of direct one-step one-pot
The novel ylide precursor 7 employed in the present work was
synthesized in five steps as shown in Figure 1a starting from
commercially available 4-iodobenzoic acid 1. In the first step,
4-iodobenzoyl chloride 2 was synthesized by heating 1 in neat
thionyl chloride at 80 °C for 2 h followed by removal of excess thio-
nyl chloride by distillation. The compound 2 was obtained in a
quantitative yield and used without any further purification. In
the second step, 2 was reacted with glycine in presence of aqueous
sodium bicarbonate and subsequent purification with solvent
extractions provided 4-iodoippuric acid 3 in 87.6% yield. In the
third step, 3 was reacted with an excess of ethanol in presence of
thionyl chloride and subsequent purification with silica gel chro-
matography provided ethyl-4-iodohippurate 4 in 64.1% yield. In
the fourth step, the iodine present on 4 was oxidized with sodium
perborate in presence of glacial acetic acid at 50 °C and subsequent
purification by recrystallization from a mixture of hexane and
methylene chloride provides 5 in a near quantitative yield. In the
final step, treatment of 5 with the auxiliary 6, which was synthe-
sized according the reported procedure,19 in aqueous sodium
carbonate and subsequent purification with silica gel chromatogra-
phy provided the ylide precursor 7 in 40.9% yield.
nucleophilic aromatic substitution reaction.5 However, biodistri-
bution and PET renography studies in healthy rats showed that
[
18F]CNPFH would not be an effective agent for renography,
because it is eliminated by both renal and hepatobiliary pathways.5
Our results obtained so far collectively indicate [18F]PFH as a
promising lead PET renal tubular secretion agent, that is, highly
suitable for PET renography. However, the high potential of [18F]
PFH for clinical translation is hampered by its current radiosynthe-
sis method, which would be difficult to be automated by majority
of the commercially available automated radiochemical
synthesizers.
Thus, here we report a new two-step, one-pot procedure for
producing a dose of [18F]PFH suitable for human use in 45 min
including HPLC purification and can be automated using any of
the commercially available synthesizers. In the present work, we
adopted a nucleophilic radiofluorination methodology recently
reported by Rotstein et al. utilizing spirocyclic iodonium(III) ylide
precursors to efficiently and regiospecifically radiofluorinate non-
activated or electron-rich arenes with no-carrier added [18F]fluo-
ride.19,20 These spirocyclic iodonium(III) ylide precursors were
developed to improve the radiochemical yields when compared
to the Meldrum’s acid derived iodonium(III) ylide precursors
initially developed by Satyamurthy and Barrio.21 Nucleophilic
radiofluorination of non-activated arenes is challenging and often
requires multistep methodologies, precursors are difficult to
The radiosynthesis of [18F]PFH using ylide precursor 7 was per-
formed manually as shown in Figure 1b. Our goal was to obtain an
injectable dose of >15 mCi [18F]PFH in <8 ml volume starting from
<75 mCi [18F]fluoride in <60 min (radiosynthesis and purification).
We selected <75 mCi [18F]fluoride as starting activity because we
O
O
(a)
COCl
O
COOH
Glycine, NaHCO3
Ethanol, SOCl2
Reflux, 18 hr
SOCl2
N
H
N
H
COOH
O
0 oC, 15 min and RT, 1 hr
80 oC, 2 hr
I
I
I
I
1
2
3
4
Acetic acid, NaBO3 50 oC, 18 hr
O
O
O
O
O
O
Na2CO3
N
H
N
H
O
+
O
O
O
RT, 18 hr
I
O
O
I
O
O
O
O
O
O
7
6
5
O
O
O
(b)
18F]F-, TEAB, DMF
130 oC, 10 min
1M NPr4OH
O
N
O
[
N
H
N
H
COOH
H
O
O
130 oC, 5 min
I
18F
18F
O
O
O
O
9
7
8
Figure 1. Synthesis of (a) iodonium ylide precursor and (b) [18F]PFH.

G. N. Nkepang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 479–483
481
Radioactivity
11.08
(a)
(b)
Collection
(c)
Absorbance
11.03
0
5
10
15
20
25 min
0
5
10
15 min
Figure 2. HPLC chromatograms of (a) [18F]PFH batch obtained while purification, (b) [18F]PFH obtained while quality control, and (c) non-radioactive PFH standard.
Table 1
can consistently produce [18F]fluoride in that range using our
Biodistribution (%ID/g) of [18F]PFH at 10 min p.i. in healthy female Sprague Dawley
cyclotron (BG-75 Biomarker Generator, ABT Molecular Imaging)
in 90 min of bombardment with a beam current of 5 A. The
rats
l
Organ
Ylide method
[
18F]SFB method4
radiosynthesis conditions were based on the previously reported
general procedure (tetraethylammonium bicarbonate (TEAB),
DMF, 120 °C, and 10 min) for the radiofluorination of the
diaryliodonium ylide precursors.19 However, increasing the tem-
perature from 120 °C to 130 °C for both radiofluorination and
hydrolysis steps almost doubled the overall radiochemical yield
of [18F]PFH. Briefly, the radiofluorination of 7 was carried out in
DMF using TEAB as a phase-transfer agent in a sealed condition.
The hydrolysis was carried out using 1 M tetrapropylammonium
hydroxide with a flow of nitrogen stream to enable DMF evapora-
tion while the reaction is proceeded to completion. After the
hydrolysis the product mixture was diluted with water and puri-
fied by semi-preparative HPLC using PBS–ethanol (90/10 v/v). Total
radiosynthesis time was 45 min including HPLC purification and
Blood
Muscle
Heart
Lung
Liver
Spleen
Stomach
Intestine
Kidney
Bone
0.23 0.08
0.04 0.01
0.09 0.03
0.14 0.05
0.07 0.02
0.08 0.03
0.10 0.07
0.07 0.02
4.21 1.69
0.04 0.03
0.26 0.01
—
0.08 0.01
0.17 0.04
0.09 0.01
0.06 0.01
0.12 0.07
0.09 0.03
2.93 1.52
—
Urine (%ID)
73.4 7.1
72.1 6.4
Values are expressed as the mean SD (n = 4). Data for [18F]SFB method was taken
from Ref. 4.
[
18F]PFH was obtained with an overall decay-corrected radiochem-
We conducted in vivo evaluation of [18F]PFH obtained by ylide
method in healthy rats to confirm its biological activity. As shown
in Table 1, the biodistribution data of [18F]PFH obtained by ylide
method at 10 min p.i. matches well with our previously reported
data for [18F]PFH obtained by [18F]SFB method indicating that both
methods provide identical product.4 Almost no bone uptake was
observed, which indicates that no free [18F]fluoride was present
in the injected dose and no defluorination occurred in vivo. Figure 3
shows the PET renogram and images obtained in a healthy rat with
ical yield of 46.4 2.9% (n = 3). As shown in Figure 2a, [18F]PFH is
clearly separated from unwanted radioactive and non-radioactive
peaks. The radiochemical purity of [18F]PFH was found to be
>99% after HPLC purification (Fig. 2b) and its retention time
matches well with the non-radioactive standard (Fig. 2c), which
confirms its radiochemical identity. The total volume of the col-
lected fraction was <6 ml and activity obtained was
17.1 1.5 mCi (n = 3) starting from 50.1 2.4 mCi (n = 3) of [18F]
fluoride. Although the specific activity was not determined, it is
[
18F]PFH produced by ylide method, which were found to be simi-
estimated to be >1 Ci/l
mol based on the [18F]PFH peak area in
lar to that of other healthy Sprague Dawley rats previously
reported.4–6
the absorbance chromatogram obtained during the purification
(Fig. 2a) in comparison to the peak area of the non-radioactive
PFH standard of know concentration and the collected activity.
Further optimization of the reaction conditions was not pursued
as our goal was achieved. This radiosynthesis and purification
can be readily automated using any commercially available syn-
thesizer as it is a single-pot reaction. The experimental details
and characterization data for compounds 3–5, 7, and [18F]PFH are
given in Reference and notes.24–26
In summary, the novel ylide precursor 7 employed in this work
produced the desired [18F]PFH in relatively high radiochemical
yield and purity. Although the reported method was performed
manually, this two-step one-pot reaction can be readily adopted
for producing [18F]PFH using any commercially available auto-
mated radiochemical synthesizer. Thus, the radiosynthesis pre-
sented here provides a very practical method for producing
clinical doses of [18F]PFH under cGMP compliance.

482
G. N. Nkepang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 479–483
Figure 3. (a) Kidney time–activity curves (renogram) and (b) lower abdominal maximum intensity projection PET/CT images at Tmax of a healthy rat injected with [18F]PFH
synthesized with new method.
acidified with 5 M H2SO4 (pH—2) and evaporated to dryness under high
Acknowledgements
vacuum. The solid residue was extracted with methanol (3 ꢁ 300 ml) and the
methanolic filtrate evaporated to afford an impure compound that was further
This work was funded by the University of Oklahoma College of
Pharmacy Startup Grant. We acknowledge the OU Nuclear Phar-
macy staff for their support. We thank Dr. Lee Collier for helpful
discussions regarding the synthesis of auxiliary 6.
extracted into diethyl ether and dried to afford 7.0 g of pale white solid (87.6%
yield). 1H NMR (300 MHz, CD3OD-d4): d 7.83 (d, 2H, J = 8.39 Hz, Ar-H), 7.59 (d,
2H, J = 8.39 Hz, Ar-H), 4.07 (m, 4H, –CH2–), 3.30 (s, 1H, NH). 13C NMR (75 MHz,
CD3OD-d4): d 171.56, 137.29, 128.64, 98.12, 40.82. HRMS (ESI): calcd for
C9H9INO3 [M+H]+:305.9627, found 305.9606. Synthesis of ethyl-4-iodohippurate
(4): 3 (4.00 g, 13.12 mmol) was suspended in 100 ml of absolute ethanol and
mixture chilled to 0 °C using an ice bath. Thionyl chloride (1.13 ml,
15.70 mmol) was added dropwise to the stirring solution. Upon addition the
solid disappeared completely. The ice bath was removed and the reaction
mixture was heated to reflux overnight. Ethanol was removed by rotary
evaporation to give a brownish solid which was further purified by a flash
column chromatography on silica gel using ethyl acetate–hexane (8:2 v/v) as
eluent to afford a yellow stick oil which was then precipitated at ꢂ25 °C using
acetone–hexane (1:7 v/v) into a 2.8 g white fluffy solid (64.1% yield). 1H NMR
(300 MHz, CDCl3-d): d 7.79 (d, 2H, J = 8.39 Hz, Ar-H), 7.53 (d, 2H, J = 8.39 Hz,
Ar-H), 6.67 (s, 1H, –NH), 4.23 (m, 4H, –CH2), 1.30 (t, 3H, J = 7.19 Hz, –CH2–CH⁄3).
13C NMR (75 MHz, CDCl3-d): d 170.01, 137.84, 128.64, 98.87, 61.79, 41.89,
14.12. HRMS (ESI): calcd for C11H12INO3Na [M+Na]+:355.9760, found 355.9755.
Synthesis of ethyl-4-(diacetoxy-iodo)hippurate (5): Sodium perborate
tetrahydrate (6.96 g, 45.03 mmol) was added in portions to a 0.15 M of 4
(1.50 g, 45.03 mmol) in glacial acetic acid (28.50 ml) and heated to 50 °C. The
reaction mixture was stirred at this temperature overnight, cooled to room
temperature, diluted with water, and extracted three times with methylene
chloride (3 ꢁ 200 ml). The combined organic extracts were dried with
anhydrous sodium sulfate, filtered, and concentrated. The product was
purified by recrystallization from hexane–methylene chloride (9:1 v/v) at
ꢂ20 °C to afford a 2.01 g white solid (99.0% yield). It should be noted that this
oxidized product does not stain with iodine and the spot is characteristically
pure white spot on silica gel TLC. 1H NMR (300 MHz, CDCl3-d): d 7.79 (d, 2H,
J = 8.38 Hz, Ar-H), 7.53 (d, 2H, J = 8.39 Hz, Ar-H), 6.69 (s, 1H, –NH), 4.23 (m, 4H,
–CH2–), 2.08 and 2.10 (S, 6H, –C(O)–CH3), 1.30 (t, 3H, J = 6.89 Hz, –CH2–CH⁄3).
13C NMR (75 MHz, CDCl3-d): d 169.97, 138.04, 129.16, 61.83, 41.84, 13.80.
Synthesis of the ylide precursor (7): To a solution of the auxiliary 6 (0.33 g,
1.95 mmol) in 10% aqueous sodium carbonate (6 ml) was added 6 ml of
ethanol. This mixture was vigorously stirred and immediately followed by the
addition of 5 (0.82 g, 1.95 mmol). The vigorously stirring solution was allowed
to react at room temperature overnight. At the end of the reaction as
determined by the TLC in ethyl acetate, the reaction mixture was diluted with
water and extracted with methylene chloride (3 ꢁ 300 ml). Combined organic
extracts were dried and concentrated. Flash column chromatography with
silica gel using first ethyl acetate and later with ethyl acetate–methanol (9:1 v/
v) as eluents afforded 400 mg of white solid (40.9% yield). This compound had
solubility problems and most of it was lost during column chromatography.
Best solvent of solubility is methanol. Also note that this compound does not
stain with iodine. 1H NMR (300 MHz, CD3OD-d4): d 8.00 (d, 2H, J = 8.39 Hz, Ar-
H), 7.88 (d, 2H, J = 8.39 Hz, Ar-H), 4.20 (q, 2H, J = 7.19 Hz, –CH⁄2–CH3), 4.10(s,
2H, –C(O)CH⁄2–), 2.09 (t, 2H, J = 7.19 Hz, –CH2⁄–CH2–) 1.78 (t, 2H, J = 7.19 Hz, –
CH2–CH⁄2–), 1.30 (t, 3H, J = 7.19 Hz, –CH2–CH3⁄). 13C NMR (75 MHz, CD3OD-d4):
d 169.78, 167.43, 165.65, 136.56, 133.25, 129.53, 118.45, 113.72, 60.98, 58.49,
41.21, 36.69, 22.71. HRMS (ESI): calcd for C19H20INO7Na [M+Na]+: 524.0182
and C38H40I2NO14Na [2M+Na]+: 1025.0467, found 524.0171 and 1025.0457.
25. Radiosynthesis of [18F]PFH using ylide precursor 7. The general reaction scheme
for the radiosynthesis of [18F]PFH is shown in Figure 1b. Briefly, [18F]fluoride
was produced by irradiating enriched [18O]-water with protons. [18F]Fluoride
References and notes
1. Rahmim, A.; Zaidi, H. Nucl. Med. Commun. 2008, 29, 193.
2. Nichols, T. E.; Qi, J.; Asma, E.; Leahy, R. M. IEEE Trans. Med. Imaging 2002, 21,
396.
3. Townsend, D. W. Semin. Nucl. Med. 2008, 38, 152.
4. Awasthi, V.; Pathuri, G.; Agashe, H. B.; Gali, H. J. Nucl. Med. 2011, 52, 147.
5. Pathuri, G.; Hedrick, A. F.; Awasthi, V.; Gali, H. Nucl. Med. Biol. 2012, 39, 1195.
6. Pathuri, G.; Sahoo, K.; Awasthi, V.; Gali, H. Nucl. Med. Commun. 2011, 32, 908.
7. Lipowska, M.; Klenc, J.; Shetty, D.; Nye, J. A.; Shim, H.; Taylor, A. T. Nucl. Med.
Biol. 2014, 41, 248.
8. Schnockel, U.; Reuter, S.; Stegger, L.; Schlatter, E.; Schafers, K. P.; Hermann, S.;
Schober, O.; Gabriels, G.; Schafers, M. Eur. J. Nucl. Med. Mol. Imaging 2008, 35,
2267.
9. Pathuri, G.; Hedrick, A. F.; January, S. E.; Galbraith, W. K.; Awasthi, V.; Arnold, C.
D.; Cowley, B. D.; Gali, H. J. Label. Compd. Radiopharm. 2015, 58, 14.
10. Hofman, M.; Binns, D.; Johnston, V.; Siva, S.; Thompson, M.; Eu, P.; Collins, M.;
Hicks, R. J. J. Nucl. Med. 2015, 56, 405.
11. Lee, J. Y.; Jeong, J. M.; Kim, Y. J.; Jeong, H.-J.; Lee, Y.-S.; Lee, D. S.; Chung, J.-K.
Nucl. Med. Biol. 2014, 41, 210.
12. Tahari, A. K.; Bravo, P. E.; Rahmim, A.; Bengel, F. M.; Szabo, Z. J. Med. Imaging
Radiat. Oncol. 2014, 58, 25.
13. Pathuri, G.; Hedrick, A. F.; Awasthi, V.; Cowley, B. D., Jr; Gali, H. Nucl. Med. Biol.
2016, 43, 1.
14. Shikano, N.; Kanai, Y.; Kawai, K.; Ishikawa, N.; Endou, H. J. Nucl. Med. 2004, 45,
80.
15. Takahara, N.; Saga, T.; Inubushi, M.; Kusuhara, H.; Seki, C.; Ito, S.; Oyama, N.;
Yokoyama, O.; Sugiyama, Y.; Fujibayashi, Y. Nucl. Med. Biol. 2013, 40, 643.
16. Taylor, A. T. J. Nucl. Med. 2014, 55, 608.
17. Yasky, J.; Volpe, R. Can. Med. Assoc. J. 1963, 88, 1055.
18. Vaidyanathan, G.; Zalutsky, M. R. Int. J. Rad. Appl. Instrum. B 1992, 19, 275.
19. Rotstein, B. H.; Stephenson, N. A.; Vasdev, N.; Liang, S. H. Nat. Commun. 2014, 5,
4365.
20. Stephenson, N. A.; Holland, J. P.; Kassenbrock, A.; Yokell, D. L.; Livni, E.; Liang, S.
H.; Vasdev, N. J. Nucl. Med. 2015, 56, 489.
21. Satyamurthy, N.; Barrio, J. R. United State Patent US 8,742,139 B2, 2014.
22. Brooks, A. F.; Topczewski, J. J.; Ichiishi, N.; Sanford, M. S.; Scott, P. J. H. Chem. Sci.
2014, 5, 4545.
23. Cardinale, J.; Ermert, J.; Humpert, S.; Coenen, H. H. RSC Adv. 2014, 4, 17293.
24. Synthesis of ylide precursor 7. The general reaction scheme for the synthesis of
ylide precursor 7 is in Figure 1a. Synthesis of 4-iodobenzoyl chloride (2): 4-
Iodobezoic acid (6.50 g, 26.21 mmol) and thionyl chloride (20 ml) were heated
at 80 °C for 2 h. Excess thionyl chloride was distilled off and directly used for
the next step without further purification. Synthesis of 4-iodohippuric acid (3):
Glycine (1.97 g, 26.21 mmol) was added to a 150 ml solution NaHCO3 (11.01 g,
131.04 mmol) dissolved in acetone–water mixture (1:3 v/v) and stirred for
10 min at 0 °C. Then 2 (26.21 mmol) dissolved in 5 ml dry acetone was added
dropwise over 15 min. The reaction temperature was raised and maintained at
room temperature for further 60 min. After completion, the mixture was
(50.1 2.4 mCi, n = 3) dissolved in [18O]-water (560
inert gas pressure to a reaction vial (5-ml serum vial) containing ꢀ8 mg of
ll) was transferred using

G. N. Nkepang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 479–483
483
tetraethylammonium bicarbonate (TEAB) and acetonitrile (200
l
l). The vial
Sonoma C18, 10
l
m, 10 ꢁ 250 mm) for separating the resulting [18F]PFH from
was then sealed with a butyl rubber stopper and an aluminum crimp. Two 21
the other compounds in the reaction mixture. A sterile mobile phase consisting
of 90% phosphate-buffered saline and 10% 200-proof absolute ethanol with a
flow rate of 4 ml/min was used for purification. A fraction from 11.6 min to
12.9 min was collected (17.1 1.5 mCi, n = 3) in a 10 ml sterile vented empty
gauge 100 needles were inserted in to the vial for nitrogen purging and venting.
All reagents were added using a 500 ll or 1 ml insulin syringe attached with a
29 gauge 0.500 needle. The solvents were evaporated under a stream of nitrogen
(ꢀ2.3 psi) at 130 °C for 12 min. Azeotropic drying was then achieved by adding
vial via passing through a 0.22 lm 33 mm PES syringe filter. The overall decay-
corrected radiochemical yield was 46.4 2.9% and radiochemical purity was
>99%. Representative HPLC chromatograms are shown in Figure 2.
anhydrous acetonitrile (500
l
l) to the reaction vial and the solvents were
evaporated under a stream of nitrogen (ꢀ2.3 psi) at 130 °C for 3 min.. Then
ylide precursor (ꢀ10 mg) dissolved in anhydrous dimethylformamide (1 ml)
was added to the dried [TEAB]18F complex and the reaction mixture was
heated in the sealed vial at 130 °C for 10 min to achieve 18F-labeling. Next 1 M
26. Analytical HPLC conditions. HPLC solvents consisted of water containing 0.1% v/
v
trifluoroacetic acid (solvent A) and acetonitrile containing 0.1% v/v
trifluoroacetic acid (solvent B). A Sonoma C18 column (ES Industries, West
Berlin, NJ, 5
tetrapropylammonium hydroxide (200
l
l) was added and the reaction mixture
lm, 100 Å, 4.6 mm ꢁ 250 mm) was used with a flow rate of 1.5 ml/
was heated at 130 °C for 5 min under a stream of nitrogen (ꢀ2.3 psi) to achieve
simultaneous deprotection and drying of the solvents. The reaction vial was
removed from the heating block, the reaction mixture was diluted with
deionized water (1.5 ml), and injected in to a semi-preparative HPLC (column:
min. The HPLC gradient system began with an initial solvent composition of
95% A and 5% B for 2 min followed by a linear gradient to 50% A and 50% B in
15 min, after which the column was reequilibrated. The absorption detector
was set at 254 nm.