Optimization of 18F-syntheses using 19F-reagents at tracer-level concentrations and liquid chromatography/tandem mass spectrometry analysis: Improved synthesis of [18F]MDL100907

Article abstract of DOI:10.1002/jlcr.3606

Traditional radiosynthetic optimization faces the challenges of high radiation exposure, cost, and inability to perform serial reactions due to tracer decay. To accelerate tracer development, we have developed a strategy to simulate radioactive ¹⁸F-syntheses by using tracer-level (nanomolar) non-radioactive ¹⁹F-reagents and LC-MS/MS analysis. The methodology was validated with fallypride synthesis under tracer-level ¹⁹F-conditions, which showed reproducible and comparable results with radiosynthesis, and proved the feasibility of this process. Using this approach, the synthesis of [¹⁸F]MDL100907 was optimized under ¹⁹F-conditions with greatly improved yield. The best conditions were successfully transferred to radiosynthesis. A radiochemical yield of 19% to 22% was achieved with the radiochemical purity >99% and the molar activity 38.8 to 53.6?GBq/ μmol (n?=?3). The tracer-level ¹⁹F-approach provides a high-throughput and cost-effective process to optimize radiosynthesis with reduced radiation exposure. This new method allows medicinal and synthetic chemists to optimize radiolabeling conditions without the need to use radioactivity.

Full text of DOI:10.1002/jlcr.3606

Optimization of 18F-Syntheses using 19F-Reagents at Tracer-level
Concentrations and LC-MS/MS Analysis: Improved Synthesis of
[18F]MDL100907
Optimization of 18F-Syntheses with Tracer-level 19F-Reagents
Xiang Zhang, Ryan Dunlow, Burchelle N. Blackman, Rolf E. Swenson
Imaging Probe Development Center, National Heart, Lung, and Blood Institute, National
Institutes of Health, Rockville, MD, USA
*Corresponding author.
Email address: rolf.swenson@nih.gov (R. E. Swenson).
Abstract
Traditional radiosynthetic optimization faces the challenges of high radiation exposure, cost and
inability to perform serial reactions due to tracer decay. To accelerate tracer development, we have
developed a strategy to simulate radioactive 18F-syntheses by using tracer-level (nanomolar) non-
radioactive 19F-reagents and LC-MS/MS analysis. The methodology was validated with fallypride
19
synthesis under tracer-level F-conditions, which showed reproducible and comparable results with
radiosynthesis, and proved the feasibility of this process. Using this approach, the synthesis of
[18F]MDL100907 was optimized under 19F-conditions with greatly improved yield. The best conditions
were successfully transferred to radiosynthesis. A radiochemical yield of 19-22% was achieved with
the radiochemical purity >99% and the molar activity 38.8 – 53.6 GBq/ µmol (n = 3). The tracer-level
19F-approach provides a high-throughput and cost-effective process to optimize radiosynthesis with
reduced radiation exposure. This new method allows medicinal and synthetic chemists to optimize
radiolabeling conditions without the need to use radioactivity.
Keywords
19F, 18F, optimization, tracer-level, fallypride, MDL100907
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/jlcr.3606
This article is protected by copyright. All rights reserved.

1. Introduction
Molecular imaging is an active research area that allows the visualization of molecular probes in
biological processes of a living subject.1, 2 In the past few decades, molecular imaging techniques have
made significant achievements in both methodological and instrumental development.3 In particular,
positron emission tomography (PET) has evolved into a powerful scientific and clinical tool for non-
invasively monitoring molecular pathways in vivo.4, 5 PET utilizes radiopharmaceuticals labeled with
positron-emitting radionuclides. Among the commonly used PET radionuclides, fluorine-18 (18F) is the
most popular PET isotope, often referred to as the radionuclide of choice in PET imaging. In the last
decade, the number of bioactive fluorine containing drugs has significantly increased, providing an
expanded pool of fluorinated bioactive candidates for PET tracer development.6,7 To produce novel
tracers and to optimize the syntheses of known tracers with low efficiency, there is an urgent need to
develop a practical and robust way to explore radiosynthetic conditions.
We propose and demonstrate an approach to optimize radiochemical reactions by tracer-level
(nanomolar) [19F]KF and quantitation using liquid chromatography/tandem mass spectrometry (LC-
MS/MS). Optimizing radiochemical reactions with natural isotope has been a known approach for
decades, which helped radiochemists to establish radiolabeling conditions.8-11 But to our knowledge no
quantitation method at no-carrier-added (NCA) level has been reported, primarily due to the lack of
available analytical methods to quantify tracer-level of product. We believe that tandem MS
spectrometry could be applied to tracer-level reactions for product quantification. Tandem quadrupole
instruments such as the triple quadrupole combine high sensitivity and high selectivity, allowing
quantitation of trace levels of analytes in very complex sample matrices. MS technique is routinely used
in research and clinical settings to quantitate drug levels for pharmacokinetic studies of various
biological matrixes.12-14 One of the key advantages to LC-MS/MS is the high-throughput ability to
evaluate numerous samples quickly. It allows medicinal and synthetic chemists to quickly assay
multiple tracer-level reactions, so that the reaction optimization can be concluded in a short time before
transferring to radiochemists for tracer production. It is especially useful when transferring synthetic
methods to radiochemistry. Although purchase of a triple quadruple mass spectrometer is a significant
investment, LC-MS/MS based assays are rountinely used in drug discovery and development.15 There
is increasing availability of LC-MS/MS for other applications such as proteomics and metabolomics
studies and clinical analysis.16-18 Labs who are routinely developing new radiochemistry will find it
indispensable for the rapid analysis of parallel reaction optimization.19,20 Another advantage of the
proposed approach is that no exposure to radioactivity is necessary as compared to the radioactive 18F-
approach, as all reaction screenings are conducted with natural 19F-reagents. Once the conditions with
18
the highest yield had been identified by LC-MS/MS, F-labeled radiosynthesis was performed for
validation purposes. This feature not only greatly reduced exposure for the radiochemists, but also
lowered the cost of purchasing radioactivity which offsets the initial investment in the LC-MS/MS
This article is protected by copyright. All rights reserved.

instrument. In addition, the proposed approach can be potentially applied to all imaging isotopes,
offering a broad scope of potential applications.
2. Results and Discussion
19
18
To evaluate the correlation of natural F to radioactive F-reaction yields, we have selected the
tracer-level synthesis of fallypride as a proof-of-concept study. [18F]Fallypride is a PET probe used to
quantify dopamine D2/D3 receptors in the brain and has been extensively employed in many clinical
PET centers.21,22 The radiosynthesis of [18F]fallypride has been optimized by several groups and well-
established with up to 70% radiochemical yield (RCY).23,24 In this study, we performed a series of
19
18
fallypride syntheses with tracer-level F, and found the yields were comparable with radioactive F-
19
19
syntheses thereby validating the F-protocol. Once the protocol was validated, the tracer-level F-
approach was applied to the optimization of MDL100907 synthesis. [18F]MDL100907 is a promising
5-HT2a receptor ligand which exhibits excellent selectivity, affinity, and favorable brain metabolism.25,26
However, the radiosynthesis of [18F]MDL100907 is limited by extremely low yield and long synthesis
time, as the electron-rich aryl group hinders the nucleophilic substitution of F-. Ren and coworkers
reported an improved radiolabeling approach using Ni-mediated fluorination. However, the overall
yield is about 3%. A more robust synthesis of this promising tracer is needed.26,27 By optimizing the
synthesis of [18F]MDL100907 under tracer-level 19F-conditions, we demonstrate the great potential of
the 19F-process for PET tracer development.
For the LC-MS/MS analysis, we chose to use two distinct MS/MS transitions for each analyte
(fallypride and MDL100907) to increase the selectivity for the desired analyte. The transition giving
the highest response was used for quantitation. The second transition was used to confirm the identity
of the analyte.
Fallypride served as the model compound to validate the feasibility of the 19F-approach. To evaluate
the possibility whether other detection methods such as UV and electrochemical detectors are sufficient
for tracer-level detection, we performed the comparison between UV and MS/MS detection. Calibration
curves were prepared for both detection methods at various fallypride concentration (Figure 1), and
limit of quantification (LOQ) was determined. For UV detection, the LOQ is 188 ng/mL which is in the
19
same level as NCA reactions. In theory it is possible to quantify F-fallypride synthesis with UV
detector since it provides sufficient sensitivity for nanomolar concentrations. The analysis of a
representative 19F-synthesis was attempted on UV detector. However, it lacks selectivity for the
nanomolar -level product in crude reactions, which is fully obscured by enormous amount of impurities
at much higher concentrations (Figure 1b). Similar outcome was predicted for electrochemical detector,
that the selectivity is insufficient for quantification purpose, but we did not have access to one to test
our hypothesis. It may be possible to quantitate other tracers with much less impurities, or in cases that
baseline separation was established for the compound of interest. However, it is case specific and not
This article is protected by copyright. All rights reserved.

applicable to all tracers. Under MS/MS condition, the LOQ for fallypride is 0.98 ng/mL, 200-fold higher
than UV method (Figure 1c). More importantly, quantification was successfully achieved on the same
crude reaction sample. In multiple reaction monitoring (MRM) mode, the trace fallypride signal was
selectively recorded among other impurity peaks in much higher concentration (Figure 1d). This mass
selection feature allows the detection and quantification of tracer-level products in complex mixtures.
MS/MS provides a more reliable and versatile approach for NCA level detection.
Next, an experiment was carried out to survey the level of background fluoride contributed from
the reaction vial (Figure 2). In this regard, fallypride synthesis was performed with the same process
19
as other tracer-level F-procedure, but no fluorine source (KF) was added. The reaction mixture was
then analyzed by LC-MS/MS. When the glass reaction vials were used without any washing, it was
found the fallypride concentration reached as high as 253 ± 34 ng/mL (n = 3), which represented ~0.7
nmol of fallypride formation attributed from background F-. However, when another group of glass
vials were pretreated by washing with K2CO3 and rinsing with water, fallypride concentration was
highly reduced by up to 70-80% (85 ± 6 ng/mL). This experiment indicated that some glassware does
contain high amount of F- which may participate in the fluorination reaction and potentially affect the
SA. A base wash of the glassware can reduce the residual F-. In all later experiments the background F-
has been subtracted for yield calculation.
The correlation between the amount of KF and yield was also studied (Figure 3). Five parallel
syntheses of fallypride were performed with various amount of KF (0.0375, 0.075, 0.15, 0.50, 1.0 µg).
However, the yield of the 5 syntheses remained in the same level (39-44%, n =3). The result suggested
that at this mass range, the amount of KF is not a major contributing factor to yield. For the non-
radioactive optimization work in this project, the amount of fluorine (0.15 µg KF) was selected to match
with the corresponding radioactive batches, considering a typical molar activity (Am) of 0.18-0.3 GBq
(5-8 mCi)/nmol for the [18F]F- produced at our institute and 0.27-0.54 GBq (10-20 mCi) of [18F]F- used
in each validation reaction.
It has been reported by Moon et al. that the amount of base plays a crucial role in improving the
yield of fallypride.24 Excess base may lead to unwanted thermal degradation of the precursor and
potentially lower the product yield and purity. The improved synthesis of [18F]fallypride under low-
base conditions gave up to 70% RCY. In our study, these conditions were evaluated as the model
reaction to validate tracer-level 19F-approach (Table 1). Initially, the high-base condition was tested for
the 19F-reaction (entry 2). A 41% yield was obtained which was considerably lower than all other low-
base conditions. This finding is consistent with literature report. For the low-base conditions (entries 3
and 4), 63% and 55% yield was obtained respectively, which are comparable with reported 18F-
radiosynthesis. Since the reaction time of 30 min is long for a typical 18F-radiosynthesis (entry 3), we
have evaluated the shortened time of 10 min (entry 5). Under these conditions, a similar yield of 64%
was achieved. Though the yield is similar to that of entry 2, it will be more practical in 18F-tracer
production due to shorter synthesis time. In addition, this condition was replicated multiple times (n =
This article is protected by copyright. All rights reserved.

4), with highly reproducible results. The use of the milder base KHCO3 also generated similar yield to
the K2CO3 and tetrabutylammonium hydrogen carbonate (TBAB) approaches (entries 6 and 7).
To evaluate the correlation between natural 19F and radioactive 18F-reactions, the radiosynthesis of
[18F]fallypride was performed under each reaction condition of Table 1, and general agreement in the
19
18
RCY between F and F-reactions was found. For example, under high-base conditions (entry 2), a
60% yield was obtained, which is substantially lower than those of low-base conditions (entries 3, 5-
7). Under the conditions which gave the highest yield (entries 3, 5-7), the isolated RCY of
[18F]fallypride reached 74-82% (n = 5), the radiochemical purity was >99%, and the Am was 47.3 –
108.4 GBq/ µmol (1.28-2.93 Ci/µmol) at the end of synthesis (Figure S1). This study has confirmed
19
the feasibility of tracer-level F-approach, in that the 19F-reactions displayed comparable results with
radiosyntheses, and the LC-MS/MS is able to provide reproducible data to analyze product in nanogram
scale.
With the protocol validated, we further evaluated the synthesis of MDL100907 using the tracer-
level 19F-approach. Since the fluorine atom is on an electron-rich aromatic system, the radiofluorination
via conventional nucleophilic approach is not practical. Recently, copper-mediated aromatic
nucleophilic radiofluorination reported by Gouverneur et. al. has provided a robust approach to achieve
fluorination on both electron-rich and electron-deficient arenes.28 Starting from the aryl boronic ester,
copper-mediated radiofluorination has successfully produced a wide range of products, including 6-
[18F]fluoro-L-DOPA and [18F]DAA1106. This method was improved by several groups so that it is
more suitable for radiochemical processes and automated synthesizers.29-31 The application of the
copper-catalyzed method may provide a superior synthetic approach of [18F]MDL100907 as compared
to previously reported synthesis.
The Cu-catalyzed synthesis of MDL100907 requires the aryl boronic ester 6 as the precursor. A
two-step synthesis for the pinacol boronate was developed (Scheme 1). Briefly, commercially available
compounds, (2,3-dimethoxyphenyl)(piperidin-4-yl)methanol (3) and (4-(2-bromoethyl)phenyl)-
boronic acid (4) were coupled under basic conditions. Without work-up or purification, the newly
generated boronic acid was treated with pinacol to yield 5. Compound 6 was prepared by protecting the
benzylic hydroxyl group with di-t-butyl dicarbonate.
Tracer-level 19F-reactions were carried out to optimize the Cu mediated synthesis (Table 2). Several
factors were evaluated in this study, such as different precursors (unprotected/protected), the relative
amounts of base and precursor, selection of catalyst, solvent, and temperature, etc. Firstly, the
unprotected alcohol 5 was tested as precursor using various catalysts and amount of base (entries 1-4).
However, no product was detected by LC-MS/MS analysis. This result is in agreement with the
literature, that unprotected hydroxyl or amino functionalities will interfere with the desired C-C
coupling.28 Next, our effort was focused on the Boc-protected pinacol boronate 6 as the precursor
(entries 5-14). Under high-base and Cu(OTf)2(py)4 condition, the reaction also did not occur which
This article is protected by copyright. All rights reserved.

confirmed low reactivity of high-base protocol (entry 5).29 To overcome this problem, the rest of
experiments were performed under low-base conditions, in which a small amount of K2CO3 (0.060 mg,
0.43 µmol) was used. Under the low-base condition (entry 6), the reactivity was dramatically increased,
a yield of 18.1% was observed by LC-MS/MS. Other approaches such as increased amount of precursor,
various temperature and solvent did not increase the yield (entries 7-10). Increasing catalyst loading has
an adverse effect, resulting in a decreased yield of 11.3% (entry 11). When the precursor loading was
reduced, the yield was also significantly dropped to 8.3% (entry 12). Other conditions such as using
Cu(OTf)2 as catalyst, or exchanging the solvent for a ACN/DMF mixture were also evaluated, and led
to no product formation (entries 13-14).
With the optimized conditions in hand (Table 2, entry 6), the tracer-level 19F-approach was
transferred to 18F-radiosynthesis (Scheme 2). The radiosynthesis was performed by the 2-step protocol.
Applying the optimized conditions, [18F]MDL100907 was obtained in 19-22% isolated RCY after
HPLC purification (n = 3). The radiochemical purity was >99%, and the Am was 38.8 – 53.6 GBq/ µmol
(1.05-1.450 Ci/µmol) at the end of synthesis (Figure S2). The synthesis time was approximately 70
min. The identity of [18F]7 was confirmed by analytical HPLC. The residual Cu content in the final
formulation was analyzed. It was found to be ≤ 1 ppm, well below the concentration limit of copper for
parenteral drug products under both US Pharmacopeia and ICH guidelines.32 Compared with the
reported yield of 3%,26 this optimized radiosynthesis offers significantly higher RCY. More importantly,
the entirely optimization process was performed under natural 19F-conditions.
19
The F-process provides high-throughput capability, allowing many conditions to be tested and
analyzed within a 24-hour period. In our lab, a batch of 10 reactions has been performed during the day,
and the LC-MS/MS analysis was completed overnight through the auto-sampling process. A generic
LC method was used for both fallypride and MDL100907 (Figure S3), which can be easily adopted to
other tracers without further modification. The efficient process of optimizing radiochemical reaction
can be used to significantly improve existing methods of radiosynthesis. In addition, for both tracers
evaluated in this study, the 19F-approach provided consistent data, which was reproduced with similar
yield in radiosynthesis. The 18F-reaction is only necessary to validate the optimized condition from the
19F-approach, which will greatly reduce the radiation exposure for the chemists. A major benefit of this
approach is that it provides techniques that can be used by medicinal or synthetic chemists to discover
new methods of preparing PET probes with no need to handle radioactivity.
3. Experimental
3.1.General
Unless otherwise noted, all chemicals and solvents were purchased from Sigma-Aldrich
(Milwaukee, WI, USA) or Fisher Scientific (Hanover Park, IL, USA) and used without further
purification. Fallypride and tosyl-fallypride were purchased from ABX GmbH (Radeberg, Germany).
(2,3-dimethoxyphenyl)(piperidin-4-yl)methanol was purchased from Aurum Pharmatech LLC (Howell,
This article is protected by copyright. All rights reserved.

NJ, USA). (4-(2-bromoethyl)phenyl)-boronic acid was purchased from Combi-Blocks, Inc (San Diego,
CA, USA). Non-carrier added [18F]fluoride was obtained from the National Institutes of Health
cyclotron facility (Bethesda, MD, USA). Glass reaction vials (catalog: 352016) were obtained from
Biotage (Charlotte, NC, USA). Ultra-pure water was produced with the Milli-Q® Integral aater
purification system (Billerica, MA, USA). The residual copper concentration in final dose was analyzed
by Robertson Microlit Laboratories (Ledgewood, NJ, USA).
Flash chromatography was performed on a Biotage Isolera™ flash system (Charlotte, NC, USA).
NMR was recorded on a Bruker 400 MHz spectrometer (Billerica, MA, USA). LC-MS/MS analysis
was performed on Agilent 6460C triple quadrupole mass spectrometry with an ESI source. The LC
inlet was Agilent 1200 series chromatographic system equipped with 1260 binary pump, 1290
thermostatted column compartment and 1260 high performance autosampler. Instrument control and
data processing were performed using Aglient’s MassHunter Software. Semi-preparative and analytical
high-pressure liquid chromatography (HPLC) were conducted on the Agilent 1260 HPLC system
equipped with multi-wavelength UV detector and radiation detector. All RCY were determined by
HPLC analysis of the crude product, unless stated otherwise.
3.2.Chemical synthesis
(R)-(2,3-dimethoxyphenyl)(1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenethyl)piperidin-4-
yl)methanol (5)
4-(2-Bromoethyl)phenylboronic acid 4 (0.91 g, 3.98 mmol) was added to a solution of (2,3-
dimethoxyphenyl)(piperidine-4-yl)methanol 3 (1.00 g, 3.98 mmol), NaHCO3 (0.50 g, 5.97 mmol) and
molecular sieves (5 – 6 g) in DMF (23 mL). The reaction solution was stirred at 85
reaction was monitored via LC-MS. Pinacol (0.470 g, 3.98 mmol) was added to the solution and stirred
at 85 C for 20 min. The solution was filtered through celite and the solvent was removed under reduced
̊C for 90 min. The
̊
pressure. The resulting oil was purified using a Biotage ACI system on a 25g KP-Sil SNAP cartridge
(450 ml 25% EtOAc in hexanes then 300 mL 1% triethylamine in EtOAc) yielding a light yellow oil
(900 mg, 47%). 1H NMR (400MHz, CDCl3): δ 7.71 (d, J = 8.1 Hz, 2H), 7.19 (d, J = 7.9 Hz, 2H), 7.04
(t, J = 7.9 Hz, 1H), 6.89 (dd, J = 7.9, 1.5 Hz, 1H), 6.84 (dd, J = 8.1, 1.5 Hz, 1H), 4.62 (d, J = 8.1 Hz,
1H), 3.86 (s, 6H), 3.07 (d, J = 11.4 Hz, 1H), 2.92 (d, J = 11.1 Hz, 1H), 2.83 – 2.77 (m, 2H), 2.57 – 2.51
(m, 2H), 2.11 – 2.04 (m, 1H), 2.01 – 1.85 (m, 2H), 1.73 – 1.61 (m, 1H), 1.33 (s, 16H). 13C NMR (101
MHz, CDCl3) δ 152.47, 146.51, 143.93, 136.39, 134.90, 128.14, 123.97, 119.70, 111.41, 99.99, 83.66,
75.02, 74.54, 60.89, 60.58, 55.71, 53.65, 42.84, 33.84, 28.79, 28.73, 24.86. HRMS (ESI): Calcd for
C28H40BNO5 (M+H)+ 482.3072, found 482.3079.
(R)-tert-butyl
((2,3-dimethoxyphenyl)(1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenethyl)
piperidin-4-yl)methyl) carbonate (6)
This article is protected by copyright. All rights reserved.

Di-tert-butyl dicarbonate (160 mg, 0.734 mmol) was added to a solution of compound 5 (334 mg,
0.668 mmol) and 4-(dimethylamino)pyridine (24.4 mg, 0.2 mmol) in pyridine (5 mL). The resulting
mixture was stirred at 60 C for 30 min. The volatile was removed under reduced pressure. The
̊
remaining oil was purified on a Biotage flash system on a 25 KP-Sil SNAP cartridge (10 - 25% EtOAc
in hexanes) yielding product as a yellow foam (85 mg, 38%) and recovered unreacted starting material
(150 mg). 1H NMR (400MHz, CDCl3): δ 7.71 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 7.03 (t, J
=8.0 Hz, 1H), 6.92 (dd, J = 7.9, 1.6 Hz, 1H), 6.83 (dd, J = 8.1, 1.5 Hz, 1H), 5.73 (d, J = 7.9 Hz, 1H),
3.89 (s, 3H), 3.85 (s, 3H), 3.02 (d, J = 10.6 Hz, 1H), 2.91 (d, J = 11.4 Hz, 1H), 2.83 – 2.72 (m, 2H),
2.57 – 2.47 (m, 2H), 1.99 – 1.83 (m, 3H), 1.77 (m, 1H), 1.47 (d, J = 6.5 Hz, 4H), 1.42 (s, 9H), 1.32 (d,
13
J = 8.0 Hz, 12H). C NMR (101 MHz, CDCl3) δ 153.10, 152.39, 146.46, 143.90, 134.90, 133.32,
128.14, 123.99, 118.77, 111.45, 83.65, 81.75, 76.89, 75.02, 60.60, 60.52, 55.63, 53.51, 53.35, 41.31,
33.81, 28.29, 27.82, 24.86. HRMS (ESI): Calcd for C33H48BNO7 (M+H)+ 582.3596, found 582.3599.
3.3.Instrumental configuration
Chromatographic condition
Chromatographic separations were carried out with gradient elution on an Agilent RRHT Zorbax
SB-C18 column (2.1×50mm, 1.8 µm) or Waters Acquity UPLC BEH-C18 (2.1x50 mm, 1.7 µm). The
mobile phase was composed of water (A) and acetonitrile (B) each containing 0.1 % formic acid. The
analytes were eluted from the column by a linear gradient which started at 5% B, held at initial
conditions for 0.30 min, then increased from 5 to 95% B within 2.95 min and held at 95% B for 1.0 min
then returned to the initial conditions. The total run time was 6.65 min. The flow rate was set at 0.6
mL/min. The column oven was kept at 40 °C throughout the analysis. The injection volume was 1 µL
and the autosampler rack temperature was 20 °C.
UV quantitation of fallypride
A calibration curve in the range of 200 – 8,000 ng/mL (ppb) was prepared for fallypride. The curve
was fitted by least square linear regression using the peak areas. The acceptable criterion for the
calibration curve was a correlation coefficient (r2) of 0.98 or better, and that each back-calculated
standard concentration must be within 20% deviation from the nominal value. The S/N ratio of the
lowest calibration level was greater than 10. The limit of detection (LOD) was estimated as 3 × SD for
the lowest calibrant. LOQ was calculated as 3 × LOD. A LOD value of 62.8 ng/mL was calculated, and
LOQ was 188 ng/mL.
Mass spectrometric conditions
Mass spectrometric data were acquired in positive ion mode with the following ESI-MS parameters:
gas temperature 350 °C; gas flow 13 L/min; nebulizer 60 psi; capillary voltage 3000 V. Nitrogen was
used as desolvation gas and collision gas, dwell time were set at 75 ms (MDL100907/Verapamil) and
This article is protected by copyright. All rights reserved.

80 ms (fallypride/verapamil) for each transition. Cell accelerator voltage was set to 2 for
fallypride/verapamil and 6 for MDL100907/verapamil. Quantification was done using MRM mode.
The product ion giving the highest response was used for quantitation (quan). The qualifier ion (qual)
was the next highest responding ion. The ratio between the quan ion and the qual ion was used, along
with retention time, to confirm the identity of the analyte in the sample. The precursor ions and MS/MS
parameters for each compound are displayed in Table 3. Yields of natural 19F-reactions were
determined by LC-MS/MS analysis. Briefly, a calibration curve in the range of 2 – 12,000 ng/mL (ppb)
was prepared for the standard compounds (fallypride and MDL100907) containing an internal standard
(verapamil). The curves were fitted by a weighted (1/x or 1/x2) least squares linear regression method
by measuring the peak area ratio of the analyte to the internal standard. The acceptable criterion for a
calibration curve was a correlation coefficient (r2) of 0.98 or better, and that each back-calculated
standard concentration must be within 20% deviation from the nominal value. S/N of the lowest
calibration level was greater than 10. All calibrators and samples were run in triplicate. LOD was
estimated as 3 × SD for the lowest calibrant. LOQ was calculated as 3 × LOD. A LOD value of 0.32
ng/mL was calculated, and LOQ was 0.98 ng/mL.
3.4.General method for fallypride optimization under 19F-condition:
For K2CO3/KHCO3 method: to a 2-mL microwave vial was added KF (0.15 μg, 2.59 nmol) in 15
μL water, K2CO3 or KHCO3 (selected amount), K222 (7-8 mg), and 1 mL of acetonitrile. For
tetrabutylammonium hydrogen carbonate (TBAB) method: KF (0.15 μg, 2.59 nmol) in 15 μL of water,
TBAB (0.18 mL, 0.075 M), and 1 mL of acetonitrile was added to the reaction vial. The solution was
azeotropically dried under vacuum and nitrogen flow at 100 ˚C. To the dried mixture, acetonitrile (1.0
mL) was added. The mixture was further azeotropically dried under nitrogen and vacuum at 100 C.
After the drying sequence was completed, the tosyl-precursor in acetonitrile (1.0 mL) was added. The
reaction vial was stirred at elevated temperature for selected time and cooled to 50 °C. The yield was
analyzed via LC-MS/MS quantification. The MS sample was prepared by diluting 10 μL of the reaction
solution with 80 μL of acetonitrile and 10 μL of verapamil (internal standard, 166 ng/mL).
3.5.Radiosynthesis of [18F]fallypride
A sample of 0.42 GBq [18F]/fluoride (11.3 mCi) in water (50-100 µL) was added to a reaction vessel
containing selected amount of base and Kryptofix 2.2.2 (7 – 8 mg) in acetonitrile (1.0 mL). The mixture
was azeotropically dried under nitrogen and vacuum at 100 C. To the dried mixture, acetonitrile (1.0
mL) was added. The mixture was further azeotropically dried under nitrogen and vacuum at 100 C.
After the drying sequence was completed, the tosyl-precursor (2 mg) in acetonitrile (1.0 mL) was added.
The reaction vial was stirred at elevated temperature for selected time and cooled to 50 °C. The reaction
mixture was quenched with 1 mL H2O (with 0.1% triethylamine) and transferred to the HPLC for
purification. HPLC conditions: Agilent XDB C18, 250 × 10 mm, 5 μ; mobile phase: 50% acetonitrile
This article is protected by copyright. All rights reserved.

and 50% water (with 0.1% triethylamine) at flow rate 4 mL/min. The labeled product was eluted
between 15-16 min, which was collected in a collection flask. The chemical identity of the product was
checked by analytical HPLC. HPLC conditions: Agilent XDB C18, 150 × 4.6 mm, 5 ; mobile phase:
60% acetonitrile and 40% H2O (with 0.1% triethylamine) at flow rate 1 mL/min. The total synthesis
time was approximately 70-75 min.
3.6.General method for MDL100907 optimization under 19F-condition:
To a 2-mL microwave vial was added KF (0.15 μg, 2.59 nmol) in 15 μL water, K2CO3 or KHCO3
(as in table) and K222 in acetonitrile (1.0 mL). The solution was azeotropically dried under vacuum and
nitrogen flow at 110 ˚C. The azeotropic drying process was repeated by adding acetonitrile (1 mL × 3).
In another vial, the boronic ester precursor was mixed with copper (II) catalyst in DMF and vortexed
for 5 s. The mixture was added to the reaction vial containing the dried F-, and the resulting mixture
was heated at elevated temperature for a selected time. TFA (200 μL) was added to the reaction vial,
which was heated at 110 ˚C for 20 min for hydrolysis. NaOH (20%, 0.5 mL) was added to the reaction
vial to quench the reaction. The yield was analyzed via LC/MS-MS quantification. The MS sample was
prepared by diluting 10 μL of the neutralized solution with 80 μL of acetonitrile and 10 μL of verapamil
(internal standard, 100 ng/mL).
3.7.Radiosynthesis of [18F]MDL100907 ([18F]7)
A sample of 0.54 GBq [18F]/fluoride (14.5 mCi) in water (50-100 µL) was added to a reaction vessel
containing K2CO3 (0.060 mg) and K222 (0.28 mg) in water (0.2 mL) and acetonitrile (1.0 mL). The
solution was azeotropically dried under vacuum and nitrogen flow at 110 ˚C. The azeotropic drying
process was repeated by adding acetonitrile (1 mL × 3). In another vial, the boronic ester precursor, 6
(5 mg) was mixed with Cu(OTf)2(py)4 (2.3 mg) in DMF (0.3 mL) and vortexed for 5 s. The mixture
was added to the reaction vial containing the dried activity, and the resulting mixture was heated on a
microwave reactor at 110 °C for 20 min. TFA (200 μL) was added to the reaction vial, which was heated
at 110 ˚C for 5 min for hydrolysis. NaOH (20%, 0.75 mL) was added to the reaction vial to adjust the
pH to basic, the product was purified by semi-preparative HPLC. Conditions: Phenomenex Luna C18
(2) column, 250 × 10 mm, 5 μ; mobile phase: 50% acetonitrile and 50% water (with 0.1% triethylamine)
at flow rate 4 mL/min. The labeled product was eluted between 21-22 min. The chemical identity of the
product was checked by analytical HPLC. HPLC conditions: Phenomenex Luna C18 (2) column, 100
× 4.6 mm, 5 ; mobile phase: 50% acetonitrile and 50% H2O (with 0.1% triethylamine) at flow rate 1
mL/min. The labeled product was eluted between 6-7 min. The total synthesis time was approximately
70 min.
This article is protected by copyright. All rights reserved.

4. Conclusions
18
A reliable 19F-approach to simulate F-radiosynthesis under similar molar activity level has been
developed. Using this strategy, the radiosynthesis of [18F]MDL100907 has been improved with 19-22%
RCY. With high-throughput ability and decreased radiation exposure, the tracer-level 19F-approach will
find great potential in 18F-tracer development process. Besides fluorine-18, this technique can be applied
on all imaging radionuclide, which will accelerate future tracer development process.
Acknowledgments
This work was supported by the Intramural Research Program of the NIH. Intramural research funds
for the Imaging Probe Development Center were administered by the National Heart, Lung, and Blood
Institute. The content of this publication does not necessarily reflect the views or policies of the
Department of Health and Human Services, nor does mention of trade names, commercial products, or
organizations imply endorsement by the U.S. Government. We thank Dr. Carolyn Woodroofe for her
assistance in proofreading.
Abbreviations
18F, fluorine-18; 19F, fluorine-19; LC-MS/MS, liquid chromatography/tandem mass spectrometry; PET,
positron emission tomography; Am, molar activity; LOD, limit of detection; LOQ, limit of quantification;
MRM, multiple reaction monitoring; NCA, no-carrier-added; RCY, radiochemical yield; S/N, signal-
to-noise; TBAB, tetrabutylammonium hydrogen carbonate; TIC, total ion chromatogram.
References
[1] Herschman HR. Molecular imaging: looking at problems, seeing solutions. Science 2003;302:605-
608.
[2] Hilderbrand SA and Weissleder R. Near-infrared fluorescence: application to in vivo molecular
imaging. Curr Opin Chem Biol 2010;14:71-79.
[3] Hagooly A, Rossin R, and Welch MJ. Small molecule receptors as imaging targets. Handb Exp
Pharmacol 2008:93-129.
[4] Elsinga PH and Dierckx RA. Small molecule PET-radiopharmaceuticals. Curr Pharm Des
2014;20:2268-2274.
[5] Miller PW, Long NJ, Vilar R, and Gee AD. Synthesis of C-11, F-18, O-15, and N-13 Radiolabels
for Positron Emission Tomography. Angew Chem Int Ed 2008;47:8998-9033.
[6] Bégué J-P and Bonnet-Delpon D. Recent advances (1995–2005) in fluorinated pharmaceuticals
based on natural products. J Fluorine Chem 2006;127:992-1012.
This article is protected by copyright. All rights reserved.

[7] Kirk KL. Fluorine in medicinal chemistry: Recent therapeutic applications of fluorinated small
molecules. J Fluorine Chem 2006;127:1013-1029.
[8] Levy S, Livni E, Elmaleh D, Curatolo W. Direct displacement with anhydrous fluoride of the C-2
trifluoromethanesulphonate
of
methyl
4,6-O-benzylidene-3-O-methyl-2-O-
trifluoromethylsulphonyl-β-D-mannopyranoside. J Chem Soc Chem Commun 1982;0:972-973.
[9] Brown LJ, Ma N, Bouvet DR, Champion S, Gibson AM, Hu Y, et al. Synthesis of the positron-
emitting radiotracer [18F]-2-fluoro-2-deoxy-d-glucose from resin-bound perfluoroalkylsulfonates.
Org Biomol Chem 2009;7:564–575
[10] Bhalla R, Levason W, Luthra SK, McRobbie G, Sanderson G, Reid G. Radiofluorination of a pre-
formed gallium(III) aza-macrocyclic complex: towards next-generation positron emission
tomography (PET) imaging agents. Chem Eur J 2015;21:4688–4694
[11] Huang X, Liu W, Hooker JM, Groves JT. Targeted fluorination with the fluoride ion by manganese-
catalyzed decarboxylation. Angew Chem Int Ed 2015;54:5241 –5245
[12] Björkhem I. Selective ion monitoring in clinical chemistry. CRC Crit Rev Clin Lab Sci 1979;1:53-
105.
[13] Axen U, Gréen K, HörlinD, Samuelsson B. Mass spectrometric determination of picomole amounts
of prostaglandins E2 and F2α using synthetic deuterium labeled carriers. Biochem Biophys Res
Commun 1971;45: 519-525.
[14] Joshi EM, Need A, Schaus J, Chen Z, Benesh D, Mitch C, et al. Efficiency gains in tracer
identification for nuclear imaging: can in vivo LC-MS/MS evaluation of small molecules screen
for successful PET tracers? ACS Chem Neurosci 2014;5:1154-1163.
[15] Di L. Kerns EH. Drug-like Propertiesꢀ: Concepts, Structure Design and Methods from ADME to
Toxicity Optimization, Second Edition. Elsevier Inc; 2016.
[16] Xiao JF, Zhou B, and Ressom HW. Metabolite identification and quantitation in LC-MS/MS-based
metabolomics. Trends Analyt Chem 2012;32:1-14.
[17] Calderón-Santiago M, Priego-Capote F, and Luque de Castro MD. Enhanced Detection and
Identification in Metabolomics by Use of LC–MS/MS Untargeted Analysis in Combination with
Gas-Phase Fractionation. Anal Chem 2014;86:7558-7565
[18] Grebe SK and Singh RJ. LC-MS/MS in the Clinical Laboratory – Where to From Here? Clin
Biochem Rev 2011;32:5-31
[19] Diagne AB, Li S, Perkowski GA, Mrksich M, and Thomson RJ. SAMDI Mass Spectrometry-
Enabled High-Throughput Optimization of a Traceless Petasis Reaction. ACS Comb Sci 2015;17:
658-662
This article is protected by copyright. All rights reserved.

[20] Bellomo A, Celebi-Olcum N, Bu X, Rivera N, Ruck RT, Welch CJ, et al. Rapid Catalyst
Identification for the Synthesis of the Pyrimidinone Core of HIV Integrase Inhibitors. Angew
Chem Int Ed 2012;51: 6912–6915.
[21] Mukherjee J, Yang ZY, Das MK, and Brown T. Fluorinated benzamide neuroleptics--III.
Development
of
(S)-N-[(1-allyl-2-pyrrolidinyl)methyl]-5-(3-[18F]fluoropropyl)-2,
3-
dimethoxybenzamide as an improved dopamine D-2 receptor tracer. Nucl Med Biol 1995;22:283-
296.
[22] Elsinga PH, Hatano K, and Ishiwata K. PET tracers for imaging of the dopaminergic system. Curr
Med Chem 2006;13:2139-2153.
[23] Christian BT, Vandehey NT, Fox AS, Murali D, Oakes TR, Converse AK, et al. The distribution
of D2/D3 receptor binding in the adolescent rhesus monkey using small animal PET imaging.
Neuroimage 2009;44:1334-1344.
[24] Moon BS, Park JH, Lee HJ, Kim JS, Kil HS, Lee BS, et al. Highly efficient production of
[18F]fallypride using small amounts of base concentration. Appl Radiat Isot 2010;68:2279-2284.
[25] Mathis CA, Mahmood K, Huang Y, Simpson NR, Gerdes JM, and Price JC. Synthesis and
preliminary in vivo evaluation of [C-11]MDL 100907: A potent and selective radioligand for the
5-HT2A receptor system. Med Chem Res 1996;6:1-10.
[26] Ren H, Wey HY, Strebl M, Neelamegam R, Ritter T, and Hooker JM. Synthesis and Imaging
Validation of [F-18]MDL100907 Enabled by Ni-Mediated Fluorination. ACS Chem Neurosci
2014;5:611-615.
[27] Mühlhausen U, Ermert J, Herth MM, and Coenen HH. Synthesis, radiofluorination and first
evaluation of (±)-[18F]MDL100907 as serotonin 5-HT2A receptor antagonist for PET. J Labelled
Compd Radiopharm 2009;52:6-12.
[28] Tredwell M, Preshlock SM, Taylor NJ, Gruber S, Huiban M, Passchier J, et al. A general copper-
mediated nucleophilic 18F fluorination of arenes. Angew Chem Int Ed 2014;53:7751-7755.
[29] Zlatopolskiy BD, Zischler J, Krapf P, Zarrad F, Urusova EA, Kordys E, et al. Copper-Mediated
Aromatic Radiofluorination Revisited: Efficient Production of PET Tracers on a Preparative Scale.
Chem Eur J 2015;21:5972-5979.
[30] Mossine AV, Brooks AF, Makaravage KJ, Miller JM, Ichiishi N, Sanford MS, et al. Synthesis of
[18F]Arenes via the Copper-Mediated [18F]Fluorination of Boronic Acids. Org Lett 2015;17:5780-
5783.
This article is protected by copyright. All rights reserved.

[31] Preshlock S, Calderwood S, Verhoog S, Tredwell M, Huiban M, Hienzsch A, et al. Enhanced
copper-mediated 18F-fluorination of aryl boronic esters provides eight radiotracers for PET
applications. Chem Commun 2016;52:8361-8364.
[32] Copper is an elemental impurity with a residual concentration limit of 30 ppm. Guideline for
Elemental Impurities Q3D. International Conference on Harmonization of Technical
Requirements for Registration of Pharmaceuticals for Human Use (2014); United States
Pharmacopeia, National Formulary 38 (2015).
This article is protected by copyright. All rights reserved.

a
b
c
d
Figure 1. Comparison between UV and MS/MS detection. a) Calibration curve for UV detection,
LOQ = 188 ng/mL; b) UV chromatogram of a representative 19F-synthesis (solid) vs. fallypride
standard at 1000 ng/mL (dot); c) Calibration curve for MS/MS detection, LOQ = 0.98 ng/mL; d)
MS/MS chromatogram of a representative 19F-synthesis: total ion chromatogram (TIC, dot), UV 254
nm (solid) and fallypride signal in MRM mode (×100 amplified, dash)
This article is protected by copyright. All rights reserved.

300
250
200
150
100
50
0
Unwashed
Washed
Figure 2. Fallypride concentration in unwashed and washed reaction vials with no KF added. When
the glass vial was washed with K2CO3 and rinsing with water, fallypride concentration was
significantly reduced by up to 70-80%.
This article is protected by copyright. All rights reserved.

100
80
60
40
20
0
0.0375
0.075
0.15
0.5
1
Amount of KF (µg)
Figure 3. The correlation between amount of KF and yield. Similar yield was found with different
amount of KF in each reaction (0.0375 – 1 µg). Reaction condition: K2CO3 (3 mg), K222 (7-8 mg),
ACN, 100 °C, 30 min.
This article is protected by copyright. All rights reserved.

Scheme 1. Synthesis of the boronic ester 6, the precursor for MDL100907
This article is protected by copyright. All rights reserved.

Scheme 2. Radiosynthesis of [18F]MDL100907 ([18F]7).
This article is protected by copyright. All rights reserved.

Table 1. Preparation of fallypride using tracer-level [19F]KF and the comparison with 18F-radiosynthesis
18F RCY
(%) a
T (°C) Time (min)
Yield (%)
n/a
Entry
Base
Solvent
ACN
n/a
1b
2
3
4
5
6
7
K2CO3 (0.8 mg)
100
30
K2CO3 (3.0 mg)
K2CO3 (0.8 mg)
ACN
ACN
100
100
30
30
41
63
60
74
68 ± 1.6c
TBAB (4.0 mg)
K2CO3 (0.8 mg)
KHCO3 (0.8 mg)
KHCO3 (1.6 mg)
ACN
ACN
ACN
ACN
100
100
100
100
10
10
10
10
55
64 ± 4d
55
77
82
79
63
a RCY was determined by HPLC analysis of the crude product.
b No KF was used to test background reaction. Glass reaction vial was washed with K2CO3. Fallypride
concentration of 85 ± 6 ng/mL was detected.
c RCY obtained from Ref. 17
d Reaction was run in multiple times (n = 4) and found good reproducibility.
This article is protected by copyright. All rights reserved.

Table 2. Optimization of MDL100907 synthesis using tracer-level [19F]KF a
K2CO3
(mg)
K222
Solvent
(mL)
T
Precursor
(mg)
Entry
Catalystb
% conv.
(mg)
(˚C)
1
2
5
5
2.8
14
Cu(OTf)2(py)4
Cu(OTf)2
DMF 0.3
DMF 0.3
DMF 0.3
DMF 0.3
DMF 0.3
DMF 0.3
DMF 0.3
DMF 0.3
DMF 0.3
DMF 0.2
DMF 0.3
DMF 0.2
DMF 0.3
110
110
110
110
110
110
110
120
100
110
110
110
110
n/a
n/a
2.8
14
R = H
3
5
0.060
0.060
2.8
0.28
0.28
14
Cu(OTf)2
n/a
4
5
Cu(OTf)2(py)4
Cu(OTf)2(py)4
Cu(OTf)2(py)4
Cu(OTf)2(py)4
Cu(OTf)2(py)4
Cu(OTf)2(py)4
Cu(OTf)2(py)4
n/a
5
5
n/a
6
5
0.060
0.060
0.060
0.060
0.060
0.060
0.060
0.060
0.060
0.28
0.28
0.28
0.28
0.28
0.28
0.28
0.28
0.28
18.1
17.0
16.9
15.5
14.8
11.3
8.3
7
10
5
8
9
5
R = Bocc
10
11
12
13
14
5
d
5
Cu(OTf)2(py)4
Cu(OTf)2(py)4
Cu(OTf)2
2.5
5
n/a
5
Cu(OTf)2(py)4
DMF:ACN 1:1 110
n/a
a.
In all experiments, 0.15 µg of KF was used as the fluorine source.
Catalyst : precursor = 1:3 (molar ratio)
b.
c.
d.
Deprotection of Boc group was carried out with TFA.
Catalyst : precursor = 1: 2
This article is protected by copyright. All rights reserved.

Table 3. Mass Spec conditions for Fallypride, MDL100907 and Verapamil
Fallypride
364.46
MDL100907
373.47
Verapamil
454.61
MW
365.5 → 225.0
88
374.2 → 123.1
36
455.3 → 165.1
20
MRM transition (quan)
Collision Energy (quan)
MRM transition (qual)
Collision Energy (qual)
Fragmentor
365.2 → 124.1
24
374.2 → 103.1
60
455.3 → 150.0
44
154
134
162
This article is protected by copyright. All rights reserved.

Optimization of 18F-Syntheses using 19F-Reagents at Tracer-level
Concentrations and LC-MS/MS Analysis: Improved Synthesis of
[18F]MDL100907
Xiang Zhang, Ryan Dunlow, Burchelle N. Blackman, Rolf E. Swenson
Graphical Table of Content
A strategy to simulate radioactive 18F-syntheses with non-radioactive 19F-reagents was developed.
Reaction optimization was performed with tracer-level (nanomolar) 19F-reagents and analyzed by high-
throughput screening with LC-MS/MS. The best conditions were then transferred to 18F-radiosynthesis.
This approach provides a high-throughput and cost-effective process to optimize radiosyntheses with
reduced radiation exposure.
This article is protected by copyright. All rights reserved.