Automated Production of ¹¹C-labeled Carboxylic Acids and Esters via “In-Loop” ¹¹C-Carbonylation using GE FX Synthesis Modules

Abstract

An in-loop ¹¹C‐carbonylation process for the radiosynthesis of ¹¹C-carboxylic acids and esters from halide precursors has been developed. The reaction proceeds at room temperature under mild conditions and enables ¹¹C‐carbonylation of both electron deficient and electron rich (hetero)aromatic halides to provide ¹¹C-carboxylic acids and esters in good to excellent radiochemical yields, high radiochemical purity and excellent molar activity. The process has been fully automated using commercial radiochemistry synthesis modules, and application to clinical production is demonstrated via validated cGMP radiosyntheses of [¹¹C]bexarotene and [¹¹C]acetoacetic acid.

1. INTRODUCTION

Positron emission tomography (PET) imaging is a non-invasive modality for imaging biological processes associated with various diseases and disorders, such as cancer, heart disease, and dementia.^1,2 PET utilizes chemical entities functionalized with radioisotopes (radiopharmaceuticals) that emit γ-radiation via ß-decay. PET scanners detect and record precise radiation signatures originating from the radiopharmaceutical to produce a PET image, which depicts the accumulation and distribution of the imaging agent in vivo.³ Carbon-11 is one such positron-emitting radionuclide with a half-life of 20.4 min that is frequently incorporated into bioactive organic molecules for PET studies.⁴ It is typically produced by the proton bombardment of the N₂ gas according to¹⁴N (p,α)¹¹C nuclear reaction. This reaction may be conducted using a cyclotron in the presence of O₂ (ca. 0.5–1%) to produce [¹¹C]CO₂, a routinely used carbon-11 synthon that can be used directly,^{5, 6} or derivatized to produce various carbon-11 labeled synthons.^{7 , 8} For example, [¹¹C]CO can be obtained via partial reduction of [¹¹C]CO₂ over a Mo, Zn, or activated charcoal surface,^9,10 and has found excellent utility in medical imaging. For example, in 1968, [¹¹C]CO was one of the first tracers used for human blood volume measurements.¹¹ In addition to such direct use, [¹¹C]CO is also a precursor to a wide array of carbonyl-containing compounds through transition metal-mediated ¹¹C-carbonylation.¹² The carbonyl group is among the most widespread functional groups in many drugs and bioactive molecules, making [¹¹C]CO an attractive synthon for preparing ¹¹C-labeled radiopharmaceuticals.¹³ Despite the wide versatility and importance of [¹¹C]CO,¹⁴ its broad application has been hampered by numerous challenges. [¹¹C]CO has been employed less frequently when compared to other carbon-11-labeled reagents (e.g. [¹¹C]MeI), because of limited availability and the need for specialized synthesis equipment. Moreover, the low reactivity and limited solubility in organic solvents [¹¹C]CO present synthetic challenges. Historical labelling procedures have therefore usually required complicated set-ups with high-pressure reactors,¹⁵ and relatively high reaction temperatures.¹⁶

Today however, with its increasing availability from modern cyclotrons, and the increasing number of radiochemistry groups developing automated radiosyntheses employing [¹¹C]CO for clinical use using standard automated radiochemistry modules,^13,17–19 the development of new ¹¹C‐carbonylation protocols that can be readily transferred between radiochemistry facilities for cGMP production of patient doses is finally becoming possible. For example, we recently published an “in-loop” method for ¹¹C-carbonylation to synthesize labeled amides at ambient pressure and temperature to address these challenges.¹⁷ This fully automated ¹¹C-carbonyl labeling process has been adapted for use with commercially available GE TracerLab FX synthetic modules, including FX_C, FX_M and FX_FN systems. The simple modification of the synthesis module enables cGMP manufacture of radiopharmaceuticals for clinical applications. Encouraged by these successes and to widen the scope of our newly developed ¹¹C‐carbonylation methodology,¹⁷ herein we report a two-step approach that involves initial ¹¹C‐carbonylation of a (hetero)aryl halide precursor and subsequent trapping with a nucleophile (HO⁻ or RO⁻) to extend the potential radiopharmaceuticals that can be accessed from [¹¹C]CO to also include carboxylic acids and esters (Scheme 1). The process has also been automated using GE TracerLab synthesis modules and applied to bioactive molecules of interest from a PET imaging perspective.

Scheme 1.

Two-step palladium mediated [¹¹C]-carbonylation for synthesizing acids and esters.

2. MATERIALS AND METHODS

See Supporting Information and Supplemental Figures 1 – 77 for full experimental details.

Production of [¹¹C]CO

[¹¹C]CO₂ was produced with a GE PETtrace cyclotron. The ¹⁴N(p,α)¹¹C nuclear reaction was performed by proton bombardment of a pressurized gas target containing high-purity nitrogen and 0.5% oxygen to generate [¹¹C]CO₂, which was delivered to a GE Process Cabinet (ProCab) via stainless steel lines. [¹¹C]CO₂ was trapped on a molecular sieve column at room temperature. The accumulated [¹¹C]CO₂ was then released into an online reduction column by heating the molecular sieve trap to 360°C. The [¹¹C]CO₂ was passed through a heated Mo column at 850°C, in a stream of helium at 50 mL/min. The gas was purified through an ascarite column and delivered from the ProCab to a sealed hot cell. The produced [¹¹C]CO was trapped on a silica trap immersed in liquid N₂, and then released and pushed by helium at 5 mL/min into the HPLC loop for reaction.

Two-step “In-loop” [¹¹C]-Carbonylation Set-Up

Modifications of GE TracerLab FX synthesis module are shown in in the Supporting Information (Supplemental Figures 4 and 5). The intermediate complex was generated from the aryl halide, N-Xantphos, and Pd(dba)₂ in THF in an external vial at room temperature for 20 minutes. Five minutes before the end of target irradiation, the reaction mixture was loaded onto the TracerLab HPLC loop, the coupling partner (nucleophile) was added to the reaction vial (either NaOH or NaOMe/NaOEt), and the hot-cell was sealed. [¹¹C]CO was delivered from the ProCab and trapped by a silica trap immersed in liquid nitrogen. After trapping, the silica trap was removed from the liquid nitrogen and the [¹¹C]CO was released and pushed by He at 5 mL/min into the HPLC loop containing the reaction mixture (Pd/Xantphos/ArX) for the first step. The loop was sealed for 5 min for the reaction to occur at room temperature. After completing the reaction, the loop was rinsed backwards using THF to transfer the reaction mixture into the reactor containing either NaOH or NaOMe/NaOEt, where the 2nd step was carried out at room temperature for 1 min. Following reaction, the crude reaction mixtures were either analyzed to determine RCC, or purified by semi-preparative HPLC and/or SPE to determine RCY, as described in the Supporting Information.

3. RESULTS AND DISCUSSION

Introductory Studies

Based on our previous studies,¹⁷ we began our investigation using iodobenzene as a model substrate for one-step “in-loop” ¹¹C‐carbonylation (Scheme 2). The intermediate complex was generated from iodobenzene 1-I, N-Xantphos, and Pd(dba)₂ in THF in an external vial at room temperature for 20 minutes. [¹¹C]CO₂ was prepared via the ¹⁴N(p,α)¹¹C and, five minutes before the end of target irradiation, the coupling partner (nucleophile) was added to the reaction vial (either NaOH or NaOMe/NaOEt). The resulting mixture was loaded onto an HPLC loop and the hot-cell was sealed. [¹¹C]CO was prepared using a molybdenum catalyst at 850 °C as previously described,¹⁷ and trapped using a SiO₂ trap (100 mg silica gel in a 1/8 inch tube) immersed in liquid nitrogen. The [¹¹C]CO was released and pushed by helium at 5 ml/min into the HPLC loop for reaction. The loop was sealed for 5 min at room temperature and the reaction mixture was then analyzed to determine radiochemical conversion (RCC). This one-step approach yielded 1-¹¹CO₂H (Nu = OH) and 1-¹¹CO₂Me (Nu = OMe) in 7% and 69% RCC, respectively (Table 1).

Scheme 2.

One-step and two-step [¹¹C]-carbonylation methods for synthesizing acids and esters.

Table 1.

Comparison of RCCs for one-step versus two-step carbonylation method

Entry	Nucleophile	One-Step RCC	Two-step RCC
1.	NaOH	7%	88 ± 6.4%
2.	NaOMe	69 ± 0.5%	74 ± 1.9%

Given that Xantphos ligated aryl palladium complexes are stable,²⁰ we also evaluated a two-step approach wherein the ¹¹C-CO complex was prepared in-loop first, and then transferred into the synthesis module reactor pre-charged with the nucleophile (Scheme 2). The second reaction was carried out at room temperature for 1 min. Under this modified protocol, increased radiochemical conversions were observed (Table 1), providing 1-¹¹CO₂H and 1-¹¹CO₂Me in 88% and 74% RCC, respectively.

Synthesis of [¹¹C]carboxylic acid derivatives:

Given higher RCCs were obtained with the two-step process, we began by examining the substrate scope for producing [¹¹C]carboxylic acid derivatives using this transformation (Scheme 3). We tested para-substituted aryl iodides, including those substituted with electron-withdrawing groups (p-NO₂ 2-I, p-CF₃ 3-I), which produced 2-¹¹CO₂H and 3-¹¹CO₂H in 90% and 89% RCC, respectively. Electron-rich groups were also tolerated. For example, 4-iodophenol (4-I) afforded 4-¹¹CO₂H in 52% RCC. Notably, 4-¹¹CO₂H was obtained while obviating a protecting group strategy, which would otherwise drastically reduce the recovery of 4-¹¹CO₂H partly due to radioactive decay. Furthermore, carbonylation of biaryl ether 5-I gave 5-¹¹CO₂H in 90% RCC.

Scheme 3.

Substrate scope for two-step [¹¹C]-carbonylation.

^aRadiochemical conversion (RCC) determined via radio-HPLC as the average of two separate reactions conducted at least two replicates. ^bUsed 25 μmol and heated at 60°C for 2 min to synthesize the intermediate complex. See supplementary information for additional details.

After successfully testing different carbocyclic iodoarenes, we next investigated ¹¹C-carbonlyation of heteroaromatic halides. For example, 4-iodopyridine analogue 6-I generated [¹¹C]isonicotinic acid 6-¹¹CO₂H in 84% RCC. Similarly, ¹¹C-carbonylation of 2-bromofuran 7-Br resulted in 7-¹¹CO₂H in 71% RCC. The reaction of 3-bromofuran 8-Br led to significant precipitation during complex formation, and resulted in low RCC to 8-¹¹CO₂H (ca. 3–4%). To improve the RCC, we prepared the intermediate complex at 60°C for 2 minutes, leading to a minor increase of RCC to 7% of 8-¹¹CO₂H. We did not try iodofurans, but in general iodo-precursors work better than their bromo-counterparts. Thiophenes were also compatible with this new methodology, and ¹¹C-carbonlyation of 2-iodothiophene (9-I) and 3-iodothiophene (10-I) produced 9-¹¹CO₂H and 10-¹¹CO₂H in 95% and 75% RCC, respectively. After successfully utilizing aryl bromides and iodides, benzylic halides bearing electron-withdrawing and -donating groups were also investigated under the new reaction conditions. We first attempted ¹¹C-carbonylation of 4-nitrobenzyl bromide 11-Br, which gave 11-¹¹CO₂H in 83% RCC. Next, we investigated ¹¹C-carbonylation of 4-methylbenzyl bromide 12-Br and similarly generated 12-¹¹CO₂H in 76% RCC.

We next wished to investigate compatibility of the new ¹¹C-carbonylation method with drug-like molecules of interest to our imaging program, beginning with unprotected indole analogs. Pleasingly, 2-iodoindole 13-I was an excellent substrate for this reaction, generating 13-¹¹CO₂H in 83% RCC. However, 3-iodoindole 14-I did not provide 14-¹¹CO₂H. Investigating this issue further, it became apparent that we failed to prepare any complex from 14-I using N-Xantphos and Pd(dba)₂ under the reaction conditions. The reasons for this poor reactivity are currently unknown, but are under further investigation. Expanding the scope further, radiolabeling of amino acids is of enormous interest to our program.²¹ Thus, to determine the applicability of the current method for radiolabeling unprotected amino acids, we evaluated unprotected haloaromatic amino acid 15-I, which gave 15-¹¹CO₂H in an excellent 91% RCC. The synthesis of bexarotene²² (Targretin) was also investigated under the optimized ¹¹C-carbonylation methodology. [¹¹C]Bexarotene (16-¹¹CO₂H) was obtained from 16-I using the current method in high (93%) RCC.

Finally, we tested expanding the substrate scope to include α-halo ketones.^{23, 24} Based on our previous experiences with vinyl iodide,¹³ the starting chloroacetone (17-Cl) was mixed with N-Xantphos and Pd(dba)₂ in THF and kept in the dark, with occasional vortex mixing for at least 20 minutes before use. [¹¹C]Acetoacetic acid (17-¹¹CO₂H) was obtained in 84% RCC using this production method.

Synthesis of [¹¹C]carboxylic ester derivatives:

The promising radiochemical yields of [¹¹C]carboxylic acid analogs as well as 1-¹¹CO₂Me led us to further investigate use of this two-step ¹¹C-carbonylation process for the synthesis of [¹¹C]carboxylic ester derivatives by employing alkoxides as the nucleophile in place of hydroxide (Scheme 4). To demonstrate proof-of-concept, sodium methoxide and sodium ethoxide were tested as nucleophiles in this study, but we expect that many other alkoxides are likely also compatible.

Scheme 4.

Substrate scope for two-step [¹¹C] carbonylation.

^aRadiochemical conversion (RCC) determined via radio-HPLC as the average of two separate reactions conducted at least 2 replicates. See supplementary information for additional details.

The intermediate [¹¹C]CO complex was generated in loop as described above, transferred to the synthesis module reactor, and then treated with either sodium methoxide (0.1 M in MeOH) or sodium ethoxide (0.1 M in EtOH) at room temperature for 1 min. 1-¹¹CO₂Me and 1-¹¹CO₂Et were obtained in 74% of 61% RCC, respectively. Precursors with additional substitution at the arene, such as 1-iodo-4-nitrobenzene 2-I, could also be treated with sodium methoxide (0.1 M in MeOH) or sodium ethoxide (0.1 M in EtOH) under the same conditions to obtain 2-¹¹CO₂Me and 2-¹¹CO₂Et in 57% and 52% RCC, respectively. Rapid decomposition was noted in the generation of 2-¹¹CO₂Et, attributed to likely radiolysis.²⁵ Next, we tested 2-iodothiophene 9-I and 2-iodoindole 13-I substrates, which afforded ethyl esters 9-¹¹CO₂Et and 13-¹¹CO₂Et in 79% and 64%, respectively. The ¹¹C-carboxylic ester derivative of bexarotene (16-¹¹CO₂Me) could be obtained from 16-I in 73% RCC upon treatment of the [¹¹C]CO complex with sodium methoxide.

Synthesis of [¹¹C]bexarotene

After successfully applying this two-step ¹¹C-carbonylation in-loop methodology to the synthesis of both functionalized acid and ester analogs, we next wished to fully automate the process for routine production and selected the synthesis of bexarotene^22,26 as a test case. Bexarotene, a retinoid X receptor RXR agonist, is an FDA-approved drug for treating T-cell lymphoma and has been investigated for treating Alzheimer’s disease.²⁷ [¹¹C]Bexarotene was previously synthesized from boronic esters²⁸ and aryl stannanes²⁹ using a copper-mediated ¹¹C-carboxylation method, which afforded [¹¹C]bexarotene in decay corrected RCYs of 32 ± 5% with A_m 38 ± 23 GBq/μmol (1.0 ± 0.6 Ci/μmol) (stannane) and 45% with A_m 11.4 GBq/μmol (310 mCi/μmol) (BPin ester).^{28, 29}

Using our ¹¹C-carbonylation conditions (Scheme 5), automated protocols were developed step-by-step. Following our standard automated [¹¹C]carbonylation protocol using an TracerLab FX_M synthesis module, the crude reaction mixture was transferred from the loop to the reactor containing 0.1 M NaOH by rinsing the loop with 0.8 mL of THF. The reactor was stirred at room temperature for 1 min to from 16-¹¹CO₂H, and quenched with 0.4 mL of HPLC mobile phase (80% acetonitrile in 0.1% TFA). Analytical HPLC analysis indicated 58.1 ± 2.7% RCC (n = 2) to [¹¹C]bexarotene (16-¹¹CO₂H).

Scheme 5.

[¹¹C]-carbonylation for synthesizing [¹¹C]bexarotene from iodide 16-I.

For semi-preparative HPLC purification, initially we used a two-loop system by adding a dedicated reaction loop to the module so as to keep the original module HPLC loop for purification.³⁰ This setup was complicated and loss of activity because of the extra transfer was observed. Thus, we designed a simplified configuration using only one HPLC loop for both reaction and semi-preparative HPLC purification. As shown in Figure S3 (see Supporting Information), [¹¹C]CO released from the silica trap enters the HPLC loop through V8 for the initial ¹¹C-carbonylation reaction. After the reaction is completed, THF (0.8 mL) from Vial V1 elutes the crude reaction mixture from the loop backwards via V30 through V8/V9 into the synthesis module reactor. By blowing He gas for 2 minutes from V16, the HPLC loop and line from V8 to the reactor (including the fluid detector) were all dried and ready for use in the subsequent semi-preparative HPLC purification. The crude reaction was then purified by semi-preparative HPLC (column: Luna, C8, 10 μ, 100 Å, 150 × 10 mm; mobile phase: 80% MeCN in 0.1%TFA; flow rate = 4 mL/min) and the collected product next needed to be reformulated into an injectable dose. The high lipophilicity of 16-¹¹CO₂H (logD_7.4 is 3.68) caused a challenge during Sep-Pak reformulation. We screened different Sep-Paks, such as Oasis HLB, C18 1cc, C18 plus, tC18 plus, tC18 3cc, tC18 light, tC18 1cc, and tC18 plus short. The retention efficiency of tC18 plus short (86%) and tC18 3cc (79%) was found to be superior in contrast to other Sep-Paks (see Supporting Information for full details). Elution from the Sep-Pak using 0.5 mL of EtOH followed by dilution with 9.5 mL of saline provided 41% recovery using tC18 3cc and 38% using tC18 plus short. Notably, when the quantity of EtOH was increased from 0.5 mL to 1.0 mL, elution efficiency increased from 41% to 75% for tC18 3cc, and 38% to 52% for tC18 plus short, respectively. The tC18 3cc was chosen for further studies given these higher recoveries.

Initial attempts at full automation produced only 614 MBq (16.6 mCi) of 16-¹¹CO₂H. Following the reaction, a significant quantity of 16-¹¹CO₂H was recovered from the dilution flask that the HPLC fraction was collected into for reformulation. This suggested that transfer loss could be reducing the final radiochemical yield, which we tentatively attributed to the high lipophilicity (and therefore reduced solubility) of 16-¹¹CO₂H. To mitigate this, we initially rinsed the apparatus with tween 80, but this resulted in recovery of only 381 MBq (10.3 mCi) of 16-¹¹CO₂H. After careful investigation of dose distribution throughout the Sep-Pak, dilution flask, filter, and waste vial(s), we noticed that concentrated tween 80 prevented 16-¹¹CO₂H from retaining on the Sep-Pak as we recovered a large amount of product from the waste. Next, we rinsed the round bottom flask with dilute tween 80 solution before the run, which gratifyingly resulted in 2331 MBq (63 mCi) of 16-¹¹CO₂H.

Following successful optimization of the automated reaction, HPLC purification and reformulation conditions for [¹¹C]bexarotene, we completed three validation runs to confirm suitability for human use (Table 2). A fully automated synthesis was accomplished, affording 2356 ± 371 MBq (64 ± 10 mCi) of [¹¹C]bexarotene (n = 3), corresponding to 8.9 ± 1.7 % decay-corrected yield (2.9 ± 0.45% non-decay corrected RCY) based upon ~74 GBq (~2 Ci) starting activity of [¹¹C]CO and 35 min synthesis time. The molar activity was 529 ± 323 GBq/μmol (14,289 ± 8735 mCi/μmol) at EOS, and the product was obtained in 97 ± 1 % radiochemical purity. Although the total RCY was not as high as previously reported CO₂ labeling methods,^28,29 in our hands the iodo-precursor was more stable and easier to handle than the corresponding Bpin and tin precursors. More importantly, the ¹¹C-carbonylation method normally produce the product in >>10 fold higher molar activity compared with the same product prepared from [¹¹C]CO₂ (because of the need for isotopic dilution to prevent side products forming when working with [¹¹C]CO₂). All other quality control data confirmed suitability of the product for clinical use.

Table 2.

Quality control data of [¹¹C]bexarotene validation radiosyntheses

QC test	Acceptance Criteria	Batch 1 Result	Batch 2 Result	Batch 3 Result	Pass / Fail
Radiochemical Purity	NLT 90%	95.66%	96.88%	97.86%	Pass
Radioactive Concentration	NLT 40 mCi/10mL @EOS	63 mCi/10mL	54 mCi/10mL	74 mCi/10mL	Pass
Active Ingredient Concentration	Report Results (μg/mL)	0.091 μg/mL	0.16 μg/mL	0.36 μg/mL	Pass
Molar Activity	≥1000mCi/μmol	24,023 mCi/μmol	11,711 mCi/μmol	7133 mCi/μmol	Pass
pH	4.5–7.5	5.0	5.0	5.0	Pass
Visual Inspection	Clear, colorless, no ppt	Clear, colorless, no ppt	Clear, colorless, no ppt	Clear, colorless, no ppt	Pass
Radiochemical Identity (HPLC)	RRT: 0.9–1.1	1.000	1.002	1.001	Pass
Radionuclide Identity	18.4–22.4 min	20.1 min	21.2 min	20.2 min	Pass
Bacterial Endotoxin	≤17.5 EU/mL	<2.00 EU/mL	<2.00 EU/mL	<2.00 EU/mL	Pass
Filter membrane Integrity	≥45psi	49 psi	46 psi	49 psi	Pass
Residual Solvent Analysis*	Acetone ≤ 5000 μg/mL MeCN ≤410 μg/mL THF ≤720 μg/mL Ethanol % reported	Acetone: 2.2 μg/mL MeCN: 112 μg/mL THF: 10.1 μg/mL Ethanol: 8.3%	Acetone: 1.5 μg/mL MeCN: 28.4 μg/mL THF: 6.4 μg/mL Ethanol: 10%	Acetone: 0.72 μg/mL MeCN: 99 μg/mL THF: 7.9 μg/mL Ethanol: 9.4%	Pass
Post-Release QC test	Release Criteria	Batch 1 Result	Batch 2 Result	Batch 3 Result	Pass / Fail
Sterility	Sterile	Sterile	Sterile	Sterile	Pass
Residual Pd (by ICP-MS)	<10 μg/batch	<n.d.†	<n.d.†	<n.d.†	Pass

^*Residual TFA should also be accounted for during QC testing. For this work, the semi-prep HPLC fraction was collected for approx. 2 mins at a flow rate of 4 mL/min, corresponding to 8 mL of HPLC mobile phase containing 0.1% (8 μL) TFA. In the unlikely event of all 8μL TFA ending up in the 10 mL dose after reformulation, this corresponds to 0.08% or 800 ppm. No limits for residual TFA are defined in the ICH guidelines, and so appropriate limits should be chosen that are consistent with local regulations (e.g. ≤1% has been used in certain instances³⁶).

^†LOD = 0.11 μg/mL

Synthesis of [¹¹C]Acetoacetic acid

[¹¹C]Acetoacetic acid (17-¹¹CO₂H) is an imaging agent used for investigating neurotherapeutics of interest to our clinical collaborators³¹ and, as such, we also wished to automate and validate the synthesis of this radiotracer for clinical production (Scheme 6). Using chloroacetone 17-Cl as a precursor was conceived, which presented a clear advantage over using the less stable and more difficult to handle organolithium precursors employed in previously described protocols.^32,33,34, Following the standard automated [¹¹C]carbonylation protocol, the loop mixture was quenched with 0.1M NaOH, forming 17-¹¹CO₂H and attenuating acid-mediated decarboxylation of the labeled product.³⁵ Analysis of the crude reaction mixture via analytical HPLC indicated co-formation of several side-products, so various purification cartridges were investigated to obtain purity levels acceptable for clinical PET imaging (see Supporting Information for details). Purification of the reaction mixture using a 6CC C18 Sep-Pak cartridge followed by a 3CC Strong Anion Exchange (SAX) Sep-Pak cartridge in series was chosen to balance product purity and radiochemical yield. The solid-phase extraction cartridge method without semi-preparative HPLC/reformulation enable straightforward set-up of the synthesis module (see Supporting Information for detail configuration). Briefly, the reaction mixture passed through two Sep-Paks (C18 and SAX), and then the SAX Sep-Pak cartridge was washed with 15 mL water. The product was simply eluted by USP saline for injection. The shorter total synthesis time gave higher yields of 17-¹¹CO₂H, and consistently provided doses in >95% RCP.

Scheme 6.

[¹¹C]-carbonylation for synthesizing [¹¹C]-acetoacetic acid from chloride 17-Cl.

Two QC HPLC methods were used to verify purity and product identity. The first was the previously reported method using a RHM-Monosaccharide column at 40°C. However, given this column is difficult to handle due to high back pressure and the product has a longer retention time (13 mins) which is not ideal given the short half-life of ¹¹C, we developed a new method used Luna SCX column at room temperature. 17-¹¹CO₂H has a retention time of 4.8 min using our new method, which is more appropriate for routine production.

We next undertook 3 process verification syntheses to validate the method for routine production. Both analytical HPLC methods were performed during all development experiments and the three validation runs. The results proved that our HPLC method with a shorter retention time showed good separation and did not miss any hidden impurities. Accordingly, 11.9 ± 0.8 GBq (322 ± 22 mCi) of 17-¹¹CO₂H was afforded, corresponding to 16.1 ± 1.1% non-decay corrected RCY based on 74 GBq (~2 Ci) of ¹¹CO and a 20–25 min synthesis time (n = 3). The molar activity was greater than 293 GBq/mmol (7919 ± 581 mCi/mmol) at EOS, and the product was obtained in 97 ± 2% radiochemical purity. Notably, the mass of 17-¹¹CO₂H was present at levels below detectable limits, and excellent molar activity values that significantly supersede previous methods^{34, 35} were obtained (ca. ~ 10-fold improvement). No degradation of 17-¹¹CO₂H was observed in USP saline for 60 min post-EOS based on analytical HPLC. All other quality control data from the three validation radiosyntheses confirmed suitability for human use (Table 3).

Table 3.

Quality control data of [¹¹C]acetoacetic acid validation radiosyntheses.

QC test	Acceptance Criteria	Batch 1 Result	Batch 2 Result	Batch 3 Result	Pass / Fail
Radiochemical Purity	NLT 90%	95.3%	97.6%	98.7%	Pass
Radioactive Concentration	NLT 40 mCi/10mL @EOS	302 mCi/10mL	318 mCi/10mL	346 mCi/10mL	Pass
Active Ingredient Concentration	Report Results (μg/mL)	<0.5 μg/mL	<0.5 μg/mL	<0.5 μg/mL	Pass
Molar Activity	≥1000mCi/μmol	>7490 mCi/μmol	>7686 mCi/μmol	>8581 mCi/μmol	Pass
pH	4.5–7.5	5	5	5.5	Pass
Visual Inspection	Clear, colorless, no ppt	Clear, colorless, no ppt	Clear, colorless, no ppt	Clear, colorless, no ppt	Pass
Radiochemical Identity (HPLC)	RRT: 0.9–1.1	1.02	1.02	1.02	Pass
Radionuclide Identity	18.4–22.4 min	20.3 min	20.4 min	20.2 min	Pass
Filter membrane Integrity	≥45psi	59 psi	61 psi	61 psi	Pass
Bacterial Endotoxin	≤17.5 EU/mL	<2.00 EU/mL	<2.00 EU/mL	<2.00 EU/mL	Pass
Residual Solvent Analysis	Acetone ≤ 5000 μg/mL THF ≤720 μg/mL	Acetone < 2 μg/mL THF 9.8 μg/mL	Acetone 1249 μg/mL THF 2 μg/mL	Acetone 368 μg/mL THF 52 μg/mL	Pass
Post-Release QC test	Release Criteria	Batch 1 Result	Batch 2 Result	Batch 3 Result	Pass / Fail
Sterility	Sterile	Sterile	Sterile	Sterile	Pass
Residual Pd (by ICP-MS)	<10 μg/batch	<n.d*	<n.d*	<n.d*	Pass

^*LOD = 0.11 μg/mL

4. CONCLUSION

In summary, this work described a two-step ¹¹C-carbonylation labeling method for preparing labeled carboxylic acids and esters at ambient pressure and room temperature. The method is amenable to various pendant electron-donating and -withdrawing functional groups of variable steric and electronic effects. The module modifications are simple and fast without requiring any specialized equipment. The synthesis was executed using commercially available radiosynthesis modules (GE TracerLab FX_M, FX_C, or FX_FN) commonly used for cGMP production of radiopharmaceuticals. The utility of the newly developed methodology is demonstrated by successfully qualified two radiotracers, [¹¹C]bexarotene and [¹¹C]acetoacetic acid, for human application. These two radiopharmaceuticals have been manufactured in high yields with great purity, especially with 10 times improved molar activities compared with previously reported methods. The current method has potential to further widen substrate scopes and promote the use of [¹¹C]carbon monoxide as a versatile precursor in ¹¹C-labeling chemistry.

Data Availability Statement

Further details of experimental methods, additional analysis, analytical data, synthesis module timelists, and additional screening and control experiments are included in the Supporting Information along with Supplemental Figures showing synthetic schemes, radiochemistry set-up, and both analytical and semi-preparative HPLC traces.