Synthesis and Evaluation of 99m Tc-Tricabonyl Labeled Isonitrile Conjugates for Prostate-Speciﬁc Membrane Antigen (PSMA) Image

: Prostate-speciﬁc membrane antigen (PSMA) is a biomarker expressed on the surface of prostate cancer (PCa). In an e ﬀ ort to improve the detection and treatment of PCa, small urea-based PSMA inhibitors have been studied extensively. In the present study, we aimed to develop 99m Tc-tricabonyl labeled urea-based PSMA conjugates containing isonitrile (CN-R)-coordinating ligands ([ 99m Tc]Tc- 15 and [ 99m Tc]Tc- 16 ). Both the PSMA conjugates were obtained at high radiochemical e ﬃ ciency ( ≥ 98.5%). High in vitro binding a ﬃ nity was observed for [ 99m Tc]Tc- 15 and [ 99m Tc]Tc- 16 ( K d = 5.5 and 0.2 nM, respectively) in PSMA-expressing 22Rv1 cells. Tumor xenografts were conducted using 22Rv1 cells and rapid accumulation of [ 99m Tc]Tc- 16 (1.87 ± 0.11% ID / g) was observed at 1 h post-injection, which subsequently increased to (2.83 ± 0.26% ID / g) at 4 h post-injection. However, [ 99m Tc]Tc- 15 showed moderate tumor uptake (1.48 ± 0.18% ID / g), which decreased at 4 h post-injection (0.81 ± 0.09% ID / g). [ 99m Tc]Tc- 16 was excreted from non-targeted tissues with high tumor-to-blood (17:1) and tumor-to-muscle ratio (41:1) at 4 h post-injection at approximately 4 times higher levels than [ 99m Tc]Tc- 15 . Uptakes of [ 99m Tc]Tc- 15 and [ 99m Tc]Tc- 16 to PSMA-expressing tumor and tissues were signiﬁcantly blocked by co-injection of 2-(Phosphonomethyl)-pentandioic acid (2-PMPA), suggesting that their uptakes are mediated by PSMA speciﬁcally. Whole-body single photon emission computed tomography imaging of [ 99m Tc]Tc- 16 veriﬁed the ex vivo biodistribution results and demonstrated clear visualization of tumors and tissues expressing PSMA compared to [ 99m Tc]Tc- 15 . In conclusion, using [ 99m Tc]Tc- 16 rather than [ 99m Tc]Tc- 15 may be the preferable because of its relatively high tumor uptake and retention.


Introduction
Prostate cancer (PCa) is a commonly diagnosed disease and is the second leading cause of cancer death in the United States. In 2018, approximately 164,690 men were diagnosed with PCa, leading to an estimated 29,430 deaths [1]. Prostate-specific membrane antigen (PSMA) is glutamate carboxypeptidase

Chemistry
Asymmetrical urea 1 was synthesized by treating l-glutamic acid di-tert-butyl ester hydrochloride with triphosgene in the presence of triethylamine (TEA) at −78 • C to produce an intermediate isocyanate, which subsequently was treated with N -Cbz-l-lysine tert-butyl ester to produce 1 after purification by silica gel chromatography (Scheme 1). The carbobenzoxy (Cbz) protecting group was removed by catalytic hydrogenation to produce 2 in quantitative yield, and subsequently reacted with Fmoc-6-Ahx-OH in the presence of a coupling agent (HBTU) to produce 3. Selective removal of the Fmoc group (20% piperidine/N,N-dimethylformamide (DMF)) produce 4, and the tert-butyl protecting group was then removed to yield 5, which was used for the synthesis of the PSMA conjugate 15.
Here, we tried to develop an asymmetrical urea-based PSMA using the isonitrile containing vectors 15 and 16 with two different spacers. Both of these were labeled with 99m Tc in a trivalent fashion using [ 99m Tc][Tc(CO)3(H2O)3] + . 99m Tc-labeled PSMA conjugates [ 99m Tc]Tc-15 and [ 99m Tc]Tc-16 were then tested in vitro for their specific PSMA binding as well as in vivo uptake in a prostate cancer 22Rv1 xenograft model as potential PSMA imaging agents.

Chemistry
Asymmetrical urea 1 was synthesized by treating L-glutamic acid di-tert-butyl ester hydrochloride with triphosgene in the presence of triethylamine (TEA) at −78 °C to produce an intermediate isocyanate, which subsequently was treated with N′-Cbz-L-lysine tert-butyl ester to produce 1 after purification by silica gel chromatography (Scheme 1). The carbobenzoxy (Cbz) protecting group was removed by catalytic hydrogenation to produce 2 in quantitative yield, and subsequently reacted with Fmoc-6-Ahx-OH in the presence of a coupling agent (HBTU) to produce 3. Selective removal of the Fmoc group (20% piperidine/N,N-dimethylformamide (DMF)) produce 4, and the tert-butyl protecting group was then removed to yield 5, which was used for the synthesis of the PSMA conjugate 15. For the synthesis of the PSMA conjugate 16, 4 was conjugated with Fmoc-Phe-OH using HBTU to generate compound 6 (Scheme 2). Selective deprotection of Fmoc group from 6 using 20% piperidine/DMF produced 7. An additional conjugation of the Fmoc-Phe-OH group to 7, followed by removing the Fmoc group generated 9. Compound 9 was then treated with 6-Boc-aminohexanoic acid in the presence of a coupling agent (HBTU) to produce 10. Subsequently, Boc and tert-butyl groups were removed using a mixture of trifluoroacetic acid (TFA)/dichloromethane (DCM) (1:1 v/v) to generate compound 11. For the synthesis of the PSMA conjugate 16, 4 was conjugated with Fmoc-Phe-OH using HBTU to generate compound 6 (Scheme 2). Selective deprotection of Fmoc group from 6 using 20% piperidine/DMF produced 7. An additional conjugation of the Fmoc-Phe-OH group to 7, followed by removing the Fmoc group generated 9. Compound 9 was then treated with 6-Boc-aminohexanoic acid in the presence of a coupling agent (HBTU) to produce 10. Subsequently, Boc and tert-butyl groups were removed using a mixture of trifluoroacetic acid (TFA)/dichloromethane (DCM) (1:1 v/v) to generate compound 11. Synthesis of TFP-activated isonitrile and conjugation with 5 and 11 was accomplished in four steps (Scheme 3). First, N-formyl-β-alanine (12) was obtained by refluxing a mixture of β-alanine, formic acid, and acetic anhydride. The carboxylic acid group was activated with TFP to produce 13. The formyl group was converted to isonitrile with a dehydrating agent triphosgene to generate 14.  Synthesis of TFP-activated isonitrile and conjugation with 5 and 11 was accomplished in four steps (Scheme 3). First, N-formyl-β-alanine (12) was obtained by refluxing a mixture of β-alanine, formic acid, and acetic anhydride. The carboxylic acid group was activated with TFP to produce 13. The formyl group was converted to isonitrile with a dehydrating agent triphosgene to generate 14. Synthesis of TFP-activated isonitrile and conjugation with 5 and 11 was accomplished in four steps (Scheme 3). First, N-formyl-β-alanine (12) was obtained by refluxing a mixture of β-alanine, formic acid, and acetic anhydride. The carboxylic acid group was activated with TFP to produce 13. The formyl group was converted to isonitrile with a dehydrating agent triphosgene to generate 14.

In Vitro Serum Stability and Distribution Coefficient (LogD)
In vitro stability of [ 99m Tc]Tc-15 and [ 99m Tc]Tc-16 were tested in human serum at 37 °C, and both showed high stability at least for 6 h ( Figure 2). LogD values were determined by partitioning [ 99m Tc]Tc-15 or [ 99m Tc]Tc-16 between 1-octanol and PBS (pH 7.4) and found to be −3.72 ± 0.05 and −2.10 ± 0.03, respectively, indicating high hydrophilicity of the compounds.

SPECT Imaging
In

Discussion
PSMA is expressed in wide variety of PCa and its expression is correlated with disease stage and plays an important role in early detection of PSMA-positive lesion in patients. It has been reported that the glutamate moiety on urea-based PSMA inhibitors interacts with the S1 binding pocket, which can interact with lipophilic spacers. The S1 binding pocket is a funnel-shaped tunnel with a depth of approximately 20 Å and a width of 8−9 Å [48][49][50]. The synergistic effect of a glutamate motif and a lipophilic spacer determines the internalization potency of PSMA inhibitor. Therefore, many efforts have been made to improve the affinity and pharmacokinetic properties by introducing various spacers into the PSMA targeting molecule [51][52][53].
Considering the importance of hydrophobic motifs in a targeting molecule, we synthesized an asymmetrical glutamate-urea-lysine-based PSMA inhibitor containing an aromatic ring (Phe-Phe) in the linker (10) and compared it to a PSMA inhibitor containing only a straight alkyl chain (5). Isonitrile residues were incorporated to PSMA inhibitors 5 and 10 to obtain 15 and 16, respectively. 15 and 16 were radiolabeled with 99m Tc using [ 99m Tc][Tc(OH2)3(CO)3] + in high radiochemical yield (≥98.5%). The labeled products were subsequently purified by radio-HPLC to a high radiochemical purity (≥99.5%) to remove remaining ligands and other 99m Tc species (Figure 1)

Discussion
PSMA is expressed in wide variety of PCa and its expression is correlated with disease stage and plays an important role in early detection of PSMA-positive lesion in patients. It has been reported that the glutamate moiety on urea-based PSMA inhibitors interacts with the S1 binding pocket, which can interact with lipophilic spacers. The S1 binding pocket is a funnel-shaped tunnel with a depth of approximately 20 Å and a width of 8−9 Å [48][49][50]. The synergistic effect of a glutamate motif and a lipophilic spacer determines the internalization potency of PSMA inhibitor. Therefore, many efforts have been made to improve the affinity and pharmacokinetic properties by introducing various spacers into the PSMA targeting molecule [51][52][53].
Considering the importance of hydrophobic motifs in a targeting molecule, we synthesized an asymmetrical glutamate-urea-lysine-based PSMA inhibitor containing an aromatic ring (Phe-Phe) in the linker (10) and compared it to a PSMA inhibitor containing only a straight alkyl chain (5). Isonitrile residues were incorporated to PSMA inhibitors 5 and 10 to obtain 15 and 16, respectively. 15 and 16 were radiolabeled with 99m Tc using [ 99m Tc][Tc(OH 2 ) 3 (CO) 3 ] + in high radiochemical yield (≥98.5%). The labeled products were subsequently purified by radio-HPLC to a high radiochemical purity (≥99.5%) to remove remaining ligands and other 99m Tc species (Figure 1) 6 h with no sign of decomposition owing to well-known fact that isonitrile complexes are highly stable and inert under physiological conditions. Hydrophilicity is usually reported in LogD, which is an important predictor to determine the pharmacokinetics of radiopharmaceutical and is usually measured by partitioning the radiotracer between n-octanol and PBS buffer (pH 7.4) under strict equilibrium conditions. The low LogD value of [ 99m Tc]Tc-15 compared to [ 99m Tc]Tc-16 suggests that it is more hydrophilic. However, the presence of two phenyl amino acid groups in the spacer did not show much influence on the LogD value compared to other reports, likely because the phenyl amino groups are hindered inside the trivalent complex [4,54].
The dissociation constants (K d ) of [ 99m Tc]Tc-15 and [ 99m Tc]Tc-16 were evaluated using the 22Rv1 cell line, which has moderate PSMA expression [55,56]. [ 99m Tc]Tc-16 showed higher binding affinity (0.2 nM) than [ 99m Tc]Tc-15 (5.5 nM). Both of them were higher than the tripeptide-based monodentate 99m Tc-labeled ligand (13.8 nM) [34] and comparable to MIP-1404 (1.07 nM) a highly potent PSMA targeting probe [57]. One reason for the high binding affinity may be because of the decreased dissociation rate from the cell surface due to the multimerization effect, which has been reported previously [17,58].
Tissue indicating that the uptakes in these organs were mainly mediated by binding to PSMA. Similar results have been reported for other PSMA-targeting agents [59][60][61][62][63]. Another reason for high kidney uptake is the hydrophilic nature of [ 99m Tc]Tc-15 and [ 99m Tc]Tc-16,which makes the kidney the primary excretion pathway. With regard to clinical translation, however, the expression of PSMA in the kidneys of nude mice is higher than expression levels in human kidneys [3,64].
The feasibility of [ 99m Tc]Tc-15 and [ 99m Tc]Tc-16 as PSMA imaging agents was tested by SPECT/CT using 22Rv1 tumor bearing BALB/c mice (Figures 3 and 4). [ 99m Tc]Tc-16 clearly allows for the visualization of tumor tissues after 1 h post-injection and showed prolonged retention of activity until 4 h (Figure 4). [ 99m Tc]Tc-16 was cleared from non-targeted tissues and showed improved images with high tumor-to-background contrast at 4 h post-injection ( Figure 4). [ 99m Tc]Tc-15 was more rapidly excreted from the blood and showed poor tumor-to-background contrast (Figure 3). Blocking experiments by co-injection of 2-PMPA demonstrated that the tumor and kidney uptakes were specifically mediated by PSMA (Figures 3 and 4).

General
All commercially available chemicals were of analytical grade and were used without further purification.

Cell Culture and Tumor Model
22Rv1 cells were cultured as monolayers in RPMI-1640 medium at 37 • C in a humidified atmosphere containing 5% CO 2 . RPMI was supplemented with 10% fetal bovine serum, 4.5 g/L d-glucose, 2 mM l-glutamine, 1 mM sodium pyruvate, and 1.5 g/L sodium bicarbonate. 22Rv1 tumor xenografts were established with male BALB/c nude mice (3-4 weeks old). Briefly, approximately, 5 × 10 6 cultured 22Rv1 cells were suspended in RPMI-640 media and subcutaneously implanted (100 µL) into the upper right flank of mice. Ex vivo biodistribution and imaging studies were performed once the tumor reached 100-400 mm 3 in volume (3-4 weeks). All animal experiments (#3520150085, approval date 9 November 2015) were performed in compliance with the Seoul National University Hospital, Seoul, Korea, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC International, 2007).

In Vitro Stability Tests in Serum
In vitro stability of the 99m Tc labeled conjugates were tested in human serum. In brief, [ 99m Tc]Tc-15 or 16 (25.9 MBq, 200 µL) was incubated with human serum (500 µL) in an incubator at 37 • C with gentle shaking. After 1, 3, and 6 h, an aliquot of the solution (50 µL) was added to methanol (100 µL). The resulting supernatant was centrifuged and radiochemical purity was determined by radio-TLC and radio-HPLC.

Determination of Distribution Coefficient (LogD Value)
The distribution coefficients (LogD values) of the 99m Tc labeled conjugates were determined by measuring the activity that partitioned between 1-octanol and PBS (50 mM, pH 7.4) under equilibrium conditions. Briefly, [ 99m Tc]Tc-15 or [ 99m Tc]Tc-16 purified by radio-HPLC and solvent was removed by rotary evaporator. The residue obtained was dissolved in 3 mL PBS (50 mM, pH 7.4) to a concentration of 2.5 MBq/mL in triplicate. Anhydrous 1-octanol was added (3 mL) and the mixture was vortexed for 5 min and centrifuged at 3300 r/min for 5 min to separate the layers. The counts in the organic and inorganic layers (100 µL) were determined using an γ-counter (Cobra II automated γ-counter). The LogD was calculated using the following equation: LogD = Log(cpm in octanol − cpm in background)/(cpm in buffer − cpm in background).

Measurement of Binding Affinity (K d ) In Vitro
To evaluate the binding affinity of [ 99m Tc]Tc-15 and [ 99m Tc]Tc-16, K d was investigated using a saturation binding assay. 22Rv1 cells (1 × 10 5 cells/well, 1 mL) were plated into a 24-well flat-bottom plate and allowed to form an adherent monolayer. The medium in each well was then replaced with HBSS supplemented with 1% bovine serum albumin, containing increasing concentrations of [ 99m Tc]Tc-15 or [ 99m Tc]Tc-16 in serial dilutions. The cells were incubated for 1 h at 37 • C with shaking. After 1 h, the media was aspirated and the cells were washed with HBSS (3 mL × 2) to remove unbound activity. Cells were lysed by adding sodium dodecyl sulfate (0.5% in PBS, 500 µL) to each well and mixed to dissolve the cells, and the lysates were transferred to plastic tubes (4 mL). The radioactivity of each sample was counted using a γ-counter (Cobra II automated γ-counter), along with reference samples which contained the total amount of added radioactivity. Nonspecific binding was determined in the presence of 2-PMPA (250 µM). Specific binding was calculated by subtracting the nonspecific bound radioactivity from that of the total binding. The K d was calculated by non-linear regression using GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, USA) using a one site binding equation.

Ex Vivo Biodistribution Study
The ex vivo biodistribution of [ 99m Tc]Tc-15 or [ 99m Tc]Tc-16 was evaluated in 22Rv1 tumor-bearing male nude mice (22-25 g). [ 99m Tc]Tc-15 or [ 99m Tc]Tc-16 (74 kBq, 100 µL) was administered via a lateral tail vein in each mice. The mice were sacrificed at 1 and 4 h post-injection. The relevant tissues and organs were excised and collected. The collected tissues and organs were washed with saline, dried, weighed, and counted using an automatic γ-counter. In order to confirm the specific uptake, mice were co-injected with 2-PMPA (100 µg). Uptake in each tissues and organ was expressed as the percentage of the injected dose per gram (% ID/g). A standard solution was additionally prepared to estimate the total dose injected per mice. Values are expressed as the mean ± standard deviation (SD).

SPECT/CT Imaging
The SPECT/CT images were taken at 1 and 3 h for [ 99m Tc]Tc-15 (7.4MBq/200 µL) and for [ 99m Tc]Tc-16 (7-6 MBq/200 µL) images were acquired at 1 and 4 h after the administration of 99m Tc-labeled conjugates to 22Rv1 tumor-bearing BALB/c male nude mice through the tail vein. For blockade experiment, mice were co-injected with potent inhibitor 2-PMPA (100 µg) to confirm uptake in the tumor was PSMA mediated. For SPECT/CT imaging, mice were anesthetized with isoflurane and scanned with a nanoScan SPECT/CT device. The scanning acquisition parameters for imaging modality are 140 keV ± 10% γ-ray energy window, 256 × 256 matrix size, 5 Section per angular step of 18 • of time acquisition, and a reconstruction algorithm of ordered subset expectation maximization with nine iterations. For CT, a tube voltage of 45 kVp, an exposure time of 1.5 Section per projection, and a reconstruction algorithm of cone-beam filtered back-projection was used. Images were processed using Invivoscope processing software. A Gauss reconstruction filter was used to the SPECT images and scale is adjusted to allow visualization of organs and tissues of interest.
Statistical Analysis. Statistical analyses were performed by Student's t-test. The difference was considered statistically significant when the p values were ≤ 0.05.

Conclusions
We successfully synthesized [ 99m Tc]Tc-15 and [ 99m Tc]Tc-16 for imaging PSMA and evaluated their binding affinities in vitro and targeting capabilities in vivo. The in vitro study results demonstrated that [ 99m Tc]Tc-16 has a higher binding affinity compared to [ 99m Tc]Tc-15, possibly because of its interaction with the S1 hydrophobic pocket and multimeric effects. Ex vivo biodistribution study for both 99m Tc-labeled conjugates demonstrated significantly improved tumor uptake and retention of [ 99m Tc]Tc-16 compared to [ 99m Tc]Tc-15 up to 4 h post-injection. The tumor uptakes were blocked by co-injection of 2-PMPA, suggesting that the uptake is PSMA mediated. Finally, whole-body SPECT/CT image demonstrated the feasibility of [ 99m Tc]Tc-16 as an efficient imaging agent for PSMA-expressing tumors with higher tumor-to-background ratio compared to [ 99m Tc]Tc-15.