Synthesis and Preclinical Evaluation of Novel 68Ga-Labeled (R)-Pyrrolidin-2-yl-boronic Acid-Based PET Tracers for Fibroblast Activation Protein-Targeted Cancer Imaging

Fibroblast activation protein (FAP) is a membrane-tethered serine protease overexpressed in the reactive stromal fibroblasts of >90% human carcinomas, which makes it a promising target for developing radiopharmaceuticals for the imaging and therapy of carcinomas. Here, we synthesized two novel (R)-pyrrolidin-2-yl-boronic acid-based FAP-targeted ligands: SB02055 (DOTA-conjugated (R)-(1-((6-(3-(piperazin-1-yl)propoxy)quinoline-4-carbonyl)glycyl)pyrrolidin-2-yl)boronic acid) and SB04028 (DOTA-conjugated ((R)-1-((6-(3-(piperazin-1-yl)propoxy)quinoline-4-carbonyl)-D-alanyl)pyrrolidin-2-yl)boronic acid). natGa- and 68Ga-complexes of both ligands were evaluated in preclinical studies and compared to previously reported natGa/68Ga-complexed PNT6555. Enzymatic assays showed that FAP binding affinities (IC50) of natGa-SB02055, natGa-SB04028 and natGa-PNT6555 were 0.41 ± 0.06, 13.9 ± 1.29 and 78.1 ± 4.59 nM, respectively. PET imaging and biodistribution studies in HEK293T:hFAP tumor-bearing mice showed that while [68Ga]Ga-SB02055 presented with a nominal tumor uptake (1.08 ± 0.37 %ID/g), [68Ga]Ga-SB04028 demonstrated clear tumor visualization with ~1.5-fold higher tumor uptake (10.1 ± 0.42 %ID/g) compared to [68Ga]Ga-PNT6555 (6.38 ± 0.45 %ID/g). High accumulation in the bladder indicated renal excretion of all three tracers. [68Ga]Ga-SB04028 displayed a low background level uptake in most normal organs, and comparable to [68Ga]Ga-PNT6555. However, since its tumor uptake was considerably higher than [68Ga]Ga-PNT6555, the corresponding tumor-to-organ uptake ratios for [68Ga]Ga-SB04028 were also significantly greater than [68Ga]Ga-PNT6555. Our data demonstrate that (R)-(((quinoline-4-carbonyl)-d-alanyl)pyrrolidin-2-yl)boronic acid is a promising pharmacophore for the design of FAP-targeted radiopharmaceuticals for cancer imaging and radioligand therapy.


Introduction
Cancer-associated fibroblasts (CAFs) found in the tumor microenvironment are functionally and phenotypically distinct from normal fibroblasts found in non-cancerous tissues [1]. Immuno-histochemical studies performed by Chesa et al. revealed that CAFs in several different primary and metastatic carcinomas, including colorectal, breast, ovarian, bladder, and lung carcinomas, overexpress a transmembrane glycoprotein called fibroblast activation protein (FAP) [2]. FAP is a cell-surface serine hydrolase shown to have dipeptidyl exopeptidase and endopeptidase activities [3]. FAP has a restricted normal tissue distribution including normal fibroblasts, but is overexpressed in the reactive stromal fibroblasts of >90% of human epithelial carcinomas (breast, lung, colorectal etc.) [2][3][4][5].  1) targeting FAP was reported to exhibit 7.05 ± 1.13 %ID/g tumor uptake and 11.18 ± 1.54 %ID/g kidney uptake in an induced Hep-G2 tumor model in mice at 30 min post injection (pi) [23]. The tumor and kidney uptake values reportedly dropped by ~27% (5.18 ± 0.82 %ID/g) and ~33% (7.46 ± 1.02 %ID/g), respectively, at 2 h pi with minimal uptake in most normal organs. In its clinical study, [ 99m Tc]Tc-iFAP was seen to accumulate in the primary tumors and lymph node metastases of patients with different cancers [24]. The mean radiation equivalent dose for the kidney (5.2 ± 0.8 mSv) was stated to be more than twice the effective dose (2.3 ± 0.4 mSv) after administrating 740 MBq of the tracer. The authors concluded the study by declaring the need for additional clinical studies to further validate the performance of [ 99m Tc]Tc-iFAP.
The development of targeted radiotracers by Haberkorn group is one of the most significant and clinically relevant applications of FAP inhibitors to date. By expanding on the framework previously outlined by Jansen et al., they generated a panel of specific and high-affinity FAP inhibitors (FAPIs), undergoing almost complete internalization of the ligand-enzyme complex [25][26][27]. These derivatives were synthesized using different chemical modifications to attach various chelators via a linker to the quinolinoyl moiety in N-(4-quinolinoyl)-Gly- (2-cyanopyrrolidine). Of these, 68 Ga-labeled FAPI-02 [28], FA-PI-04 ( Figure 1) and FAPI-46 [29], 99m Tc-labeled FAPI-34 [30] and 18 F-labeled FAPI-42 [31] and FAPI-74 [32] are the most advanced and validated in clinical studies for cancer imaging.
Most recently, [ 68 Ga]Ga/[ 177 Lu]Lu/[ 225 Ac]Ac-PNT6555 were presented as highly potent FAP-targeted theranostic pairs in two different conference abstracts [33,34]. PNT6555 ( Figure 1) is a DOTA-conjugated FAP-targeted ligand based on a (R)-(1-((4-(aminomethyl) benzoyl)-D-alanyl)pyrrolidin-2-yl)boronic acid scaffold. There are varying preclinical and clinical reports on the association between the uptake intensity of radiolabeled FAPIs and FAP expression. For example, while [ 68 Ga]Ga-FAPI-04 is observed to have a high uptake in the bone and muscle in mice, the former is not observed in humans [26,[36][37][38][39][40]. However, the non-tumor-specific uptake of [ 68 Ga]Ga-FAPI-04 and [ 68 Ga]Ga-FAPI-46 was recently reported in muscles and degenerative lesions mostly associated with the joints and vertebral bones [39]. Although having additional clinical studies would help ascertain these ambiguous associations, efforts towards the development of FAP-targeted inhibitors with improved pharmacokinetic properties and enhanced FAP specificity compared to the already existing FAPIs is warranted.
Compared to nitrile, the boronic acid warhead has been reported to have a higher FAP binding affinity [14,19], possibly due to its stronger electrophilic character, and it could have a faster pharmacokinetics in vivo due to its highly hydrophilic nature. Furthermore, previous findings confirm that FAP can tolerate a P2 D-alanine apart from a P2 glycine [20,21]. Lastly, a P3 quinolinoyl moiety has been previously optimized for developing radioligands for FAP targeting by Haberkorn et al. [25,27]. With this aim in mind, we designed two novel boronic acid-based DOTA-conjugated FAP-targeted ligands; [ 68 Ga]Ga-SB02055 and [ 68 Ga]Ga-SB04028 (Figure 1), bearing a N-(4-quinolinoyl)-Gly-boroPro and N-(4-quinolinoyl)-D-Ala-boroPro pharmacophores, respectively. The addition of chelator DOTA via a piperazine-based linker (Figure 1) allowed the formation of stable complexes with the PET isotope 68 Ga. We posited that these novel boronic acid-based tracers may exhibit a higher tumor uptake by virtue of their more potent FAP inhibition and demonstrate superior pharmacokinetics by undergoing quick clearance from all the non-target organs/tissues and serve as PET imaging agents with excellent imaging contrast. Both tracers were subjected to a thorough preclinical evaluation including in vitro FAP binding assays, in vivo stability testing and, PET/CT imaging and ex vivo biodistribution studies using HEK293T:hFAP tumor xenograft mouse model. The results were then compared with those obtained from [ 68 Ga]Ga-PNT6555, which is currently been evaluated in the clinic [35].

Synthesis of 68 Ga-and nat Ga-Complexed DOTA-Conjugated FAP-Targeted Ligands
Detailed information on the synthesis, purification and characterizations of all intermediates, precursors and nat Ga/ 68 Ga-complexed ligands is provided in the Supplementary Materials. The identities of intermediates, precursors and nonradioactive Ga-complexed standards were confirmed by MS and/or NMR analyses (Figures S1-S11). For the synthesis of SB02055 (Scheme 1), compound 1 [41] was coupled overnight with compound 2 [20,42] to obtain compound 3 in a 35% yield. Compound 3 was Boc-deprotected by stirring it overnight with diethyl ether (Et 2 O):4M HCl/dioxane (v/v) in a 1:1 ratio. The mixture was then evaporated before reacting it with DOTA-NHS to afford compound 4 as a pinanediol ester of precursor SB02055 in 11% yield over two steps. Compound 4 was treated with a cleavage cocktail consisting of 95% trifluoroacetic acid (TFA)/2.5% H 2 O/2.5% triisopropylsilane (TIS) for 4 h at room temperature (RT) to finally afford the desired precursor SB02055 in 55% yield. For the synthesis of SB04028 (Scheme 2), compound 5 prepared following the literature procedures [23] was coupled with compound 1 overnight to afford compound 6 in a 47% yield. Compound 6 was then treated overnight with Et₂O:4M HCl/dioxane (v/v) in a 1:1 ratio for removing the Boc-group. The resulting mixture was evaporated before reacting it with DOTA-NHS to obtain compound 7 as a pinanediol ester of SB04028 in 14% yield over two steps. Finally, compound 7 was treated with a cleavage cocktail consisting of 95% TFA/2.5% H2O/2.5% TIS for 4 h at RT to yield the desired precursor SB04028 in a 63% yield. For the synthesis of SB04028 (Scheme 2), compound 5 prepared following the literature procedures [23] was coupled with compound 1 overnight to afford compound 6 in a 47% yield. Compound 6 was then treated overnight with Et 2 O:4M HCl/dioxane (v/v) in a 1:1 ratio for removing the Boc-group. The resulting mixture was evaporated before reacting it with DOTA-NHS to obtain compound 7 as a pinanediol ester of SB04028 in 14% yield over two steps. Finally, compound 7 was treated with a cleavage cocktail consisting of For the synthesis of SB04028 (Scheme 2), compound 5 prepared following the literature procedures [23] was coupled with compound 1 overnight to afford compound 6 in a 47% yield. Compound 6 was then treated overnight with Et₂O:4M HCl/dioxane (v/v) in a 1:1 ratio for removing the Boc-group. The resulting mixture was evaporated before reacting it with DOTA-NHS to obtain compound 7 as a pinanediol ester of SB04028 in 14% yield over two steps. Finally, compound 7 was treated with a cleavage cocktail consisting of 95% TFA/2.5% H2O/2.5% TIS for 4 h at RT to yield the desired precursor SB04028 in a 63% yield.

Ex Vivo Biodistribution and PET/CT Imaging Studies
The PET/CT image acquired at 1 h pi with [ 68 Ga]Ga-SB04028 showed a clear visualization of the HEK293T:hFAP tumor xenograft [44], with the tumor uptake being comparatively higher than [ 68 Ga]Ga-PNT6555 ( Figure 3). Moreover, [ 68 Ga]Ga-SB04028 displayed a faster clearance from most normal organs/tissues, with a relatively lower kidney uptake and slightly superior tumor/background contrast ratios compared to [ 68 Ga]Ga-PNT6555. On the other hand, while the uptake of [ 68 Ga]Ga-SB02055 in most normal organs/tissues was minimal, the tumor uptake of [ 68 Ga]Ga-SB02055 was also

Ex Vivo Biodistribution and PET/CT Imaging Studies
The PET/CT image acquired at 1 h pi with [ 68 Ga]Ga-SB04028 showed a clear visualization of the HEK293T:hFAP tumor xenograft [44], with the tumor uptake being comparatively higher than [ 68 Ga]Ga-PNT6555 ( Figure 3). Moreover, [ 68 Ga]Ga-SB04028 displayed a faster clearance from most normal organs/tissues, with a relatively lower kidney uptake and slightly superior tumor/background contrast ratios compared to [ 68 Ga]Ga-PNT6555. On the other hand, while the uptake of [ 68 Ga]Ga-SB02055 in most normal organs/tissues was minimal, the tumor uptake of [ 68 Ga]Ga-SB02055 was also negligible on the PET/CT image ( Figure 3). A high accumulation of all the three radiotracers in the urinary bladder indicated excretion via the renal pathway. The co-injection of [ 68 Ga]Ga-SB04028 with FAPI-04 (0.5 mg/mouse) reduced the tumor uptake to the background level ( Figure 3).

Ex Vivo Biodistribution and PET/CT Imaging Studies
The PET/CT image acquired at 1 h pi with [ 68 Ga]Ga-SB04028 showed a clear visualization of the HEK293T:hFAP tumor xenograft [44], with the tumor uptake being comparatively higher than [ 68 Ga]Ga-PNT6555 ( Figure 3). Moreover, [ 68 Ga]Ga-SB04028 displayed a faster clearance from most normal organs/tissues, with a relatively lower kidney uptake and slightly superior tumor/background contrast ratios compared to [ 68 Ga]Ga-PNT6555. On the other hand, while the uptake of [ 68 Ga]Ga-SB02055 in most normal organs/tissues was minimal, the tumor uptake of [ 68 Ga]Ga-SB02055 was also negligible on the PET/CT image (Figure 3). A high accumulation of all the three radiotracers in the urinary bladder indicated excretion via the renal pathway. The co-injection of [ 68 Ga]Ga-SB04028 with FAPI-04 (0.5 mg/mouse) reduced the tumor uptake to the background level ( Figure 3).  Data from biodistribution studies ( Figure 4 and Table S1) for all three tracers conducted at 1 h pi in HEK293T:hFAP tumor-bearing mice were found to be concordant with their respective PET/CT images. The tumor uptake values were found to be 1.08 ± 0.37, 10.1 ± 0.42 and 6.38 ± 0.45 %ID/g for [ 68 Ga]Ga-SB02055, [ 68 Ga]Ga-SB04028 and [ 68 Ga]Ga-PNT6555, respectively. All the tracers were excreted rapidly via the renal pathway with a low but comparable kidney uptake (1.74 ± 0.95 %ID/g for [ 68 Ga]Ga-SB02055, 2.10 ± 0.33 %ID/g for [ 68 Ga]Ga-SB04028 and 2.29 ± 0.43 %ID/g for [ 68 Ga]Ga-PNT6555).

In Vivo Stability Studies
The in vivo stability studies revealed [ 68 Ga]Ga-SB04028 to be considerably stable in the blood plasma of male NRG mice (n = 3) at 15 min pi compared to [ 68 Ga]Ga-SB02055. While no intact [ 68 Ga]Ga-SB02055 was present in the mouse plasma at 15 min pi, 46.6 ± 0.21% of radioactivity from [ 68 Ga]Ga-SB04028 was found to be present as an intact tracer. The fractions of tracers remaining intact in the urine at 15 min pi for [ 68 Ga]Ga-SB02055 and [ 68 Ga]Ga-SB04028 were 93.5 ± 0.91% and 53.6 ± 7.11%, respectively ( Figures S1-S4).
Surprisingly, findings from the in vitro enzymatic assay for nat Ga-SB02055 were not in concordance with the data from the PET/CT imaging and biodistribution analysis of its radioactive counterpart obtained from HEK293T:hFAP tumor-bearing mice at 1 h pi.
We initially suspected the lack of in vivo stability of [ 68 Ga]Ga-SB02055 to be a reason for its nominal tumor uptake. Upon subjecting it to in vivo stability testing at 15 min pi, we found no intact [ 68 Ga]Ga-SB02055 in the mouse plasma. Interestingly, the urine samples collected at the same time presented with >93% intact [ 68 Ga]Ga-SB02055 (Figures S1 and  S2). The low tumor uptake at 1 h pi could possibly be a consequence of [ 68 Ga]Ga-SB02055 undergoing quick clearance from the blood and rapid renal excretion into the urine in ≤15 min of injecting the tracer in vivo. Furthermore, an unidentified highly hydrophilic 68 Ga-labeled species accounting for >99% plasma fraction was observed at 15 min pi during the stability testing. The higher than background level uptake in the blood could supposedly be attributed to this uncharacterized 68 Ga-labeled species. Further investigation of [ 68 Ga]Ga-SB02055 in this regard is currently underway.
The imaging and biodistribution analysis of mice co-injected [ 68 Ga]Ga-SB04028 with FAPI-04 (0.5 mg/mouse) revealed a~97% reduction in the tumor uptake (10.1 ± 0.42 %ID/g vs. 0.30 ± 0.04 %ID/g) substantiating in vivo FAP specificity of our lead candidate [ 68 Ga]Ga-SB04028 (Figures 3 and 4, Table S1). Moreover, as anticipated since there was no significant reduction observed in the kidney uptake of [ 68 Ga]Ga-SB04028 (2.10 ± 0.33 %ID/g vs. 2.53 ± 0.57 %ID/g), we can conclusively state that retention in the kidneys is by virtue of the tracer's propensity to undergo renal elimination and is not FAP mediated.
Unlike its P2 glycine analogue [ 68 Ga]Ga-SB02055, [ 68 Ga]Ga-SB04028 with a P2 Dalanine residue was found to be relatively more stable during in vivo stability testing. Strikingly, >47% of [ 68 Ga]Ga-SB04028 remained intact in the mouse plasma at 15 min pi ( Figures S3 and S4). The remaining fractions corresponded to currently uncharacterized but highly hydrophilic 68 Ga-labeled species. The urine samples from the mouse presented with >54% intact tracer, with the remaining portions co-eluting more or less with the uncharacterized 68 Ga-labeled species found in the plasma samples for [ 68 Ga]Ga-SB04028.
The exact nature and identity of the 68 Ga-labeled species eluting prior to each of the intact tracers during in vivo stability testing has not been characterized. However, it has been shown that boronic acids under basic (physiological) conditions could be converted to other species. These include: (1) the degradation products formed under physiological pH conditions due to the inherent susceptibility of boronic acids to undergo oxidation [46,47]; the empty p orbital of boron in boronic acid is prone to attack by nucleophilic oxygen of reactive oxygen species (ROS), resulting in the formation of labile boric esters and/or oxidative de-boronation products; notably, it is the same attribute that allows boronic acid-based ligands to react with the nucleophilic hydroxyl of the catalytic serine residue of FAP and form reversible covalent adducts; (2) the anionic boronic acid/hydroxyboronate anion formed under more basic conditions as a result of hydroxyl (OH − ) attack on the highly electrophilic boron atom [48]; naturally, these ionic species will have a tendency to elute before the neutral boronic acid forms; and (3) boronate esters formed under aqueous conditions at a physiological pH of~7.4 [48]. Efforts are underway to characterize these species.
The goal of the present work was to develop novel boroPro-based PET imaging agents with superior pharmacokinetics properties and excellent imaging contrast for easy and reliable diagnosis of FAP-overexpressing carcinomas. Although our lead candidate [ 68 Ga]Ga-SB04028 demonstrated superior tumor/background contrast ratios compared to [ 68 Ga]Ga-PNT6555, there is still some scope for further optimization. (1) A 4,4-difluoro substituent at the (2S)-2-cyanopyrrolidine ring was previously reported to enhance FAP inhibitory activity compared to that of unsubstituted analogue [12][13][14]. It would be interesting to generate a difluorinated derivative of [ 68 Ga]Ga-SB04028 for preclinical investigation, although its synthesis might be a bit challenging. (2) In terms of diagnostic radiopharmaceuticals, 99m Tc is the most widely used with SPECT imaging accounting for >70% of all the imaging procedures performed in the field of nuclear medicine. Replacing DOTA with a suitable chelator to allow radiolabeling with 99m Tc is definitely worth exploiting.

Cell Culture
HEK293T cells were obtained from American Type Culture Collection (Manassas, VA, USA). The IMPACT Rodent Pathogen Test (IDEXX BioAnalytics, Columbia, MO, USA) confirmed that the cells were pathogen free. Detailed information on the generation of HEK293T:hFAP cells has been previously reported by our group [44]. HEK293T:hFAP cells were cultured in DMEM GlutaMAX™ medium supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 µg/mL) at 37 • C in a Panasonic Healthcare (Tokyo, Japan) MCO-19AIC humidified incubator containing 5% CO 2 . Cells were grown until 80-90% confluence, washed with sterile PBS (pH 7.4) and collected.

In Vitro Fluorescence-Based Binding Assay
The half maximal inhibitory concentration (IC 50 ) values of the FAP-targeted ligands were measured using in vitro enzymatic assay following previously reported procedures [41,44]. Briefly, rhFAP (Biolegend, San Diego, CA, USA; 0.2 µg/mL, 50 µL) was added into a Costar clear bottom 96-well plate. Varied concentrations (25 pM to 1 µM) of nat Ga-complexed ligands were added to each well (in duplicate) containing the rhFAP. PBS was added to the control well (in duplicate). After incubating for 30 min at 37 • C, the fluorescence substrate Suc-Gly-Pro-AMC (Bachem, Bubendorf, Switzerland; 20 mM, 50 µL) was added to each well. The velocities of AMC release were measured kinetically at λ ex = 380 nm, λ em = 460 nm at 60 min at 37 • C using a FlexStation 3 Multi-Mode Microplate Reader.

Ex Vivo Biodistribution and PET/CT Imaging Studies
Imaging and biodistribution studies were performed using immunodeficient male NRG (NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ) mice following previously reported procedures [41,44]. All experiments were conducted according to the guidelines established by the Canadian Council on Animal Care and approved by Animal Ethics Committee of the University of British Columbia. The mice were subcutaneously inoculated with 7.5 × 10 6 HEK293T:hFAP cells in the left dorsal flank. When the tumors grew to 6-8 mm in diameter, the mice were used for PET/CT imaging and biodistribution studies.
The PET/CT imaging experiments were carried out using a Siemens Inveon micro PET/CT scanner (Knoxville, TN, USA). Tumor-bearing mice were injected with~4 to 6 MBq of 68 Ga-labeled tracer through a lateral caudal tail vein under 2.5% isoflurane in oxygen anesthesia, followed by recovery and free roaming in their cage during the uptake period. A 10 min localization CT scan was acquired using 3 overlapping positions to cover the entire mouse. The CT scan was used for the attenuation and scatter correction, and anatomical localization. A list mode acquisition was then performed for 15 min at 1 h pi with the mouse under isoflurane sedation. The images were reconstructed using 3D OSEM/MAP iterative methods.
For the biodistribution studies, the mice were injected with~1-2 MBq of the 68 Galabeled tracer, using the exact procedures as described above. At 1 h pi, the mice were euthanized by CO 2 inhalation. Blood was withdrawn by a cardiac puncture, and the organs/tissues of interest were collected, weighed and counted using a Perkin Elmer For blocking studies, HEK293T:hFAP tumor-bearing mice were co-injected [ 68 Ga]Ga-SB04028 with an excess of FAPI-04 (0.5 mg/mouse) as a competitor. Imaging and biodistribution studies were performed at 1 h pi similar to the unblocked mice.

In Vivo Stability Studies
In vivo stability studies were performed using healthy (non-tumor-bearing) male NRG mice following previously reported procedures [43,49]. Briefly, [ 68 Ga]Ga-SB02055 (6.32 ± 0.16 MBq) and [ 68 Ga]Ga-SB04028 (9.90 ± 0.26 MBq) were injected via the lateral caudal vein into the mice (n = 3). At 15 min pi, the mice were sedated and euthanized, and urine and blood were collected. The blood was processed to obtain plasma by adding 0.5 mL of acetonitrile to 0.5-0.8 mL of whole blood, then vortexing and centrifuging the resulting mixture on a benchtop mini-centrifuge (Fisherbrand™ Mini-Centrifuge 100-240 V, 50/60 Hz Universal Plug) for 20 min. The supernatant was collected and filtered to give the blood plasma. The plasma and urine samples were analyzed via radio-HPLC by using the same conditions as those used for quality control of these 68 Ga-labeled radioligands.

Statistical Analysis
Data were reported as mean ± standard deviation (SD) and analyzed with GraphPad Prism, version 9.5.1. Two-way ANOVA and multiple t-tests were performed for all the tumor and organ/tissue uptake values in the biodistribution studies of [ 68 Ga]Ga-SB02055, [ 68 Ga]Ga-SB04028, and [ 68 Ga]Ga-PNT6555 in the HEK293T:hFAP tumor model. Statistical significance was defined at p < 0.05 using the Holm-Sidak method.

Conclusions
We successfully synthesized and evaluated two novel 68 Ga-labeled (R)-pyrrolidin-2yl-boronic acid-derived PET tracers for FAP-targeted cancer imaging. While the P2 glycinecontaining [ 68 Ga]Ga-SB02055 displayed a minimal tumor uptake, its P2 D-alanine congener [ 68 Ga]Ga-SB04028 demonstrated a significantly higher tumor uptake and superior imaging contrast compared to [ 68 Ga]Ga-PNT6555. (R)-(((quinoline-4-carbonyl)-D-alanyl)pyrrolidin-2-yl)boronic acid is a highly promising pharmacophore that can be used to develop PET imaging agents with superior pharmacokinetics properties and excellent imaging contrasts for visualization of FAP-overexpressing carcinomas. The replacement of DOTA with suitable chelators/prosthetic groups to allow radiolabeling with two of the most widely used imaging isotopes, 99m Tc and 18 F, is warranted in the future.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ph16060798/s1. Detailed synthetic procedures and results for the preparation of FAP-targeted ligands and their nat Ga/ 68 Ga-complexed analogues; Table S1: Biodistribution and tumor/organ uptake ratios of [ 68 Ga]Ga-SB02055, [ 68 Ga]Ga-SB04028 and [ 68 Ga]Ga-PNT6555 in HEK239T:hFAP tumor-bearing mice; Figure S1: A representative MS spectrum of compound 3; Figure S2: A representative NMR spectrum of compound 3; Figure S3: A representative MS spectrum of compound 4; Figure S4: A representative MS spectrum of SB02055; Figure S5: A representative MS spectrum of nat Ga-SB02055; Figure S6: A representative MS spectrum of compound 6; Figure S7: A representative NMR spectrum of compound 6; Figure S8: A representative MS spectrum of compound 7; Figure S9: A representative MS spectrum of SB04028; Figure S10: A representative MS spectrum of nat Ga-SB04028; Figure S11: A representative MS spectrum of nat Ga-PNT6555; Figure  S12: Representative radio-HPLC chromatograms from an in vivo stability study performed to determine the percent of the intact fraction of [ 68 Ga]Ga-SB02055 in mouse plasma and urine samples collected at 15 min post-injection; Figure S13