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Synthesis of ¹⁸F-radiolabeled LLP2A for use in PET imaging via aryltrifluoroborate formation Walker, Daniel 2012

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18F PET Tracer +e -e+e positron range 511 keVO OH 18F HO HO OHHN O HN O N H N H O NH O NH OHO OH N N N NH O NH2 N H N H O HO O H N O H N OH O N H O OH H N S O HN O NH2 H N O N H NH O H N S O N H O NH NH H2N HOO O2N K[18F]-Kryptofix DMSO 130 °C O 18F F3C F3C H N Peptide H N Peptide O N K[18F]-Kryptofix DMSO 150 °C 8 min O 18F KMnO4 NaOH 150 °C 3 min O 18F OH NHS DCC THF 25 °C 3 min O 18F O N O OO 18F H N H2N 25 °C 15 minOTos K[18F]-Kryptofix CH3CN 100 °C 10 min 18F Peptide O N3 Peptide O N N N 18FCuSO4 AscorbateB O O Biomolecule KH18F2/HCl B F F Biomolecule F RR PGPGR B F F F R B F F F + H2O HF R B F OH H2O HF R B HO OHB F F F N OO B F F F N O O B F F F O B F F F O B F F N OO B F F N O O δ+ δ+ B F Fδ+ O B F F OB HO OH FF F O OH F FF O OH B O O Nu F FF O OH B O O F FF O OH B O O Nu F FF O OH B O O Nu + H-B F FF O OH ✔O H N HO N H H N O OH O O N H O F F F B FF F C-11 dramatically reduces the imaging time window (19), therefore reducing image quality compared with ligands la- beled with other PET isotopes (20). Our labeling technique captures the advantages of a single-step labeling in the case of F-18 to afford ligands with specific activities that are potentially useful for imaging.7 As this approach is conceptually very different from other radiolabeling methods, it is essential to briefly address some of the chemical attributes of an 18F-labeled ArBF3. The first is the question of chemical purity. Although labeling must pro- ceed through mono- and difluoronated intermediates en route to the labeled ArBF3, the mono- and difluoroboranes/ boronates are unstable at pH 7 (21), and as such, the ArBF3 is the only labeled species isolated. The second concern relates to specific activity. Although high specific activity is always preferable, there is no universally accepted value as to what minimal specific activity represents a threshold of utility and often a value of ∼1 Ci/!mol is sufficient for imaging. Tradi- tionally, radiosyntheses are performed under no carrier– added conditions to guarantee the highest possible specific activities. A prevailing misconception in such work is that no carrier–added fluoride has a specific activity close to that of carrier-free, that is, 1,720 Ci/!mol; in practice, however, the specific activity of “no carrier–added” [18F]fluoride gener- ally falls in the range of 3 to 10 Ci/!mol. As such, there is a significant amount of carrier [19F]fluoride present in all no carrier–added syntheses. Furthermore, the decay that ac- companies multistep radiosyntheses of mid-size molecules further reduces the final specific activities, which commonly fall in the range of 1 to 2 Ci/!mol, or less. Because three fluo- ride ions condense with one arylboronate to give an ArBF3, the law of mass action ensures that the resulting ArBF3 has a decay-corrected specific activity that is thrice that of the source fluoride; therefore, activities as high as 30 Ci/!mol may be envisaged if no carrier–added fluoride with a specific Figure 4. In vivo PET imaging of MMPs in murine breast carcinomas. A, IVIS image of 67NR/CMV-Luciferase derived primary tumor (left) is from the same mouse imaged next day by MicroPET with 50 !Ci of marimastat-ArBF3 (middle). Control mouse injected with 440 !Ci control-ArBF3 (right). Pictures were reconstructed from a scan taken 50 to 80 min after tracer injection. B, MicroPET images of 67NR breast tumor mice with 100 !Ci of marimastat-ArBF3 (5) injected either in an unblocked tumor mouse (left) or in a tumor mouse preblocked with 300 nmol of marimastat (right). C, biological replicate of B using tumors established on different dates and imaged on different days. D, time–activity curves of the primary tumor of unblocked and preblocked marimastat-ArBF3 (5) injected 67NR mice, respectively. 7 Li Y, Ting R, Harwig C, et al. Kit-like 18F-labeling of small molecules with specific activities suitable for in vivo PET imaging: toward imaging cancer as- sociated matrix metalloproteases. 2010; submitted for publication. In vivo PET Imaging of Proteases in Cancer Cancer Res; 70(19) October 1, 2010www.aacrjournals.org 7567  American Association for Cancer Research Copyright © 2010  on May 12, 2011cancerres.aacrjournals.orgDownloaded from Published OnlineFirst August 20, 2010; DOI:10.1158/0008-5472.CAN-10-1584X2 X3 X4 X5N H N H O R X1 L    D    VN H H N O N H O O HO H N O HN O N H N H O O N RDiscussion The emergence of molecular imaging has a significant effect on basic research and potentially on clinical care in the future. Development of high-affinity targeting probes is one of the key prerequisites for imaging of specific molecular targets in living systems. With the advances in genomic and proteomics, many potential therapeutic targets have been identified. Discovery of novel imaging agents against these targets enables in vivo molecular target assessment and, therefore, facilitates the under- standing of their roles in disease progression and the development of targeted therapeutics. Targeting ligands must have the ability to reach the targets with sufficient concentration and retention time to be detectable in vivo. An ideal molecular imaging agent should have the following properties: high-affinity and specificity against targeted molecules, metabolically stable, rapid excretion, and very low nonspecific binding. High-affinity cancer targeting ligands can be developed rationally based on the structural information of natural lig- ands and targeted receptors, or they can be developed through screening random and focused combinatorial libraries (35, 36). In the last few years, we have successfully used the one bead– one compound combinatorial library approach to identify several cancer-specific targeting ligands (24, 35–38). We pre- viously reported that a one bead–one compound combinato- rial library-derived a4h1 integrin targeting peptidomimetic (LLP2A), when conjugated to fluorochrome-labeled strepta- vidin in a tetravalent form, could image lymphoma xenograft with high specificity (24). Here, we show that LLP2A, when Figure 2. Specific binding of LLP2A-Cy5.5 to a4h1 integrin. Confocal microscopy images of Molt-4 (A and D), a4-transfected K562 (B and E), and parental K562 (C and F) cells incubated for 1 h at 37jC in the presence of 1 nmol/L LLP2A-Cy5.5 with (D–F) or without (A–C) blocking dose of unlabeled LLP2A (100 Amol/L). Note that only a4h1-expressing Molt-4 (A) and a4-transfected K562 cells (B) efficiently bind the conjugate, and excess LLP2A completely blocked the NIR fluorescence signals (D and E), demonstrating the high specificity of LLP2A-Cy5.5. Figure 3. NIR fluorescence imag- ing of subcutaneous Molt-4 tumor- bearing mice. The LLP2A-Cy5.5 was given at a dose of 2 nmol (A) or 0.5 nmol (B and C) per mouse via tail vein. All NIR fluorescence images were acquired with 30 s exposure time at different time points postin- jection. A, in vivo fluorescence images of subcutaneous Molt-4 tu- mor-bearing mice received 2 nmol LLP2A-Cy5.5 conjugates. Fluores- cence signals from Cy5.5 were pseudocolored. B, in vivo NIR fluo- rescence images of mice at 3 h after injection of 0.5 nmol LLP2A-Cy5.5 without blocking (left ) or with block- ing (right ) by injecting 200 nmol LLP2A 30 min before probe adminis- tration. C, ex vivo images of excised tumors and organs 6 h after injection of 0.5 nmol LLP2A-Cy5.5. Molecular Cancer Therapeutics 435 Mol Cancer Ther 2008;7(2). February 2008  American Association for Cancer Research Copyright © 2008  on May 1, 2012mct.aacrjournals.orgDownloaded from Published OnlineFirst February 1, 2008; DOI:10.1158/1535-7163.MCT-07-0575 Discussion The emergence of molecular imaging has a significant effect on basic research and potentially on clinical care in the future. Development of high-affinity targeting probes is one of the key prerequisites for imaging of specific molecular targets in living systems. With the advances in genomic and proteomics, many potential therapeuti targets have been identified. Discovery of novel imaging agents against these targets enables in vivo molecular arget assessment and, therefore, facilitates the under- standing of their roles in disease progression and the development of targeted therapeutics. Targeting ligands must have the ability to reach the targets with sufficient concentration and retention time to be detectable in vivo. An ideal molecular imaging agent should have the following properties: high-affinity and specificity against targeted molecules, metabolically table, rapid excretion, and very low nonspecific binding. High-affinity cancer targeting ligands can be developed rationally based on the structural information of natural lig- ands and targeted receptors, or they can be developed through screening random and focused combinatorial libraries (35, 36). In the last few years, we have successfully used the one bead– one compound combinatorial library approach to identify several cancer-specific targeting ligands (24, 35–38). We pre- viously reported that a one bead–one compound combinato- rial library-derived a4h1 integrin targeting peptidomimetic (LLP2A), when conjugated to fluorochrome-labeled strepta- v din in a tetravalent form, could image lymphoma xenograft with high specificity (24). Here, we show that LLP2A, when Figure 2. Specific binding of LLP2A-Cy5.5 to a4h1 integrin. Confocal microscopy images of Molt-4 (A and D), a4-transfected K562 (B and E), and parental K562 (C and F) cells incubated for 1 h at 37jC in the presence of 1 nmol/L LLP2A-Cy5.5 with (D–F) or without (A–C) blocking dose of unlabeled LLP2A (100 Amol/L). Note that only a4h1-expressing Molt-4 (A) and a4-transfected K562 cells (B) efficiently bind the conjugate, and excess LLP2A completely blocked the NIR fluorescence signals (D and E), demonstrating the high specificity of LLP2A-Cy5.5. Figure 3. NIR fluorescence imag- ing of subcutaneous Molt-4 tumor- bearing mice. The LLP2A-Cy5.5 was given at a dose of 2 nmol (A) or 0.5 nmol (B and C) per mouse via tail vein. All NIR fluorescence images were acquired with 30 s exposure time at different time points postin- jection. A, in vivo fluorescence images of subcutaneous Molt-4 tu- mor-bearing mice received 2 nmol LLP2A-Cy5.5 conjugates. Fluores- cence signals from Cy5.5 were pseudocolored. B, in vivo NIR fluo- rescence images of mice at 3 h after injection of 0.5 nmol LLP2A-Cy5.5 without blocking (left ) or with block- ing (right ) by injecting 200 nmol LLP2A 30 min before probe adminis- tration. C, ex vivo images of excised tumors and organs 6 h after injection of 0.5 nmol LLP2A-Cy5.5. Molecular Cancer Therapeutics 435 Mol Cancer Ther 2008;7(2). February 2008  American Association for Cancer Research Copyright © 2008  on May 1, 2012mct.aacrjournals.orgDownloaded from Published OnlineFirst February 1, 2008; DOI:10.1158/1535-7163.MCT-07-0575N H H N O N H O O HO H N O HN O N H N H O O N O O H N O FF F B O O HO H N O N H O O HO H N O HN O N H N H O O N NH2 O O H2N O FF F B O O OHN H O N H H N O N H O H N O HN O HN N O O O H N O HO O N3 F FF O N H B N H O N H H N O N H O H N O HN O HN N O O O H N O HO O N NNF F F O NH B 18F F F F FF O N H B O O F18F F N H O N H H N O N H O H N O HN O HN N O O O H2N O HO O N3 OH HO H N O N H O O HO H N O HN O N H N H O O N NH2 O O H2NH N O O NH2 a HN O O N H O H N Fmoc R1 H N O O N H O NH2 R1 H N O O N H O H N R1 O R2 N H Fmoc b cR H N O O H H N CO2 R NH2 +N H O O NH2N C N N C N N C N NN N N O PF6 N N N N N N O PF6 N N N N N O PF6 N N ClN H H N O O O H N NH2 O O H N a O OH N N H O R + N N H N NH2 O R + λmax = 304 nm O ON H H N O O O H N N H H N O N H O O O O O H N a, b O O O ON H H N O N H O O O O O H N a, b N H H N O N H O O O H N O O O H N HN OO O O O OH2N OH O N C O N H N H O O OH + aN H H N O N H O O O H N O O O H N HN OO N H H N O N H O O O H N O HN O N H N H O O O H N OO a, b O ON H H N O N H O O O H N O HN O N H N H O O O H N OO N H H N O N H O O O H N O HN O N H N H O O N O O H N a, bN H H N O N H O O O H N O HN O N H N H O O N O O H N a, b N H H N O N H O O O H N O HN O N H N H O O N O O H2Na, b, c F FF O OH B O O F FF O OHN H H N O N H O O O H N O HN O N H N H O O N O O H2N + a, b N H H N O N H O O HO H N O HN O N H N H O O N O O H N O FF F B O O F FF O OH B O OBr O O N3 OH O a, bN H H N O N H O O O H N O HN O N H N H O O N O O H2N N3 OH O + a, b N H O N H H N O N H O H N O HN O HN N O O O H N O HO O N3N H H N O N H O O HO H N O HN O N H N H O O N O O H N O FF F B O O N H H N O N H O O HO H N O HN O N H N H O O N O O H N O FF F BF F F a N H H N O N H O O HO H N O HN O N H N H O O N O O H N O FF FN H H N O N H O O O H N O HN O N H N H O O N O O H2N + O O HO O OH H N C N H O N H H N O N H O H N O HN O HN N O O O HN O HO S O O HO O OH N C S a, bN H H N O N H O O HO H N O HN O N H N H O O N O O H N O FF F BF18F FF FF O N H B NHHN F FF O N H B F18F F aF FF O N H B F18F F N H O N H H N O N H O H N O HN O HN N O O O H N O HO O N NNF F F O NH B 18F F F N H O N H H N O N H O H N O HN O HN N O O O H N O HO O N3 + aN H H N O O O H N O ON H H N O N H O O O O O H N O ON H H N O N H O O O H N O O O H N HN OO O ON H N H O OH OF FF O OH B O O N3 OH ON H H N O N H O O HO H N O HN O N H N H O O N O O H N O FF F BF F FO O HO O OH H N C N H O N H H N O N H O H N O HN O HN N O O O HN O HO S   

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