A Trifunctional Theranostic Ligand Targeting Fibroblast Activation Protein-α (FAPα).

PURPOSE: Fibroblast activation protein-α (FAPα) is uniquely expressed in activated fibroblasts, including cancer-associated fibroblasts that populate tumor stroma and contribute to proliferation and immunosuppression. Radiolabeled FAPα inhibitors enable imaging of multiple human cancers, but time-dependent clearance from tumors currently limits their utility as FAPα-targeted radiotherapeutics. We sought to increase the area under the curve (AUC) by constructing a trifunctional ligand that binds FAPα with high affinity and also binds albumin and theranostic radiometals. PROCEDURES: RPS-309 comprised a FAPα-targeting moiety, an albumin-binding group, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Inhibition of recombinant human FAPα (rhFAPα) was determined by colorimetric assay. Affinity for human serum albumin (HSA) was determined by high-performance affinity chromatography. The tissue distribution of [68Ga]Ga-RPS-309 in SW872 tumor xenograft-bearing mice was imaged by microPET/CT and quantified by biodistribution studies performed from 30 min to 3 h post injection (p.i.). The biodistribution of [177Lu]Lu-RPS-309 was determined at 4, 24, and 96 h p.i. RESULTS: RPS-309 inhibits rhFAPα with IC50 = 7.3 ± 1.4 nM. [68Ga]Ga-RPS-309 is taken up specifically by FAPα-expressing cells and binds HSA with Kd = 4.6 ± 0.1 µM. Uptake of the radiolabeled ligand in tumors was evident from 30 min p.i. (> 5 %ID/g) and was significantly reduced by co-injection of RPS-309. Specific skeletal uptake was also observed. Activity in tumors was constant through 4 h p.i., but cleared significantly by 24 h. The AUC in this period was 127 (%ID/g) × h. CONCLUSIONS: RPS-309 is a high-affinity FAPα inhibitor with prolonged plasma residence. Introduction of the albumin-binding group did not compromise FAPα binding. Although initial tumor uptake was high and FAPα-specific, RPS-309 also progressively cleared from tumors. Nevertheless, RPS-309 incorporates multiple sites in which structural diversity can be introduced, and therefore serves as a platform for future structure-activity relationship studies.

Preclinical Evaluation of a High-Affinity Sarcophagine-Containing PSMA Ligand for 64Cu/67Cu-Based Theranostics in Prostate Cancer.

The application of small molecules targeting prostate-specific membrane antigen (PSMA) has emerged as a highly promising clinical strategy for visualization and treatment of prostate cancer. Ligands that integrate the ability to both quantify the distribution of radioactivity and treat disease through the use of a matched pair of radionuclides have particular value in clinical and regulatory settings. In this study, we describe the development and preclinical evaluation of RPS-085, a ligand that binds PSMA and serum albumin and exploits the 64/67Cu radionuclide pair for prostate cancer theranostics. RPS-085 was synthesized by conjugation of a PSMA-targeting moiety, an Nε-(2-(4-iodophenyl)acetyl)lysine albumin binding group, and a bifunctionalized MeCOSar chelator. The IC50 of the metal-free RPS-085 was determined in a competitive binding assay. The affinity for human serum albumin of the radiolabeled compound was determined by high-performance affinity chromatography. Radiolabeling was performed in NH4OAc buffer at 25 °C. The stability of the radiolabeled compounds was assessed in vitro and in vivo. The biodistribution of [64/67Cu]Cu-RPS-085 was determined following intravenous administration to male BALB/c mice bearing LNCaP tumor xenografts. The radiochemical yields of [64/67Cu]Cu-RPS-085 were nearly quantitative after 20 min. The metal-free complex is a potent inhibitor of PSMA (IC50 = 29 ± 2 nM), and the radiolabeled compound has moderate affinity for human serum albumin (Kd = 9.9 ± 1.7 µM). Accumulation of the tracer in mice was primarily evident in tumor and kidneys. Activity in all other tissues, including blood, was negligible, and the radiolabeled compounds demonstrated high stability in vitro and in vivo. Tumor activity reached a maximum at 4 h post injection (p.i.) and cleared gradually over a period of 96 h. By contrast, activity in the kidney cleared rapidly from 4 to 24 h p.i. As a consequence, by 24 h p.i., the tumor-to-kidney ratio exceeds 2, and the predicted dose to tumors is significantly greater than the dose to kidneys. [64Cu]Cu-RPS-085 combines rapid tissue distribution and clearance with prolonged retention in LNCaP tumor xenografts. The pharmacokinetics should enable radioligand therapy using [67Cu]Cu-RPS-085. By virtue of its rapid kidney clearance, the therapeutic index of [67Cu]Cu-RPS-085 likely compares favorably to its parent structure, [177Lu]Lu-RPS-063, a highly avid PSMA-targeting compound. On this basis, [64/67Cu]Cu-RPS-085 show great promise as PSMA-targeting theranostic ligands for prostate cancer imaging and therapy.

A general 11C-labeling approach enabled by fluoride-mediated desilylation of organosilanes.

Carbon-11 (11C) is one of the most ideal positron emitters for labeling bioactive molecules for molecular imaging studies. The lack of convenient and fast incorporation methods to introduce 11C into organic molecules often hampers the use of this radioisotope. Here, a fluoride-mediated desilylation (FMDS) 11C-labeling approach is reported. This method relies on thermodynamically favored Si-F bond formation to generate a carbanion, therefore enabling the highly efficient and speedy incorporation of [11C]CO2 and [11C]CH3I into molecules with diversified structures. It provides facile and rapid access to 11C-labeled compounds with carbon-11 attached at various hybridized carbons as well as oxygen, sulfur and nitrogen atoms with broad functional group tolerance. The exemplified syntheses of several biologically and clinically important radiotracers illustrates the potentials of this methodology.

Improved synthesis of the bifunctional chelator p-SCN-Bn-HOPO.

The bifunctional ligand p-SCN-Bn-HOPO, which has four 1,2-hydroxypyridinone groups on a spermine backbone with an isothiocyanate linker, has been shown to be an efficient and stable chelator for Zr(iv) and, more importantly, the radioisotope 89Zr for use in radiolabeling antibodies for positron emission tomography (PET) imaging. Previous studies of 89Zr-HOPO-trastuzumab in mice showed low background, good tumor to organ contrast, and very low bone uptake which show p-SCN-Bn-HOPO to be an important next-generation bifunctional chelator for radioimmunoPET imaging with 89Zr. However, the reported synthesis of p-SCN-Bn-HOPO involves nine steps and multiple HPLC purifications with an overall yield of about 1.4%. Herein we report an improved and efficient synthesis of p-SCN-Bn-HOPO in four steps with 14.3% overall yield which will improve its availability for further biological studies and wider application in PET imaging. The new synthetic route also allows variation in linker length and chemistries which may be helpful in modifying in vivo clearance behaviors of future agents.

[18F]Fluoroethyltriazolyl Monocyclam Derivatives as Imaging Probes for the Chemokine Receptor CXCR4.

Determining chemokine receptor CXCR4 expression is significant in multiple diseases due to its role in promoting inflammation, cell migration and tumorigenesis. [68Ga]Pentixafor is a promising ligand for imaging CXCR4 expression in multiple tumor types, but its utility is limited by the physical properties of 68Ga. We screened a library of >200 fluorine-containing structural derivatives of AMD-3465 to identify promising candidates for in vivo imaging of CXCR4 expression by positron emission tomography (PET). Compounds containing fluoroethyltriazoles consistently achieved higher docking scores. Six of these higher scoring compounds were radiolabeled by click chemistry and evaluated in PC3-CXCR4 cells and BALB/c mice bearing bilateral PC3-WT and PC3-CXCR4 xenograft tumors. The apparent CXCR4 affinity of the ligands was relatively low, but tumor uptake was CXCR4-specific. The tumor uptake of [18F]RPS-534 (7.2 ± 0.3 %ID/g) and [18F]RPS-547 (3.1 ± 0.5 %ID/g) at 1 h p.i. was highest, leading to high tumor-to-blood, tumor-to-muscle, and tumor-to-lung ratios. Total cell-associated activity better predicted in vivo tumor uptake than did the docking score or apparent CXCR4 affinity. By this metric, and on the basis of their high yielding radiosynthesis, high tumor uptake, and good contrast to background, [18F]RPS-547, and especially [18F]RPS-534, are promising 18F-labeled candidates for imaging CXCR4 expression.

A Single Dose of 225Ac-RPS-074 Induces a Complete Tumor Response in an LNCaP Xenograft Model.

Promising biochemical responses to 225Ac-prostate-specific membrane antigen (PSMA) 617, even in patients who are refractory to ß-particle radiation, illustrate the potential of targeted α-therapy for the treatment of metastatic castration-resistant prostate cancer. However, side effects such as xerostomia are severe and irreversible. To fully harness the potential of targeted α-therapy, it is necessary to increase the therapeutic index of the targeted radioligands. One emerging strategy is to increase clearance half-life through enhanced binding to serum albumin. We have evaluated the albumin-binding PSMA-targeting ligand RPS-074 in a LNCaP xenograft model to determine its potential value to the treatment of prostate cancer. Methods:225Ac-RPS-074 was evaluated in male BALB/c mice bearing LNCaP xenograft tumors. A biodistribution study was performed over 21 d to determine the dosimetry in tumors and normal tissue. The dose response was measured in groups of 7 mice using 37, 74, and 148 kBq of 225Ac-RPS-074 and compared with positive and negative control groups. Mice were sacrificed when tumor volume exceeded 1,500 mm3Results:225Ac-RPS-074 was labeled in greater than 98% radiochemical yield and showed high (>10% injected dose/g) and sustained accumulation in LNCaP tumors from 24 h to beyond 14 d. Signal in blood and highly vascularized tissues was evident over the first 24 h after injection and cleared by 7 d. The tumor-to-kidney ratio was 4.3 ± 0.7 at 24 h and 62.2 ± 9.5 at 14 d. A single injection of 148 kBq induced a complete response in 6 of 7 tumors, with no apparent toxic effects. Treatment with 74 kBq induced a partial response in 7 of 7 tumors, but from 42 d, 6 of 7 experienced significant regrowth. The 37-kBq group experienced a survival benefit relative to the negative control but not compared with the positive control group. Conclusion: A single dose of 148 kBq of 225Ac-RPS-074 induced a complete response in 86% of tumors, with tumor-to-normal-tissue ratios that predict an improved therapeutic index. The use of the macropa chelator enabled quantitative radiolabeling and may facilitate the clinical translation of this promising targeted α-therapeutic.

Albumin-Binding PSMA Ligands: Implications for Expanding the Therapeutic Window.

Despite significant gains in the treatment of metastatic castration-resistant prostate cancer by radioligands targeting prostate-specific membrane antigen (PSMA), 30% of patients never respond to therapy. One possible explanation is insufficient dose delivery to the tumor because of suboptimal pharmacokinetics. We have recently described RPS-063, a trifunctional ligand targeting PSMA with high uptake in LNCaP xenograft tumors but also in kidneys. We aimed to use structural modifications to increase the tumor-to-kidney ratio through increased albumin binding and tumor uptake and reduction of kidney activity. Methods: Four structurally related trifunctional PSMA-targeting small molecules were prepared by either varying the albumin-binding group or inserting a polyethylene glycol 8 linker into a common structure. The compounds were ranked by PSMA affinity and albumin affinity and were radiolabeled with 68Ga and 177Lu. Tissue kinetics were determined in male BALB/C nu/nu mice bearing LNCaP xenograft tumors. Results: Each of the compounds binds PSMA with a half-maximal inhibitory concentration of no more than 10 nM. The albumin-binding group had a minimal effect on PSMA affinity but changed albumin affinity by an order of magnitude. However, the addition of a polyethylene glycol 8 spacer weakened affinity for albumin in each case. Increased affinity for albumin corresponded with delayed blood clearance and modified uptake kinetics in the tumor and kidney. Uptake of 177Lu-RPS-072 (34.9 ± 2.4 %ID/g) and 177Lu-RPS-077 (27.4 ± 0.6 %ID/g) increased up to 24 h after injection, and washout by 96 h was not significant. As a result, the area under the curve (AUC) in the tumor was in the following order: 177Lu-RPS-072 > 177Lu-RPS-077 > 177Lu-RPS-063 > 177Lu-RPS-071. Increased linker length corresponded to more rapid clearance from kidneys. Consequently, the ratio of tumor AUC and kidney AUC was 4.7 ± 0.3 for 177Lu-RPS-072. Conclusion: The tumor AUC and tumor-to-kidney ratio of 177Lu-RPS-072 are significantly enhanced compared with any small molecule investigated in a LNCaP xenograft model to date. In comparison to other PSMA-targeting radioligands that have been evaluated in a PC3-PIP model, activity in kidneys is reduced and activity in tumors compares favorably when the different PSMA expression levels in LNCaP and PC3-PIP cells are considered. RPS-072 therefore exhibits an increased therapeutic index, shows the potential to increase the dose delivered to tumors, and is a highly promising candidate for targeted radioligand therapy.

66Ga: A Novelty or a Valuable Preclinical Screening Tool for the Design of Targeted Radiopharmaceuticals?

Emerging interest in extending the plasma half-life of small molecule radioligands warrants a consideration of the appropriate radionuclide for PET imaging at longer time points (>8 h). Among candidate positron-emitting radionuclides, 66Ga (t1/2 = 9.5 h, ß+ = 57%) has suitable nuclear and chemical properties for the labeling and PET imaging of radioligands of this profile. We investigated the value of 66Ga to preclinical screening and the evaluation of albumin-binding PSMA-targeting small molecules. 66Ga was produced by irradiation of a natZn target. 66Ga3+ ions were separated from Zn2+ ions by an optimized UTEVA anion exchange column that retained 99.99987% of Zn2+ ions and allowed 90.2 ± 2.8% recovery of 66Ga3+. Three ligands were radiolabeled in 46.4 ± 20.5%; radiochemical yield and >90% radiochemical purity. Molar activity was 632 ± 380 MBq/µmol. Uptake in the tumor and kidneys at 1, 3, 6, and 24 h p.i. was determined by µPET/CT imaging and more completely predicted the distribution kinetics than uptake of the [68Ga]Ga-labeled ligands did. Although there are multiple challenges to the use of 66Ga for clinical PET imaging, it can be a valuable research tool for ligand screening and preclinical imaging beyond 24 h.

Trifunctional PSMA-targeting constructs for prostate cancer with unprecedented localization to LNCaP tumors.

PURPOSE: Treatment of late-stage prostate cancer by targeted radiotherapeutics such as 131I-MIP-1095 and 177Lu-PSMA-617 has shown encouraging early results. Lu-177 is preferred to I-131 in clinical settings, but targeted radioligand therapy (RLT) with 177Lu-PSMA-617 has not reached its full potential due to insufficient dose delivery to the tumor. We recently developed a dual-targeting radioiodinated ligand, RPS-027, that targets PSMA and uses albumin binding to enable good tumor uptake and significantly reduced kidney uptake in a preclinical model. Further development of this ligand is limited by the inability to independently modify PSMA and albumin binding and the requirement of I-131 for therapeutic application. We therefore sought to devise a new class of trifunctional ligands for RLT with (1) a high-affinity PSMA-binding domain, (2) an albumin-binding group (ABG), and (3) a chelator for radiometals such as 68Ga3+, 177Lu3+ and 225Ac3+. METHODS: Ligands incorporating a triazolylphenylurea-containing PSMA-targeting group, an Nε-(2-(4-iodophenyl)acetyl)lysine ABG and the bifunctional chelator p-SCN-Bn-DOTA linked by a PEG-containing polymer containing 0,3,4,6,8 or 12 repeats were prepared. PSMA affinity was determined in LNCaP cells and uptake and tissue distribution was studied in mice bearing LNCaP tumor xenografts and compared to 177Lu-PSMA-617. Imaging studies were performed up to 24 h post-injection (p.i.) using 66Ga3+ and biodistribution studies at 4 h, 24 h and 96 h p.i. with 177Lu3+. RESULTS: PSMA affinity was high (IC50 = 1-10 nM) and inversely proportional to the linker length. Tumor uptake correlated with binding affinity and was significantly greater than for 177Lu-PSMA-617 over 96 h. The highest uptake was achieved with 177Lu-RPS-063 (30.0 ± 6.9 %ID/g; 4 h p.i.). Kidney uptake was generally high, with the exception of the lowest affinity ligand 177Lu-RPS-067. Each of the compounds showed slower blood clearance than 177Lu-PSMA-617, with clearance proportional to linker length. CONCLUSIONS: The high tumor uptake achieved with these trifunctional ligands predicts larger (up to 4×) doses delivered to the tumor than can be achieved with 177Lu-PSMA-617. Although PSMA-mediated kidney uptake was also observed, the exceptional area under the curve (AUC) in the tumor warrants further investigation of these novel ligands as candidates for RLT.

[18F]RPS-544: A PET tracer for imaging the chemokine receptor CXCR4.

INTRODUCTION: CXCR4 specific [18F]-labeled positron emission tomography (PET) imaging agents are needed which would enable general distribution of the radiotracer for clinical investigation. We sought to synthesize, radiolabel and evaluate [18F]RPS-544, a novel non-peptide CXCR4 antagonist as a CXCR4 specific probe. We compared [18F]RPS-544 with the previously published [18F]-3 ([18F]RPS-510 in this paper) in a bi-lateral tumor model of differential CXCR4 expression for its ability to selectively target CXCR4 expression. METHODS: Radiolabeling of [18F]RPS-544 and [18F]RPS-510 was performed by aromatic substitution on a 6-nitropyridyl group using no-carrier-added [18F]fluoride under basic conditions. 18F incorporation was determined by radioHPLC. Semi-preparative HPLC was used to purify the final product prior to reformulation. Imaging and biodistribution was performed in nude mice with bilateral PC3 (CXCR4+ and WT) xenograft tumors at 1, 2 and 4 h post injection. RESULTS: RPS-544 bound CXCR4 with an IC50 of 4.9 ±â€¯0.3 nM. [18F]RPS-544 showed preferential uptake in CXCR4+ tumors, with a CXCR4/WT ratio of 3.3 ±â€¯1.3 at 1 h p.i. and 2.3 ±â€¯0.5 at 2 h p.i. Maximum uptake in the CXCR4+ tumors was 3.4 ±â€¯1.2%ID/g at 1 h p.i., significantly greater (p = 0.003) than the uptake in the WT tumor. Tumor/blood ratios were 2.5 ±â€¯0.4 and 3.6 ±â€¯0.3 at 1 and 2 h p.i. Tumor/muscle ratios were >4 at all time-points. Tumor/lung ratios were >2 at 1 h and 2 h p.i. Substantial uptake was observed in the liver (15-25%ID/g), kidneys (25-35%ID/g), the small intestine (1-7%ID/g) and the large intestine (1-12%ID/g). Blood concentrations varied over time (0.5-2%ID/g). All other organs showed uptake of <1%ID/g at all time points studied with clearance profiles similar to blood clearance. CONCLUSIONS: Here we present, to the best of our knowledge, the first high affinity [18F]-labeled tracer, suitable for in vivo PET imaging of CXCR4. [18F]RPS-544 displayed high affinity for CXCR4 and good tumor uptake with a maximum uptake at 1 h p.i.. CXCR4 dependent uptake was demonstrated using bilateral tumors with differential CXCR4 expression as well as pharmacological blockade using the known CXCR4 antagonist, AMD-3100. Tissue contrast as judged by tumor to normal tissue ratios was positive in several key tissues. The structural and pharmacological similarities between [18F]RPS-544 and the approved drug AMD-3465, combined with the ease of synthesis and high molar activity (>185 GBq/µmol) achieved during radiosynthesis could lead to accelerated translation into the clinic.

An Eighteen-Membered Macrocyclic Ligand for Actinium-225 Targeted Alpha Therapy.

The 18-membered macrocycle H2 macropa was investigated for 225 Ac chelation in targeted alpha therapy (TAT). Radiolabeling studies showed that macropa, at submicromolar concentration, complexed all 225 Ac (26 kBq) in 5 min at RT. [225 Ac(macropa)]+ remained intact over 7 to 8 days when challenged with either excess La3+ ions or human serum, and did not accumulate in any organ after 5 h in healthy mice. A bifunctional analogue, macropa-NCS, was conjugated to trastuzumab as well as to the prostate-specific membrane antigen-targeting compound RPS-070. Both constructs rapidly radiolabeled 225 Ac in just minutes at RT, and macropa-Tmab retained >99 % of its 225 Ac in human serum after 7 days. In LNCaP xenograft mice, 225 Ac-macropa-RPS-070 was selectively targeted to tumors and did not release free 225 Ac over 96 h. These findings establish macropa to be a highly promising ligand for 225 Ac chelation that will facilitate the clinical development of 225 Ac TAT for the treatment of soft-tissue metastases.

Synthesis and pre-clinical evaluation of a new class of high-affinity 18F-labeled PSMA ligands for detection of prostate cancer by PET imaging.

PURPOSE: Current clinical imaging of PSMA-positive prostate cancer by positron emission tomography (PET) mainly features 68Ga-labeled tracers, notably [68Ga]Ga-PSMA-HBED-CC. The longer half-life of fluorine-18 offers significant advantages over Ga-68, clinically and logistically. We aimed to develop high-affinity PSMA inhibitors labeled with fluorine-18 as alternative tracers for prostate cancer. METHODS: Six triazolylphenyl ureas and their alkyne precursors were synthesized from the Glu-urea-Lys PSMA binding moiety. PSMA affinity was determined in a competitive binding assay using LNCaP cells. The [18F]triazoles were isolated following a Cu(I)-catalyzed click reaction between the alkynes and [18F]fluoroethylazide. The 18F-labeled compounds were evaluated in nude mice bearing LNCaP tumors and compared to [68Ga]Ga-PSMA-HBED-CC and [18F]DCFPyL. Biodistribution studies of the two tracers with the highest imaged-derived tumor uptake and highest PSMA affinity were undertaken at 1 h, 2 h and 4 h post-injection (p.i.), and co-administration of PMPA was used to determine whether uptake was PSMA-specific. RESULTS: F-18-labeled triazolylphenyl ureas were prepared with a decay-corrected RCY of 20-40 %, >98 % radiochemical and chemical purity, and specific activity of up to 391 GBq/µmol. PSMA binding (IC50) ranged from 3-36 nM. The position of the triazole influenced tumor uptake (3 > 4 > 2), and direct conjugation of the triazole with the phenylurea moiety was preferred to insertion of a spacer group. Image-derived tumor uptake ranged from 6-14 %ID/g at 2 h p.i., the time of maximum tumor uptake; uptake of [68Ga]Ga-PSMA-HBED-CC and [18F]DCFPyL was 5-6 %ID/g at 1-3 h p.i., the time of maximum tumor uptake. Biodistribution studies of the two most promising compounds gave maximum tumor uptakes of 10.9 ± 1.0 % and 14.3 ± 2.5 %ID/g, respectively, as compared to 6.27 ± 1.44 %ID/g for [68Ga]Ga-PSMA-HBED-CC. CONCLUSIONS: Six [18F]triazolylphenyl ureas were prepared in good radiochemical yield. Compounds showed PSMA-specific uptake in LNCaP tumors as high as 14 % ID/g, more than a 2-fold increase over [68Ga]Ga-PSMA-HBED-CC. The facile and high-yielding radiosynthesis of these 18F-labeled triazoles as well as their promising in vitro and in vivo characteristics make them worthy of clinical development for PET imaging of prostate cancer.

p-SCN-Bn-HOPO: A Superior Bifunctional Chelator for (89)Zr ImmunoPET.

Zirconium-89 has an ideal half-life for use in antibody-based PET imaging; however, when used with the chelator DFO, there is an accumulation of radioactivity in the bone, suggesting that the (89)Zr(4+) cation is being released in vivo. Therefore, a more robust chelator for (89)Zr could reduce the in vivo release and the dose to nontarget tissues. Evaluation of the ligand 3,4,3-(LI-1,2-HOPO) demonstrated efficient binding of (89)Zr(4+) and high stability; therefore, we developed a bifunctional derivative, p-SCN-Bn-HOPO, for conjugation to an antibody. A Zr-HOPO crystal structure was obtained showing that the Zr is fully coordinated by the octadentate HOPO ligand, as expected, forming a stable complex. p-SCN-Bn-HOPO was synthesized through a novel pathway. Both p-SCN-Bn-HOPO and p-SCN-Bn-DFO were conjugated to trastuzumab and radiolabeled with (89)Zr. Both complexes labeled efficiently and achieved specific activities of approximately 2 mCi/mg. PET imaging studies in nude mice with BT474 tumors (n = 4) showed good tumor uptake for both compounds, but with a marked decrease in bone uptake for the (89)Zr-HOPO-trastuzumab images. Biodistribution data confirmed the lower bone activity, measuring 17.0%ID/g in the bone at 336 h for (89)Zr-DFO-trastuzumab while (89)Zr-HOPO-trastuzumab only had 2.4%ID/g. We successfully synthesized p-SCN-Bn-HOPO, a bifunctional derivative of 3,4,3-(LI-1,2-HOPO) as a potential chelator for (89)Zr. In vivo studies demonstrate the successful use of (89)Zr-HOPO-trastuzumab to image BT474 breast cancer with low background, good tumor to organ contrast, and, importantly, very low bone uptake. The reduced bone uptake seen with (89)Zr-HOPO-trastuzumab suggests superior stability of the (89)Zr-HOPO complex.

Synthesis and evaluation of aporphine analogs containing C1 allyl isosteres at the h5-HT(2A) receptor.

A series of C1 aporphine analogs related to compound 5 and that contain substituted allylic, alkynyl, nitrile, ester and benzyl groups was synthesized and evaluated for affinity at h5HT2A and α1A receptors in functional activity assays that measure calcium release. The presence of branched allylic substituent groups diminished affinity for the h5HT2A receptor. Likewise, the alkynyl, nitrile and ester derivatives evaluated displayed lower 5-HT2A receptor affinity as compared to 5. Hydrophobic, steric and electronic effects impact the affinity of p-substituted benzyl derivatives 8i-8k but in different ways. High hydrophobicity and size favor 5-HT2A affinity whereas, high electronegativity disfavors 5-HT2A affinity. p-Bromobenzyl analog 8k was identified as a 5-HT2A receptor selective ligand, with the highest 5-HT2A receptor affinity of any aporphine known to date. Most of the other analogs were selective for the 5-HT2A versus the α1A receptor. ChemScore binding energies from docking studies correlated qualitatively with the observed trends in affinity for 8i-8k, although the binding energies were not well differentiated quantitatively.

A divergent route to 9, 10-oxygenated tetrahydroprotoberberine and 8-oxoprotoberberine alkaloids: synthesis of (±)-isocorypalmine and oxypalmatine.

A new route which is germane to the synthesis of 9,10-oxygenated tetrahydroprotoberberines and 8-oxoprotoberberines is described. The route features the use of a diester (14) generated from reaction of dimethylmalonate with an aryl halide in the presence of n-butyllithium. The amide 17 prepared in subsequent steps is a versatile precursor for the synthesis of tetrahydroprotoberberine and 8-oxoprotoberberine scaffolds using standard high-yielding reactions. In this manner, (±)-isocorypalmine and oxypalmatine have been synthesized in 23% and 22 % yields respectively.

Alternative chelator for 89Zr radiopharmaceuticals: radiolabeling and evaluation of 3,4,3-(LI-1,2-HOPO).

Zirconium-89 is an effective radionuclide for antibody-based positron emission tomography (PET) imaging because its physical half-life (78.41 h) matches the biological half-life of IgG antibodies. Desferrioxamine (DFO) is currently the preferred chelator for (89)Zr(4+); however, accumulation of (89)Zr in the bones of mice suggests that (89)Zr(4+) is released from DFO in vivo. An improved chelator for (89)Zr(4+) could eliminate the release of osteophilic (89)Zr(4+) and lead to a safer PET tracer with reduced background radiation dose. Herein, we present an octadentate chelator 3,4,3-(LI-1,2-HOPO) (or HOPO) as a potentially superior alternative to DFO. The HOPO ligand formed a 1:1 Zr-HOPO complex that was evaluated experimentally and theoretically. The stability of (89)Zr-HOPO matched or surpassed that of (89)Zr-DFO in every experiment. In healthy mice, (89)Zr-HOPO cleared the body rapidly with no signs of demetalation. Ultimately, HOPO has the potential to replace DFO as the chelator of choice for (89)Zr-based PET imaging agents.

Aporphinoid antagonists of 5-HT2A receptors: further evaluation of ring A substituents and the size of ring C.

A series of ring A-modified analogs of nantenine as well as structural variants in ring C were synthesized and evaluated for antagonist activity at 5-HT2A and α1A receptors. Halogenation improves 5-HT2A antagonist potency in molecules containing a C1 methoxyl/C2 methoxyl or C1 methoxyl/C2 hydroxyl moiety. Bromination or iodination (but not chlorination) with the latter moiety also significantly increased α1A antagonist potency. Homologation or contraction of ring C adversely affected antagonist activity at both receptors, implying that a six-membered ring C motif is beneficial for high antagonist potency at both receptors. Molecular docking studies suggest that the improved antagonist activity (by virtue of improved affinity) of C3-halogenated aporphines in this study is attributable to favorable interactions with the C3 halogen and F339 and/or F340.

Evaluation of structural effects on 5-HT(2A) receptor antagonism by aporphines: identification of a new aporphine with 5-HT(2A) antagonist activity.

A set of aporphine analogs related to nantenine was evaluated for antagonist activity at 5-HT2A and α1A adrenergic receptors. With regards to 5-HT2A receptor antagonism, a C2 allyl group is detrimental to activity. The chiral center of nantenine is not important for 5-HT2A antagonist activity, however the N6 nitrogen atom is a critical feature for 5-HT2A antagonism. Compound 12b was the most potent 5-HT2A aporphine antagonist identified in this study and has similar potency to previously identified aporphine antagonists 2 and 3. The ring A and N6 modifications examined were detrimental to α1A antagonism. A slight eutomeric preference for the R enantiomer of nantenine was observed in relation to α1A antagonism.

A New Route to Azafluoranthene Natural Products via Direct Arylation.

Microwave-assisted direct arylation was successfully employed in the synthesis of azafluoranthene alkaloids for the first time. Direct arylation reactions on a diverse set of phenyltetrahydroisoquinolines produces the indeno[1,2,3-ij]isoquinoline nucleus en route to a high yielding azafluoranthene synthesis. The method was used as a key step in the efficient preparation of the natural products rufescine and triclisine. As demonstrated herein, this synthetic approach should be generally applicable to the preparation of natural and un-natural azafluoranthene alkaloids as well as "azafluoranthene-like" isoquinoline alkaloids.

New aporphinoid 5-HT2A and α1A antagonists via structural manipulations of nantenine.

A series of C1, C2, C3 and N6 analogs of nantenine (2) was synthesized and evaluated in 5-HT(2A) and α(1A) receptor functional assays. Alkyl substitution of the C1 and N6 methyl groups of nantenine provided selective 5-HT(2A) and α(1A) antagonists, respectively. The C2 alkyloxy analogs studied were generally selective for α(1A) versus 5-HT(2A). The C3 bromo analog 15 is one of the most potent aporphinoid 5-HT(2A) antagonists known presently.