Synthesis and evaluation of a novel (68)Ga-labeled DOTA-benzamide derivative for malignant melanoma imaging

Malignant melanoma displays a highly aggressive metastasis. Thus, early diagnosis of malignant melanoma is important for patient survival. We designed and synthesized a novel (68)Ga-labeled benzamide derivative that specifically binds to melanoma as demonstrated by its ability to bind to melanin. (68)Ga-SCN-DOTA-PCA was synthesized with a radiochemical yield of ～80% and a radiochemical purity of >97% by analytical HPLC. The in vitro binding of (68)Ga-SCN-DOTA-PCA to melanin and its cellular uptake demonstrated the selective uptake in melanin. In addition, the biodistribution and micro-PET imaging of (68)Ga-SCN-DOTA-PCA in B16F10 tumor models showed the specific accumulation in melanoma. These results suggest that (68)Ga-SCN-DOTA-PCA would be a promising agent for melanoma diagnosis.     

Synthesis and characterization of a (68)Ga-labeled N-(2-diethylaminoethyl)benzamide derivative as potential PET Probe for malignant melanoma

Radiolabeled benzamides have been reported to be attractive agents for targeting malignant melanoma as they bind melanin and display high accumulation in melanoma cells. Herein, we report the synthesis and bioevaluation of a novel (68)Ga-labeled benzamide as a potential PET agent for malignant melanoma. The novel radiotracer was synthesized in good radiochemical yields (80% decay corrected yield) and high specific radioactivity (10GBq/μmol). Cellular uptake of (68)Ga-SCN-NOTA-BZA was significantly higher in B16F10 cells (mouse melanoma) treated with L-tyrosine. Biodistribution and micro-PET studies of (68)Ga-SCN-NOTA-BZA in B16F10-bearing mice showed selective uptake into the tumor. The radiotracer was cleared via renal excretion without further metabolism. These results demonstrate that (68)Ga-SCN-NOTA-BZA is a potential PET probe for malignant melanoma.     

Synthesis and evaluation of novel tropane derivatives as potential PET imaging agents for the dopamine transporter

A novel series of tropane derivatives containing a fluorinated tertiary amino or amide at the 2β position was synthesized, labeled with the positron-emitter fluorine-18 (t(1/2)=109.8 min), and tested as potential in vivo dopamine transporter (DAT) imaging agents. The corresponding chlorinated analogs were prepared and employed as precursors for radiolabeling leading to the fluorine-18-labeled derivatives via a one-step nucleophilic aliphatic substitution reaction. In vitro binding results showed that the 2β-amino compounds 6b, 6d and 7b displayed moderately high affinities to DAT (K(i)<10nM). Biodistribution studies of [(18)F]6b and [(18)F]6d showed that the brain uptakes in rats were low. This is likely due to their low lipophilicities. Further structural modifications of these tropane derivatives will be needed to improve their in vivo properties as DAT imaging agents.     

Development and characterization of a 68Ga-labeled A20FMDV2 peptide probe for the PET imaging of αvβ6 integrin-positive pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is known to be one of the most lethal cancers. Since the majority of patients are diagnosed at an advanced stage, development of a detection method for PDAC at an earlier stage of disease progression is strongly desirable. Integrin α_Vβ6 is a promising target for early PDAC detection because its expression increases during precancerous changes. The present study aimed to develop an imaging probe for positron emission tomography (PET) which targets α_Vβ6 integrin-positive PDAC. We selected A20FMDV2 peptide, which binds specifically to αvβ6 integrin, as a probe scaffold, and ⁶⁸Ga as a radioisotope. A20FMDV2 peptide has not been previously labeled with ⁶⁸Ga. A cysteine residue was introduced to the N-terminus of the probe at a site-specific conjugation of maleimide-NOTA (mal-NOTA) chelate. Different numbers of glycine residues were also introduced between cysteine and the A20FMDV2 sequence as a spacer in order to reduce the steric hindrance of the mal-NOTA on the binding probe to αVβ6 integrin. In vitro, the competitive binding assay revealed that probes containing a 6-glycine linker ([^natGa]CG6 and [^natGa]Ac-CG6) showed high affinity to αVβ6 integrin. Both probes could be labeled by ^67/68Ga with high radiochemical yield (>50%) and purity (>98%). On biodistribution analysis, [⁶⁷Ga]Ac-CG6 showed higher tumor accumulation, faster blood clearance, and lower accumulation in the surrounding organs of pancreas than did [⁶⁷Ga]CG6. The α_Vβ6 integrin-positive xenografts were clearly visualized by PET imaging with [⁶⁸Ga]Ac-CG6. The intratumoral distribution of [⁶⁸Ga]Ac-CG6 coincided with the α_Vβ6 integrin-positive regions detected by immunohistochemistry. Thus, [⁶⁸Ga]Ac-CG6 is a useful peptide probe for the imaging of α_Vβ6 integrin in PDAC.     

Synthesis and evaluation of novel N-fluoropyridyl derivatives of tropane as potential PET imaging agents for the dopamine transporter

A series of novel N-fluoropyridyl-containing tropane derivatives were synthesized and their binding affinities for the dopamine transporter (DAT), serotonin transporter (SERT) and norepinephrine (NET) were determined via competitive radioligand binding assays. Among these derivatives, compound 6d showed the highest binding affinity to DAT (K_i = 4.1 nM), and selectivity for DAT over SERT (5-fold) and NET (16-fold). Compound 6d was radiolabeled with Fluorine-18 in two steps. Regional brain distribution and ex vivo autoradiography studies of [¹⁸F]6d demonstrated that the ligand was selectively localized in the striatum region, where DAT binding sites are highly expressed. [¹⁸F]6d may be useful as a potential radioligand for imaging DATs with PET.     

多巴胺神经系统显像分子探针研究

多巴胺神经系统在神经退行性疾病和精神紊乱中充当了主要角色,比如帕金森病、亨廷顿病、迟发性运动障碍、精神分裂症。以多巴胺能神经系统为靶点的PET显像可以了解多巴胺合成、受体密度和状态改变,为神经系统疾病的早期诊断、疗效监测、发病机制以及脑认知功能的研究等方面提供客观、科学的观察手段。本文综述了以多巴胺受体、多巴胺转运体及囊泡单胺转运体为靶点的PET显像剂的研究进展。     

Synthesis and amine transporter affinities of novel phenyltropane derivatives as potential positron emission tomography (PET) imaging agents

A series of novel fluoroalkyl-containing tropane derivatives (6-8, 10-14, 17, and 18) were synthesized from cocaine. Novel compounds were evaluated for affinity and selectivity in competitive radioligand binding assays selective for cerebral serotonin (5-HT), dopamine (DA), and norepinephrine (NE) transporters (SERT, DAT, and NET). The nortropane-fluoroalkyl esters (7, 10, 11) were most potent for SERT (K(i): 0.18, 0.24, and 0.30 nM, respectively). Tosylate esters 17 and 18, synthesized as precursors for [(18)F]-labeled, Positron Emission Tomography (PET) imaging agents, also showed high affinity for DAT.     

Synthesis and characterization of styrylchromone derivatives as beta-amyloid imaging agents

Several promising agents have been synthesized and evaluated for in vivo imaging probes of beta-amyloid plaques in Alzheimer's disease (AD) brain. Recently, we have developed flavone derivatives, which possess the basic structure of the 2-phenylchromone, as useful candidates for amyloid imaging agents. In an attempt to further develop novel tracers, we synthesized and evaluated a series of 2-styrylchromone derivatives, which replace the 2-phenyl substituent of flavone backbone with the 2-styryl. A series of radioiodinated styrylchromone derivatives were designed and synthesized. The binding affinities for amyloid plaques were assessed by in vitro binding assay using pre-formed synthetic Abeta(1-40) aggregates. The new series of styrylchromone derivatives showed high binding affinity to Abeta aggregates at the K(d) values of 32.0, 17.5 and 8.7nM for [(125)I]6, [(125)I]9, and [(125)I]12, respectively. In biodistribution studies using normal mice, [(125)I]6 and [(125)I]9 examined in normal mice displayed high brain uptakes with 4.9 and 2.8%ID/g at 2min post injection. The radioactivity washed out from the brain rapidly (1.6 and 1.0%ID/g at 60min post injection for [(125)I]6 and [(125)I]9, respectively). But [(125)I]12 did not show marked brain uptake, and the washout rate from the brain was relatively slow throughout the time course (1.1 and 1.4%ID/g at 2 and 30min post injection, respectively). Although additional modifications are necessary to improve the brain uptake and rapid clearance of non-specifically bound radiotracer, the styrylchromone backbone may be useful as a backbone structure to develop novel beta-amyloid imaging agents.     

Biodistribution of 68Ga-labeled LNA-DNA mixmer antisense oligonucleotides for rat chromogranin-A

In vivo monitoring of gene expression may be accomplished using a most advanced imaging technology such as positron emission tomography (PET). However, a range of methodological and biological hurdles needs exploration. In the present study, 20-mer DNA-LNA (locked nucleic acid) mixmer oligonucleotides specific for rat Chromogranin-A (Chg-A) mRNA were labeled with 68Ga and their biodistribution were investigated in rats; namely, two Antisense (LNA1, LNA2--differing only in the positioning of LNA modification), Mismatched, and Sense sequences. In addition, in vivo and in vitro metabolite analysis of LNA1 and LNA2 was compared, and hybridization in solution was performed to verify the hybridization ability after labeling. Furthermore, semiquantitative polymerase chain reaction was carried out to find organs expressing Chg-A mRNA in the rat. The biodistribution patterns altered according to the sequence and the positioning of LNA modification. The pattern of Mismatched--differing only in two nucleotides from the two Antisenses--was similar to that of Sense, whereas the pattern of LNA1 and LNA2 showed differences. Uptake in the adrenal gland was twofold higher with LNA2 compared to the other three oligonucleotides. Intact LNA2 could be observed in the 60-minute sample in vivo, whereas in vitro, the intact compound of both Antisenses could also be detected after 2 hours. Hybridization in solution revealed that the two Antisenses retained their hybridization abilities after 68Ga-labeling. With decreasing magnitude, Chg-A mRNA was expressed in the adrenal gland, intestine, testis, and pancreas. This study further supported LNA-DNA mixmer to be a favorable modification for antisense targeting approach with respect to hybridization and longer plasma residence; however, the organ uptake was dominated by processes irrelevant to specific hybridization.     

Synthesis of benzamide derivatives and their evaluation as antiprion agents

A new set of 5-(2-(pyrrolidin-1-yl)acetamido)-N-butyl-2-(substituted)benzamide and 5-(2-(piperidin-1-yl)acetamido)-N-butyl-2-(substituted) benzamide derivatives were synthesized in which as structural features the 2-(1-pyrrolidinyl)- or 2-(1-piperidyl)acetylamino group or a diphenylether moiety are associated to a benzamide scaffold. Their binding affinity for human PrP(C) and inhibition of its conversion into PrP(Sc) were determined in vitro; moreover, the antiprion activity was assayed by inhibition of PrP(Sc) accumulation in scrapie-infected mouse neuroblastoma cells (ScN2a) and scrapie mouse brain (SMB) cells. The results clearly indicate the benzamide derivatives as attractive lead compounds for the development of potential therapeutic agents against prion disease.     

Labeled 3-aryl-4-indolylmaleimide derivatives and their potential as angiogenic PET biomarkers

In a continued effort to find a suitable PET tracer for visualization of angiogenic processes, we explored the 3,4-diarylmaleimide family, known to have high affinity and selectivity towards the VEGFR-TKs. One previously reported agent and three new halogen-containing 3,4-diarylmaleimide derivatives were synthesized. The four maleimide derivatives were evaluated for their affinity and selectivity towards the VEGFRs and exhibited promising results. An automated carbon-11 radiolabeling route with a total synthesis time of 50 min successfully labeled the lead compound, resulting in 1.55 ± 0.15 GBq of tracer with a radiochemical yield of 20 ± 2%, 96% radiochemical purity and a SA of 111 ± 22 GBq/μmol (EOB, n = 5). The tracer possessed high stability in in vitro blood stability tests and specific VEGFR-TK binding profiles in intact cell binding experiments. Tracer lipophilicity was evaluated in an n-octanol/phosphate buffer system giving a Log D_7.4 of 1.99 ± 0.04. For the in vivo experiments, two animal models were used. The first was a U87 glioma tumor model, frequently reported in the literature and the second, a newly developed 293/KDR tumor model. Both models were validated for VEGFR-2 expression and used in in vivo biodistribution studies. These studies revealed low accumulation and rapid washout of the tracer from tumor tissue. High accumulation of activity in the liver prompted us to examine the tracer’s in vitro stability to liver microsomes, revealing low resistance to P450 metabolism. In spite of encouraging in vitro results, the labeled lead tracer failed to accumulate in VEGFR-2 overexpressing tumors. It is possible that poor resistance to P450 metabolism reduces tracer’s circulation leading to low tumor accumulation.     

Synthesis and evaluation of macrocyclic amino acid derivatives for tumor imaging by gallium-68 positron emission tomography

(68)Ga PET imaging in clinical oncology represents a notable development because the availability of (68)Ga is not dependent on a cyclotron. Furthermore, labeled amino acid derivatives have been proven to be useful for the imaging many tumor types. In the present study, we synthesized β-aminoalanine, γ-aminohomoalanine, and lysine conjugates of macrocyclic bifunctional chelating agents, such as, NOTA (1a-c) and DOTA (2a-c). The compounds produced were found to be potential useful as (68)Ga-PET imaging agents. In particular, they showed high tumor uptakes in vitro and in vivo, and had high labeling yields and excellent stabilities. The co-ordination chemistry of NOTA-monoamide compound 1a was studied by multinuclear NMR. In vitro studies showed that the synthesized compounds were taken up by cancer cells more than controls ((68)Ga-NOTA and (68)Ga-DOTA). Furthermore, in vivo studies showed that they have high tumor to muscle and tumor to blood ratios, and small-animal PET imaging revealed high tumor uptakes as compared with other organs, and high bladder activities, indicating rapid renal excretion. These results might motivate the use of (68)Ga amino acid PET for tumor diagnosis.     

Synthesis of carbon-11-labeled aminoalkylindole derivatives as new candidates of cannabinoid receptor radioligands for PET imaging of alcohol abuse

Carbon-11-labeled aminoalkylindole derivatives (1-butyl-7-[¹¹C]methoxy-1H-indol-3-yl)(naphthalene-1-yl)methanone ([¹¹C]3), 1-butyl-7-[¹¹C]methoxy-3-(naphthalene-1-ylmethyl)-1H-indole ([¹¹C]5), and 1-butyl-7-[¹¹C]methoxy-3-(naphthalene-2-yl)-1H-indole ([¹¹C]8) were prepared by O-[¹¹C]methylation of their corresponding precursors with [¹¹C]CH₃OTf under basic condition (2 N NaOH) and isolated by a simplified solid-phase extraction (SPE) method in 50–60% radiochemical yields based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The overall synthesis time from EOB was 23 min, the radiochemical purity was >99%, and the specific activity at end of synthesis (EOS) was 185–555 GBq/μmol.     

Synthesis and in vitro evaluation of 18F labeled tyrosine derivatives as potential positron emission tomography (PET) imaging agents

Three new ¹⁸F labeled fluoroalkyl tyrosine derivatives, O-(2-[¹⁸F]fluoroethyl)-α-methyltyrosine (FEMT, [¹⁸F]2), O-(2-[¹⁸F]fluoroethyl)-2-l-azatyrosine (FEAT, [¹⁸F]3), O-(2-[¹⁸F]fluoroethyl)-l-tyrosineamide (FETA, [¹⁸F]4) have been synthesized and radiofluorinated with 5–34% decay-corrected yield. In vitro studies were carried out in U-138 MG human glioblastoma. Cellular uptake of new tracers was compared to clinically utilized imaging agent O-(2-[¹⁸F]fluoroethyl)-l-tyrosine (FET, [¹⁸F]1). The uptake of tracers followed the order of FET ([¹⁸F]1) > FEAT([¹⁸F]3) > FEMT ([¹⁸F]2) ≈ FETA ([¹⁸F]4).     

Synthesis and evaluation of indolinyl- and indolylphenylacetylenes as PET imaging agents for beta-amyloid plaques

Two new phenylacetylene derivatives, 5-((4-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)phenyl)ethynyl)indoline 8 and 5-((4-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)phenyl)ethynyl)-1H-indole 14, targeting β-amyloid (Aβ) plaques have been prepared. In vitro binding carried out in tissue homogenates prepared from postmortem AD brains with [¹²⁵I]IMPY (6-iodo-2-(4′-dimethylamino-)phenyl-imidazo[1,2-a]pyridine) as the radioligand indicated good binding affinities (K_i = 4.0 and 1.5 nM for 8 and 14, respectively). Brain penetration of the corresponding radiofluorinated ligands, evaluated in the normal mice, showed good initial brain penetration (4.50 and 2.43% ID/g (injected dose/gram) for [¹⁸F]8 and [¹⁸F]14 at 2 min after injection) with moderate to low washout rates from the brain (1.71% ID/g at 2 h and 2.10% ID/g at 3 h, respectively). Autoradiography and homogenate binding studies demonstrated the high specific binding of [¹⁸F]14 to the Aβ plaques; however, [¹⁸F]8 showed low specific binding. These preliminary results identified that indolylphenylacetylene, 14, may be a good lead for further structural modification to develop a useful Aβ plaque imaging agent.     

Synthesis of aminosterols and their derivatives

Reactions of steroidal epoxides such as 5,6 alpha-epoxy-5 alpha-cholestane (I) and its 3 beta-chloro (II) and 3 beta-acetoxy (III) analogs with urea in dimethylformamide afforded 6 beta-amino-5 alpha-cholestan-5-ol (IV-VI), 6 beta-amino-N-formyl-5 alpha-cholestan-5-ol (VII-IX), and 6 beta-amino-N-amido-5 alpha-cholestan-5-ol (X-XII), along with the 5 alpha-cholestane-5,6 beta-diol (XIII-XV). In addition to these compounds, the 3 beta-acetoxy analog also afforded the N-carboxyl derivative (XVI).     

Synthesis of sulfur containing dihydro-pyrrolo derivatives and their biological evaluation as antioxidants

The 1,3-dipolar cycloaddition to N-phenylmaleimide of azomethine ylides, generated in situ from sulfanyl-substituted imines of glycine esters, yields 5H-dihydro-pyrrolo products with syn diastereoselectivity. The syn (major) and anti (minor) products were isolated chromatographically and fully characterized by spectroscopic methods and in two cases also by X-ray analysis. The diastereomeric cycloadducts were tested for their antioxidant activity with good results.