Synthesis and biological evaluation of a novel 99mTc labeled 2-nitroimidazole derivative as a potential agent for imaging tumor hypoxia

Tumor hypoxia plays a major role in reducing the efficacy of therapeutic modalities like chemotherapy and radiation therapy in combating cancer. In order to target hypoxic tissues, a tripeptide ligand having a 2-nitroimidazole moiety, as a bioreductive species, was synthesized. The latter was radiolabeled with ^99mTc for imaging hypoxic regions of tumors and was characterized by means of its rhenium analogue. The biodistribution and scintigraphic image of the corresponding ^99mTc-complex showed accumulation in tumor and these results suggest that it could be a marker for imaging tumor hypoxia.     

Identification of novel 4-anilinoquinazoline derivatives as potent EGFR inhibitors both under normoxia and hypoxia

A novel series of 4-anilinoquinazoline derivatives (19a–19t) were designed and synthesized through incorporation of the 2-nitroimidazole moiety into the 4-anilinoquinazoline scaffold of EGFR inhibitors. The most promising compound 19h displayed potent EGFR inhibitory activity with the IC₅₀ value of 0.47 nM. It also strongly suppressed the proliferation of A549 and HT-29 cells with sub-micromolar IC₅₀ values both under normoxia and hypoxia, which were several folds more potent than gefitinib and erlotinib. Further reductive mimic investigation revealed that 19h could be reductive activated under hypoxia and was fully consistent with the results of cell apoptotic assay and in vitro metabolism evaluation. Our results suggest that the incorporation of hypoxia-activated moiety into EGFR inhibitor scaffold might be a tractable strategy to overcome the tumor hypoxia.     

Improving bioorthogonal protein ubiquitylation by click reaction

Posttranslational modification of proteins with ubiquitin (ubiquitylation) regulates numerous cellular processes. Besides functioning as a signal for proteasomal degradation, ubiquitylation has also non-proteolytic functions by altering the biochemical properties of the modified protein. To investigate the effect(s) of ubiquitylation on the properties of a protein, sufficient amounts of homogenously and well-defined ubiquitylated proteins are required. Here, we report on the elaboration of a method for the generation of high amounts of site-specifically mono-ubiquitylated proteins. Firstly, a one-step affinity purification scheme was developed for ubiquitin containing the unnatural amino acid azidohomoalanine at the C-terminal position. This ubiquitin was conjugated in a click reaction to recombinant DNA polymerase β, equipped with an alkyne function at a distinct position. Secondly, addition of defined amounts of SDS to the reaction significantly improved product formation. With these two technical improvements, we have developed a straight forward procedure for the efficient generation of site-specifically ubiquitylated proteins that can be used to study the effect(s) of ubiquitylation on the activities/properties of a protein.     

Synthesis and preliminary evaluation of 18F-icotinib for EGFR-targeted PET imaging of lung cancer

Epidermal growth factor receptor (EGFR) has emerged as an attracting target in the field of imaging and treatment for non-small cell lung cancer (NSCLC). Radiolabeled EGFR-tyrosine kinase inhibitors (EGFR-TKIs) specifically targeting EGFR are deemed as promising probes for the imaging of NSCLC. This study aimed to label icotinib (one kind of EGFR-TKI) with ¹⁸F through click reaction to develop a new EGFR-targeting PET probe-¹⁸F-icotinib. ¹⁸F-icotinib was obtained in 44.81% decay-corrected yield in 100?min synthesis time with 34?GBq/μmol specific activity and >99% radiochemical purity at the end of synthesis. The identity of the product was confirmed by co-injection with ¹⁸F-icotinib and ¹⁹F-icotinib. The Log P was 1.28?±?0.04 (n?=?6). The tracer displayed excellent stability after incubation for 4?h in vitro. ¹⁸F-icotinib showed satisfying binding ability to A549 NSCLC cells, which could be inhibited by icotinib. PET imaging studies demonstrated a specific uptake of the radiotracer (0.90?±?0.24% ID/g) in A549 tumor-bearing mice, while lower uptake was observed in heart, lung and spleen at 1.5?h post injection. Inmunohistochemical staining confirmed that the A549 tumor was EGFR-positive. Therefore, we considered that ¹⁸F-icotinib was a highly promising compound for EGFR-based tumor PET imaging.     

Synthesis and evaluation of a novel Tc-labeled bioreductive probe for tumor hypoxia imaging

Tumor hypoxia is closely associated with the malignant progression and/or the high metastatic ability of tumors and often induces resistance to chemo- and/or radiotherapy. Thus, the detection and evaluation of hypoxia is important for the optimization of cancer therapy. We designed a novel ^99mTc-labeled probe for tumor hypoxia imaging that utilizes bioreductive reactions in hypoxic cells. This probe, which contains a 4-nitrobenzyl ester group, is reduced in hypoxic cells to produce a corresponding carboxylate anion that cannot penetrate cell membranes because of its hydrophilicity and negative charge; therefore, it is expected to be trapped inside hypoxic cells. Based on this unique strategy, we synthesized the Technetium-99m (^99mTc)-labeled probe ^99mTc-SD32. The uptake of ^99mTc-SD32 in tumor cells was investigated under normoxic and hypoxic conditions. ^99mTc-SD32 showed sufficient accumulation and good retention in hypoxic cells. In addition, we demonstrated that ^99mTc-SD32 was subjected to bioreduction in hypoxic cells and was trapped as the corresponding carboxylate anion. These results indicated that ^99mTc-SD32 would be a promising agent for in vivo hypoxia imaging.     

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

ABSTRACT

Antibody pretargeting is a promising strategy for improving molecular imaging, wherein the separation in time of antibody targeting and radiolabeling can lead to rapid attainment of high contrast, potentially increased sensitivity, and reduced patient radiation exposure. The inverse electron demand Diels-Alder ‘click’ reaction between trans-cyclooctene (TCO) conjugated antibodies and radiolabeled tetrazines presents an ideal platform for pretargeted imaging due to rapid reaction kinetics, bioorthogonality, and potential for optimization of both slow and fast clearing components. Herein, we evaluated a series of anti-human epidermal growth factor receptor 2 (HER2) pretargeting antibodies containing distinct molar ratios of site-specifically incorporated TCO. The effect of stoichiometry on tissue distribution was assessed for pretargeting TCO-modified antibodies (monitored by ¹²⁵I) and subsequent accumulation of an ¹¹¹In-labeled tetrazine in a therapeutically relevant HER2+tumor-bearing mouse model. Single photon emission computed tomography (SPECT) imaging was also employed to assess tumor imaging at various TCO-to-monoclonal antibody (mAb) ratios. Increasing TCO-to-mAb molar ratios correlated with increased in vivo click reaction efficiency evident by increased tumor distribution and systemic exposure of ¹¹¹In-labeled tetrazines. The pharmacokinetics of TCO-modified antibodies did not vary with stoichiometry. Pretargeted SPECT imaging of HER2-expressing tumors using ¹¹¹In-labeled tetrazine demonstrated robust click reaction with circulating antibody at ~2 hours and good tumor delineation for both the 2 and 6 TCO-to-mAb ratio variants at 24 hours, consistent with a limited cell-surface pool of pretargeted antibody and benefit from further distribution and internalization. To our knowledge, this represents the first reported systematic analysis of how pretargeted imaging is affected solely by variation in click reaction stoichiometry through site-specific conjugation chemistry.     

Catalyst-free cycloaddition of 1,3-diene-1-carbamates with azodicarboxylates: A rapid click reaction

Novel click reactions are of continued interest in many scientific research areas and applications. Herein, we report a novel practical, catalyst-free, azo-Diels-Alder reaction between dienecarbamates and azodicarboxylates exhibiting a remarkable functional group tolerance. The availability of starting materials, mild reaction conditions, chemoselectivity and scalability make this cycloaddition a viable supplement to the other reactions in “click” chemistry.     

Synthesis and evaluation of radioactive/fluorescent peptide probes for imaging of legumain activity

Legumain or asparaginyl endopeptidase is an enzyme overexpressed in some cancers and involved in cancer migration, invasion, and metastasis. We have developed radioiodine- ([¹²⁵I]I-LCP) or fluorescein-labeled peptides (FL-LCP) with a cell-permeable d-Arg nonamer fused to an anionic d-Glu nonamer via a legumain-cleavable linker, to function as peptide probes that measure and monitor legumain activity. Non-cleavable probes of FL-NCP and [¹²⁵I]I-NCP were similarly prepared and evaluated as negative control probes by altering their non-cleavable sequence. Model peptides with the legumain-cleavable or non-cleavable sequence (LCP and NCP, respectively) reacted with recombinant human legumain, and only LCP was digested by this enzyme. [¹²⁵I]I-LCP uptake in legumain-positive HCT116 cells was significantly higher than that of [¹²⁵I]I-NCP (11.2 ± 0.44% vs 1.75 ± 0.06% dose/mg). The accumulation of FL-LCP in the HCT116 cells was rather low (4.75 ± 0.29% dose/mg protein), but not significantly different from the levels of FL-NCP. It is possible that low concentrations of [¹²⁵I]I-LCP (40 pM) can be effectively internalized after legumain cleavage. On the other hand, the cellular uptake of much higher concentrations of the FL-LCP derivative (1 mM) may be restricted by high concentrations of polyanions. The in vivo biodistribution studies in tumor-bearing mice demonstrated that the tumor uptake of [¹²⁵I]I-LCP was 1.34% injected dose per gram (% ID/g) at 30 min. The tumor/blood and tumor/muscle ratios at 30 min were 0.63 and 1.77, respectively, indicating that the [¹²⁵I]I-LCP accumulation in tumors was inadequate for in vivo imaging. Although further structural modifications are necessary to improve pharmacokinetic properties, [¹²⁵I]I-LCP has been demonstrated to be an effective scaffold for the development of nuclear medicine imaging probes to monitor legumain activity in living subjects.     

Proflavine derivatives as fluorescent imaging agents of amyloid deposits

A series of proflavine derivatives for use to further image Aβ amyloid deposits were synthesized and characterized. Aged 3xTg-AD (23 months old) mice hippocampus sections incubated with these derivatives revealed preferential labeling of amyloid plaques. Furthermore an in vitro binding study showed an inhibitory effect, although moderate, of these compounds on Aβ₄₀ fibril formation. This study highlights the potential of proflavine as a molecular scaffold for designing new Aβ imaging agents, its native fluorescence allowing in vitro neuropathological staining in AD damaged brain sections.     

Radiosynthesis and biological evaluation of an fluorine-18 labeled galactose derivative [18F]FPGal for imaging the hepatic asialoglycoprotein receptor

The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the surface of hepatocytes where it recognizes and endocytoses glycoproteins with galactosyl and N-acetylgalactosamine groups. Given its hepatic distribution, the asialoglycoprotein receptor can be targeted by positron imaging agents to study liver function using PET imaging. In this study, the positron imaging agent [¹⁸F]FPGal was designed to specifically target hepatic asialoglycoprotein receptor and its effectiveness was assessed in in vitro and in vivo models. The radiosynthesis of [¹⁸F]FPGal required 50 min with total radiochemical yields of [¹⁸F]FPGal from [¹⁸F]fluoride as 10% (corrected radiochemical yield). The K_d of [¹⁸F]FPGal to ASGPR in HepG2 cells was 1.99 ± 0.05 mM. Uptake values of 0.55% were observed within 30 min of incubation with HepG2 cells, which could be blocked by 200 mM d(+)-galactose (<0.1%). In vivo biodistribution analysis showed that the liver accumulation of [¹⁸F]FPGal at 30 min was 4.47 ± 0.96% ID/g in normal mice compared to 1.33 ± 0.07% ID/g in hepatic fibrotic mice (P < 0.01). Reduced uptake in the hepatic fibrosis mouse models was confirmed through PET/CT images at 30 min. Compared to normal mice, the standard uptake value (SUV) in the hepatic fibrosis mice was significantly lower when assessed through dynamic data collection for 1 h. Therefore, [¹⁸F]FPGal is a feasible PET probe that provide insight into ASGPR related liver disease.     

Synthesis and tubulin-binding properties of non-symmetrical click C5-curcuminoids

A click-type entry into shortened curcuminoids of the diarylpentanoid type has been developed. The reaction is ideally suited to generate non-symmetrical analogues of curcumin, a class of natural products difficult to access but of growing biomedical relevance and special mechanistic interest to investigate the unique binding mode of curcumin to tubulin. Investigation of a series of click diarylpentane curcuminoids and their pyrazole adducts in various cellular tubulin functional assays validated this class of compounds as a novel type of anti-mitotic agents, evidencing structure–activity relationships, and identifying the pyrazole adduct 4k as a promising lead.     

Nitroimidazole conjugates of bis(thiosemicarbazonato)Cu(II) - Potential combination agents for the PET imaging of hypoxia

Combination agents comprising two different pharmacophores with the same biological target have the potential to show additive or synergistic activity. Bis(thiosemicarbazonato)copper(II) complexes (e.g. ⁶⁴Cu-ATSM) and nitroimidazoles (e.g. ¹⁸F-MISO) are classes of tracer used for the delineation of tumor hypoxia by positron emission tomography (PET). Three nitroimidazole-bis(thiosemicarbazonato)copper(II) conjugates were produced in order to investigate their potential as combination hypoxia imaging agents. Two were derived from the known bifunctional bis(thiosemicarbazone) H₂ATSM/A and the third from the new precursor diacetyl-2-(4-N-methyl-3-thiosemicarbazone)-3-(4-N-ethylamino-3-thiosemicarbazone) - H₂ATSM/en. Oxygen-dependent uptake studies were performed using the ⁶⁴Cu radiolabelled complexes in EMT6 carcinoma cells. All the complexes displayed appreciable hypoxia selectivity, with the nitroimidazole conjugates displaying greater selectivity than a simple propyl derivative used as a control. Participation of the nitroimidazole group in the trapping mechanism is indicated by the increased hypoxic uptake of the 2- vs. the 4-substituted ⁶⁴Cu-ATSM/A derivatives. The 2-nitroimidazole derivative of ⁶⁴Cu-ATSM/en demonstrated superior hypoxia selectivity to ⁶⁴Cu-ATSM over the range of oxygen concentrations tested. Biodistribution of the radiolabelled 2-nitroimidazole conjugates was carried out in EMT6 tumor-bearing mice. The complexes showed significantly different uptake trends in comparison to each other and previously studied Cu-ATSM derivatives. Uptake of the Cu-ATSM/en conjugate in non-target organs was considerably lower than for derivatives based on Cu-ATSM/A.     

Synthesis and characterization of radioiodinated 3-phenethyl-2-indolinone derivatives for SPECT imaging of survivin in tumors

Survivin, overexpressed in most cancers, is associated with poor prognosis and resistance to radiation therapy and chemotherapy. Herein, we report the synthesis of three 3-phenethyl-2-indolinone derivatives and their application as in vivo imaging agents for survivin. Of these, 3-(2-(benzo[d][1,3]dioxol-5-yl)-2-oxoethyl)-3-hydroxy-5- iodoindolin-2-one (IPI-1) showed the highest binding affinity (K_d?=?68.3?nM) to recombinant human survivin, as determined by quartz crystal microbalance (QCM). In vitro studies demonstrated that the [¹²⁵I]IPI-1 binding in survivin-positive MDA-MB-231 cells was significantly higher than that in survivin-negative MCF-10A cells. In addition, uptake of [¹²⁵I]IPI-1 by MDA-MB-231 cells decreased in a dose-dependent manner in the presence of the high-affinity survivin ligand S12; this is indicative of specific binding of [¹²⁵I]IPI-1 to cellular survivin protein in vitro. Biodistribution studies in MDA-MB-231 tumor-bearing mice demonstrated the moderate uptake of [¹²⁵I]IPI-1 in the tumor tissue (1.37%?ID/g) at 30?min that decreased to 0.32%?ID/g at 180?min. Co-injection of S12 (2.5?mg/kg) slightly reduced tumor uptake and the tumor/muscle ratio of [¹²⁵I]IPI-1. Although further structural modifications are necessary to improve pharmacokinetic properties, our results indicate that PI derivatives may be useful as tumor-imaging probes targeting survivin.     

Synthesis and bioevaluation of substituted chalcones,coumaranones and other flavonoids as anti-HIV agents

A series of chalcone, flavone, coumaranone and other flavonoid compounds were screened for their anti HIV-1 activity in two cell culture models using TZM-bl and PM1 cells. Within the systems evaluated, the most promising compounds contained either an α- or β-hydroxy-carbonyl motif within their structure (e.g., 8 and 9). Efficacious substituents were identified and used to design new HIV inhibitors with increased potency and lower cytotoxicity. Of the scaffolds evaluated, specific chalcones were found to provide the best balance between anti-HIV potency and low host cell toxicity. Chalcone 8l was shown to inhibit different clinical isolates of HIV in a dose-dependent manner (e.g., IC₅₀ typically ⩽ 5 μM). Inhibition of HIV infection experiments using TZM-bl cells demonstrated that chalcone 8l and flavonol 9c had IC₅₀ values of 4.7 μM and 10.4 μM, respectively. These insights were used to design new chalcones 8o and 8p. Rewardingly, chalcones 8o and 8p (at 10 μM) each gave >92% inhibition of viral propagation without impacting PM1 host cell viability. Inhibition of viral propagation significantly increased (60–90%) when PM1 cells were pre-incubated with chalcone 8o, but not with the related flavonol 9c. These results suggested that chalcone 8o may be of value as both a HIV prophylactic and therapy. In summary, O-benzyl-substituted chalcones were identified as promising anti-HIV agents for future investigation.     

Systematic screening of potential beta-cell imaging agents

The beta-cell loss seen in diabetes mellitus could be monitored clinically by positron emission tomography (PET) if imaging agents were sufficiently specific for beta-cells to overcome the high ratio of non-beta-cell to beta-cell tissue in pancreas. In this report, we present a screening assay for identifying beta-cell-specific compounds that is based on the relative accumulation and retention by islet, INS-1, and exocrine (PANC-1) cells of candidate molecules. Molecules thought to have a high affinity for beta-cells were tested and included glibenclamide, tolbutamide, serotonin, L-DOPA, dopamine, nicotinamide, fluorodeoxyglucose, and fluorodithizone. Glibenclamide and fluorodithizone were the most specific, but the specificity ratios fell well below those needed to attain robust signal to background ratio as a PET imaging agent for quantifying beta-cell mass. In vivo tests of the biodistribution of glibenclamide and fluorodithizone in rats indicated that the compounds were not specifically associated with pancreas, bearing out the predictions of the in vitro screen.     

Synthesis and application of cNGR-containing imaging agents for detection of angiogenesis

Angiogenesis is a multi-step process regulated by pro- and anti-angiogenic factors. Inhibition of angiogenesis is a potential anti cancer treatment strategy that is now investigated clinically. In addition, advances in the understanding of the angiogenic process have led to the development of new angiogenesis therapies for ischemic heart disease.Currently, researchers search for objective measures that indicate pharmacological responses to pro- and anti-angiogenic drugs and therefore, there is a great interest in techniques to visualize angiogenesis noninvasively. As CD13 is selectively expressed in angiogenic blood vessels, it can serve as a target for molecular imaging tracers to noninvasively visualize angiogenic processes in animal models and patients. Here, an overview on the currently used CD13 targeted molecular imaging probes for noninvasive visualization of angiogenesis is given.     

Synthesis and evaluation of Gd-DTPA-labeled arabinogalactans as potential MRI contrast agents

Arabinogalactan derivatives conjugated with gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA) by ethylenediamine (Gd-DTPA-CMAG-A2) or hexylamine (Gd-DTPA-CMAG-A6) have been synthesized and characterized by means of Fourier transform infrared spectra (FTIR), 13C nuclear magnetic resonance (13C NMR), size exclusion chromatography (SEC), and inductively coupled plasma atomic emission spectrometry (ICP-AES). Relaxivity studies showed that arabinogalactan-bound complexes possessed higher relaxation effectiveness compared with the clinically used Gd-DTPA, and the influence of the spacer arm lengths on the T1 relaxivities was studied. Their stability was investigated by competition study with Ca2+, EDTA, and DTPA. MR imaging of Wistar rats showed remarkable enhancement in rat liver and kidney after i.v. injection of Gd-DTPA-CMAG-A2 (0.079+/-0.002 mmol/kg Gd3+): The mean percentage enhancement of the liver parenchyma and kidney was 38.7+/-6.4% and 69.4+/-4.4% at 10-30 min. Our preliminary in vivo and in vitro study indicates that the arabinogalactan-bound complexes are potential liver-specific contrast agents for MRI.     

Synthesis of 3'-azido-3'-deoxythymidine (AZT)--Cinchona alkaloid conjugates via click chemistry: Toward novel fluorescent markers and cytostatic agents

Novel nucleoside-Cinchona alkaloid conjugates were synthesized using ‘click’ chemistry approach based on the copper(I) catalyzed Huisgen azide-alkyne cycloaddition. Two series of conjugates were prepared employing 3′-azido-3′-deoxythymidine (AZT) as the azide component and the four 10,11-didehydro Cinchona alkaloids as well as their 9-O-propargyl ethers as the alkyne components. All obtained conjugates showed strong fluorescence emission and some of them exhibited marked cytotoxic activity in vitro.     

Synthesis and cytotoxic activity of novel acyclic nucleoside analogues with functionality in click chemistry

We describe synthesis of novel acyclic nucleoside analogues which are building blocks for CuAAC reaction and their activity against two types of human cancer cell lines (HeLa, KB). Three of chosen compounds show promising cytotoxic activity. Synthesis pathway starting from simple and easily accessible substrates employing DMT or TBDPS protective groups is described. Adenosine and thymidine analogues containing alkyne moiety and adenosine analogue containing azido group were synthesized. The obtained units showed ability of forming triazole motif under the CuAAC reaction conditions.     

Preparation and bioevaluation of 99mTc-HYNIC-annexin B1 as a novel radioligand for apoptosis imaging

Annexin B1, a novel Ca²⁺-dependent PS-binding protein, has been shown to have a high affinity for PS exposed on the surface of apoptotic cells. To develop and bioevaluate an annexin B1 based PS-targeting radiotracer, annexin B1 was radiolabeled with ^99mTc using HYNIC as a bifunctional chelator. Binding assays with activated platelets and apoptotic SP2/0 cells were carried out to evaluate the in vitro biological activity of ^99mTc-HYNIC-annexin B1. Biodistribution of this radioligand was studied in normal mice. Dexamethasone-induced murine thymus apoptosis and fas-mediated murine liver apoptosis models were used to investigate the ability of radiolabeled annexin B1 to detect apoptosis in vivo. The labeling procedure yielded a compound with up to 98% radiochemical purity and good in vitro stability. The in vitro binding assays indicated that ^99mTc-HYNIC-annexin B1 retain its PS-binding activity. Biodistribution of the compound in mice showed that ^99mTc-HYNIC-annexin B1 is rapidly cleared from the blood and predominantly accumulates in the kidney. The marked increase in dexamethasone-treated murine thymus uptake and fas-mediated murine liver uptake correlated with histologic evidence of apoptosis. These data suggested that ^99mTc-HYNIC-annexin B1 retain its in vitro and in vivo biological activities. This radiotracer may therefore be useful as a novel radioligand for the noninvasive detecting of PS externalization associated with apoptosis.