Synthesis and Biological Evaluation of New Compounds with Nitroimidazole Moiety.

Heterocyclic compounds, particularly those containing azole rings, have shown extensive biological activity, including anticancer, antibacterial, and antifungal properties. Among these, the imidazole ring stands out due to its diverse therapeutic potential. In the presented study, we designed and synthesized a series of imidazole derivatives to identify compounds with high biological potential. We focused on two groups: thiosemicarbazide derivatives and hydrazone derivatives. We synthesized these compounds using conventional methods and confirmed their structures via nuclear magnetic resonance spectroscopy (NMR), MS, and elemental analysis, and then assessed their antibacterial and antifungal activities in vitro using the broth microdilution method against Gram-positive and Gram-negative bacteria, as well as Candida spp. strains. Our results showed that thiosemicarbazide derivatives exhibited varied activity against Gram-positive bacteria, with MIC values ranging from 31.25 to 1000 µg/mL. The hydrazone derivatives, however, did not display significant antibacterial activity. These findings suggest that structural modifications can significantly influence the antimicrobial efficacy of imidazole derivatives, highlighting the potential of thiosemicarbazide derivatives as promising candidates for further development in antibacterial therapies. Additionally, the cytotoxic activity against four cancer cell lines was evaluated. Two derivatives of hydrazide-hydrazone showed moderate anticancer activity.

ε-Poly-l-lysine: A Naturally Occurring Biodegradable Polypeptide for Selective Detection of 5-Nitroimidazole Antibiotics in Animal Products and Living Cells via Fluorescence.

The 5-nitroimidazole (5-NI) class of antibiotics, such as metronidazole, ornidazole, secnidazole, and tinidazole, are widely used to prevent bacterial infection in humans and livestock industries. However, their overuse contaminates the farmed animal products and water bodies. Hence, a selective, sensitive, and cost-effective method to detect 5-NI antibiotics is the need of the hour. Herein, we report a rapid, inexpensive, and efficient sensing system to detect 5-NI drugs using an as-prepared solution of ε-poly-l-lysine (ε-PL), a naturally occurring and biodegradable homopolypeptide that has an intrinsic fluorescence via clustering-triggered emission. The low nanomolar detection limit (3.25-3.97 nM) for the aforementioned representative 5-NI drugs highlights the sensitivity of the system, outperforming most of the reported sensors alike. The resulting fluorescence quenching was found to be static in nature. Importantly, excellent recovery (100.26-104.41%) was obtained for all real samples and animal products tested. Visual detection was demonstrated by using paper strips and silica gel for practical applications. Furthermore, ε-PL could detect 5-NI antibiotics in living 3T3-L1 mouse fibroblast cells via cellular imaging. Taken together, the present work demonstrates the detection of 5-NI antibiotics using a biocompatible natural polypeptide, ε-PL, and represents a simple and inexpensive analytical tool for practical application.

Structural Evaluation of a Nitroreductase Engineered for Improved Activation of the 5-Nitroimidazole PET Probe SN33623.

Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell ablation models. We recently engineered a nitroreductase (E. coli NfsB F70A/F108Y) for the substantially enhanced reduction of the 5-nitroimidazole PET-capable probe, SN33623, which permits the theranostic imaging of vectors labeled with oxygen-insensitive bacterial nitroreductases. This mutant enzyme also shows improved activation of the DNA-alkylation prodrugs CB1954 and metronidazole. To elucidate the mechanism behind these enhancements, we resolved the crystal structure of the mutant enzyme to 1.98 Å and compared it to the wild-type enzyme. Structural analysis revealed an expanded substrate access channel and new hydrogen bonding interactions. Additionally, computational modeling of SN33623, CB1954, and metronidazole binding in the active sites of both the mutant and wild-type enzymes revealed key differences in substrate orientations and interactions, with improvements in activity being mirrored by reduced distances between the N5-H of isoalloxazine and the substrate nitro group oxygen in the mutant models. These findings deepen our understanding of nitroreductase substrate specificity and catalytic mechanisms and have potential implications for developing more effective theranostic imaging strategies in cancer treatment.

Activity of pyridyl-pyrazolone derivatives against Trypanosoma cruzi.

New affordable drugs are needed for the treatment of infection with the protozoan parasite Trypanosoma cruzi responsible for the Chagas disease (CD). Only two old drugs are currently available, nifurtimox and benznidazole (Bz) but they exhibit unwanted side effects and display a weak activity in the late chronic phase of the disease. In this context, we evaluated the activity of a series of aryl-pyrazolone derivatives against T cruzi, using both bloodstream trypomastigote and intracellular amastigote forms of the parasite. The test compounds originate from a series of anticancer agents targeting the immune checkpoint ligand PD-L1 and bear an analogy with known anti-trypanosomal pyrazolones. A first group of 6 phenyl-pyrazolones was tested, revealing the activity of a single pyridyl-pyrazolone derivative. Then a second group of 8 compounds with a common pyridyl-pyrazolone core was evaluated. The in vitro testing process led to the identification of two non-cytotoxic and highly potent molecules against the intracellular form of T. cruzi, with an activity comparable to Bz. Moreover, one compound revealed an activity largely superior to that of Bz against bloodstream trypomastigotes, while being non-cytotoxic (selectivity index >1000). Unfortunately, the compound showed little activity in vivo, most likely due to its very limited plasma stability. However, the study opens novel perspectives for the design of new anti-trypanosomal products and the mechanism of action of the compounds is discussed.

New 2-nitroimidazole-N-acylhydrazones, analogs of benznidazole, as anti-Trypanosoma cruzi agents.

Chagas disease is a neglected tropical parasitic disease caused by the protozoan Trypanosoma cruzi. Worldwide, an estimated 8 million people are infected with T. cruzi, causing more than 10,000 deaths per year. Currently, only two drugs, nifurtimox and benznidazole (BNZ), are approved for its treatment. However, both are ineffective during the chronic phase, show toxicity, and produce serious side effects. This work aimed to obtain and evaluate novel 2-nitroimidazole-N-acylhydrazone derivatives analogous to BNZ. The design of these compounds used the two important pharmacophoric subunits of the BNZ prototype, the 2-nitroimidazole nucleus and the benzene ring, and the bioisosterism among the amide group of BNZ and N-acylhydrazone. The 27 compounds were obtained by a three-step route in 57%-98% yields. The biological results demonstrated the potential of this new class of compounds, since eight compounds were potent and selective in the in vitro assay against T. cruzi amastigotes and trypomastigotes using a drug-susceptible strain of T. cruzi (Tulahuen) (IC50 = 4.3-6.25 µM) and proved to be highly selective with low cytotoxicity on L929 cells. The type I nitroreductase (TcNTR) assay suggests that the new compounds may act as substrates for this enzyme.

Anti-trypanosomal Lignans Isolated from Salvadoran Peperomia pseudopereskiifolia.

A search for anti-trypanosomal natural compounds from plants collected in El Salvador, a country particularly endemic for Chagas disease, resulted in the isolation of five lignan-type compounds (1-5) from Peperomia pseudopereskiifolia. The lignan derivatives 1, 2, and 4 are new. Their absolute configuration was determined by chemical derivatization. Compounds 1, 5, 6, and 8 exhibited anti-trypanosomal activity against the amastigote form of T. cruzi comparable to that of the existing drug benznidazole.

Novel 4-nitroimidazole analogues: synthesis, in vitro biological evaluation, in silico studies, and molecular dynamics simulation.

A new series of 4-nitroimidazole bearing aryl piperazines 7-16, tetrazole 17 and 1,3,4-thiadiazole 18 derivatives was synthesized. All derivatives were screened for their anticancer activity against eight diverse human cancer cell lines (Capan-1, HCT-116, LN229, NCI-H460, DND-41, HL-60, K562, and Z138). Compound 17 proved the most potent compound of the series inhibiting proliferation of most of the selected human cancer cell lines with IC50 values in the low micromolar range. In addition, compound 11 exhibited IC50 values ranging 8.60-64.0 µM against a selection of cancer cell lines. These findings suggest that derivative 17 can potentially be a new lead compound for further development of novel antiproliferative agents. Additionally, 17-18 were assessed for their antibacterial and antituberculosis activity. Derivatives 17 and 18 were the most potent compounds of this series against both Staphylococcus aureus strain Wichita and a methicillin resistant strain of S. aureus (MRSA), as well as against Mycobacterium tuberculosis strain mc26230. The antiviral activity of 7-18 was also evaluated against diverse viruses, but no activity was detected. The docking study of compound 17 with putative protein targets in acute myeloid leukemia had been studied. Furthermore, the molecular dynamics simulation of 17 and 18 had been investigated.

Preparation and bioevaluation of a novel 99mTc-labelled propylene amine oxime (PnAO) containing two 4-methyl-2-nitroimidazole groups as a promising tumor hypoxia imaging agent.

Hypoxia is a common phenomenon in solid tumors, and its presence inhibits the efficacy of tumor chemotherapy and radiotherapy. Accurate measurement of hypoxia before tumor treatment is essential. Three propylene amine oxime (PnAO) derivatives with different substituents attached to 2-nitroimidazole were synthesized in the work, they are 3,3,9,9-tetramethyl-1,11-bis(4-bromo-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dione dioxime (Br2P2), 3,3,9,9-tetramethyl-1,11-bis(4-methyl-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dione dioxime (Me2P2) and 3,3,9,9-tetramethyl-1,11-bis(4,5-dimethyl-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dione dioxime (2Me2P2). The three compounds were radiolabeled with 99mTc to give three complexes([99mTc]Tc-Br2P2, [99mTc]Tc-Me2P2 and [99mTc]Tc-2Me2P2) with good in vitro stability. [99mTc]Tc-Me2P2 with a more suitable reduction potential had the highest hypoxic cellular uptake, compared with [99mTc]Tc-2P2 that have been previously reported, [99mTc]Tc-Br2P2 and [99mTc]Tc-2Me2P2. Biodistribution results in S180 tumor-bearing mice demonstrated that [99mTc]Tc-Me2P2 had the highest tumor-to-muscle (T/M) ratio (12.37 ± 1.16) at 2 h in the four complexes. Autoradiography and immunohistochemical staining results revealed that [99mTc]Tc-Me2P2 specifically targeted tumor hypoxic regions. The SPECT/CT imaging results showed that [99mTc]Tc-Me2P2 could target the tumor site. [99mTc]Tc-Me2P2 may become a potential hypoxia imaging agent.

Pimonidazole-alkyne conjugate for sensitive detection of hypoxia by Cu-catalyzed click reaction.

Hypoxia is involved in various diseases, such as cancers. Pimonidazole has often been used as the gold-standard marker to visualize hypoxic regions. Pimonidazole labels hypoxic regions by forming a covalent bond with a neighboring protein under hypoxic conditions in the body, which is detected by immunohistochemistry performed on tissue sections. To date, some pimonidazole-fluorophore conjugates have been reported as fluorescent probes for hypoxia imaging that do not require immunostaining. They are superior to pimonidazole because immunostaining can produce high background signals. However, large fluorophores in the conjugates may alter the original biodistribution and reactivity. Here, we report a new hypoxia marker, Pimo-yne, as a pimonidazole-alkyne conjugate. Pimo-yne has a similar hypoxia detection capability as pimonidazole because the alkyne tag is small and can be detected by Cu-catalyzed click reaction with azide-tagged fluorescent dyes. We successfully visualized hypoxic regions in tumor tissue sections using Pimo-yne with reduced background signals. The detected regions overlapped well with those detected by pimonidazole immunohistochemistry. To further reduce the background, we employed a turn-on azide-tagged fluorescent dye.

Heterobifunctional Cross-Linker with Dinitroimidazole and N-Hydroxysuccinimide Ester Motifs for Protein Functionalization and Cysteine-Lysine Peptide Stapling.

A heterobifunctional cross-linker with one sulfhydryl-reactive dinitroimidazole end and another amine-reactive N-hydroxysuccinimide (NHS) ester end was designed and synthesized. The two motifs of this cross-linker, dinitroimidazole and NHS ester, proved to react with thiol and amine, respectively, in an orthogonal way. The cross-linker was further applied to construct stapled peptides of different sizes and mono- and dual functionalization (including biotinylation, PEGylation, and fluorescence labeling) of protein.

Comparison of three 18F-labeled 2-nitroimidazoles for imaging hypoxia in breast cancer xenografts: [18F]FBNA, [18F]FAZA and [18F]FMISO.

BACKGROUND: Tumour hypoxia is associated with increased metastasis, invasion, poor therapy response and prognosis. Most PET radiotracers developed and used for clinical hypoxia imaging belong to the 2-nitroimidazole family. Recently we have developed novel 2-nitroimidazole-derived PET radiotracer [18F]FBNA (N-(4-[18F]fluoro-benzyl)-2-(2-nitro-1H-imidazol-1-yl)-acet-amide), an 18F-labeled analogue of antiparasitic drug benznidazole. The present study aimed to analyze its radio-pharmacological properties and systematically compare its PET imaging profiles with [18F]FMISO and [18F]FAZA in preclinical triple-negative (MDA-MB231) and estrogen receptor-positive (MCF-7) breast cancer models. METHODS: In vitro cellular uptake experiments were carried out in MDA-MB321 and MCF-7 cells under normoxic and hypoxic conditions. Metabolic stability in vivo was determined in BALB/c mice using radio-TLC analysis. Dynamic PET experiments over 3 h post-injection were performed in MDA-MB231 and MCF-7 tumour-bearing mice. Those PET data were used for kinetic modelling analysis utilizing the reversible two-tissue-compartment model. Autoradiography was carried out in tumour tissue slices and compared to HIF-1α immunohistochemistry. Detailed ex vivo biodistribution was accomplished in BALB/c mice, and this biodistribution data were used for dosimetry calculation. RESULTS: Under hypoxic conditions in vitro cellular uptake was elevated in both cell lines, MCF-7 and MDA-MB231, for all three radiotracers. After intravenous injection, [18F]FBNA formed two radiometabolites, resulting in a final fraction of 65 ± 9 % intact [18F]FBNA after 60 min p.i. After 3 h p.i., [18F]FBNA tumour uptake reached SUV values of 0.78 ± 0.01 in MCF-7 and 0.61 ± 0.04 in MDA-MB231 tumours (both n = 3), representing tumour-to-muscle ratios of 2.19 ± 0.04 and 1.98 ± 0.15, respectively. [18F]FMISO resulted in higher tumour uptakes (SUV 1.36 ± 0.04 in MCF-7 and 1.23 ± 0.08 in MDA-MB231 (both n = 4; p < 0.05) than [18F]FAZA (0.66 ± 0.11 in MCF-7 and 0.63 ± 0.14 in MDA-MB231 (both n = 4; n.s.)), representing tumour-to-muscle ratios of 3.24 ± 0.30 and 3.32 ± 0.50 for [18F]FMISO, and 2.92 ± 0.74 and 3.00 ± 0.42 for [18F]FAZA, respectively. While the fraction per time of radiotracer entering the second compartment (k3) was similar within uncertainties for all three radiotracers in MDA-MB231 tumours, it was different in MCF-7 tumours. The ratios k3/(k3 + k2) and K1*k3/(k3 + k2) in MCF-7 tumours were also significantly different, indicating dissimilar fractions of radiotracer bound and trapped intracellularly: K1*k3/(k2 + k3) [18F]FMISO (0.0088 ± 0.001)/min, n = 4; p < 0.001) > [18F]FAZA (0.0052 ± 0.002)/min, n = 4; p < 0.01) > [18F]FBNA (0.003 ± 0.001)/min, n = 3). In contrast, in MDA-MB231 tumours, only K1 was significantly elevated for [18F]FMISO. However, this did not result in significant differences for K1*k3/(k2 + k3) for all three 2-nitroimidazoles in MDA-MB231 tumours. CONCLUSION: Novel 2-nitroimidazole PET radiotracer [18F]FBNA showed uptake into hypoxic breast cancer cells and tumour tissue presumably associated with elevated HIF1-α expression. Systematic comparison of PET imaging performance with [18F]FMISO and [18F]FAZA in different types of preclinical breast cancer models revealed a similar tumour uptake profile for [18F]FBNA with [18F]FAZA and, despite its higher lipophilicity, still a slightly higher muscle tissue clearance compared to [18F]FMISO.

Improving tumor/muscle and tumor/blood ratios of 99mTc-labeled nitroimidazole propylene amine oxime (PnAO) complexes with ethylene glycol linkers.

Three nitroimidazole propylene amine oxime (PnAO) derivatives with different lengths of ethylene glycol chain were synthesized and radiolabeled with 99mTc. The radiochemical purities of three 99mTc-labeled complexes, oxo[[6,6,12,12-tetramethyl-1,17-bis(2-nitro-1H-imidazol-1-yl)-3,15-dioxa-7,11-diazaheptadecane-5, 13-dione dioximato] (3-)-N,N',N'',N''']-technetium-99m (99mTc-2P2O1), oxo[[9,9,15,15-tetramethyl-1,23-bis(2-nitro-1H-imidazol-1-yl)-3,6,18,21-tetraoxa-10, 14-diazatricosane-8,16-dione dioximato] (3-)-N,N',N'',N''']-technetium-99m (99mTc-2P2O2) and oxo[[15,15,21,21-tetramethyl-1,35-bis(2-nitro-1H-imidazol-1-yl)-3,6,9,12,24,27,30,33-octaoxa-16,20-diazapentatriacontane-14,22-dione dioximato] (3-)-N,N',N'',N''']-technetium-99m (99mTc-2P2O4), were above 90%, and they were all stable both in vitro and in vivo. The hypoxia/normoxia uptake ratios of the three complexes were 2.92 ± 0.61, 2.63 ± 0.64 and 2.29 ± 0.67 in S180 cellular uptake assay (4 h). All of these complexes presented good hypoxia selectivity. The results of biodistribution studies in S180 tumor-bearing mice revealed that the tumor/muscle (T/M) ratios (7.20 ± 2.37, 7.19 ± 1.75, 5.56 ± 1.10) and tumor/blood (T/B) ratios (1.66 ± 0.34, 1.73 ± 0.25, 2.13 ± 0.19) at 4 h of three complexes were significantly higher than those of 99mTc-2P2 (3.24 ± 0.65, 0.81 ± 0.34) without the ethylene glycol chains. Among them, 99mTc-2P2O4 had the best T/B ratio. The new complexes have higher tumor/blood and tumor/muscle ratios by adding suitable length of ethylene glycol chain. It is helpful for the design and optimization of hypoxic imaging agents.

Molecular dynamic assisted investigation on impact of mutations in deazaflavin dependent nitroreductase against pretomanid: a computational study.

In the past decade, TB drugs belonging to the nitroimidazole class, pretomanid and delamanid, have been authorised to treat MDR-TB and XDR-TB. With a novel inhibition mechanism and a reduction in the span of treatment, it is now being administered in various combinations. This approach is not the ultimate remedy since the target protein Deazaflavin dependent nitroreductase (Ddn) has a high mutation frequency, and already pretomanid resistant clinical isolates are reported in various studies. Ddn is essential for M.tuberculosis to emerge from hypoxia, and point mutations in critical residues confer resistance to Nitro-imidazoles. Among the pool of available mutants, we have selected seven mutants viz DdnL49P, DdnY65S, DdnS78Y, DdnK79Q, DdnW88R, DdnY133C, and DdnY136S, all of which exhibited resistance to pretomanid. To address this issue, through computational study primarily by MD simulation, we attempted to elucidate these point mutations' impact and investigate the resistance mechanism. Hence, the DdnWT and mutant (MT) complexes were subjected to all-atom molecular dynamics (MD) simulations for 100 ns. Interestingly, we observed the escalation of the distance between cofactor and ligand in some mutants, along with a significant change in ligand conformation relative to the DdnWT. Moreover, we confirmed that mutations rendered ligand instability and were ejected from the binding pocket as a result. In conclusion, the results obtained provide a new structural insight and vital clues for designing novel inhibitors to combat nitroimidazole resistanceCommunicated by Ramaswamy H. Sarma.

Reactive Molecular Dynamics Simulation of the Structural Damages of the B-DNA Induced by the Oxidation/Nitration of Guanine.

Reactive molecular dynamics simulations (RMD) have been carried out to investigate structural alterations of the dodecamer double-strand B-DNA due to the oxidation/nitration modifications introduced to its guanine bases, including 8-oxoguanine, 8-nitroguanine, and 5-guanidino-4-nitroimidazole, considering two distribution patterns. These modifications may arise in the case of cancer treatment using oxidative/nitrosative reactive nitrogen species as anticancer agents. Results show that these mutations affect structural characteristics of the B-DNA dodecamer in the order 8-nitroguanine > 5-guanidino-4-nitroimidazole ≫ 8-oxoguanine. For instance, the base-pair per turn for these modified B-DNA are changed respectively to 9.79, 10.88 and 10.58 from 10.51 in the native defect-free B-DNA, which is compatible with the experimental value of 10.10. In addition, these mutations allow more water molecules to diffuse into the dodecamer structure and consequently increase the possibility of the penetration of reactive and nonreactive species toward constituting nucleic base-pairs. The largest variation of the B-DNA structure is observed for the mutated B-DNA with 8-nitroguanine modifications applied to its separated CG base-pairs along the dodecamer chain. The structural changes introduced by these nitro-/oxo-modified guanine bases can be considered as a critical step in the damage of the DNA structure and alterations of its function.

Indolin-2-one Nitroimidazole Antibiotics Exhibit an Unexpected Dual Mode of Action.

Nitroimidazoles such as metronidazole are used as anti-infective drugs against anaerobic bacteria. Upon in vivo reduction of the nitro group, reactive radicals damage DNA and proteins in the absence of oxygen. Unexpectedly, a recent study of nitroimidazoles linked to an indolin-2-one substituent revealed potent activities against aerobic bacteria. This suggests a different, yet undiscovered mode of action (MoA). To decipher this MoA, we first performed whole proteome analysis of compound-treated cells, revealing an upregulation of bacteriophage-associated proteins, indicative of DNA damage. Since DNA binding of the compound was not observed, we applied activity-based protein profiling (ABPP) for direct target discovery. Labeling studies revealed topoisomerase IV, an essential enzyme for DNA replication, as the most enriched hit in pathogenic Staphylococcus aureus cells. Subsequent topoisomerase assays confirmed the inhibition of DNA decatenation in the presence of indolin-2-one nitroimidazole with an activity comparable to ciprofloxacin, a known inhibitor of this enzyme. Furthermore, we determined significantly increased redox potentials of indolin-2-one nitroimidazoles compared to classic 5-nitroimidazoles such as metronidazole, which facilitates in vivo reduction. Overall, this study unravels that indolin-2-one-functionalized nitroimidazoles feature an unexpected dual MoA: first, the direct inhibition of the topoisomerase IV and second the classic nitroimidazole MoA of reductive bioactivation leading to damaging reactive species. Importantly, this dual MoA impairs resistance development. Given the clinical application of this compound class, the new mechanism could be a starting point to mitigate resistance.

Observation of Transient Anions That Do Not Decay through Dissociative Electron Attachment: New Pathways for Radiosensitization.

Low-energy electrons (LEEs) can very efficiently induce bond breaking via dissociative electron attachment (DEA). While DEA is ubiquitous, the importance of other reactions initiated by LEEs remains much more elusive. Here, we looked into this question by measuring highly accurate total cross sections for electron scattering from 1-methyl-5-nitroimidazole (1M5NI), a model radiosensitizer. The small uncertainty and high energy resolution allow us to identify many resonant features related to the formation of transient anions. In addition to novel insights about DEA reactions through the lower-lying resonances, our key finding is that the higher-lying resonances do not undergo DEA, implying alternative decay channels with significant cross sections. In particular, dissociation into two neutral fragments is probably involved in the case of 1M5NI. This finding has direct implications for the understanding of LEE-induced chemistry, particularly in the fundamental processes underlying the radiosensitization activity.

Mitochondria-targeting multifunctional nanoplatform for cascade phototherapy and hypoxia-activated chemotherapy.

Despite considerable progress has been achieved in hypoxia-associated anti-tumor therapy, the efficacy of utilizing hypoxia-activated prodrugs alone is not satisfied owing to the inadequate hypoxia within the tumor regions. In this work, a mitochondrial targeted nanoplatform integrating photodynamic therapy, photothermal therapy and hypoxia-activated chemotherapy has been developed to synergistically treat cancer and maximize the therapeutic window. Polydopamine coated hollow copper sulfide nanoparticles were used as the photothermal nanoagents and thermosensitive drug carriers for loading the hypoxia-activated prodrug, TH302, in our study. Chlorin e6 (Ce6) and triphenyl phosphonium (TPP) were conjugated onto the surface of the nanoplatform. Under the action of TPP, the obtained nanoplatform preferentially accumulated in mitochondria to restore the drug activity and avoid drug resistance. Using 660 nm laser to excite Ce6 can generate ROS and simultaneously exacerbate the cellular hypoxia. While under the irradiation of 808 nm laser, the nanoplatform produced local heat which can increase the release of TH302 in tumor cells, ablate cancer cells as well as intensify the tumor hypoxia levels. The aggravated tumor hypoxia then significantly boosted the anti-tumor efficiency of TH302. Both in vitro and in vivo studies demonstrated the greatly improved anti-cancer activity compared to conventional hypoxia-associated chemotherapy. This work highlights the potential of using a combination of hypoxia-activated prodrugs plus phototherapy for synergistic cancer treatment.

Synthesis and bioevaluation of the cyclopentadienyl tricarbonyl technetium-99m 2-nitroimidazole derivatives for tumor hypoxia imaging.

Hypoxia imaging agents can play an important role in the tumor treatment by avoiding the worse effect of radiotherapy and chemotherapy due to the tumor hypoxia. Due to the small size and easy coordination, tricarbonyl technetium-99m can be used to label a wide range of imaging agents. In this work, the tricarbonyl 99mTc labeled small-sized hypoxia imaging agents containing 2-nitroimidazoles were prepared, which have different carbon chain lengths between cyclopentadienyl and 2-nitroimidazole, and which have one or two 2-nitroimidazole groups. The results of S180 cell experiment and biodistribution indicated that these molecules have different hypoxic selectivity. When contains one 2-nitroimidazole, as the carbon chain lengthens, which means the molecular volume becomes larger, hypoxia cellular uptake and selectivity decrease in S180 cell uptake experiment. In biodistribution study in mice bearing S180 tumor, Tc-2 (1-cyclopentadienyl-5-(2-nitro-1H-imidazol-1-yl)-pentan-1-one tricarbonyl 99mTc complex), which has intermediate carbon chain, is better due to the more complex factors. Its tumor/blood (T/B) ratio is 3.56 ± 0.25, tumor/muscle(T/M) ratio is 1.73 ± 0.29 and tumor uptake is 2.23 ± 0.24%ID/g at 2 h. Comparing to other tricarbonyl technetium complexes containing one 2-nitroimidazole, the complexes in this work have an advantage in tumor/blood ratio and tumor uptake. This suggests that the small-volume cyclopentadienyl may have an advantage when used as a ligand. When contains two 2-nitroimidazole groups, the complex, 1-cyclopentadienyl-5-di(2-(2-nitro-1H-imidazol-1-yl)ethyl)amino-pentan-1-one tricarbonyl 99mTc complex (Tc-4), has the better results in the cell experiment than those which contain one 2-nitroimidazole group. Thus the hypoxia imaging agent contains two 2-nitroimidazole groups is more advantageous, but further modifications of Tc-4 are needed to improve its clearance rate in the blood, because the increased lipophilicity leads to a decrease in the T/B ratio of Tc-4. In conclusion, small volume hypoxia imaging agents with two 2-nitroimidazole groups may be the trend of development.

Noncanonical Amino Acids for Hypoxia-Responsive Peptide Self-Assembly and Fluorescence.

Design of endogenous stimuli-responsive amino acids allows for precisely modulating proteins or peptides under a biological microenvironment and thereby regulating their performance. Herein we report a noncanonical amino acid 2-nitroimidazol-1-yl alanine and explore its functions in creation of the nitroreductase (NTR)-responsive peptide-based supramolecular probes for efficient hypoxia imaging. On the basis of the reduction potential of the nitroimidazole unit, the amino acid was synthesized via the Mitsunobu reaction between 2-nitroimidazole and a serine derivate. We elucidated the relationship between the NTR-responsiveness of the amino acid and the structural feature of peptides involving a series of peptides. This eventually facilitates development of aromatic peptides undergoing NTR-responsive self-assembly by rationally optimizing the sequences. Due to the intrinsic role of 2-nitroimidazole in the fluorescence quench, we created a morphology-transformable supramolecular probe for imaging hypoxic tumor cells based on NTR reduction. We found that the resulting supramolecular probes penetrated into solid tumors, thus allowing for efficient fluorescence imaging of tumor cells in hypoxic regions. Our findings demonstrate development of a readily synthesized and versatile amino acid with exemplified properties in creating fluorescent peptide nanostructures responsive to a biological microenvironment, thus providing a powerful toolkit for synthetic biology and development of novel biomaterials.

Synthesis and evaluation of multivalent nitroimidazole-based near-infrared fluorescent agents for neuroblastoma and colon cancer imaging.

Hypoxia is one of key characteristics of microenvironments of solid tumors, and evaluation of hypoxia status in solid tumors is important to determine cancer stage and appropriate treatment. In the present study, novel, multivalent, near-infrared (NIR) fluorescent imaging agents were developed to measure tumor hypoxia. These agents were synthesized using an amino acid as a backbone to connect mono-, bis-, or tris-2-nitroimidazole as a hypoxia-sensitive moiety to enhance uptake by the tumor and to attach sulfo-Cyanine 5.5 as an NIR fluorophore to visualize tumor accumulation. Studies of physical characteristics demonstrated that the novel NIR imaging agents showed suitable optical properties for in vitro and in vivo imaging and were stable in serum. In vitro cellular uptake studies in SK-N-BE(2) and SW620 cell lines demonstrated that NIR imaging agents bearing 2-nitroimidazole structures showed significantly higher tumor uptake in hypoxic cells than in normoxic cells. Moreover, in vivo optical imaging studies using SK-N-BE(2) and SW620 xenografted mice demonstrated that novel, multivalent, 2-nitroimadazole NIR imaging agents with two or three 2-nitroimidazole moieties showed higher uptake in tumor than the control agents with only one 2-nitroimidazole. These observations suggest that novel, multivalent, NIR agents could serve as potential optical imaging agents for evaluating tumor hypoxia.