Synthesis and Characterization of Sulfonamide-Containing Naphthalimides as Fluorescent Probes.

A tumor-targeting fluorescent probe has attracted increasing interest in fluorescent imaging for the noninvasive detection of cancers in recent years. Sulfonamide-containing naphthalimide derivatives (SN-2NI, SD-NI) were synthesized by the incorporation of N-butyl-4-ethyldiamino-1,8-naphthalene imide (NI) into sulfonamide (SN) and sulfadiazine (SD) as the tumor-targeting groups, respectively. These derivatives were further characterized by mass spectrometry (MS), nuclear magnetic resonance spectroscopy (1H NMR), Fourier transform infrared spectroscopy (FT-IR), ultraviolet-visible spectroscopy (UV), and a fluorescence assay. In vitro properties, including cell cytotoxicity and the cell uptake of tumor cells, were also evaluated. Sulfonamide-containing naphthalimide derivatives possessed low cell cytotoxicity to B16F10 melanoma cells. Moreover, SN-2NI and SD-NI can be taken up highly by B16F10 cells and then achieve good green fluorescent images in B16F10 cells. Therefore, sulfonamide-containing naphthalimide derivatives can be considered to be the potential probes used to target fluorescent imaging in tumors.

Synthesis and Biological Properties of Fluorescent Strigolactone Mimics Derived from 1,8-Naphthalimide.

Strigolactones (SLs) have potential to be used in sustainable agriculture to mitigate various stresses that plants have to deal with. The natural SLs, as well as the synthetic analogs, are difficult to obtain in sufficient amounts for practical applications. At the same time, fluorescent SLs would be useful for the mechanistic understanding of their effects based on bio-imaging or spectroscopic techniques. In this study, new fluorescent SL mimics containing a substituted 1,8-naphthalimide ring system connected through an ether link to a bioactive furan-2-one moiety were prepared. The structural, spectroscopic, and biological activity of the new SL mimics on phytopathogens were investigated and compared with previously synthetized fluorescent SL mimics. The chemical group at the C-6 position of the naphthalimide ring influences the fluorescence parameters. All SL mimics showed effects similar to GR24 on phytopathogens, indicating their suitability for practical applications. The pattern of the biological activity depended on the fungal species, SL mimic and concentration, and hyphal order. This dependence is probably related to the specificity of each fungal receptor-SL mimic interaction, which will have to be analyzed in-depth. Based on the biological properties and spectroscopic particularities, one SL mimic could be a good candidate for microscopic and spectroscopic investigations.

Design, synthesis and structure-activity relationship of 1,8-naphthalimide derivatives as highly potent hCYP1B1 inhibitors.

Human cytochrome P450 1B1 enzyme (hCYP1B1), a member of hCYP1 subfamily, plays a crucial role in multiple diseases by participating in many metabolic pathways. Although a suite of potent hCYP1B1 inhibitors have been previously reported, most of them also act as aryl hydrocarbon receptor (AhR) agonists that can up-regulate the expression of hCYP1B1 and then counteract their inhibitory potential in living systems. This study aimed to develop novel efficacious hCYP1B1 inhibitors that worked well in living cells but without AhR agonist effects. For these purposes, a series of 1,8-naphthalimide derivatives were designed and synthesized, and their structure-activity relationships (SAR) as hCYP1B1 inhibitors were analyzed. Following three rounds SAR studies, several potent hCYP1B1 inhibitors were discovered, among which compound 3n was selected for further investigations owing to its extremely potent anti-hCYP1B1 activity (IC50 = 0.040 nM) and its blocking AhR transcription activity in living cells. Inhibition kinetic analyses showed that 3n potently inhibited hCYP1B1 via a mix inhibition manner, showing a Ki value of 21.71 pM. Docking simulations suggested that introducing a pyrimidine moiety to the hit compound (1d) facilitated 3n to form two strong interactions with hCYP1B1/heme, viz., the C-Br⋯π halogen bond and the N-Fe coordination bond. Further investigations demonstrated that 3n (5 µM) could significantly reverse the paclitaxel (PTX) resistance in H460/PTX cells, evidenced by the dramatically reduced IC50 values, from 632.6 nM (PTX alone) to 100.8 nM (PTX plus 3n). Collectively, this study devised a highly potent hCYP1B1 inhibitor (3n) without AhR agonist effect, which offered a promising drug candidate for overcoming hCYP1B1-associated drug resistance.

Identification of naphthalimide-derivatives as novel PBD-targeted polo-like kinase 1 inhibitors with efficacy in drug-resistant lung cancer cells.

Targeting polo-box domain (PBD) small molecule for polo-like kinase 1 (PLK1) inhibition is a viable alternative to target kinase domain (KD), which could avoid pan-selectivity and dose-limiting toxicity of ATP-competitive inhibitors. However, their efficacy in these settings is still low and inaccessible to clinical requirement. Herein, we utilized a structure-based high-throughput virtual screen to find novel chemical scaffold capable of inhibiting PLK1 via targeting PBD and identified an initial hit molecule compound 1a. Based on the lead compound 1a, a structural optimization approach was carried out and several series of derivatives with naphthalimide structural motif were synthesized. Compound 4Bb was identified as a new potent PLK1 inhibitor with a KD value of 0.29 µM. 4Bb could target PLK1 PBD to inhibit PLK1 activity and subsequently suppress the interaction of PLK1 with protein regulator of cytokinesis 1 (PRC1), finally leading to mitotic catastrophe in drug-resistant lung cancer cells. Furthermore, 4Bb could undergo nucleophilic substitution with the thiol group of glutathione (GSH) to disturb the redox homeostasis through exhausting GSH. By regulating cell cycle machinery and increasing cellular oxidative stress, 4Bb exhibited potent cytotoxicity to multiple cancer cells and drug-resistant cancer cells. Subcutaneous and oral administration of 4Bb could effectively inhibit the growth of drug-resistant tumors in vivo, doubling the survival time of tumor bearing mice without side effects in normal tissues. Thus, our study offers an orally-available, structurally-novel PLK1 inhibitor for drug-resistant lung cancer therapy.

Identification of a novel antifungal backbone of naphthalimide thiazoles with synergistic potential for chemical and dynamic treatment.

Aim: The high incidence and prevalence of fungal infections call for new antifungal drugs. This work was to develop naphthalimide thiazoles as potential antifungal agents. Results & methodology: These compounds showed significant antifungal potency toward some tested fungi. Especially, naphthalimide thiazole 4h with excellent anti-Candida tropicalis efficacy possessed good hemolysis level, low toxicity and no obvious resistance. Deciphering the mechanism showed that 4h interacted with DNA and disrupted the antioxidant defense system of C. tropicalis. Compound 4h also triggered membrane depolarization, leakage of cytoplasmic contents and LDH inhibition. Simultaneously, 4h rendered metabolic inactivation and eradicated the formed biofilms of C. tropicalis. Conclusion: The multifaceted synergistic effect initiated by naphthalimide thiazoles is a reasonable treatment window for prospective development.

Naphthalimide-phenanthroimidazole incorporated new fluorescent sensor for "turn-on" Cu2+ detection in living cancer cells.

In recent years, fluorescent sensors have emerged as attractive imaging probes due to their distinct responses toward bio-relevant metal ions. However, the bioimaging application main barrier is the 'turn-off' response toward paramagnetic metal ions such as Cu2+ under physiological conditions. Herein, we report a new sensor (2-methyl(4-bromo-N-ethylpiperazinyl-1,8-naphthalimido)-4-(1H-phenanthro[9,10-d]imidazole-2-yl) phenol)NPP with multifunctional (Naphthalimide, Piperazine, Phenanthroimidazole) units for fluorescent and colourimetric detection of Cu2+ in an aqueous medium. Both absorption and fluorescence spectral titration strategies were used to monitor the Cu2+-sensing property of NPP. The NPP displays a weak emission at ca. 455 nm, which remarkably enhances (⁓3.2-fold) upon selective binding of Cu2+ over a range of metal ions, including other paramagnetic metal ions (Mn2+, Fe3+, Co2+). The stoichiometry, binding constant (Ka) and the LOD (limit of detection) of NPP toward Cu2+ ions were found to be 1:1, 5.0 (± 0.2) × 104 M-1 and 6.5 (± 0.4) × 10-7 M, respectively. We have also used NPP as a fluorescent probe to detect Cu2+ in live (human cervical HeLa) cancer cells. The emission intensity of NPP was almost recovered in HeLa cells by incubating 'in situ' the derived Cu2+ complex (NPP-Cu2+) in the presence of a benchmark chelating agent such as EDTA (ethylenediaminetetraacetate). The fluorescent emission of NPP was reverted significantly in each cycle upon sequencial addition of Cu2+ and EDTA to the NPP solution. Overall, NPP is a novel, simple, economic and portable sensor that can detect Cu2+ in biological and environmental scenarios.

Design, Synthesis, and Evaluation of Novel 3-Carboranyl-1,8-Naphthalimide Derivatives as Potential Anticancer Agents.

We synthesized a series of novel 3-carboranyl-1,8-naphthalimide derivatives, mitonafide and pinafide analogs, using click chemistry, reductive amination and amidation reactions and investigated their in vitro effects on cytotoxicity, cell death, cell cycle, and the production of reactive oxygen species in a HepG2 cancer cell line. The analyses showed that modified naphthalic anhydrides and naphthalimides bearing ortho- or meta-carboranes exhibited diversified activity. Naphthalimides were more cytotoxic than naphthalic anhydrides, with the highest IC50 value determined for compound 9 (3.10 µM). These compounds were capable of inducing cell cycle arrest at G0/G1 or G2M phase and promoting apoptosis, autophagy or ferroptosis. The most promising conjugate 35 caused strong apoptosis and induced ROS production, which was proven by the increased level of 2'-deoxy-8-oxoguanosine in DNA. The tested conjugates were found to be weak topoisomerase II inhibitors and classical DNA intercalators. Compounds 33, 34, and 36 fluorescently stained lysosomes in HepG2 cells. Additionally, we performed a similarity-based assessment of the property profile of the conjugates using the principal component analysis. The creation of an inhibitory profile and descriptor-based plane allowed forming a structure-activity landscape. Finally, a ligand-based comparative molecular field analysis was carried out to specify the (un)favorable structural modifications (pharmacophoric pattern) that are potentially important for the quantitative structure-activity relationship modeling of the carborane-naphthalimide conjugates.

Synthesis, electrochemistry, DNA binding and in vitro cytotoxic activity of tripodal ferrocenyl bis-naphthalimide derivatives.

A series of tripodal ferrocenyl bis-naphthalimide derivatives were synthesized and characterized. All of the bis-naphthalimide derivatives exhibited good DNA binding ability which was confirmed by ethidium bromide (EB) displacement experiment and ultraviolet (UV)-visible absorption titration. And the binding mode of these compounds was proved to be a hybrid binding mode by experiments. The cytotoxicity of synthesized compounds against 4 different human cancer cell lines (EC109, BGC823, SGC7901 and HEPG2) was evaluated by thiazolyl blue tetrazolium bromide (MTT) assay. All of the bis-naphthalimide derivatives exhibited good anticancer activity than the positive control drug (amonafide), which was due to the promotion of reactive oxygen species (ROS) level in test cancer cells by the reversible one-electron redox process of ferrocenyl bis-naphthalimide derivatives. Although there was no obvious relationship between the binding constants and the chain length, the structure cytotoxicity relationship revealed that the linker of n = 3, m = 1 was the best choice for the tested tripodol bis-naphthalimide derivatives. SYNOPSIS: A series of tripodal ferrocenyl bis-naphthalimide derivatives were synthesized to study the DNA binding ability and the cytotoxicity induced by reactive oxygen species. All of the compounds exhibited good DNA binding ability. And the structure cytotoxicity relationship revealed that the structure of 5h was the best choice.

Design, synthesis and antitumor evaluation of new 1,8-naphthalimide derivatives targeting nuclear DNA.

Four series of new 3-nitro naphthalimides derivatives, 4(4a‒4f), 5(5a‒5i), 6(6a‒6e) and 7 (7a‒7j), were designed and synthesized as antitumor agents. Methyl thiazolyl tetrazolium (MTT) screening assay results revealed that some compounds displayed effective in vitro antiproliferative activity on SMMC-7721, T24, SKOV-3, A549 and MGC-803 cancer cell lines in comparison with 5-fluorouracil (5-FU), mitonafide and amonafide. Nude mouse xenotransplantation model assay results indicated that compounds 6b and 7b exhibited good in vivo antiproliferative activity in MGC-803 xenografts in comparison with amonafide and cisplatin, suggesting that compounds 6b and 7b could be good candidates for antitumor agents. Gel electrophoresis assay indicated that DNA and Topo I were the potential targets of compounds 6b and 7b, and comet assay confirmed that compounds 6b and 7b could induce DNA damage, while the further study showed that the 6b- and 7b-induced DNA damage was accompanied by the upregulation of p-ATM, P-Chk2, Cdc25A and p-H2AX. Cell cycle arrest studies demonstrated that compounds 6b and 7b arrested the cell cycle at the S phase, accompanied by the upregulation of the expression levels of the antioncogene p21 and the down-regulation of the expression levels of cyclin E. Apoptosis assays indicated that compounds 6b and 7b caused the apoptosis of tumor cells along with the upregulation of the expression of Bax, caspase-3, caspase-9 and PARP and the downregulation of Bcl-2. These mechanistic studies suggested that compounds 6b and 7b exerted their antitumor activity by targeting to DNA, thereby inducing DNA damage and Topo I inhibition, and consequently causing S stage arrest and the induction of apoptosis.

Directing charge transfer in perylene based light-harvesting antenna molecules.

Directing energy and charge transfer processes in light-harvesting antenna systems is quintessential for optimizing the efficiency of molecular devices for artificial photosynthesis. In this work, we report a novel synthetic method to construct two regioisomeric antenna molecules (1-D2A2 and 7-D2A2), in which the 4-(n-butylamino)naphthalene monoimide energy and electron donor is attached to the perylene monoimide diester (PMIDE) acceptor at the 1- and 7-bay positions, respectively. The non-symmetric structure of PMIDE renders a polarized distribution of the frontier molecular orbitals along the long axis of this acceptor moiety, which differentiates the electron coupling between the donor, attached at either the 1- or the 7-position, and the acceptor. We demonstrate that directional control of the photo-driven charge transfer process has been obtained by engineering the molecular structure of the light-harvesting antenna molecules.

Investigation of a Sensing Strategy Based on a Nucleophilic Addition Reaction for Quantitative Detection of Bisulfite (HSO3-).

A reaction-based sensor (NAS-1) showed a high affinity and sensitivity to HSO3- via a nucleophilic addition reaction in the aqueous media, giving dual signals from absorption and emission spectra. NAS-1 was successfully applied in RK13 epithelial cells to detect HSO3- in a cellular environment.

Synthesis of Nitro-Aryl Functionalised 4-Amino-1,8-Naphthalimides and Their Evaluation as Fluorescent Hypoxia Sensors.

Fluorescent sensors are a vital research tool, enabling the study of intricate cellular processes in a sensitive manner. The design and synthesis of responsive and targeted probes is necessary to allow such processes to be interrogated in the cellular environment. This remains a challenge, and requires methods for functionalisation of fluorophores with multiple appendages for sensing and targeting groups. Methods to synthesise more structurally complex derivatives of fluorophores will expand their potential scope. Most known 4-amino-1,8-naphthalimides are only functionalised at imide and 4-positions, and structural modifications at additional positions will increase the breadth of their utility as responsive sensors. In this work, methods for the incorporation of a hypoxia sensing group to 4-amino-1,8-naphthalimide were evaluated. An intermediate was developed that allowed us to incorporate a sensing group, targeting group, and ICT donor to the naphthalimide core in a modular fashion. Synthetic strategies for attaching the hypoxia sensing group and how they affected the fluorescence of the naphthalimide were evaluated by photophysical characterisation and time-dependent density functional theory. An extracellular hypoxia probe was then rationally designed that could selectively image the hypoxic and necrotic region of tumour spheroids. Our results demonstrate the versatility of the naphthalimide scaffold and expand its utility. This approach to probe design will enable the flexible, efficient generation of selective, targeted fluorescent sensors for various biological purposes.

Investigation of endogenous malondialdehyde through fluorescent probe MDA-6 during oxidative stress.

The malondialdehyde (MDA)-specific detection probe (MDA-6) was successfully synthesised through the photoinduced electron transfer (PET) mechanism which possesses many biological applications. In vivo biological applicability of this probe was proved in different cell lines, zebrafish and mice. In these models, the MDA was produced by oxygen stress injury and the relationship between MDA and probe were evaluated in vitro as well as in vivo under different stress conditions. After comparing evaluated results with commercial MDA kit, MDA-6 was concluded with high specificity, low limit of detection (0.03 µM), and can achieve micro-detection of MDA with low cytotoxicity, demonstrating MDA-6 enables safe and effective detection.

Live cell imaging by 3-imino-(2-phenol)-1,8-naphthalimides: The effect of ex vivo hydrolysis.

A series of 3-amino-N-substituted-1,8-naphthalimides and their salicylic Schiff base derivatives were synthesized. The structure of the obtained compounds was confirmed using 1H and 13C NMR, FT-IR spectroscopy and elemental analysis and COSY and HMQC for the representative molecules. The photophysical (UV-Vis, PL) and biological properties of all of the prepared compounds were studied. It was found that the amine with the n-hexyl group in EtOH had the highest PL quantum yield (Ф = 85%) compared to the others. Moreover, the chelating properties of the azomethines with the n-hexyl group (1a, 1b, 1c) were tested against various cations (Al3+, Ba2+, Co2+, Cu2+, Cr3+, Fe2+, Fe3+, Mn2+, Ni2+, Pb2+, Sr2+ and Zn2+) in an acetonitrile, acetone and PBS/AC mixture. Compounds that contained the electron withdrawing groups (-Br, -I) had the ability to chelate most of the studied cations, while the unsubstituted derivative chelated only the trivalent cations such as Al3+, Cr3+ and Fe3+ in acetonitrile. The effect of the environment on the keto-enol tautomeric equilibrium was also demonstrated, especially in the case of the derivative with a bromine atom. The biological studies showed that the tested molecules had no cytotoxicity. Additionally, the ability to image intracellular organelles such as the mitochondria and endoplasmic reticulum was revealed. The crucial role of the hydrolysis of imines for cellular imaging was presented.

A naphthalimide derivative can release COS and form H2S in a light-controlled manner and protect cells against ROS with real-time monitoring ability.

As an important gasotransmitter, hydrogen sulfide having multiple biological roles cannot be easily probed in cells. In this study, a light controllable H2S donor, Nap-Sul-ONB, derived from naphthalimide was developed. Under the irradiation of 365 nm light, a readily controlled stimulus, the donor could release COS to form H2S and exhibit turn on fluorescence to indicate the release of payload and its cellular location. Besides, the ROS scavenging ability and cell protective effect of Nap-Sul-ONB against endogenous and exogenous ROS were studied. The results showed that upon 365 nm light irradiation, Nap-Sul-ONB could reduce the cellular ROS level and increase the survival rate of PMA-treated cells.

Naphthalimide Dyes with Orthogonal Functional Groups for "Click" Chemistry: Attachment to Solid Supports and Applications in Drug Allergy Diagnosis.

The preparation and characterization of new functional materials for sensing have an important role in clinical diagnosis. Monitoring the surface functionalization of functional material is crucial because the final sensing properties are affected by how the (bio)molecules are immobilized on the surface of solid supports. Here, a new approach for the preparation of functional materials for biomedical diagnosis was developed. This method employs a fluorescent dye comprising 4-amino-1,8-naphthalimide with two orthogonal functional groups suitable for click chemistry. The orthogonal reactivity of these groups allows the sequential functionalization of the fluorophore, firstly with (bio)molecules, and then binding of the (bio)molecule-naphthalimide macrostructure onto the surface of a solid support. The fluorescent properties confirm the immobilization of the (bio)molecule on the surface of the solid support, without requiring other indirect methods to verify the immobilization. These functional materials were tested successfully with sera of patients, thus proving their potential application for allergic drug diagnosis.

Synthesis of naphthalimide derivatives with potential anticancer activity, their comparative ds- and G-quadruplex-DNA binding studies and related biological activities.

Two new cytotoxic 1,8-naphthalimide derivatives have been synthesized and characterized. Their biological activities as cytotoxicity and antimicrobial activities and inhibitory activities against DNA-polymerase were evaluated. The interactions of compounds with double-stranded- and quadruple-DNA have been studied by UV-Vis, fluorescent intercalator displacement, competition dialysis, circular dichroism and the findings were compared with the parent naphthalimide and the other compounds. The results show that both compounds (1 and 2) and the parent compound NI have strong cytotoxic activities against Beas-2B, MCF-7, HepG2 and MDA-MB-231 cancer cell lines, antimicrobial activities against Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and inhibitory activities towards Taq-polymerase and transcriptase. These novel cationic compounds 1 and 2 can stabilize G-quadruplexes DNA according to thermal denaturation experiments, they change the 3D structure of the DNA (see details in CD experiments) and they exhibit different binding affinities for q-DNA and ds-DNA revealed by spectrophotometric titrations and competitive dialysis studies.

Selective Recognition of Herbicides in Water Using a Fluorescent Molecularly Imprinted Polymer Sensor.

Fluorescent molecularly imprinted polymer (FMIP) optosensor was utilized for the selective identification of 2,4-dichlorophenoxacetic acid (2,4-D) due to worldwide pollution caused by using herbicides in agricultural industry. In this regards, two derivatives of polymerizable 1,8-naphthalimide namely, 1,8-naphthalimide containing thiourea (NI) and diethyl amine tagged 1,8-naphthalimide (NII) were used as the receptors and 2,4-D was applied as a template. Also, precipitation polymerization was applied to prepare the fluorescent molecularly imprinted polymer (FMIP). The morphological, structural and thermal analysis was carried out using SEM, TEM, EDS, BET, FTIR, DSC and TGA for characterizing the fluorescent optosensor. The adsorption efficiency of FMIP and FNIP was studied using Langmuir, Freundlich, BET and Redlich Peterson isotherms. The results represented that the adsorption of 2,4-D on FMIP and FNIP agreed the Freundlich adsorption isotherm with correlation coefficient of 0.9935 and 0.9801, respectively. The prepared sensor was able for the selective determination of 2,4-D salt in the linear range of 5 × 10-7-1 × 10-3 M with a limit of detection of 16.8 nM. The present study revealed that the FMIP prepared by 1,8-naphthalimide derivative (NI) could potentially recognize the trace concentration of 2,4-D. Graphical Abstract Graphical abstract of flourescene switching mechanism in a fluorescent molecularly imprinted polymer sensor.

Dual-site fluorescent probe for multi-response detection of ClO- and H2O2 and bio-imaging.

Hypochlorite (ClO-) and hydrogen peroxide (H2O2) commonly coexist in organism and are involved in the same physiological and pathological processes. So it is of great importance to develop fluorescent probes to detect both simultaneously. Herein, we reported the first dual-site fluorescent probe (Geisha-1) for the quantitative detection of ClO- and H2O2. This probe is constructed by chemically grafting N,N-dimethylthiocarbamate and borate to a fluorescence resonance energy transfer (FRET) platform. As a result, Geisha-1 not only presents three different responses to ClO-, H2O2, and ClO- + H2O2 (the coexistence of ClO- and H2O2) with high sensitivity and selectivity, but also exhibits low toxicity and cell membrane and tissue permeability, and it was further successfully applied to image ClO- and H2O2 in living cells and tissues. Thus, Geisha-1 provides a promising application prospect in biological systems and an alternative strategy for the construction of dual-site fluorescent probes aiming at the multi-response detection of other biologically relevant analytes.

Decrease of Protein Vicinal Dithiols in Parkinsonism Disclosed by a Monoarsenical Fluorescent Probe.

Vicinal dithiol-containing proteins (VDPs) play an important role in maintaining the structures and functions of proteins mainly through the conversion between dithiols and disulfide bonds. The content of VDPs also reflects the redox status of an organism. To specifically and expediently detect VDPs, we developed a turn-on monoarsenical fluorescent probe (NEP) based on the intramolecular charge transfer mechanism. Naphthalimide was chosen as a fluorophore and linked with the receptor moiety (cyclic dithiarsolane) via carbamate segment. In the presence of VDPs, NEP displays a strong green fluorescence signal produced by the cyclic dithiarsolane cleavage and subsequent intramolecular cyclization to liberate the fluorophore. Furthermore, NEP exhibits high selectivity toward VDPs over other protein thiols and low molecular weight thiols. The favorable properties of NEP enable it readily to detect VDPs in live cells and in vivo. In addition, a remarkable decrease of VDPs in parkinsonism was disclosed for the first time, highlighting that regulating VDPs level has a therapeutic potential for parkinsonism.