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• Cancer is still an important factor endangering the safety of people all over the world, and there were nearly 20 million new cases in 2020 [1-4]. Early detection and treatment of cancer are important for improving the survival rate and the quality of life of tumor patients [5-8]. Fluorescent probes have prospects of being applied in early diagnosis because of their advantages in sensitivity and in situ detection [9-15]. Therefore, it is worthwhile to develop fluorescent reagents that can diagnose and treat tumor lesions at the same time [16-24].

  On one hand, the antitumor activities of naphthalimide derivatives was firstly discovered by Braña and his colleagues [25-28]. The representative compound Amonafide exhibited great antitumor activities both in vitro and in vivo (Fig. 1). However, due to the N-acetylation metabolism of the side chain in humans, Amonafide would cause high variable and unpredictable toxicity, which leads to great obstacles in phase III clinical trials [29]. Cyclization was an important structural modification strategy to avoid N-acetylation [30-36]. For example, R16 could inhibit the proliferation of a panel of human cancer cell lines and suppress tumor growth in mice implanted with S-180 sarcoma and H22 hepatoma. Though the antitumor activities in vitro were improved significantly, the solubility of these derivatives were reduced greatly, which would induce low yield of the target compounds and affect the antitumor efficacy in vivo [37].

  Figure 1

  

  Figure 1.  Structures of Amonafide, R16 and BODIPY derivatives 1 and 2.

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  On the other hand, naphthalimide and its analogues are conventional fluorescent dyes for fibers and wildly used as fluorescent probes [38-40]. The conjugated polycyclic systems of naphthalimide derivatives are the structural basis of chromophores and fluorophores, which have many applications in fluorescence sensing. As reported, rigidifying the structure of fluorophore can reduce the energy consumption from rotation and vibration, which can efficiently improve the fluorescence properties [41]. Many similar modified strategies are applied in BODIPY fluorescent dyes. For example, Mei et al. reported a selective and sensitive chemosensor (1) for Cu²⁺, which has longer wavelength through extending the rigidity of the BODIPY core [42]. Zhao and Carreira also reported a highly fluorescent (Φ), photostable aza-dipyrromethene dye (2) with very sharp and intense absorption (full width at half maximum height, fwhm = 30.4 nm; ε = 159 000) in the NIR region (λ_max = 740 nm) [43].

  Recently, our research group developed a class of naphthalimide derivatives modified with seven-membered heterocycles at the 4- and 5- positions [44]. These compounds exhibited excellent optical properties. Besides, we observed that these compounds have good solubility, which may overcome the solubility problems of cyclization. Therefore, we designed and synthesized more derivatives with different nitrogen-containing substituent groups. The newly-synthesized compounds were evaluated of their antitumor activities against leukemia and solid tumor cell lines compared with Amonafide. Besides, their potentials as theranostic agents were primarily explored.

  As shown in Scheme 1, the synthesis of the target molecule was similar to the scheme previously reported by our group. The intermediate 4-bromo-5-nitro-1,8-naphthalic anhydride was synthesized as we described before [44]. It was then substituted with different nitrogen-containing substituents and subjected to a ring-forming reaction at the 4,5-position with diethanolamine to yield Y1-Y8 and N,N'-bis(2-hydroxyethyl)ethylenediamine to yield Y9-Y16.

  Scheme 1

  

  Scheme 1.  Syntheses of target compounds Y1-Y16. (a) C₂H₅OH, reflux, 6 h; (b) CH₃OCH₂CH₂OH, reflux, 8 h; (c) K₂CO₃, CH₃OCH₂CH₂OH, reflux, 8 h.

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  The antiproliferative activity of compounds Y1-Y16 were evaluated against human cancer cell lines A549 (human non-small cell lung tumor cell), HL60 (Human promyelocytic leukemia cells). Amonafide were tested as a reference compound. In HL60 cells, N',N'-dimethylethane-1,2-diamine derivatives Y1 and Y9 exhibited great antitumor activities, which was consistent with previous reported naphthalimide derivatives (3.1 and 1.4 µmol/L, Table 1). The N',N'-diethyl analogues of Y1 and Y9 (Y2, Y10) exhibited relatively weaker antiproliferative activities (6.2 and 2.2 µmol/L). However, extention of the linker between the imide and the amino group to three carbon chain (Y3, Y4, Y11 and Y12) greatly decreased their antiproliferative activities. No obvious antitumor activities were observed for the four compounds in HL60 cells. Compounds increasing the rigidity of the amino group (Y5, Y6, Y13 and Y14) also exhibited potent antiproliferative activities. For example, the pyrrolidine and piperidine derivatives (Y5 and Y6) showed potent antitumor activities with IC₅₀ values at 1.9 and 3.4 µmol/L. Furthermore, compounds without amino side chain (Y7 and Y15) or with the methylpiperazine side chain (Y8 and Y16) did not display obvious antitumor activities. Similar structure-activity relationship was observed in A549 cells.

  Table 1

  

  Table 1.  The antiproliferative activities of compounds Y1-Y16.

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  Compounds Y1, Y5 and Y9 were then chosen and evaluated against more solid tumor cell lines, including Eca-109 (human esophageal cancer cell), HCT116 (human colon cancer cell), HT29 (human colon cancer cell), MDA-MB-231 (triple negative breast cancer cell). Amonafide was also tested as reference compounds. As shown in Table 2, compound Y9 was the most potent one of these compounds. It showed similar antiproliferative activities with Amonafide in HCT116, HT29 and MDA-MB-231 cells. Compounds Y1 and Y5 also displayed great antiproliferative activity with IC₅₀ at single digital micromolars in these tested cells except for Y5 in HT29 cells.

  Table 2

  

  Table 2.  The antiproliferative activities of compounds Y1, Y5 and Y9.

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  In our previous report, we found that the water solubility of the seven-membered ring-fused naphthalimide derivatives was greatly improved compared to previous heterocycle-fused naphthalimide derivatives [44]. Besides, according to the spectroscopic tests, the fluorescence quantum yields and molar extinction coefficients of these derivatives in aqueous solution were found to be much higher. As shown in Table 3 and Fig. S49 (Supporting information), it was found that the absorption wavelength of the asymmetric derivatives (Y1 and Y5) in organic phases was around 450 nm and the emission wavelength was around 500 nm. Therefore, the Stokes shift was around 50 nm. Besides, the fluorescence quantum yield was between 0.4 and 0.89. While in water, the absorption wavelength shifted to about 470 nm and the emission wavelength shifted to about 530 nm. What is more, the fluorescence quantum yield in water was still about 0.4–0.5, which was a very rare spectral property.

  Table 3

  

  Table 3.  The fluorescence properties of compounds Y1, Y5 and Y9.^a

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  The absorption wavalength of the symmetric compounds (Y9) in organic phases was around 470 nm, and the emission length had no obvious changes with its asymmetric analogues. The Stokes shift decreased to around 30 nm. The fluorescence quantum yield in organic solvents of Y9 was close to that of its asymmetric analogues. However, the fluorescence quantum yield of Y9 in the aqueous phase decreased to 0.12. Therefore, the representative compounds of the asymmetric series could exert green fluorescence in aqueous medium, which have advantages for cell staining and labelling in biological system. We hypothesize symmetric structures tend to form aggregates in aqueous solution more easily, so the fluorescence quantum yield of Y9 were much lower than that of Y1 and Y5 in aqueous solution.

  The representative compounds of the asymmetric derivatives were selected for further study. Based on the growth inhibitory activities of Y1 and Y5 in A549 and HL-60 cells, the effects of compounds on cell cycle progression were then investigated by analyzing cellular DNA content by flow cytometric.

  As shown in Fig. 2, cell cycle arrest in G2/M phase was found after treated with compounds at different concentrations (0, 1, 5, 10 µmol/L). Compared with the control group (4.57%), the proportion of A549 cells arrested at the G2/M phase increased to 7.21% and 6.81% with 10 µmol/L Y1 and Y5, respectively. Similarly, the percentage of HL-60 cells in the G2/M phase for control group is 6.45%, while treated with 10 µmol/L Y1 and Y5, the percentage of cells arrested at the G2/M phase increased to 15.4% and 17.56%.

  Figure 2

  

  Figure 2.  Compounds Y1 and Y5 induced G2/M arrest in A549 and HL-60 cells. A549 cells (A) and HL60 cells (B) were treated with 0, 1, 5, 10 µmol/L Y1 and Y5 for 24 h and analyzed by DNA flow cytometry.

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  To further validate the antitumor mechanism of action of compounds Y1 and Y5, the apoptosis rates were assessed. After incubated with different concentrations of compounds, cells were stained with Annexin V-Alexa Fluor 647 and PI which were used to assess the early and late apoptotic cell population, respectively. Apoptotic cells were observed in a dose-dependent manner when treated with increasing concentrations of Y1 and Y5 compared with the control group (Fig. 3). As illustrated in Fig. 3A, 20 µmol/L Y1 led to 32.18% of apoptotic A549 cells while 20 µmol/L Y5 induced 30.77% apoptosis cells, in comparison with 15.78% apoptosis rate for control group. Likewise, as shown in Fig. 3B, 20 µmol/L Y1 and Y5 induced the apoptosis of 78.11% and 81.98% HL-60 cells, respectively.

  Figure 3

  

  Figure 3.  Apoptosis ratio of the A549 and HL-60 cells. (A) A549 cells were treated with Y1 and Y5 at the indicated concentrations for 24 h. (B) HL-60 cells were treated with Y1 and Y5 at different concentrations. (C) Representative flow cytometric quantification of apoptotic cells.

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  In this paper, a series of seven-membered ring-fused naphthalimide derivatives were designed and synthesized. We evaluated their effects to inhibit A549 and HL60 tumor cell growth and summarized their structure-activity relationship. The N′,N′-dimethylethane-1,2-diamine derivatives Y1 and Y9 and their N-(2-aminoethyl)pyrrolidine analogue Y5 were proved to exhibit great antiproliferative activities in A549 and HL60 cells. They could also effectively inhibit growth of human esophageal, colon and breast cancer cells at single digital micromolar, which was similar with Amonafide. Besides, Y1, Y5 and Y9 were tested of their photophysical properties. The asymmetric derivatives (Y1 and Y5) have high fluorescence quantum yields in water, which is a rare property among naphthalimide derivatives. Furthermore, compounds Y1 and Y5 exerted their antitumor effects via G2/M phase cell cycle arrest and apoptosis induction in HL60 and A549 in dose dependent manner. Since seven-membered ring-fused naphthalimide derivatives possess great fluorescence quantum yields in water and potent anti-tumor activites at the same time, they have great potential to be used as theranostic agents for cancer.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  Acknowledgments

  We thank the National Natural Science Foundation of China (Nos. 21878088, 21476077), and Key Project of the Shanghai Science and Technology Committee (No. 18DZ1112703) for financial support.

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2022.07.039.

  
  
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• 

  Figure 1  Structures of Amonafide, R16 and BODIPY derivatives 1 and 2.

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  Scheme 1  Syntheses of target compounds Y1-Y16. (a) C₂H₅OH, reflux, 6 h; (b) CH₃OCH₂CH₂OH, reflux, 8 h; (c) K₂CO₃, CH₃OCH₂CH₂OH, reflux, 8 h.

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  Figure 2  Compounds Y1 and Y5 induced G2/M arrest in A549 and HL-60 cells. A549 cells (A) and HL60 cells (B) were treated with 0, 1, 5, 10 µmol/L Y1 and Y5 for 24 h and analyzed by DNA flow cytometry.

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  Figure 3  Apoptosis ratio of the A549 and HL-60 cells. (A) A549 cells were treated with Y1 and Y5 at the indicated concentrations for 24 h. (B) HL-60 cells were treated with Y1 and Y5 at different concentrations. (C) Representative flow cytometric quantification of apoptotic cells.

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  Table 1.  The antiproliferative activities of compounds Y1-Y16.

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  Table 2.  The antiproliferative activities of compounds Y1, Y5 and Y9.

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  Table 3.  The fluorescence properties of compounds Y1, Y5 and Y9.^a

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•