Synthesis and Biological Evaluation of Some 1,8-Naphthalimide-Acridinyl Hybrids

Guangxi University of Chinese Medicine, Nanning 530222, China School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China Center for Excellence in Post-Harvest Technologies, North Carolina A&T State University, )e North Carolina Research Campus, 500 Laureate Way, Kannapolis, NC 28081, USA Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization, Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences, Guilin, Guangxi 541006, China Guangxi Medical University, Nanning 530021, China


Introduction
Treatment of cancer remains a challenge for the global population, researchers, and the medical field. Antitumor agents with DNA intercalative properties have been widely studied. ey are characterized by the presence of a planar chromophore, a tri-or tetracyclic ring system, and one or two flexible substituent groups [1]. Typically, planar structure of DNA-intercalating agents can strongly bind to DNA, resulting in the death of cancer cells [2]. Acridine derivatives are extensively investigated as DNA intercalators because of their planar structure. Examples include nitracrine 1, m-AMSA 2, and DACA 3. Some of them are reported to be clinically useful as antitumor agents (Figure 1) [3][4][5]. Naphthalimides have been extensively investigated as anticancer agents. is is because they possess the desirable π-conjugated backbone with double amide moieties which can readily interact with various active targets. Significant examples include compounds such as amonafide 3, mitonafide 4, and DMP 840-bisnafide 5 ( Figure 2) [6]. is encourages our strong interest in modifying acridine ring or naphthalimide, for identifying potential novel antitumor drugs [7,8].
Topoisomerases have been indicated to be the necessary DNA targeting enzymes, that initially induce a cleavage of DNA strand, and follow by the reorganization and reconnection of the damaged DNA strand [9]. erefore, most useful DNA-intercalating agents were also related to the inhibition of DNA-topoisomerase I or II [1,[10][11][12]. Some acridine derivatives [13][14][15] and 1,8-naphthalene imide derivatives [16] could exert their cytotoxicity through suppressing DNA or RNA synthesis, thus causing the inhibition of DNA-topoisomerase I (Topo I) or topoisomerase II (Topo II). Reports indicate that the bis-derivatives which are constituted by two planar cores connected by one bridge possessed DNA binding capacity and were effective in killing cancerous cells [2]. In order to develop a new topoisomerase inhibitor, we developed new acridinenaphthalimide hybrids with thiocarbamide as the linkage (Figure 3). e anticancer and DNA-Topo I inhibitory activities of the synthesized derivatives were evaluated at the same time. Besides, tacrine is the best-known acetylcholinesterase (AChE) inhibitor, and its analogue acridine derivative also exhibits potent anti-AChE activity [17,18]. us, the anti-AChE activities of all compounds were also investigated in this study. e possible pharmacological mechanisms of all compounds with two targets (Topo I and AChE) were investigated by molecular docking, in order to identify lead Topo I or AChE inhibitors.

Chemistry
2.1.1. General. Acridine isothiocyanate derivatives were synthesized according to our previously published method [19]. Other reagents and solvents were of reagent grade and were obtained from Aladdin Chemical Reagent Limited Company (Shanghai, China). All yields mentioned refer to the yields of isolated products after purification. e intermediates and the synthesized products were fully characterized by spectroscopic data. e Bruker DRX-500 ( 1 H: 600 MHz; 13 C: 150 MHz) equipment was used to record the NMR spectra, with DMSO-d 6 as the solvent. Chemical shifts (d) were presented in the form of parts per million (ppm), while the J values were presented in hertz (Hz). Mass spectra were generated using a ermo Fisher LCQ Fleet (ESI) machine. Agilent 1200 high-performance liquid chromatograph (HPLC) with DAD detector was used for chromatographic detection. XT-4A melting point apparatus was employed to measure the melting points, with no correction. (1) and N-Amido-4-bromine-1, 8-naphthalimide (3). Compounds 1 and 3 were synthesized according to the procedure found in the literature [20,21] after minor modifications.

Synthesis of N-Amido-1,8-naphthalimide
About 0.99 g of anhydride naphthalene (5 mmol) was dissolved in 50 mL DMF. Later, an excess of 85% w/w hydrazine hydrate (10 mL) was added, followed by refluxing for 4 h. e mixture was then cooled, and the precipitated solids were filtered and recrystallized from ethanol to obtain a solid yellow compound (1). About 2.5 mL of 85% w/w hydrazine hydrate was added dropwise into 25 mL suspension of 95% ethanol and 1.39 g 4bromo-1,8-naphthalic anhydride (5 mmoL). After mechanical stirring for 5 h under ambient temperature, the precipitate was collected through filtration, rinsed with 95% ethanol, and dried under vacuum. e resulting solid was then recrystallised with chlorobenzene to obtain the pure product (3).

Synthesis of N-Amido-4-propyl-1,8-naphthalimide (4).
According to the method reported in the literature [20], 15 mL ethylene glycol monomethyl ether containing 0.15 g  Journal of Chemistry (0.5 mmoL) of compound 3 and 1 mL propylamine (0.72 mmol) was placed into a 50 mL round-bottom flask. e reaction mixture was then refluxed for 3.5 h. After cooling to room temperature, 50 mL ice water was added, and the precipitate was collected by filtration, washed with water and dried under vacuum. e crude solid was recrystallized from chloroform to give yellow needles (4) with a yield of 65.43%. (2) or (5). About 0.46 mmoL of N-amido-1,8-naphthalimide (1) or N-amido-4-propyl-1,8naphthalimide (4) was dissolved in 20 mL acetonitrile by heating to reflux, followed by the addition of 0.46 (for the synthesis of compound 2) or 0.92 mmoL (for the synthesis of compound 5) acridine derivatives (a, b) into the resultant solution. e reaction mixture was then refluxed for 12-24 h at 80°C, and the progress of the reaction was monitored by thin-layer chromatography (TLC). e resultant precipitate was filtered and washed with hot chloroform to obtain pure product (2) or (5).  [23]. Briefly, cells (4 * 10 3 cells/ well) were inoculated into the 96-well plates at 37°C for  Journal of Chemistry

Topo I Inhibitory Activity.
e catalytic activity of Topo I was determined in accordance with the literature reports, with supercoiled pBR32 DNA as the substrate [24]. Topo I and pBR322 were provided by Takara Bio Inc. One enzyme unit is referred to as the amount of enzyme that could totally relax 0.5 μg supercoiled pBR322 DNA at 37°C within 30 min. Briefly, 20 μL reaction mixture was added into the mixture containing 2 μL of the 10X reaction buffer solution (supplemented with 500 mM of KAc, 200 mM of Tris-Ac, 100 mM of Mg (Ac) 2 ), 1 mg/mL of BSA, pBR322 DNA (0.5 μg), and various compounds (2 μL, 0.5 mM). is was then diluted with distilled water to 19 μL. Later, one unit of Topo I (diluted to 1 unit/μL) was added into the mixture and incubated for 30 min at 37°C. e reaction was terminated by the addition of 0.5% SDS, bromophenol blue (0.25 μg/mL), and 15% glycerol. e reaction products were isolated after 50 min of horizontal 0.8% agarose gel electrophoresis in 1X Tris-acetate/EDTA buffer, at 80 V under ambient temperature. e gel was then stained using ethidium bromide (5 μg/mL), and the images were recorded by the Gel Documentation System (Bio-Rad, USA).

Molecular
Docking. Molecular docking between target proteins and ligands was performed using Surflex-Dock in Sybyl 2.0, which was according to our reported method in the literature [26]. is system is performed by generation of an idealized active site (Protocol) consisting of dummy atoms that guide the docking process. e crystal structures of both DNA-Topo I and AChE complex were downloaded based on RCSB website (http://www.rcsb.com) (PDB ID: 1T8I as well as 1UT6, respectively). e proteins were then imported into Surflex-Dock and prepared according to the following criteria using the biopolymer preparation tool: H-Addition, H-Bond; removal of water molecules; termini treatment, charged; and protonation type of histidines. e structures of the compounds were drawn using ChemDraw. Molecules in the training set were aligned by the FlexS in SYBYL. All values were assigned with valence, and the Gasteiger-Marsili charges were calculated for each compound. Ultimately, ligand docking under the Surflex-Dock GeomX precision was performed to dock the generated grid of protein and the ligands. e docking results were then imported into the LigPlot+ 2.1, and the combination between compounds and proteins was discussed regarding H-Bond, conjugate action, and hydrophilic or hydrophobic action.

Synthesis.
ree 1,8-naphthalimide-acridinyl hybrids (2a, 2b, and 5b) were synthesized according to the procedure shown in scheme 1. e structures assigned to 2a, 2b, and 5b on the basis of MS, 1 H NMR, and 13 C NMR spectroscopic data are in accordance with the proposed molecular structures.
Intermediate N-amido-1,8-naphthalimide (1 or 3) was prepared according to the method reported in the literature [20,21]. e target products of aroyl thiourea derivatives (2 or 5) were synthesized by nucleophilic addition reaction of N-amido-1,8-naphthalimide derivatives (1 or 4), with isothiocyanate (R 1 = a, b). However, the molar ratio of them influences obviously the yield of the target product. Compounds 2a and 2b were synthesized successfully with intermediate 1 and isothiocyanate (R 1 = a, b) in a molar ratio of 1 : 1. Compound 5b was obtained by intermediate 4 and benz[c]acridine isothiocyanate (R 1 = b) in a higher molar ratio of 1 : 2. e solvent CH 3 CN was found to be the most suitable solvent in this study based on the yield and purity obtained. However, the practical yields of 2b and 5b were still found to be less than 30%, which may be related to steric hindrance of benz[c]acridine. e structures of compounds 2a, 2b, and 5b were confirmed by their 1 H NMR spectra, which displayed two characteristic NH signals of thiourea at a range of δ 10.62-11.47. eir 13 C NMR spectra showed the characteristic carbon signals at a range of δ 182.85-162.43, attributed to two C�O and one C�S. e ESI-MS indicated that the molecular weights were in accordance with the calculated value.

Cytotoxicity Test.
e in vitro cytotoxicities of the synthesized compounds were evaluated through the MTS assay. Six types of human cancer cells, two types of human normal cells, and one type of neurocytoma cells were used. ese included HL-60 human leukemia, MT-4 human acute lymphoblast leukemia, HepG2 human hepatocarcinoma, HeLa human cervical cancer, SK-OV-3 human ovarian cancer, MCF-7 human breast carcinoma cells, human normal liver cells LO2, human normal lung epithelial cells BEAS-2B, and human neuroblastoma SH-SY5Y cells. e IC 50 value was calculated according to the Reed and Muench method [27], with the consistence being used as the X-axis and the cell viability as the Y-axis.
e assay results are shown in Table 1.
As shown in Table 1, 2b exhibited significant anticancer activity to most cell lines. e IC 50 for MT-4, HepG2, HeLa, and SK-OV-3 cells were 14.66 ± 0.31, 27.32 ± 2.67, 17.51 ± 0.34, and 32.26 ± 1.74 μM, respectively. But it was not better than the three positive controls in all cell lines. Both benz[c]acridine ring and naphthalene anhydride structure may have contributed to its anticancer activity of 2b. However, 5b had no obvious toxic effect on any cancer cell lines, which may be related to the increase of the steric hindrance from the structure. It should be noted that all target compounds have no damage to normal cells LO2 and BEAS-2B with IC 50 > 100 μM, and they were less toxic than positive control. Compound 2b can thus be suggested as a potential candidate for development into antitumor drugs with high efficiency and low toxicity. In the meantime, the proliferation of human neuroblastoma SH-SY5Y cells was used to determine the neurotoxicity of all target compounds. e results indicated that compound 2a has no significant influence on the growth of SH-SY5Y cells with IC 50 > 100 μM. However, 2b and 2c have certain neurotoxicity with the IC 50 value of 22.34 ± 0.98 and 57.72 ± 2.14 μM, respectively.

Topo I Inhibitory Activity of Compounds.
Literature indicates that stabilization of Topo I by topoisomerase poison is detrimental to cells. is is due to the disruption of DNA uncoiling, increased strand breaks, and unstable RNA transcripts, as well as incomplete DNA replication [28]. Topo I inhibitors such as camptothecin (CPT) have long been used for clinical cancer treatment. However, a lot of side effects of CPT have been reported, and it has been replaced by more effective and safer Topo I inhibitors [29]. To explore new inhibitors and their underlying mechanisms, Topo I inhibitory activity of all synthesized compounds was tested using the Topo I-mediated DNA cleavage assay [30]. Typically, the anti-Topo I potency of the compound would be associated with the number as well as intensity of gel bands related to the DNA fragments. As shown in Figure 4, all compounds could suppress the transformation  e compound 2b was found to be a promising Topo I inhibitor, whose activity was similar to that of CPTat the concentration of 0.5 mM. However, compounds 2a and 5b only displayed medium Topo I inhibitory activities. No obvious intensities of the bands corresponding to DNA below 0.5 mM of all compounds were observed. e enzyme activities of compounds nearly confirmed to the MTS results. ese results indicate that the anticancer mechanism of these compounds might be related to Topo I inhibitory.

Molecular
Docking with Topo I. Molecular docking was carried out using Surflex-Dock in Sybyl 2.0, to investigate the pattern by which potent inhibitor binds with the human DNA-Topo I complex.
e 1T8I (PDB code) structure in Protein Data Bank was improved and used in this study. To validate the molecular docking approach employed in this study, the crystallographic pose of CTP obtained from the structure of the DNA-Topo I complex (PDB ID: 1T8I) was compared with the top docking pose acquired in this study. Figure 5 depicts the superposition of the two binding poses of CTP, which are located within the DNA-Topo I binding site. Such superposition would result in a root-mean-square deviation (RMSD) of superposition of 0.54Å. e obtained RMSD value was far lower than the well-established tolerance level of 2.0Å [31,32], thus validating the adopted docking methodology.
Further, the interactions of 2a, 2b, and 5b, with Topo I at the active site were analyzed and compared. e obtained results are indicated in Table 2 and Figure 6. Of them, the ligand-receptor complex that had the greatest total score was regarded as the most stable binding conformation. ese conformations were then chosen to further explore the underlying binding mechanisms relying on hydrogen bond, hydrophobic interaction, and π-π stacking [33]. As shown in Table 2, all tested compounds exhibited good inhibitory activities with the total score ranging between 8.98 and 9.53, which were close to that of CPT (10.27). It was evident that 2a, 2b, and 5b could interact with various hydrophobic residues of Topo I, among which TGP11 and DC112 were common. e binding of 2a, 2b, and 5b with Topo I was found to involve the H-bonds with residues like ARG364, LYS425, GLU356, DT10, and DA113. In addition, the planar conjugated parts of 2a, 2b, and 5b were also effective for the same amino acid residue TGP11 by the π-π stacking force. All these interactions would assist these compounds in anchoring within the protein binding site. As predicted in this study, 2a, 2b, and 5b had exhibited similar Topo I       Journal of Chemistry inhibition activities as CPT, but the order of activity was not exactly the same as the docking study. ere might be related to other pharmacological mechanisms.

Anti-AChE Activity.
In the past decades, evaluation of the level of acetylcholine (Ach) based on cholinergic hypothesis has remained the main strategy for the drug design for the treatment of Alzheimer's disease (AD) [34]. In fact, most of the currently prescribed AD drugs are acetylcholinesterase inhibitors (AChEI), such as tacrine and rivastigmine [35]. Tacrine is a well-known tetrahydroacridine, which exhibits potent anti-AChE activity and is recognized to be clinically useful. e acridine groups of the synthesized compounds (2a, 2b, and 5b) share a similar structure with tacrine. us, the anti-AChE activity and related molecular docking studies were performed in this study. eir inhibitory effects on AChE at 0.4 mM were evaluated according to the procedure described in the experimental section. As shown in Figure 7, only 2a (58.65%) had exhibited significant inhibitory effect, compared to tacrine (78.82%), with the IC 50 values of 0.32 ± 0.04, and 0.06 ± 0.01 mM, respectively. But 2b (12.10%) and 5b (16.06%) had weak inhibitory effects on AChE. is may be due to the inability of the conjugated plane of benz[c]acridine ring, in insertion into the hydrophobic pocket of AChE.

Molecular Docking with Acetylcholinesterase (AChE).
To understand the binding interactions between molecules and AChE, molecular docking was carried out, and AChE (PDB code: 1UT6) was chosen for this study. According to the validation method described in Section 2.2.3, the docked ligands were discovered to have similar binding poses, to those of the co-crystallized ligand, with the RMSD of superposition of 1.53Å (Figure 8). ese results validate the adopted docking methodology. Interactions between AChE and the selected compounds are presented in Table 3 and Figure 9. We found that the docking results were not exactly consistent with the AChE activities in the experiment. e total scores of 2b and 5b were found to be 7.49 and 9.40, respectively. However, both of them exhibited the lowest AChE inhibitory activity, which might be related to the low water solubility on account of the large rigid structure of the compound. In addition, compound 2a had the lowest total score of 5.41, but it exhibited better inhibitory activity than other compounds. As observed in the docking study, compound 2a was located at the hydrophobic pocket and  was surrounded by the residues TUR70, TYR334, PHE330,  TRP432, TYR442, TRP84, GLY118, GLY117, ASP72,  TYR121, and TRP279. In addition, the 1,8-naphthalimide structure also formed the π-π stacking with TYR84 (bond lengths of 3.54, 3.38, and 3.98).

Conclusions
In summary, in the current research work, three 1,8naphthalimide-acridinyl thiourea hybrids were synthesized. Among all these compounds, 2a and 2b exhibited relatively good anticancer activity against six human cancer cell lines, especially MT-4 cell lines. Besides, compound 2b can be considered as a possible Topo I inhibitor, exhibiting the best anticancer activity. Compound 2a displayed much better AChE inhibitory activity than the other two compounds. us, we infer that 2a and 2b can be selected as the representative compounds for further investigation as potential Topo I or anti-AChE inhibitors.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare no conflicts of interest in association with this manuscript.