Study on the Synthesis, Biological Activity and Spectroscopy of Naphthalimide-Diamine Conjugates

Eleven novel naphthalimide-diamine conjugates were synthesized and their structures were confirmed by elemental analysis, 1H-NMR, 13C-NMR and MS. Their in vitro antitumor activities were assessed using MTT assays on two cancerous cell lines K562, HCT116, and one normal hepatoma cell line QSG 7701. Compound 7f exhibited potent antitumor activity on HCT116 cells and favorable cell selectivity toward QSG 7701 compared with the positive control, amonafide. Moreover, 7f could block HeG2 cells in the G2/M phase and induce HeG2 cells apoptosis. The interaction of compound 7f with herring sperm DNA was studied by UV/vis absorption and fluorescence spectroscopy under physiological conditions (pH = 7.4). The observed spectral quenching of compound 7f by DNA and the displacement of EB from DNA-EB complex by compound 7f indicated that compound 7f could intercalate into DNA base pairs, which was also corroborated by the effect of KI on compound-DNA interaction. Further caloric fluorescent tests revealed that the quenching mechanism was a static type. Meanwhile, the binding constants, thermodynamic parameters and the effect of NaCl on compound-DNA interaction showed that the type of interaction force was mainly hydrogen bonds and the binding process was driven by hydrogen and van der Waals bonding.

Previous studies revealed that polyamines are a kind of promising carriers to transport cytotoxic agents into cancer cells [26]. Polyamines are important for tumor cell growth and function, the biosynthetic pathway of native polyamines (putrescine, spermidine and spermine) has been a popular target for therapeutic intervention during the last decades [27].
Our previous work proved that conjugates 1-3 (Figure 1), composed of 1,8-naphthalimide units covalently attached to a polyamine such as spermidine or homospermidine, possessed remarkable cell selectivity through to human hepatoma Bel-7402 and human normal hepatocyte QSG-7701 trials [28,29]. In addition, naphthalimide-polyamine conjugates have been proved to induce cancer cell apoptosis via different pathways [28][29][30]. These results encouraged us to screen more substituted naphthalimide-polyamine conjugates in order to assess their antitumor activity. DNA as carrier of genetic information is a major target for drug interaction due to its abilities to interfere with transcription (gene expression and protein synthesis) and DNA replication, a major step in cell growth and division. Generally, a variety of small molecules interact reversibly with DNA in three primary ways, including intercalation of planar or approximately planar aromatic ring systems between base-pairs [31], groove binding in which the small molecules bound on nucleic acids are located in the major or minor groove [18,19] and binding along the exterior of DNA helix through interactions which are generally nonspecific and are primarily electrostatic. The 1,8-naphthalimide derivatives are the DNA intercalating agents because they consist of a flat, generally p-π deficient aromatic system of which binds to DNA by insertion between the base pairs of the double helix. However, there are rare reports on the interaction mechanism of naphthalimide-polyamine conjugates and DNA. In this work, naphthalimide-diamine conjugates were synthesized and their antitumor activity assessed in vitro. The interactions between a representative compound 7f and herring sperm DNA were first studied by UV and fluorescence spectroscopy. The binding constants and main sorts of binding force were also investigated. Moreover, mechanism of how the novel conjugate 7f killed HeG2 cells 7f was reported.

Cytotoxic Effects
The cytotoxicities of the novel conjugates were assessed in vitro by the MTT assay in the presence of aminoguanidine (an inhibitor of amine oxidase) against three cell lines, human leukemia K562, cancer of colon HCT116 and human normal hepatocyte QSG-7701 [34]. As is shown in Table 1, most of the naphthalimide-diamine conjugates 7a-k showed good antitumor activities against K562, HCT116 cells, in which compound 7f seemed to have equipotent antitumor activity against HCT116 and cell selectivity toward QSG-7701 compared to the control, amonafide. At the same time, compounds 7f-h, which have putrescine backbones, exhibited good antitumor activities against K562, HCT116 cells. In addition, compounds 7j-k, which have longer side chains than the others, also exhibited good antitumor activities. Moreover, The compound 7f with a primary terminal amino group in the polyamine motif exhibited better biological properties than the corresponding compounds with a terminal tertiary amino group [35]. However, the dose-responsive curves shown in Figure 2 indicated that compound 7f displayed much better selectivity between HCT116 and QSG-7701 than amonafide, especially at the concentrations around the IC 50 values (5-10 μM). Thus, compound 7f was selected for further investigation. All data are expressed as means ± SD from three separate determinations. IC 50 values were given only if they were less than 50 µM, which was the maximum concentration tested.

Cell Cycle Analysis and Apoptosis
To reveal the cytotoxic mechanism of compound 7f, we first examined its effects on the cell cycle perturbation. The DNA content analysis by High Content Screening (HCS) confirmed that 7f could induce HepG2 cell cycle perturbation (Figure 3). The exposure of HepG2 cells to 7f resulted in the dose-dependent accumulation of cells in G2/M phase from 29.7% of the control to 49.8% of 7f. Meanwhile, the corresponding reduction of G0/G1 phase from 52.6% of the control to 29.7% of 7f was also observed, accompanying by the little change in the S phase. The results showed that compound 7f could block HepG2 cells in the G2/M phase. Apoptosis, or namely programmed cell death, can be triggered by several stimuli. Both naphthalimides and polyamine derivatives could trigger cell apoptosis [36]. In order to determine whether the antitumor-proliferative effect of compound 7f was associated with cell apoptosis, the HepG2 cell apoptosis was detected by the staining of DNA with Hoechst 33342, and the apoptotic cells were counted by selecting 200 cells randomly ( Figure 4). The apoptotic bodies were observed clearly in 7f-treated groups by laser scanning confocal microscope. 7f could trigger apoptosis in a dose-dependent manner with the 47.0% apoptotic ratio of 7f at 20 μM compared with the less than five percent in the control group.

UV Spectroscopy
As is shown in Figure 5, the UV spectrum of compound 7f in the absence and presence of herring sperm DNA was measured by an ultraviolet visible range spectrophotometer. It was observed that a continuous decrease in the absorbance of compound 7f followed with the increasing concentration of DNA, implying compound 7f could insert into the base pairs of DNA. The spectral effects have been rationalized that the empty π*-orbital of the small molecule couples with the π*-orbital of the DNA base pairs, which causes an energy decrease and a decrease of the π-π* transition energy [37][38][39]. At the same time, the empty π*-orbital is partially filled with electrons to reduce the transition probability. Therefore, the absorption of small molecules should exhibit hypochromism.  Utilizing the absorption spectrum obtained by UV, we could also calculate compound 7f's apparent binding constant according to the following formula [40]: (1) where A 0 and A denote the absorbance in the absence and presence of DNA, respectively and where ε G and ε H-G denote the molar absorption coefficient of compound and its formed complex with DNA. The value of apparent binding constant could be measured from the intercept and slope by plotting A 0 /(A − A 0 ) against c DNA , and the corresponding value of K was 9.806 × 10 3 ( Figure 6).

Fluorescence Quenching
To evaluate the DNA binding properties of naphthalimide homospermidine conjugate, the inherent fluorescence of compound 7f allowed us to investigate its interaction with herring sperm DNA by fluorescence spectrometry.

Relative Fluorscence Wavelength/nm
As is shown in Figure 7, the fluorescence of compound 7f was quenched upon the addition of DNA. This indicated that DNA is one potential target of compound 7f as expected.
Ethidium bromide (EB) is a well known DNA intercalator, which is often used as a spectral probe to establish the mode of binding of small molecules to double-helical DNA [41]. The fluorescence of EB increases after binding with DNA due to intercalation. Like EB, if naphthalimides intercalate into the helix of DNA, it would compete with EB for its intercalation sites in DNA, and the displacement of EB from the DNA-EB complex leads to a significant decrease in the fluorescence intensity of the DNA-EB complex [42][43][44]. Therefore, herring sperm DNA-EB complex in the presence of increasing concentrations of naphthalimide-diamine conjugate 7f was also measured. As is shown in Figure 8, the fluorescence intensity of DNA-EB complex was decreased by gradually growing concentrations of compound 7f, suggesting that compound 7f could intercalate into DNA and a new complex was possibly formed between compound 7f and DNA [45].

Fluorescence Quenching Mechanism
Fluorescence quenching can occur by different mechanisms, which are usually classified as dynamic quenching and static quenching. Dynamic quenching refers to a process whereby the fluorophore and the quencher come into contact during the transient existence of the excited state, so the bimolecular quenching constants would be larger at higher temperatures. Static quenching, however, results from the formation of a ground state complex between the fluorophore and the quencher, which decreases [46]. To elucidate the quenching mechanism of the interaction between naphthalimidediamine conjugate and DNA (or DNA-EB), fluorescence quenching tests were also performed at 298, 303 and 310 K, which could be described by Stern-Volmer equation [47][48][49].
The Stern-Volmer equation is the following: where F 0 and F are the fluorescence intensities in the absence and presence of quencher (DNA for compound 7f or compound 7f for DNA-EB, respectively), K SV is the Stern-Volmer quenching constant, [c] is the concentration of DNA (or compound 7f), K q is the biomolecule quenching rate constant and Kq = K SV /τ 0 . τ 0 is the average lifetime of the molecule without any quencher and the fluorescence lifetime of the biopolymer is 10 −8 s [50]. The Stern-Volmer plots of F 0 /F versus [c] at the three temperatures were shown in Figures 9 and 10, and the calculated K SV and K q values were presented in Tables 2 and 3.   Table 3. Quenching constants of the interaction between compound 7f and herring sperm DNA-EB at different temperatures. The values of the quenching constant K SV decreased with increasing temperature and the values of K q were much greater than that of the maximum scattering collision quenching constant (2.000 × 10 10 L·mol −1 ), indicating that the fluorescence quenching of compound 7f initiated by DNA or DNA-EB complex initiated by compound 7f was static quenching [51].

Interaction Mode between Compounds and DNA
When small molecules bind independently to a set of equivalent sites in a macromolecule, the binding constant (K b ) can be determined by the following equation [40,52]: where K b denotes the binding constant for interaction of naphthalimide-DNA, and F 0 , F, [c] have the same meanings as in Equation (2).  Table 4. Binding constants and thermodynamic parameters of the interaction between compound 7f and herring sperm DNA at different temperatures.   (Figures 11 and 12), and the corresponding values of K b were listed in Tables 4 and 5. The down-regulated trend of K b with increasing temperature was in accordance with K SV 's dependence on temperature as mentioned above, implying that the binding between naphthalimide and DNA was moderate, and a reversible naphthalimide-DNA complex might be formed [53].
There are several acting forces between a small organic molecule and biomacromolecules, such as hydrophobic force, hydrogen bond, van der Waals force, electrostatic interactions, etc. It is assumed that the interaction enthalpy change (∆H°) does not vary significantly over the limited temperature range studied, thus the thermodynamic parameters can be calculated from the van't Hoff equation: In Equations (4) and (5), K is analogous to the binding constant at the corresponding temperature and R is gas constant. The enthalpy change (∆H°) and entropy change (∆S°) were calculated from the Equations (4) and (5), and the corresponding results were listed in Tables 4 and 5. From Tables 4 and 5, it can be seen that the negative ∆H° and negative ∆S° values showed that the hydrogen bond and weak van der Waals played a dominant role in the interactions between compound 7f and DNA [54].
Kenaka [55] found that hydrogen bonding was the main feature of DNA intercalating agents, and the present evidences indicated that compound 7f was a DNA intercalator. Therefore, it was inferred that the process of interaction between compound 7f and DNA was driven by hydrogen bonding and van der Waals forces. It was also inferred that planar structure of naphthalene ring intercalated into the DNA base pairs when compound 7f bound to DNA between the double helix and that the hydrogen bond and weak van der Waals played a dominant role in the interactions between compound 7f and DNA.

Iodide Quenching Studies
A highly negatively charged quencher is expected to be repelled by the negatively charged phosphate backbone of DNA, therefore an intercalative bound drug molecules should be protected from being quenched by anionic quencher, but the free aqueous complexes or groove binding drugs should be quenched readily by anionic quenchers. At the same time, whether the quencher accesses to fluorophore also plays a role in free and bound one [56].   Figure 13). It was apparent that iodide quenching effect was decreased when compound 7f was bound to DNA, which suggested that the compound 7f is likely intercalated into the base pairs of DNA.

Effect of Ionic Intensity on the Compound 7f and DNA Interaction
DNA is an anionic polyelectrolyte with phosphate groups and monitoring the spectral change with different ionic strength is an efficient method to distinguish the binding modes between molecules and DNA. NaCl is used to control the ionic strength of the solutions. The addition of Na + would attenuate the electrostatic interaction between molecules and DNA because of its competition for phosphate groups in DNA [57]. Hence the effect of NaCl on the fluorescence of DNA-compound 7f system was studied. As is seen from Figure 14, the fluorescence intensity of compound 7f-DNA complex was basically unchanged with increasing concentration of NaCl. The results revealed that interaction between compound 7f and DNA could exclude the electrostatic interaction mode and was prompted by hydrogen bond and van der Waals force.

General Information
All chemicals (reagent grade) used were commercially available. All the 1 H-NMR and 13 C-NMR spectra were recorded on a Bruker AV-400 model spectrometer in D 2 O. Chemical shifts (δ) for 1 H-NMR spectra were reported in parts per million to residual solvent protons. ESI-MS spectra were recorded on an ESQUIRE-LC Mass spectrometer. Elemental analyses were performed on a Gmbe VarioEL Elemental instrument and were within 0.4% of the theoretical values.
The respective N-Boc-protected amine (1.2 mmol) was dissolved in EtOH (20 mL) and stirred at 0 °C for 10 min. 4 M HCl (15 mL) was added dropwise at 0 °C. The reaction mixture was stirred at room-temperature overnight. The solution typically gave a bright white solid as a precipitate. The solid was filtered, washed several times with absolute ethanol and dried under vacuum to give the pure target compounds 7.

Biological Materials and Methods
All chemicals used in bioassay were purchased from Sigma (Beijing, China), unless otherwise indicated. RPMI1640 and fetal calf serum (FCS) were purchased from Gibco (Shanghai, China). Stock solution (10 mM) was prepared in DMSO and diluted to various concentrations with serum-free culture medium.

Cell Culture
Cell lines, K562, 7721 and HCT116 were obtained from American Type Culture Collection (ATCC, Shanghai, China). Cells were cultured in RPMI1640, supplemented with 10% heat-inactivated fetal calf serum (FCS), antibiotics (penicillin, 100 units/mL; streptomycin sulfate, 100 μg/mL) at 37 °C, in an atmosphere of 95% air and 5% CO 2 under humidified conditions. Aminoguanidine (1 mM) was added as an inhibitor of amine oxidase derived from FCS and had no effect on the various parameters of the cell measured in this study.

MTT Assay
Chemosensitivity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, exponentially growing K562 cells were seeded into 96-well plates at 4000 cells/well and treated with indicated concentrations of samples for 48 h, and then 10 μL of MTT (10 mg/mL) was added. After incubation for 4 h at 37 °C, the purple formazan crystals (i.e., a reduced form of MTT) generated from viable cells were dissolved by adding 100 μL 10% sodium dodecyl sulphate (SDS) in each well. The absorbance of each well was then read at 570 nm.
In addition, exponentially growing HCT116 or 7721 cells were seeded into 96-well plates at 5000 cells/well and allowed to attach overnight, and then 100 μL of MTT (1 mg/mL) was added. After incubation for 4 h at 37 °C, the MTT solution was removed and the remaining formazan crystals were dissolved with 150 μL DMSO in each well. The absorbance of each well was then read at 570 nm.

Cell Cycle Analysis
Exponentially growing HepG2 cells were seeded in 96 well plates (4 × 10 5 cells/well), cultured for 24 h and then treated with different concentrations of compound 7f for 48 h. After incubation for 48 h, cells were washed twice with ice-cold 10% PBS, fixed and permeabilized with ice-cold 70% ethanol at −20 °C overnight. The cells were treated with 50 μg/mL RNase A at room temperature for 30 min after washed with ice-cold PBS, and finally stained with 50 μg/mL propidium iodide (PI) in the dark at 4 °C for 30 min. The distribution of cell cycle phases with different DNA contents was read in image analysis system of high content screening living cells.

Apoptosis
Exponentially growing HepG2 cells were seeded in 96 well plates (6 × 10 4 cells/well) and treated with indicated concentrations of 7f. After incubated for 48 h, cells were washed three times with PBS, and finally stained with with 10 μM Hoechst 33342 in the dark at 37 °C for 20 min. They were detected by laser scanning confocal microscope and the apoptotic cells were count by selecting 200 cells randomly.

Conclusions
A series of naphthalimide-diamine conjugates were synthesized and their in vitro antitumor activities were evaluated. Compound 7f was found to have potent antitumor activity and good cell selectivity with amonafide as a control. Moreover, compound 7f could arrest HepG2 cells in the G2/M phase and induce HepG2 cells apoptosis. The interaction of compound 7f with DNA was first studied by spectroscopic methods. The binding of compound 7f to DNA resulted in a series of changes in the spectral characteristics. The absorption spectra of compound 7f with added DNA showed a hypochromic effect and the fluorescence emission of DNA-EB was efficiently quenched by compound 7f. These observed spectral data and the iodide quenching effect suggested that compound 7f interacts with DNA through an intercalative mode. Further fluorescent assays at different temperatures disclosed that the quenching mechanism of both compound 7f with DNA and compound 7f with DNA-EB was a static type. Meanwhile, the binding constant, thermodynamic parameters obtained from the same caloric fluorescent tests and the effect of NaCl on compound 7f-DNA interaction suggested that the binding process was driven by hydrogen bonding and van der Waals forces.