3.1 Synthesis and characterization
The general synthetic procedure was described in Scheme 1, and the more detailed description was included in the supporting information. Compounds 1–3 were synthesized as shown in scheme 1. The synthesis of p-nitrophenylacetonitrile adopts polyphosphoric acid and mixed acid (sulfuric acid and nitric acid) system to synthesize, using polyphosphoric acid as positioning catalyst and mixed acid as nitrating agent according to the corresponding literature[17–19]. Preparation of 3- (4-tert-butylphenyl) -2- (4-nitrophenyl) acrylonitrile 2 was obtained by refluxing 4-nitrophenyl acetonitrile with p-tert-butylbenzaldehyde in ethanol solution. The 2,4-bis(4-(tert-butyl)phenyl)-1,3-bis(4-nitrophenyl)cyclobutene- 1,3-dicarbonitrile 3 subsequently obtained by [2 + 2] cycloaddition under 350 nm illumination by 3- (4-tert-butylphenyl) -2- (4-nitrophenyl) acrylonitrile 2. The structures of Compounds 1–3 were confirmed by NMR spectra and HRMS. The molecular ion peaks appeared at m/z 306.17 and 612.27 in HRMS (Figure S1-S2, see ESI), which were exactly in accordance with the molecular weight of compounds 2 and 3. In the 1H NMR spectra of compounds 2 and 3, compared with the 1H NMR image of compound 2, it can be seen that the hydrogen of compound 3 at 5.2 ppm is caused by [2 + 2] cycloaddition to produce cyclobutane (Figure S5-S6, see ESI).
2.1 X-ray Crystal Structures
The suitable single crystals of 2 (CCDC No. 2262860) and 3 (CCDC No. 2262861) for X-ray structure determination were obtained from CH2Cl2 solutions of the purified products upon layering with n-Hexane. A suitable size crystal of the complex was selected and placed on a Bruker D8 VENTURE X-ray diffractometer with a graphite monochromator, and the data were collected by ω scanning with Mo Kα (λ = 0.071073 nm) rays in a certain θ range. The ORTEP structures of compound 2 and 3 are presented in Fig. 1, and crystallographic data see Table 1. As shown in Fig. 1, compound 2 has a linear molecular structure with unit cell parameters: a = 17.634 (4) Å, b = 6.8371(14) Å, c = 13.936(3) Å, α = 90 °, β = 96.95(3) °, γ = 90°. In addition, compound 3 has clearly shown the four-membered [2 + 2] photocyclized push-pull type quaternary cyclobutene structure with nitrophenyl-units and tBu-units were clockwise arranged around quaternary cyclobutene core. Also, the cell parameters were determined as: a = 29.568 (6) Å, b = 29.568 (6) Å, c = 10.199 (4) Å, α = 90 °, β = 90 °, γ = 120°.
Table 1
The crystallographic data of compound 2 and 3.
Complex | 2 | 3 |
Empirical formula | C19H18N2O2 | C38 H36N4O4 |
CCDC number | 2262860 | 2262861 |
Formula weight | 306.35 | 612.71 |
Temperature/K | 293 | 293 |
Crystal system | Monoclinic | Hexagonal |
Space group | P21/c | R− 3 |
a / nm | 17.634(4) | 29.568(6) |
b / nm | 6.8371(14) | 29.568(6) |
c / nm | 13.936(3) | 10.199(4) |
β / (o) | 96.95(3) | 90 |
Volume / nm3 | 1667.9(6) | 7722(14) |
Z | 4 | 9 |
Dc / (Mg·m− 3) | 1.220 | 1.186 |
µ / mm− 1 | 0.080 | 0.078 |
θ range / (°) | 0.999 ~ 25.990 | 1.001 ~ 24.990 |
F(000) | 648 | 2916 |
R1, wR2 | 0.1011,0.3529 | 0.0760,0.2517 |
Goodness-of-fit on F2 | 0.991 | 1.045 |
3.2 Thermal Stability
Thermal properties were evaluated by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) carried out under a nitrogen atmosphere. Figure 3 showed the weight loss of compound 2 and compound 3. The onset temperature with 5% weight loss was about 297 oC for compound 2 and 282 oC for compound 3, respectively, which indicates that compound 2 and compound 3 possess outstanding thermal stability. The DSC data was investigated at a scanning rate of 15 K min− 1 (Figure S7) of compound 2 and the scanning rate of compound 3 was 20 K min− 1 (Figure S8). The melting temperatures (Tm) of compound 2 and compound 3 were 190 oC and 269.5 oC while crystallization temperatures (Tc) of compound 2 and compound 3 were 171 oC and 146.1 oC, respectively, indicating the two compounds are crystalline.
3.3 Electronic Structure
The UV-visible absorption spectra of all chromophores were recorded in DCM as shown in Fig. 3. It can be seen from the UV-Vis spectrum of compound 2 that there are three characteristic peaks, which are 239 nm, 274 nm and 346 nm, respectively. When the [2 + 2] cycloaddition reaction occurs, the original characteristic peak at 346 nm disappears, and the remaining two characteristic peaks have a slight blue shift. It can be seen that the original existing double bond is destroyed due to the [2 + 2] cycloaddition reaction, which reduces the conjugation of the molecule. Additionally, solvatochromism is commonly used in many fields of chemical and biological research to study bulk and local polarity in macrosystems. When we use a series of organic solvents including acetonitrile, methanol, tetrahydrofuran, dichloromethane and toluene. As shown in Fig. 4, when lower polar solvent was used, such as toluene, the main absorption band was red-shifted to the longer wavelength region. Comparing with compound 2, the [2 + 2] photocyclized quaternary clobutane 3 has the much clear solvatochromism properties. In order to further in-depth understand the electronic structure of cyanide-contained compounds 2 and 3, the electrochemical characterizations including CV and DPV measurements were carried out, and all redox potentials were derived from both CV and DPV measurements. In Fig. 5, compound 2 has two reversible reduction curves at E1/2 = -0.92 and − 1.48V, respectively. In the case of compound 3, the first reversible reduction curve was appeared at more negative potential values at E1/2 = -1.00V, and further reductions became irreversible.
3.4 Interactions with ctDNA
Figure 7 Calculated interaction constants of compounds 2–3 based on (a) Determination of quenching mode by Stern-Volmer equation; (b) The Calculated binding constants by Lineweaver-Burk dual reciprocal equation; (c)
Since cyano-contained small organic molecules have great electronic static interactions with various molecules, such as deoxyribonucleic acid (DNA), it is worthy to test the interaction between these cyano-contained small organic molecules. Considering the fluorescence spectroscopy is one of the most useful techniques for DNA binding studies [16–21], we tested the fluorescence changes upon titration of different amount of compounds 2–3. Also, all of the experiments involving the interaction of 4a-b with ctDNA were conducted in buffer solution (50 mM NaCl, 5 mM trisHCl, pH = 7.1) at room temperature. Before fluorescence titration, a solution of ctDNA in trisHCl buffer solution gave ratios of UV absorbance at 260 and 280 nm of ca. 1.9, indicating that the DNA was sufficiently free of protein. This is because of the DNA concentration was determined by absorption spectroscopy using a molar absorption coefficient (6600 M) at 260 nm. The titration measurements were carried out by varying the concentration of ctDNA-buffer solution upon addition of 50 mL of a 1×10− 2 M DMSO solutions of 2–3 and keeping a fixed complex concentration in tris-HCl buffer (pH = 7.11). For fluorescence quenching experiments, the ctDNA was firstly pretreated with ethidium bromide (EB) for 30 min and 2–3 were then added to this mixture so their effect on the emission intensity could be measured at room temperatures. The quenching properties of the DNA complex in the presence of EB was analyzed by using the Stern–Volmer equation. Additionally, Fluorescence studies were used to quantify the interaction between EBctDNA and compounds 2–3 (Fig. 7) to judge the quenching mode by using the Stern–Volmer static equation:
F0/F = 1 + Ksv[Q] = 1 + Kqτ0[Q],
Also, the quenching constants Ksv and Kq can be obtained from the slope of the line by fitting F0/F to [Q]. As shown in Fig. 7a, the quenching curve is basically a straight line, indicating that there is only one quenching method, and Kq = 3.28×1011L/(mol·s) for 3 and Kq = 2.84×1011L/(mol·s) for 2. Since these two values are greater than 2×1010L/(mol·s) (maximum collision rate constant), it has been indicated that the type of quenching of EB-DNA fluorescence intensity is static quenching. According to the static quenching selection Lineweaver-Burk double reciprocal equation, the binding constants are calculated as:
1/(F-F) = 1/F + 1/KF[Q],
After Ploting (F0-F)−1 vs. [Q]−1, the intercept/slope is the binding constant Ka (Fig. 7b). Herein, the binding constant were calculated as Ka = 2.01×104 for 3 and Ka = 0.614×104 for 2. Then in Fig. 7c, the equation are uesd to exclude the effect of DNA concentration on binding capacity of two compounds:
F0/F = 1 + Ksqr,
The r is the ratio of the concentration of the compound to the ctDNA. Ploting r against F0/F, then Ksq = 0.08 for 3 and Ksq = 0.075 for 2. Although these bonding-constant is smaller than regular high-valent metallocorroles[22], the observed phenomena could be explained as the molecular hinderance of axial pyridine ligands and meso-pentafluorophenyl-substituents. Importantly, there are three possible reasons for the observed fluorescence decrease. Firstly, the formation of a chemical bonded cyclobutene-EB complex causes fluorescence quenching; secondly, cyclobutane compounds binding with EBctDNA forms a new nonfluorescent EBctDNA-cyclobutane complex, which decreased the fluorescence intensity of EBctDNA. Thirdly, the cyclobutane compounds competes with EB in binding with DNA and the intercalated EB was excluded from the DNA double helix, resulting in decreases in the observed fluorescence intensity. Considering two compounds have similar electronic structures, the difference on the fluorescence quenching properties could be explained as the molecular hinderance of [2 + 2] structure.
3.5 Antitumor behaviors
Firstly, Oral squamous cell carcinoma (OSCC) from Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and all cells were cultured in RPMI-1640 medium with 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL Streptomycin. The cells were cultured at 37°C in an atmosphere of 5% CO2. Then, OSCC cells were seeded in 96-well plates at a density of 1.2×104 cells/well. Cells were incubated with various concentrations of compound 3 conjugates for 24h at 37˚C. Then 10 µL of MTT (3-(4,5-dimethylthia-zol-2- yl)-2,5-diphenyltetrazolium bromide) (5 mg/mL) were added to each well. Unreacted dye was removed after an incubation of 1.5h. 100 µL of DMSO was added per well to dissolve the purple formazan. Absorbance was measured at 590 nm with background correction at 630 nm using a microplate reader. All studies were performed in triplicates. As shown in Fig. 8, the IC50 values of compound 3 in OSCC cells is 1.944M, respectively.