Synthesis,anti-ovarian cancer activity evaluation and docking studies of pyran derivatives

2.1 Apparatus and materials

IR spectra (400–4000 cm^–1) were obtained using a Brucker Equinox-55 spectrophotometer. ¹H NMR spectra were obtained using a Varian Inova-400 spectrometer (at 400 MHz). Mass spectra were obtained using a micrOTOF-Q II mass spectrometer. The melting points were taken on a XT-4 micro melting apparatus, and the thermometer was uncorrected.

2.2 Synthesis and characterization of compounds 1–3

Compounds 1–3 were synthesized according to the methods of a previous report [6]. A mixture of benzaldehyde (3-cyanobenzaldehyde or 3,4-dimethylbenzaldehyde, 99%) (10 mmol) and 1,3-cyclohexanedione (20 mmol, 98%) was dissolved in 100 mL of EtOH (99.8%). A few drops of piperidine were added, and the mixture was stirred for 3 h at room temperature. After reaction completion as determined by TLC (petroleum ether: dichloromethane = 1 : 1), water was added until precipitation occurred. After filtering the precipitates, they were sequentially washed with ice-cooled water and ethanol and then dried in a vacuum. The yields of the three new compounds 1–3 are 44.2%, 37.6% and 49.8%, respectively. These data of the yields are based on 1,3-cyclohexanedione.

2-Amino-5-oxo-4-phenyl-4,5,6,7-tetrahydro-cyclopenta[b]pyran-3-carbonitrile (1): 200–201°C. IR (KBr pellet cm^–1): 3329, 1976, 1711 cm^–1. ¹H NMR (DMSO-d₆, δ, ppm): 7.292–7.328 (t, 2H), 7.186–7.240 (q, 5H), 4.196 (s, 1H), 2.681–2.816 (m, 2H), 2.368–2.389 (t, 2H). HRMS (ESI⁺): m/z: calcd for C₁₅H₁₂N₂O₂: 275.0796 [M + Na⁺]; found: 275.0776.

2-Amino-4-(3-cyano-phenyl)-5-oxo-4,5,6,7-tetrahydro-cyclopenta[b]pyran-3-carbonitrile (2): 203–204°C. IR (KBr pellet cm^–1): 3543, 2765, 1986 cm^–1. ¹H NMR (DMSO-d₆, δ, ppm): 7.714–7.731 (d, 2H), 7.540–7.599 (q, 2H), 7.341 (s, 2H), 4.349 (s, 1H), 2.689–2.821 (m, 2H), 2.377–2.398 (t, 2H). HRMS (ESI⁺): m/z: calcd for C₁₆H₁₁N₃O₂: 300.0749 [M + Na⁺]; found: 300.0721.

2-Amino-4-(3,4-dimethyl-phenyl)-5-oxo-4,5,6,7-tetrahydro-cyclopenta[b]pyran-3-carbonitrile (3): 210–211°C. IR (KBr pellet cm^–1): 3424, 1912, 1720 cm^–1. ¹H NMR (DMSO-d₆, δ, ppm): 7.170 (s, 2H), 7.041–7.060 (d, 1H), 6.885–6.932 (t, 2H), 4.100 (s, 1H), 2.663–2.806 (m, 2H), 2.351–2.374 (t, 2H), 2.172–2.188 (d, 6H). HRMS (ESI⁺): m/z: calcd for C₁₇H₁₆N₂O₂: 303.1109 [M + Na⁺]; found: 303.1121.

2.3 Crystal structure determination

Suitable single crystals of compounds 1–3 were obtained by evaporation of chloroform solution. The diffraction data were collected on a Bruker Smart Apex CCD area detector using a graphite monochromated Mo Kα radiation (λ = 0.71073Å) at room temperature. The structure was solved by using the program SHELXL-97 [7] and Fourier difference techniques, and refined by full-matrix least-squares method on F². All hydrogen atoms were added theoretically. Crystallographic data for compounds 1–3 is listed in Table 1.

To detect the anti-proliferation activity of compounds 1–3 on CAOV3 cell line, the CCK-8 detection kit was performed in this experiment. All the preformation in this research was finished under the instruction of the protocols with some modifications [8]. In brief, human ovarian cancer CAOV3 cell in the logarithmic growth phase were collected and seeded into 96 well plates at the concentration of 1×10⁴ cells per well and cultured with Dulbecco’s modified Eagle’s medium (DMEM, 100μL) supplemented with 10% fetal bovine serum in each well. All the cells were incubated for 24 h at 37°C in a CO₂ incubator, then compounds 1–3 was added into wells for indicated treatment for 24 h, followed by the 10% CCK-8 solution addition for cell proliferation detection. The absorbance of each well was measured at 570 nm. This experiment was repeated at least three times, and the results were presented with mean±SD.

2.5 RT-PCR

After treated with compounds 1–3, the relative expression level of the Foxm1b in the CAOV3 human ovarian cancer cell was determined with RT-PCR. This experiment was conducted in accordance with the instructions with a little modification. In short, the human ovarian cancer CAOV3 cell in the logarithmic growth phase were collected and seeded into 96 well plates at the concentration of 1×10⁴ cells per well and treated with 50μg/mL compounds 1–3 for 24 h. After treatment, the total RNA in the ovarian cancer CAOV3 cells was extracted with TRIzol Reagent under the instruction of the manufacturer’s protocols. The concentration of the total RNA was determined with OD260/OD280 ratio, and the total RNA was reverse transcripted into cDNA with a high-capacity cDNA reverse transcription kit. Ultimately, the Foxm1b in the CAOV3 human ovarian cancer cell was determined by SYBR Green Master Mix after compound treatment. 2^–ΔΔCt method was used for relative quantification from triplicate preformation.

2.6 Simulation details

Autodock Vina v1.2 has been utilized to study the binding mode of compounds 1–3 with tubulin. In order to explain the experimental results and probe deeper investigations, the FOXM1B was used as downloaded from the protein data bank (PDB ID: 3G73). The geometry structures of compounds 1–3 were optimized through quantum chemistry calculations by Gaussian 09, B3LYP theory of level along with 6–31 g* basic set were used. The AutoDockTools v1.5.6 has been adopted to transfer 3G73 and optimized structures of 1–3 to autodock vina input files. Only polar hydrogens on these structures are considered. The center coordinates of search grid of tubulin were set to –4.328, 36.034 and 25.808, respectively, the length of the search grid was 30. All other parameters needed by autodock vina are used as default if not mentioned specifically. The calculated results were analyzed and visualized by PyMoL v1.8.6.

3.1 Molecular structure

The structures of the three compounds were measured by X-ray diffraction crystal structural analysis. The results revealed that compounds 1 (C₁₅H₁₂N₂O₂) and 3 (C₁₉H₂₂N₂O₃) belonged to the monoclinic crystal system with the space group of P2₁/c and P2₁/n, respectively and compound 2 (C₁₆H₁₁N₃O₂) belonged to triclinic crystal system with the space group of P-1.

The three molecular structural units of the compounds 1, 2 and 3 were given in Fig. 1. Due to the fact that the three compounds are isostructures, we selected compound 1 (C₁₅H₁₂N₂O₂) as example for introducing their structures. Within the structure of compound 1, the dihedral angle of six-membered ring containing one oxygen atoms [C(4), C(3), O(1), C(2), C(1) and C(5)] and the adjacent six-membered carbon ring [C(9), C(10), C(11), C(12), C(13) and C(14)] is 77.43°. The six-membered ring containing one oxygen atoms [C(4), C(3), O(1), C(2), C(1) and C(5)] and the adjacent five-membered carbon ring [C(4), C(3), C(8), C(7) and C(6)] are in the same planner. The bond lengths of C-C, C-N and C-O are in the range of 1.329–1.525 Å, 1.352(10)–1.449(11) Å and 1.149 Å–1.339 Å, respectively. These bond distances are all in their normal ranges and they are comparable with those previously reported known compounds. For compound 2, the -CN group is connected to the carbon atom (C2) from six-membered carbon ring [C(2), C(3), C(4), C(5), C(6) and C(7)]. For compound 3, the two –CH₃ groups are linked to two carbon atoms [C(13) and C(14)] from six-membered carbon ring [C(10), C(11), C(12), C(13), C(14) and C(15)]. The other structural features of compounds 2 and 3 are similar with those in compound 1. The structural features of compound 1 are similar as the previously reported compound [9] and the major difference between them is the kinds of the functional groups.

Moreover, the three compounds all exhibited layered supra-molecular structures under the interactions of hydrogen bonds. The N–H⋯O, N–H⋯N and N–H⋯O, N–H⋯N, N–H⋯O and O–H⋯O hydrogen bonds could be found in the packing structure of the compounds 1–3. The detailed informations of hydrogen bonds were provided in Table 2. By means of these interactions of hydrogen bonding, the three two-dimensional layered supra-molecular structures were generated. These hydrogen bonds of compounds 1–3 played important roles for the stabilities of the three compounds.

We selected the single crystals of compounds 1–3 to use in the biological studies. Their purities of the three compounds are all 100%. The CCK-8 detection kit was performed to detect the anti-proliferation activity of compounds 1–3 against human ovarian cancer cells CAOV3 in vitro, using doxorubicin as the positive control drug. As the results showed in Fig. 2, compound 2 showed the best inhibitory effect on the proliferation of the human ovarian cancer cells CAOV3 compared with the compounds 1 and 3, as well as the positive control drug doxorubicin. Compound 2 has the 50% inhibition of cell viability (IC₅₀) of 4.12±0.11μM, and compounds 1 and 3 have the IC₅₀ over 80μM. In this research, we got this information that compound 2 has the best anti-proliferation activity on human ovarian cancer cells CAOV3 in the compounds 1–3.

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Fig. 2

Reduced proliferation activity of human ovarian cancer cells CAOV3 and normal cells after compounds treatment. The human ovarian cancer cells CAOV3 and normal cells were seeded into 96 well plates and treated with compounds 1–3. Proliferation activity of human ovarian cancer cells CAOV3 (A) and normal cells (B) was determined with CCK-8 and the IC₅₀ was calculated. ^***means p < 0.005.

In the previous research, we have confirmed the excellent inhibitory effect of compounds on the CAOV3 human ovarian cancer cells proliferation. And the previous researches indicated that the Foxm1b plays a vital important role in the CAOV3 human ovarian cancer cells. Thus, to explore the specific mechanism of the compound on CAOV3 human ovarian cancer cells, the relative expression level of the Foxm1b in the CAOV3 human ovarian cancer cells was measured with RT-PCR. From the results showed in Fig. 3, after compound 2 treatment, the expression level of the Foxm1b in the CAOV3 human ovarian cancer cells was significantly reduced. However, in consistence with the above experiment, compounds 1 and 3 have weaker inhibitory effect on the expression level of the Foxm1b in the CAOV3 human ovarian cancer cells.

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Fig. 3

Inhibited expression level of the Foxm1b in the CAOV3 human ovarian cancer cells after compounds treatment. The human ovarian cancer cells CAOV3 were seeded into 96 well plates and treated with compounds 1–3. The RT-PCR was performed to detect the expression level of the Foxm1b in the CAOV3 human ovarian cancer cells. Ns means p > 0.05, ^*means p < 0.05, ^**means p < 0.01 and ^***means p < 0.005.

The molecular docking measures the potential interactions between chemical compounds and protein receptor, as well as the corresponding strength. Such interactions are generally formed between either polar atoms (oxygen, nitrogen and chloride, etc.) on ligand or on protein to the polar hydrogens on the protein or on the ligands. Before doing the autodocking calculation the quantum chemistry calculation has been performed to provide the stable structure, as a starting point, the structure from the X-ray measurement has been optimized by density functional theory under the B3LYP/6-31 g^* theory of level. The comparison between experiment and calculation has been shown in Table 3 for selected structural parameters. As can be seen from the results, the maximum deviation for bond is 0.02 angstrom, the maximum deviation for angle is a little large which is about 3 degree, for dihedral the deviation is significant (about 15 degrees), such large deviations for angle and dihedral can been attributed to the different constrains in experiment and calculation, for experiment, each molecule is surrounded by neighbouring molecules while in calculation only single molecule has been considered in the gas phase. Considering during the autodock calculation, only single molecule will be inserted into the receptor protein, so the optimized structure from DFT calculation will be used as the input configuration for the autodock calculation.

A maximum number of binding modes has been set as 25 for the autodock calculation, the lowest affinity energies are –5.7, –6.3 and –6.1 kcal/mol for the three compounds. The results can be rationalized by the compound and receptor protein have different binding strengths. As shown in Fig. 4, all three compounds could form polar interactions, the polar interaction presented by compound 1 is between the O atom on five-membered ring group and -OH group on residue SER-306 from the receptor, the polar interaction presented by compound 2 is between the hydrogen on the -NH₂ group and oxygen on the -OH from residue SER-240, and the polar interaction presented by compound 3 is between the hydrogen on the -NH₂ group and oxygen on the carbonyl from residue ASP-261. The corresponding binding distances are 3.0, 2.4, and 2.7 angstrom for three compounds, which explains the differences of binding energies and suggests that the anti-cancer capability has the trend of compound 2 > compound 3 > compound 1. Such result has good agreement to the experimental observations.

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Fig. 4

The polar interactions of compounds 1 (left), 2 (middle) and 3 (right) to the protein receptor, each of the compounds could form two polar interactions and their corresponding distances are 3.0, 2.4, and 2.7 angstrom, respectively.