Design, biological evaluation and 3D QSAR studies of novel dioxin-containing pyrazoline derivatives with thiourea skeleton as selective HER-2 inhibitors

A series of novel dioxin-containing pyrazoline derivatives with thiourea skeleton have been designed, synthesized and evaluated for their EGFR/HER-2 inhibitory and anti-proliferation activities. A majority of them displayed selective HER-2 inhibitory activity against EGFR inhibitory activity. Compound C20 displayed the most potent activity against HER-2 and MDA-MB-453 human breast cancer cell line (IC50 = 0.03 μM and GI50 = 0.15 μM), being slightly more potent than the positive control Erlotinib (IC50 = 0.16 μM and GI50 = 1.56 μM) and comparable with Lapatinib (IC50 = 0.01 μM and GI50 = 0.03 μM). It is a more exciting result that C20 was over 900 times more potent against HER-2 than against EGFR while this value was 0.19 for Erlotinib and 1.00 for Lapatinib, indicating high selectivity. The results of docking simulation indicate that the dioxin moiety occupied the exit of the active pocket and pushed the carbothioamide deep into the active site. QSAR models have been built with activity data and binding conformations to begin our work in this paper as well as to provide a reliable tool for reasonable design of EGFR/HER-2 inhibitors in future.

According to the studies above, a novel series of 4,5-dihydro-1H-pyrazole derivatives containing thiourea skeleton and dioxin structure was designed as potential HER-2 selective inhibitors and predicted to have a positive progress with a sound cancer therapeutic benefit.

Results
Preliminary Calculation. Before synthesis, the target series was amplified into four series (Fig. 2) to exam their binding energy with EGFR and HER-2, respectively. Molecular docking was conducted to predict the binding energy (CDOCKER INTERACTION ENERGY in CDOCKER protocol in Discovery Studio 3.5) between the four series and EGFR/HER-2. EGFR with Erlotinib (PDB code: 1M17) 25 and HER-2 with ligand 03Q (PDB code: 3PP0) 26 were used respectively. The average EGFR binding energy of series I, II, III, IV were − 36.3199 kcal/mol, − 36.4076 kcal/mol, − 40.7584 kcal/mol, − 40.4444 kcal/mol. Meanwhile, the top HER-2 binding energy of series I, II, III, IV were − 52.8093 kcal/mol, − 51.7253 kcal/mol, − 50.8926 kcal/mol, − 49.7947 kcal/mol. Aiming at acquiring selective HER-2 inhibitors, we prefer Series I after we additionally compared the bottom HER-2 binding energy of series I and II (− 40.9818 kcal/mol vs − 37.4009 kcal/mol).
Since ADMET properties are important parameters of pharmacokinetics 27,28 , the ADMET prediction of all 80 compounds was provided (Fig. 3). The results were satisfactory. Thus, we chose Series I ( Fig. 4 and Table 1) as target compounds after the preliminary analysis above. The general synthesis method was also organized in Fig. 4. Figure 1. 3D-QSAR of 4,5-dihydro-1H-pyrazole derivatives containing naphthalin moiety for EGFR (A,B) and for HER-2 (C,D). Red contours mean high electron density is expected to increase activity while blue contours mean low electron density is better. Green areas mean steric bulk is better while yellow areas mean small groups are helpful. Kinase inhibitory activity and cell proliferation. All the synthesized compounds C1-C20 were evaluated for their EGFR and HER-2 inhibitory activity. The results were expressed as concentrations of IC 50 (the half maximal inhibitory concentration of EGFR and HER-2 mediated autophosphorylation) and GI 50 (the half maximal inhibitory concentration of MCF-7 human breast cancer cell line and MDA-MB-453 human breast cancer cell line growth), presented in Table 2. A general glance of the bioactivity as well as SI (the selectivity index of EGFR/ HER-2) suggested that most of this series were potential HER-2 inhibitors with merely mediocre EGFR inhibitory activity.   Linear regression (Fig. S1) was conducted to ensure the GI50 values of these compounds shared a similar tendency with their relevant IC50 values, checking that the anti-proliferative effect was produced by inhibitory action against HER-2. As a result, MDA-MB-453 cell line was notably positive correlated to HER-2 (R square = 0.924) while MCF-7 cell line showed no obvious correlation with HER-2 (R square = 0.239). This inference agreed with the relative expression levels of HER-2 in the two cell lines.
Out of the twenty synthesized compounds, C20 displayed the most potent activity against HER-2 and MDA-MB-453 cell line (IC50 = 0.03 μM and GI50 = 0.15 μM), being comparable with the positive control Erlotinib (IC50 = 0.16 μM and GI50 = 1.56 μM) and Lapatinib (IC50 = 0.01 μM and GI50 = 0.03 μM). A more exciting result was that C20 was 952 times more potent against HER-2 than against EGFR, because this value was 0.19 for Erlotinib and 1.00 for Lapatinib. This fact indicated that our modification led to favorable effect in both inhibitory activity and selectivity. Flow Cytometry. We conducted the flow cytometry (FCM) (Fig. 5) to explain the inhibitory activity of the lead compound C20. The results indicated that the compound could induce apoptosis of activated MDA-MB-453 cell line in a dose-dependent manner. With the cells treated with 0.05, 0.1 and 0.2 μM of compounds for 24 h, the percentage of apoptosis by Annexin V-FITC/PI staining was increased correspondingly in a dose-dependent manner.
Western Blot. The corresponding p-HER-2 and p-EGFR levels of MDA-MB-453 dealt by C20 were measured by Western blot (Fig. 6). With the increasing of C20 levels, p-HER-2 levels showed gradually trend of decrease. A corresponding increase of Caspase3 levels indicated that this effect could induce apoptosis. Consequently, compound C20 could directly bind to HER-2, and lower its phosphorylation level. The variation of p-EGFR levels is unconspicuous because the tested concentrations of compound is far below that could cause inhibitory activity of EGFR. BLI Assay. BLI assay was conducted between labeled compound C20 and HER-2. The result was shown in Fig. 7. The calculated K D by the system itself was 1.87 ± 0.14 × 10 −7 M, indicating a relatively strong interaction on small molecule level.
Broadened kinase selectivity. To further expand the discussion of selectivity, we tested the inhibitory activities of representative compounds (C9, C10, C13, C15, C17 and C20) against three other related kinases. The three kinases chosen were: VEGFR2 (activating similar pathways and often studied together), HER-3 (also called erbB-3, belonging to EGFR family but activating different pathway), FAK (a downstream kinase of EGFR but in a unique pathway). The results were expressed as IC 50 values, presented in Table 3. Thus, these results further validated the selectivity of the compounds against HER-2 as well as the positive correlation between bioactivities against HER-2 and MDA-MB-453 (Fig. S2).  Table 2. EGFR and HER-2 inhibitory activity and selectivity index with anti-proliferation activity of the synthesized compounds (C1-C20).
Toxicity test. Besides, the cytotoxic activity of the compounds were evaluated on a mouse embryonic fibroblast cell line (NIH-3T3) using the MTT assay 29 . The results were summarized in Table 4 as well as hemolytic activities. It can be concluded that the selected compounds with potent inhibitory activity and high selectivity were low toxic, which was comparable to the positive control DDCP 30 .  Docking Study. Molecular docking is a procedure in which molecular modeling techniques are used to predict how a protein (enzyme) interacts with small molecules (ligands) 31 . Although the CDOCKER protocol in Discovery Studio 3.5 (Discovery Studio 3.5, Accelrys, Inc. San Diego, CA) was used before synthesis to help design our basic backbone, the results could also be used to explore the interaction between compounds and EGFR/HER-2 as well as to visualize the probable binding mode after bioassay. After preparing the receptor and ligands, the site sphere was selected based on the ligand binding location. The binding models of the most potent compounds with corresponding targets were depicted in Fig. 8 (C20 with 3PP0; C20 and C2 with 1M17, respectively). Not a surprise, the CDocker Interaction Energy (interaction energy between the ligand and the receptor) and the CDocker Energy (energy of the ligand -receptor complexes) agreed with the inhibitory trend for all the synthesized compounds. In the binding model ( Fig. 8), compound C20 is nicely bound to 3PP0 via two hydrogen bonds. One of the amino hydrogen of carbothioamide contributes to both hydrogen bonding interaction with the main chain of ALA751 (N-H … O: 2.235 Å, 113.632°) and the main chain of LEU796 (N-H … O: 2.242 Å, 132.736°), being a probable explanation for its nice activity. This result indicated that the molecular interactions between both rings of compound and surrounding residues contributed to the strong interaction between the carbothioamide moiety and corresponding residues. When the rings changed, the interactions on the carbothioamide varied. In the EGFR model, compound C2 is nicely bound to 1M17 via two hydrogen bonds and two π -cation interactions. The sulfur atom of carbothioamide contributes to one of the hydrogen bonding interaction (S … H-O: 2.314 Å, 160.269°) with the hydrogen atom on the side chain of THR766. Besides, one of the amino hydrogen of carbothioamide contributes to the hydrogen bonding interaction with the main chain of GLN767 (N-H … O:     Fig. 9) was conducted to reveal the possible situation of the compounds embedded in the active pocket. The dioxin moiety occupied the exit of the active pocket including VAL734, ALA751, SER783, LEU796, CYS805, LEU852 and THR862, pushed the carbothioamide deep into the active site, confirming the success of structural modification. QSAR model. By using the Create 3D QSAR protocol of Discovery Studio 3.5, twenty synthesized compounds with definite IC 50 values against HER-2 were selected as the model dataset. By convention, we used the pIC 50 scale (− log IC 50 ), in which higher value indicates exponentially greater potency, to measure the inhibitory activity. The training set and testing set were chosen by the Diverse Molecules method in Discovery Studio 3.5. To ensure a good alignment, we chose the alignment conformation of each molecule with lowest energy in the docked results of CDOCKER protocol. Besides, we applied the alignment by the substructure C1 before building the QSAR model. With the correlation coefficient r 2 between observed activity of testing set and training set found to be 0.722, the QSAR model we built is acceptable ( larger than 0.4). Compounds C6 and C16 with -OBn  group should be involved otherwise this value could be much higher (Fig. S3). As shown in Fig. 10, the molecules aligned with the iso-surfaces of the 3D QSAR model coefficients on electrostatic potential grids and Van der Waals grids are listed. Electrostatic map indicates red contours around regions where high electron density (negative charge) is expected to increase activity, and blue contours represent areas where low electron density (partial positive charge) is expected to increase activity. Similarly, steric map indicates areas where steric bulk is predicted to increase (green) or decrease (yellow) activity. As for the dioxin moiety and carbothioamide, they both are in apropriate size, the only point for further modification is that one of the metheylene on dioxin can introduce electron-withdrawing substitute. Meanwhile, the other benzene ring with substituent is more complex. The para-position asks for larger group and an electron-donating one is sightly better. Although the meta-and ortho-positions need electron-withdrawing groups, the size should be more strictly controlled within two carbon unit. The 3D QSAR model agrees with the inhibitory activity well and provide us the direction of further modification.

Discussion
To conclude, a series of novel dioxin-containing pyrazoline derivatives with thiourea skeleton have been designed, synthesized and evaluated for their EGFR/HER-2 inhibitory and anti-proliferation activities. A majority of them displayed selective HER-2 inhibitory activity against EGFR inhibitory activity. Compound C20 displayed the most potent activity against HER-2 and MDA-MB-453 human breast cancer cell line (IC50 = 0.03 μM and GI50 = 0.15 μM), being slightly more potent than the positive control Erlotinib (IC50 = 0.16 μM and GI50 = 1.56 μM) and comparable with Lapatinib (IC50 = 0.01 μM and GI50 = 0.03 μM). It is a more exciting result that C20 was over 900 times more potent against HER-2 than against EGFR while this value was 0.19 for Erlotinib and 1.00 for Lapatinib, indicating high selectivity. The results of docking simulation indicated that the dioxin moiety occupied the exit of the active pocket and pushed the carbothioamide deep into the active site. QSAR models were built with activity data and binding conformations to begin our work in this paper as well as to provide a reliable tool for reasonable design of EGFR/HER-2 inhibitors in future.
To deduce how the HER-2 inhibitory activity was affected by the structure variation and modification, subsequently preliminary SAR (Structure Activity Relationship) studies were performed. Initially, we fixed the R group with substituted phenyl group to analyze the substitutes. Benzyloxyphenyl group was temporarily skipped for the extra benzene ring extruded the steric structure backbone but phenyl group was included. For single substitute, para-position generally showed better effect than meta-position while ortho-position nearly ruined the HER-2 inhibitory activity (C4, IC50 = 125.3 μM). As for para-position, we could perceive the tendency that -OMe > -CF 3 16.01 μM). Thus we could conjecture that appropriate size might be like -OMe or -CF 3 , for the order was -OMe > -CF 3 > -SMe > -Me instead of -OMe > -SMe > -Me > -CF 3 , and -I was too large, for the order was -Br > -Cl > -I > -F instead of -I > -Br > -Cl > -F. As for meta-position, the inferred order was -Br > -Cl > -OMe, indicating that the electronic factor might be more important than the steric factor. The corresponding compounds were C8 (IC50 = 0.45 μM) > C7 (IC50 = 62.85 μM) > C5 (IC50 = 86.55 μM). Meanwhile, for multi substitutes, only two hints could be inferred due to the sample size. One was that 6-F could make up the disadvantage of 2-F to a large extent with the corresponding result C18 (IC50 = 0.96 μM) > C4 (IC50 = 125.3 μM). The other was that a meta-group with suitable size could indeed contribute to the bioactivity when there already existed a suitable para-group, with the corresponding result C20 (IC50 = 0.03 μM) > C10 (IC50 = 0.08 μM). Secondly, Naphthalen-1-yl group showed better effect than Naphthalen-2-yl group as C2 (IC50 = 0.56 μM) > C3 (IC50 = 7.18 μM). Considering the discussion of substitutes above, the inference was that Naphthalen-2-yl group exceeded the steric limit of para-and meta-positions while Naphthalen-1-yl group overcome the disadvantage of ortho-position by stretching into a larger plane. Thirdly, although a heterocyclic ring (here it was furan) indicated  2 (A,B). Red contours mean high electron density is expected to increase activity while blue contours mean low electron density is better. Green areas mean steric bulk is better while yellow areas mean small groups are helpful.
Scientific RepoRts | 6:27571 | DOI: 10.1038/srep27571 better effect than benzene ring with the corresponding result C19 (IC50 = 12.04 μM) > C1 (IC50 = 16.01 μM), the difficulty in introducing substitutes made this feeble superiority fade away. Finally, we came to benzyloxyphenyl group. This group distorted the original backbone no matter the situation was 3-OBn or 4-OBn. Although compounds with benzyloxyphenyl group were not that good as the others in the ADMET properties, both 3-OBn and 4-OBn showed admirable activity accidentally with the corresponding results C6 (IC50 = 0.06 μM) and C16 (IC50 = 0.13 μM) respectively. A possible explanation might be the conformational inversion according to the molecular overlap result. That might be an interesting direction in future research.

Materials and Methods
Chemistry section. The synthesized compounds C1-C20 were all prepared in two steps. Firstly, 1-(2,3-dihy- [1,4]dioxin-6-yl)ethan-1-one on treatment with different substituted benzaldehydes in presence of 50% NaOH were stirred at room temperature till reactions completed, yielding different analogues of chalcones (B). Secondly, thiosemicarbazide was added to participate the cyclization of the obtained analogues of chalcones (B), leading to the corresponding target compounds C1-C20. All of the synthetic compounds gave satisfactory analytical and spectroscopic data, which were in full accordance with their depicted structures.
(The detailed information is in Supplementary Information) 6 was located at the 5′ upstream to the HER-2 and EGFR sequences. Sf-9 cells were infected for 3 days for protein expression. Sf-9 cell pellets were solubilized at 0 °C in a buffer at pH 7.4 containing 50 mM HEPES, 10 mM NaCl, 1% Triton, 10 μM ammonium molybdate, 100 μM sodium vanadate, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 10 μg/mL pepstatin, and 16 μg/mL benzamidine, HCl for 20 min followed by 20 min centrifugation. Crude extract supernatant was passed through an equilibrated Ni-NTA superflow packed column and washed with 10 mM and then 100 mM imidazole to remove nonspecifically bound material. Histidine-tagged proteins were eluted with 250 and 500 mM imidazole and dialyzed against 50 mM NaCl, 20 mM HEPES, 10% glycerol and 1 μg/mL each of aprotinin, leupeptin, and pepstatin for 2 h. The entire purification procedure was performed at 4 °C or on ice. Both EGFR and HER-2 kinase assays were set up to assess the level of autophosphorylation based on DELFIA/ Time-Resolved Fluorometry. Compounds C1-C20 were dissolved in 100% DMSO and diluted to the appropriate concentrations with 25 mM HEPES at pH 7.4. In each well, 10 μL of compound was incubated with 10 μL (12.5 ng for HER-2 or 5 ng for EGFR) of recombinant enzyme (1:80 dilution in 100 mM HEPES) for 10 min at room temperature. Then, 10 μL of 5× buffer (containing 20 mM HEPES, 2 mM MnCl 2 , 100 μM Na 3 VO 4 , and 1 mM DTT) and 20 μL of 0.1 mM ATP-50 mM MgCl 2 was added for 1 h. Positive and negative controls were included in each plate by incubation of enzyme with or without ATP-MgCl 2 . At the end of incubation, liquid was aspirated and plates were washed three times with wash buffer. A 75 μL (400 ng) sample of europium-labeled anti-phosphotyrosine antibody was added to each well for another 1 h of incubation. After washing, enhancement solution was added and the signal was detected by Victor (Wallac Inc.) with excitation at 340 nm and emission at 615 nm. The percentage of autophosphorylation inhibition by the compounds was calculated using the following equation: 100% -[(negative control)/(positive control -negative control)]. The IC 50 was obtained from curves of percentage inhibition with eight concentrations of compound. As the contaminants in the enzyme preparation are fairly low, the majority of the signal detected by the anti-phosphotyrosine antibody is from EGFR or HER-2. The experiment was performed for three independent times and in triplicate each time.
Flow Cytometry. MDA-MB-453 cell line was seeded per well in 24-well plates and incubated overnight. Then they were treated with compound C20 at three different concentrations (0.05 μM, 0.1 μM and 0.2 μM, separately). DMSO was chosen as the negative control with a regular dosage of 0.2%. After 24 h, cells were harvested for the apoptosis detection. In brief, collected cells were washed once with PBS and subsequently washed once with binding buffer, and then stained with Annexin V-FITC and propidium iodide (PI) in the binding buffer for 20 min at room temperature in the dark. Apoptotic cells were quantified using a FACScan cytofluorometer (PT. Madagasi Scientific RepoRts | 6:27571 | DOI: 10.1038/srep27571 Brosa Inc. JI. Batang Hari NO. 73, Propinsi Sumatera Utara, Indonesia) plotting at least 10,000 events per sample. To quantify the data, the frequencies in all quadrants were analyzed using flowjo software. We regarded cells in the lower right quadrant (Annexin V positive/PI negative) as early apoptotic cells, and cells in upper right quadrant (Annexin V positive/PI positive) as late apoptotic cells and necrotic cells.
Western Blot. After incubation with compound, cells were harvested and washed with PBS, then lysed in lysis buffer (30 mm Tris, pH 7.5, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na 3 VO 4 , 1% Nonidet P-40, 10% glycerol, and phosphatase and protease inhibitors). After centrifugation at 12,000 g for 5 min, the supernatant was collected as total protein. The concentration of the protein was determined by a BCATM protein assay kit (Pierce, Rockford, IL, USA). The protein samples were separated by 10% SDS-PAGE and subsequently electrotransferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% non-fat milk for 2 h at room temperature. The blocked membrane was probed with the indicated primary antibodies overnight at 4 °C, and then incubated with a horse radish peroxidase (HRP)-coupled secondary antibody. P-HER-2 and p-EGFR levels were measured as shown in Fig. 6.
BLI assay. Bio-layer interferometry (BLI). Binding of compounds (labeled; 0.01, 0.05, 0.1, 0.5 μM) to the HER-2 protein (truncated) was measured and analyzed on an Octet Red96 instrument (ForteBio) at room temperature. The buffer-equilibrated streptavidin biosensors were loaded with 100 μg/mL protein. A duplicate set of sensors was incubated in the buffer without protein for a background binding control. The assay was performed in black 96-well plates (Thermo Fisher Scientific) with the total working volume of 0.21 mL per well. The signal was analyzed using a double reference subtraction protocol to subtract the non-specific binding, background, and signal drift caused by sensor variability. The binding event was quantified by the shift of interference pattern of the light. The result was shown in Fig. 7 and the K D was calculated by the Octet Red96 instrument.  Safety test section. Cytotoxicity test. The cytotoxic activity in vitro was measured against mouse fibroblast NIH-3T3 cell using the MTT assay. Cells were cultured in a 96-well plate at a density of 5 × 10 5 cells and different concentrations of compounds were respectively added to each well. The incubation was permitted at 37 °C, 5% CO 2 atmosphere for 24 h before the cytotoxicity assessments. 20 μL MTT reagent (4 mg/mL) was added per well 4 h before the end of the incubation. Four hours later, the plate was centrifuged at 1200 rcf for 5 min and the supernatants were removed, each well was added with 200 μL DMSO. The absorbance was measured at a wavelength of 490 nm (OD 490 nm) on an ELISA microplate reader. Three replicate wells were used for each concentration and each assay was measured three times, after which the average of IC 50 was calculated. The cytotoxicity of each compound was expressed as the concentration of compound that reduced cell viability to 50% (IC 50 ). The results were summarized in Table 4.

HER-3 Inhibitory
Hemolysis test. Hemolytic activity was assayed using fresh capillary human blood. Erythrocytes were collected by centrifuging the blood three times in chilled phosphate buffered saline (PBS at 4 °C) at 1000 × g for 10 min. The final pellet was resuspended in PBS to give a 2% w/v solution. Using a microtitre plate, 100 μL of the erythrocyte solution was added to dextran, PLL, stearyl-PLL or stearyl-PLL+ LDL (1-1000/μg/mL) in a volume of 100 mL. Samples were then incubated for 3 h and the microtitre plate was centrifuged then at 1000 × g for 10 min and the supernatants (100 μL) transferred into a new microtitre plate. Hemoglobin release was determined spectrophotometrically using a microtitre plate reader (absorbance at 550 nm). Results were expressed as the amount of released hemoglobin induced by the compounds as a percentage of the total. Hemolysis test was tested according to the guide of biological evaluation of medical device (SFDA, China). compounds was then carried out using the Discovery Stutio (version 3.5) as implemented through the graphical user interface CDocker protocol.

Molecular
CDOCKER is an implementation of a CHARMm based molecular docking tool using a rigid receptor, including the following steps: (1) A series of ligands conformations are generated using high temperature molecular dynamics with different random seeds.
(2) Random orientations of the conformations are generated by translating the center of the ligand to a specified position within the receptor active site, and making a series of random rotations. A softened energy is calculated and the orientation is kept when it is less than a specified limit. This process repeats until either the desired number of low-energy orientations is obtained, or the test times of bad orientations reached the maximum number.
(3) Each orientation is subjected to simulated annealing molecular dynamics. The temperature is heated up to a high temperature then cooled to the target temperature. A final energy minimization of the ligand in the rigid receptor using non-softened potential is performed.
(4) For each of the final pose, the CHARMm energy (interaction energy plus ligand strain) and the interaction energy alone are figured out. The poses are sorted according to CHARMm energy and the top scoring (most negative, thus favorable to binding) poses are retained. The whole kinase domain defined as a receptor and the site sphere was selected based on the original ligand binding location, then the original ligand was removed and the ligands prepared by us were placed during the molecular docking procedure. CHARMm was selected as the force field. The molecular docking was performed with a simulated annealing method. The heating steps were 2000 with 700 of heating target temperature. The cooling steps were 5000 with 300 cooling target temperature. Ten molecular docking poses saved for each ligand were ranked according to their dock score function. The pose with the highest -CDocker energy was chosen as the most suitable pose.
ADMET Prediction. Absorption, distribution, metabolism, excretion, and toxicity properties (ADMET) of the 20 novel compounds were calculated using the DS software. The aqueous solubility, blood brain barrier penetration, cytochrome P450 2D6 inhibition, hepatotoxicity, human intestinal absorption and plasma protein binding were predicted using this software.
QSAR Model. In the model, 80% (that is 16) of the 20 compounds were utilized as a training set for QSAR modeling. The remaining 20% (that is 4) were chosen as test subset using the same protocol. The selected test compounds were: C5, C8, C10 and C15.
The inhibitory activity of the compounds in literatures [IC 50 (mol/L)] was initially changed into the minus logarithmic scale [pIC 50 (mol/L)] and then used for subsequent QSAR analysis as the response variable.
In Discovery Studio, the CHARMm force field is applied and the electrostatic potential together with the Van der Waals potential are treated as separate terms. As the electrostatic potential probe, A + le point change is used while distance-dependent dielectric constant is used to mimic the solvent effect. As for the Van der Waals potential, a carbon atom with a radius of 1.73 Å is used as a probe.
A Partial Least-Squares (PLS) model is built using energy grids as descriptors. QSAR models were built by using the Create 3D QSAR Model protocol in Discovery Studio 3.5.
Statistical analysis. Statistical analysis was performed with SPSS Version 13.0 statistic software package.
Data were expressed as means ± standard deviation (SD). Comparisons between groups were performed with analysis of non-parametric test. A value of P < 0.05 was considered statistically significant.