Design, Synthesis, and Cytotoxic Analysis of Novel Hederagenin–Pyrazine Derivatives Based on Partial Least Squares Discriminant Analysis

Hederagenin (He) is a novel triterpene template for the development of new antitumor compounds. In this study, 26 new He–pyrazine derivatives were synthetized in an attempt to develop potent antitumor agents; they were screened for in vitro cytotoxicity against tumor and non-tumor cell lines. The majority of these derivatives showed much stronger cytotoxic activity than He. Remarkably, the most potent was compound 9 (half maximal inhibitory concentration (IC50) was 3.45 ± 0.59 μM), which exhibited similar antitumor activities against A549 (human non-small-cell lung cancer) as the positive drug cisplatin (DDP; IC50 was 3.85 ± 0.63 μM), while it showed lower cytotoxicity on H9c2 (murine heart myoblast; IC50 was 16.69 ± 0.12 μM) cell lines. Compound 9 could induce the early apoptosis and evoke cell-cycle arrest at the synthesis (S) phase of A549 cells. Impressively, we innovatively introduced the method of cluster analysis modeled as partial least squares discriminant analysis (PLS-DA) into the structure–activity relationship (SAR) evaluation, and SAR confirmed that pyrazine had a profound effect on the antitumor activity of He. The present studies highlight the importance of pyrazine derivatives of He in the discovery and development of novel antitumor agents.


Introduction
Natural molecules are an indispensable source of lead compounds for drug discovery [1][2][3].Pentacyclic triterpenes are widely present in medicinal plants, and they show a unique range of pharmacological activities, including great antitumor, anti-inflammatory, and antiviral activities [4][5][6].They attracted the attention of scholars due to their enormous chemopreventive and antineoplastic potential [7,8].In view of this, the structural modification of pentacyclic triterpenoids is always a focus in drug research [9][10][11].Hederagenin (He, 3b,23-dihydroxyurs-12-ene-28-oic acid; Figure 1) is a member of the pentacyclic triterpenoid family, which is extracted mainly from the medicinal plant ivy (Hedera helix L.) and Dipsacus asper [12,13].A variety of pharmacological activities were ascribed to He, such as anticancer [14], anti-inflammatory [15], neuroprotective [16], and antiatherosclerosis effects [17].Notably, He exhibits potential antitumor activity in several cancer cell lines.He could induce apoptotic cell death in LoVo and breast cancer cells through regulation of the mitochondrial apoptosis pathway [18,19], as well as evoke apoptosis in cisplatin-resistant head and neck cancer As shown in Schemes 1-4, all the designed derivatives were synthesized [20,24].In Scheme 1, six kinds of chloromethyl pyrazines were produced by N-chlorosuccinimide (NCS) and benzoyl peroxide (BPO) in carbon tetrachloride (CCl4) under light conditions with nitrogen protection, and the yield ranged from 40-60%.Then, compounds 1-6 were obtained through a combination of He and chloromethyl pyrazines in N,N-dimethylformamide (DMF) containing K2CO3 at 85 °C, and the yield was 35-90%.As seen in Scheme 2, compound 7 was obtained from He with 2-bromoethanol in DMF containing K2CO3 at 85 °C with 95% yield.Then, compounds 8-13 were produced with 20-90% yield through a combination of He and pyrazinic acid with methyl groups using 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) in dry dichloromethane (DCM).In addition, pyrazinic acid with methyl groups was obtained from pyrazine with a methyl group in H2O with KMnO4, and the yield was 70-90%.Interestingly, compounds 20-26 were obtained with 30-90% yield using a similar method to that shown in Scheme 2. In this reaction, the hydroxyl group of 4-chlorobutanol reacted with the carboxyl group of He instead of methyl chloride.As described in Scheme 3, the intermediate was obtained by combining pyrazinic acid with methyl groups with 4-chlorobutanol in EDCI and DMAP in DCM, and the yield was 80-95%; then, compounds 14-19 were produced with 30-90% yield through a combination of He and the intermediate under K2CO3 in DMF at 85 °C.Interestingly, according to the original routine, we planned on gaining compounds 14-19 with the synthesis conditions of Scheme 4; however, compounds 21-26 were obtained unexpectedly.Based on the data, we redesigned the experimental steps as Scheme 3, and obtained compounds 14-19 successfully.All reactions were carried out as detailed in the experimental section, which also describes the spectra for all compounds (spectra data can be found in Supplementary Materials) [20,[32][33][34].As shown in Schemes 1-4, all the designed derivatives were synthesized [20,24].In Scheme 1, six kinds of chloromethyl pyrazines were produced by N-chlorosuccinimide (NCS) and benzoyl peroxide (BPO) in carbon tetrachloride (CCl 4 ) under light conditions with nitrogen protection, and the yield ranged from 40-60%.Then, compounds 1-6 were obtained through a combination of He and chloromethyl pyrazines in N,N-dimethylformamide (DMF) containing K 2 CO 3 at 85 • C, and the yield was 35-90%.As seen in Scheme 2, compound 7 was obtained from He with 2-bromoethanol in DMF containing K 2 CO 3 at 85 • C with 95% yield.Then, compounds 8-13 were produced with 20-90% yield through a combination of He and pyrazinic acid with methyl groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) in dry dichloromethane (DCM).In addition, pyrazinic acid with methyl groups was obtained from pyrazine with a methyl group in H 2 O with KMnO 4 , and the yield was 70-90%.Interestingly, compounds 20-26 were obtained with 30-90% yield using a similar method to that shown in Scheme 2. In this reaction, the hydroxyl group of 4-chlorobutanol reacted with the carboxyl group of He instead of methyl chloride.As described in Scheme 3, the intermediate was obtained by combining pyrazinic acid with methyl groups with 4-chlorobutanol in EDCI and DMAP in DCM, and the yield was 80-95%; then, compounds 14-19 were produced with 30-90% yield through a combination of He and the intermediate under K 2 CO 3 in DMF at 85 • C. Interestingly, according to the original routine, we planned on gaining compounds 14-19 with the synthesis conditions of Scheme 4; however, compounds 21-26 were obtained unexpectedly.Based on the data, we redesigned the experimental steps as Scheme 3, and obtained compounds 14-19 successfully.All reactions were carried out as detailed in the experimental section, which also describes the spectra for all compounds (spectra data can be found in Supplementary Materials) [20,[32][33][34].

Cytotoxicity Assay
The cytotoxicity of hederagenin derivatives in vitro was evaluated on three tumor cell lines (A549: human non-small-cell lung cancer; MCF-7: human mammary cancer; HepG2: human hepatocellular carcinoma) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.In addition, their toxicity evaluations were tested on H9c2 (murine heart myoblast) and MDCK (Madin-Darby canine kidney) cells.The IC 50 values of these compounds are summarized in Table 1.Many of the derivatives displayed more significant growth inhibition effects than He.Remarkably, as shown in Figure 2, the most promising was compound 9, which exhibited similar antitumor activities against A549 as the positive drug cisplatin (DDP), while it showed lower cytotoxicity than DDP on MDCK and H9c2 cell lines.Many of the derivatives displayed more significant growth inhibition effects than He.Remarkably, as shown in Figure 2, the most promising was compound 9, which exhibited similar antitumor activities against A549 as the positive drug cisplatin (DDP), while it showed lower cytotoxicity than DDP on MDCK and H9c2 cell lines.

Cluster Analysis Principal Component Analysis (PCA)
A PCA study was designed to verify the adaptability of experimental data to the model.For the initial overview of the dataset and the detection of trends and outliers, PCA was carried out.Analysis showed no samples being outside the Hotelling T 2 95% confidence ellipse that could influence the analyses, and high values of explained variation and predictive ability were obtained (Table 2).All data were analyzed using SIMACA 13.0.To further explore the structure-activity relationship, PLS-DA was performed, comparing all designed pairs of groups of He derivatives.Analyses revealed an antitumor activity discrimination between the different types of He derivatives compared in pairs.As Figures 3 and 4 depict, He derivatives combined with the same pyrazine exhibited similar in vitro antitumor activities, and the length of the carbon chain at the attachment site caused a difference in activity.In different tumor cell models, He derivatives linked with one carbon, two carbons, and four carbons were divided into three groups according to the difference in in vitro antitumor activity (Figure 3).As for the effect of the structure modification site of He, He derivatives modified at C-28 and C-23 were divided into two groups according to the difference in in vitro antitumor activity (Figure 4).It is worth noticing that this put forward a reference point for the selection of structural modification sites for He.Through data analysis, we found that the number of methyl groups on the pyrazines also showed a certain degree of regularity in their effect on activity.There was no law between the position of methyl groups on pyrazines and antitumor activity.Pyrazine derivatives with different numbers of methyl groups were divided into six groups according to the difference in in vitro antitumor activity.Incredibly, the analysis results of cluster analysis were consistent with the results of direct observation.For example, compounds 21-26 (similar activities on HepG2, IC 50 ranging from 6.42 to 7.99 µM) could be separated from the others (IC 50 > 8 µM on HepG2) by direct observation.Similarly, the results of cluster analysis of PLS-DA supported this result (Figure 4).The cluster analysis of PLS-DA might provide us with further directions for further analysis of He derivatives.A PCA study was designed to verify the adaptability of experimental data to the model.For the initial overview of the dataset and the detection of trends and outliers, PCA was carried out.Analysis showed no samples being outside the Hotelling T 2 95% confidence ellipse that could influence the analyses, and high values of explained variation and predictive ability were obtained (Table 2).All data were analyzed using SIMACA 13.0.
Table 2.The evaluation of explained variation (R 2 X) and predictive ability (Q 2 (cum)).To further explore the structure-activity relationship, PLS-DA was performed, comparing all designed pairs of groups of He derivatives.Analyses revealed an antitumor activity discrimination between the different types of He derivatives compared in pairs.As Figures 3 and 4 depict, He derivatives combined with the same pyrazine exhibited similar in vitro antitumor activities, and the length of the carbon chain at the attachment site caused a difference in activity.In different tumor cell models, He derivatives linked with one carbon, two carbons, and four carbons were divided into three groups according to the difference in in vitro antitumor activity (Figure 3).As for the effect of the structure modification site of He, He derivatives modified at C-28 and C-23 were divided into two groups according to the difference in in vitro antitumor activity (Figure 4).It is worth noticing that this put forward a reference point for the selection of structural modification sites for He.Through data analysis, we found that the number of methyl groups on the pyrazines also showed a certain degree of regularity in their effect on activity.There was no law between the position of methyl groups on pyrazines and antitumor activity.Pyrazine derivatives with different numbers of methyl groups were divided into six groups according to the difference in in vitro antitumor activity.Incredibly, the analysis results of cluster analysis were consistent with the results of direct observation.For example, compounds 21-26 (similar activities on HepG2, IC50 ranging from 6.42 to 7.99 µM) could be separated from the others (IC50 > 8 µM on HepG2) by direct observation.Similarly, the results of cluster analysis of PLS-DA supported this result (Figure 4).The cluster analysis of PLS-DA might provide us with further directions for further analysis of He derivatives.

Structure-Activity Relationship Analysis
Combining the data analysis from Section 2.2.1 and Section Partial Least Squares Discriminant Analysis (PLS-DA), we could easily find that the structural modification site of He, the length of the carbon chain, and the type of pyrazine had an effect on the in vitro antitumor activity of the He derivatives.In general, as observed for compounds 14-19 and 21-26, structural modification at positions C-28 and C-23 could improve antitumor biological activity in vitro, while structural transformation of C-23 might have more potential to enhance cytotoxicity on the same series of tumor cells; it was confirmed that the IC50 values of compounds 21-26 were usually lower than those of compounds 14-19.In general, compounds with chains containing two and four carbons were superior to those with one carbon for improving the antitumor activity of the compounds.For example, compounds 14-19 with a chain containing four carbons generally exhibited superior antitumor activity to others, especially on HepG2.He derivatives 2, 9 and 15 with 2,6dimethylpiperazine showed better antitumor activity in vitro than others.In addition, comparing compounds 7 and 20 with others, we knew that the introduction of the pyrazine structure was essential for improving the in vitro antitumor activity of He.Notably, compound 9 (He combined with 2,6-dimethylpiperazine via a chain containing two carbon atoms), the drug with the most potential, was also in line with this law.It is worth mentioning that compounds 14-19 and 21-26 exhibited good antitumor activity and some cytotoxic selectivity toward HepG2 cells in vitro (the selective inhibition (SI = IC50 MCF-7 /IC50 HepG2 ) value of compound 14 was 4.43).What these compounds had in common was that they were modified by four-carbon chains.This meant that changes in the length of the carbon chain at C-28 and C-23 might alter the selectivity of compounds for different cell lines; we speculated that this result might be caused by the combination of the compound and the tumor cell target.

Analyses of Apoptosis Morphological Detection of Apoptosis Using Giemsa Staining
To characterize the morphological detection of apoptosis induced by compound 9 on A549, the nuclear and cytoplasmic morphological changes in compound-9-treated A549 were observed with Giemsa staining.As shown in Figure 5, the number of A549 cells in the control group was higher than other groups, and the cells appeared polygonal with a normal cell shape.With an increase in dose, the phenomenon of cell shrinkage and cell disruption became more obvious.At the dose of 2 µM, the cells had a lot of puffing and fragmentation.When the concentration was 6 µM, there was almost no normal cell morphology.

Structure-Activity Relationship Analysis
Combining the data analysis from Section 2.2.1 and Section Partial Least Squares Discriminant Analysis (PLS-DA), we could easily find that the structural modification site of He, the length of the carbon chain, and the type of pyrazine had an effect on the in vitro antitumor activity of the He derivatives.In general, as observed for compounds 14-19 and 21-26, structural modification at positions C-28 and C-23 could improve antitumor biological activity in vitro, while structural transformation of C-23 might have more potential to enhance cytotoxicity on the same series of tumor cells; it was confirmed that the IC 50 values of compounds 21-26 were usually lower than those of compounds 14-19.In general, compounds with chains containing two and four carbons were superior to those with one carbon for improving the antitumor activity of the compounds.For example, compounds 14-19 with a chain containing four carbons generally exhibited superior antitumor activity to others, especially on HepG2.He derivatives 2, 9 and 15 with 2,6-dimethylpiperazine showed better antitumor activity in vitro than others.In addition, comparing compounds 7 and 20 with others, we knew that the introduction of the pyrazine structure was essential for improving the in vitro antitumor activity of He.Notably, compound 9 (He combined with 2,6-dimethylpiperazine via a chain containing two carbon atoms), the drug with the most potential, was also in line with this law.It is worth mentioning that compounds 14-19 and 21-26 exhibited good antitumor activity and some cytotoxic selectivity toward HepG2 cells in vitro (the selective inhibition (SI = IC 50 MCF-7 /IC 50 HepG2 ) value of compound 14 was 4.43).What these compounds had in common was that they were modified by four-carbon chains.This meant that changes in the length of the carbon chain at C-28 and C-23 might alter the selectivity of compounds for different cell lines; we speculated that this result might be caused by the combination of the compound and the tumor cell target.

Analyses of Apoptosis Morphological Detection of Apoptosis Using Giemsa Staining
To characterize the morphological detection of apoptosis induced by compound 9 on A549, the nuclear and cytoplasmic morphological changes in compound-9-treated A549 were observed with Giemsa staining.As shown in Figure 5, the number of A549 cells in the control group was higher than other groups, and the cells appeared polygonal with a normal cell shape.With an increase in dose, the phenomenon of cell shrinkage and cell disruption became more obvious.At the dose of 2 µM, the cells had a lot of puffing and fragmentation.When the concentration was 6 µM, there was almost no normal cell morphology.To characterize the effects of apoptosis inducted by compound 9 on A549, the nuclear morphological changes in compound-9-treated A549 were observed with DAPI staining.After treating with compound 9 for 72 h, A549 cells showed nuclear morphological changes typical of apoptosis in a dose-dependent manner.As pictured in Figure 6, in the control group, the nucleus was intact and evenly colored.The light-blue fluorescence was diffuse and the cells did not show the characteristics of apoptosis.When the concentration of the drug increased, nuclear condensation, nuclear fragmentation, and the formation of apoptotic bodies appeared.When treated at 6 µM, the number of cells decreased drastically, the shape of the cells became irregular, and nuclear fragmentation occurred.

,6-Diamidino-2-phenylindole (DAPI) Staining
To characterize the effects of apoptosis inducted by compound 9 on A549, the nuclear morphological changes in compound-9-treated A549 were observed with DAPI staining.After treating with compound 9 for 72 h, A549 cells showed nuclear morphological changes typical of apoptosis in a dose-dependent manner.As pictured in Figure 6, in the control group, the nucleus was intact and evenly colored.The light-blue fluorescence was diffuse and the cells did not show the characteristics of apoptosis.When the concentration of the drug increased, nuclear condensation, nuclear fragmentation, and the formation of apoptotic bodies appeared.When treated at 6 µM, the number of cells decreased drastically, the shape of the cells became irregular, and nuclear fragmentation occurred.

Detection of Apoptosis Using Annexin V Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Staining
To further depict the apoptosis induced by compound 9, apoptotic rates were analyzed by flow cytometry using Annexin V-FITC/PI staining.As depicted in Figure 7, with different concentrations (0, 2, 5, and 10 µM) of compound 9 treatment, the percentage of apoptotic cells (including early and late apoptosis ratios) increased from 3.7% of the control to 17.4%, 72.0%, and 91.5%, respectively.The result indicates that compound 9 had the potential to induce the apoptosis of A549 cells.

Detection of Apoptosis Using Annexin V Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Staining
To further depict the apoptosis induced by compound 9, apoptotic rates were analyzed by flow cytometry using Annexin V-FITC/PI staining.As depicted in Figure 7, with different concentrations (0, 2, 5, and 10 µM) of compound 9 treatment, the percentage of apoptotic cells (including early and late apoptosis ratios) increased from 3.7% of the control to 17.4%, 72.0%, and 91.5%, respectively.The result indicates that compound 9 had the potential to induce the apoptosis of A549 cells.To further depict the apoptosis induced by compound 9, apoptotic rates were analyzed by flow cytometry using Annexin V-FITC/PI staining.As depicted in Figure 7, with different concentrations (0, 2, 5, and 10 µM) of compound 9 treatment, the percentage of apoptotic cells (including early and late apoptosis ratios) increased from 3.7% of the control to 17.4%, 72.0%, and 91.5%, respectively.The result indicates that compound 9 had the potential to induce the apoptosis of A549 cells.

Detection of the Effect of Compound 9 on Cell-Cycle Progression
The remarkable activity of compound 9 against the A549 cell line prompted us to investigate its effect on the cell cycle of A549 by flow cytometry.As shown in Figure 8, when the concentration increased (0, 2, 5, and 10 µM), the percentage of A549 cells in the synthesis (S) phase overtly increased (from 16.67% to 60.46%) which clearly indicated that compound 9 arrested the cells in the S phase in a concentration-dependent manner.
The remarkable activity of compound 9 against the A549 cell line prompted us to investigate its effect on the cell cycle of A549 by flow cytometry.As shown in Figure 8, when the concentration increased (0, 2, 5, and 10 µM), the percentage of A549 cells in the synthesis (S) phase overtly increased (from 16.67% to 60.46%) which clearly indicated that compound 9 arrested the cells in the S phase in a concentration-dependent manner.

Discussion
A series of different esters of He were designed and synthesized.All these compounds were screened for cytotoxic activity employing a panel of five cell lines, including HepG2, A549, MCF-7, H9c2, and MDCK cells, using the MTT assay.From these data analyzed with PLS-DA, it was evident that almost all derivatives exhibited higher cytotoxicity for all tested cancer cell lines compared to He. Compound 9 was found to be the most likely drug candidate, showing IC50 values ranging from 3.22-4.48µM.As shown by Giemsa, DAPI, and Annexin V-FITC/PI staining, it was found that compound 9 mainly acted by inducing early apoptosis.Moreover, the present research found that compound 9 could induce cell-cycle arrest at the S phase.After exploring the structure-activity relationship, we found that the introduction of a 2,6-dimethyl pyrazine structure with a linker containing 2-4 carbon atoms at position 23 of He might be more effective in enhancing the antitumor

Discussion
A series of different esters of He were designed and synthesized.All these compounds were screened for cytotoxic activity employing a panel of five cell lines, including HepG2, A549, MCF-7, H9c2, and MDCK cells, using the MTT assay.From these data analyzed with PLS-DA, it was evident that almost all derivatives exhibited higher cytotoxicity for all tested cancer cell lines compared to He. Compound 9 was found to be the most likely drug candidate, showing IC 50 values ranging from 3.22-4.48µM.As shown by Giemsa, DAPI, and Annexin V-FITC/PI staining, it was found that compound 9 mainly acted by inducing early apoptosis.Moreover, the present research found that compound 9 could induce cell-cycle arrest at the S phase.After exploring the structure-activity relationship, we found that the introduction of a 2,6-dimethyl pyrazine structure with a linker containing 2-4 carbon atoms at position 23 of He might be more effective in enhancing the antitumor effect of the compound in vitro.Further intensive modifications at C-23 and studies are currently being performed in our research group, and the results will be reported in due course.

General Aspects
Chemical shifts (δ) are given in ppm and coupling constants (J) in Hz.Reagents were bought from commercial suppliers without any further purification.NMR spectra were recorded on a Bruker-500 or -400 spectrometer (Bruker, Dresden, Germany) with tetramethylsilane (TMS; TCI, Tokyo, Japan) as an internal standard; high-resolution mass spectra (HRMS) were acquired using a Thermo Sientific TMLTQ Orbitrap XL hybrid FTMS instrument (Thermo Technologies, New York, NY, USA), and an X-5 micro melting point apparatus (Beijing, China).

Isolation of Hederagenin (He)
Dried Dipsacus asper Wall.ex Henry was extracted with 70% ethanol; three extractions at refluxed temperature were carried out, and the solvent was kept in contact with the sample for 3-9 h at a concentration ratio of 10 to 20 times the amount of ethanol than medicinal herbs.The combined ethanol extracts were concentrated under reduced pressure in a rotary evaporator.Then, 7% hydrochloric acid solution in water (20 times the residue) was added to this residue, and the mixture was heated under reflux temperature for 4-8 h.After this hydrolysis, the residue was filtered off, and the precipitate was obtained, washed with water to neutrality, and dried at 65 • C to obtain a crude product of He.The crude product was purified by column chromatography on a silica gel with solvent (DCM:MeOH = 70:1), and the mixture was concentrated and evaporated to dryness.After standing overnight under 25-50 mL of anhydrous ethanol, the residue was filtered off, and He was obtained.Hederagenin (He): yield <10%; white powder, melting point (m.p.) >300 • C; HRMS (electrospray ionization (ESI)) m/z: [M + Cl] − 507.3274, calculated for C 30 H 48 ClO 4 507.3247.All spectroscopic data (NMR and MS) were in agreement with the literature.

Preparation of Pyrazinic Acid
Powdered potassium permanganate (3.3 g, 20.90 mmol) was dissolved in water (50 mL), then dropped into the aqueous solution of methyl pyrazine within 20 min.The mixture was stirred for 2 h at 75 • C. The reaction was monitored with thin-layer chromatography (TLC) until the end of the reaction.The mixture was cooled down, filtered, and washed with 50 mL of water; then, the filtrate was adjusted to pH 1.5 with nitric acid, slowly warmed to 50 • C, and maintained for 10 min, before being cooled down, extracted with 3 × 20 mL of ethyl acetate, and dried over anhydrous sodium sulfate.The product was left untreated.Other methyl pyrazines included 2,3-dimethylpyrazine, 2,6-dimethylpyrazine, 2,3,5-trimethylpyrazine and 2,3,5,6-tetramethyl pyrazine, carried out as 2,6-dimethylpyrazine [35,36].

Preparation of Compound 7
He (1.246 g, 2.64 mmol) and K 2 CO 3 (728 mg, 5.28 mmol) were added to a solution of 2-bromoethanol (1.649 g, 13.20 mmol) in DMF (20 mL), and the mixture was stirred for 4 h at 85 • C. The reaction mixture was then diluted with ethyl acetate (30 mL), washed with water and brine successively, dried over anhydrous sodium sulfate, and purified by column chromatography to yield the white powder.

Biology Evaluation
MCF-7, A549, HepG2, H9c2, and MDCK cells were obtained from the Chinese Academy of Medical Sciences and Peking Union Medical College.All of the cell lines were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 1% (v/v) penicillin/streptomycin and 10% (v/v) fetal bovine serum (FBS; Thermo Technologies, New York, NY, USA) under a humidified atmosphere containing 5% CO 2 at 37 • C. The stock solutions of He derivatives were dissolved in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA) and added at various concentrations to the cell culture.Cellular morphologies were observed using an inverted fluorescence microscope (Olympus IX71, Tokyo, Japan), a plate reader (BIORAD 550 spectrophotometer, Bio-rad Life Science Development Ltd., Beijing, China), and a Canton 2 flow cytometer (BD, New York, NY, USA).

Antitumor Activity
The antitumor activity of these compounds were evaluated on MCF-7, A549, HepG2, MDCK, and H9c2 cell lines using the MTT assay.Cells at a density of 3 × 10 3 cells/well were plated in a 96-multiwell plate in DMEM containing 10% FBS for 24 h at 37 • C with 5% CO 2 .Then, cells were treated for 72 h with the required concentrations (3.125, 6.25, 12.5, 25, or 50 µM) of the compounds and reference drugs (He and DDP) dissolved with the vehicle DMSO.After that, the culture substrate was replaced with 200 µL of DMEM containing 20 µL of MTT solution at 5 mg/mL and incubated for 4 h.The supernatant was removed and the dissolved formazans precipitated with 120 µL of DMSO.Wells without drugs were used as blanks.A plate reader was used to determine the absorbance at 490 nm.The above steps were repeated three times.The IC 50 values were defined as the concentration of the compound which gave 50% growth inhibition, and they were calculated using Graph Pad Prism 5.The inhibition rate was calculated using the following formula, where OD is the optical density: % inhibition = 1 − (Sample group OD/Control group OD) × 100%. (1)

Giemsa Staining
A549 cells in the logarithmic growth phase were seeded in 12-well plates (1.2 × 10 4 cells/well).After incubation for 24 h at 37 • C with 5% CO 2 , various concentrations (0, 2, 4, or 6 µM) of compound 9 were added to the cultures, and the plate was incubated for further 72 h.Then, we discarded the cell culture fluid, washed twice with phosphate-buffered saline (PBS), and fixed the cells with cold methanol.The A549 cells were stained with 6% Giemsa solution for 5 min, washed with water, and dried.The cell morphological changes were observed under an inverted microscope.

DAPI Staining
A549 cells in the logarithmic growth phase were seeded in 12-well plates (1.2 × 10 4 cells/well).After incubation for 24 h at 37 • C with 5% CO 2 , various concentrations (0, 2, 4, or 6 µM) of compound 9 were added to the cultures, and the plate was incubated for further 72 h.Then, we discarded the cell culture fluid, washed twice with PBS, and fixed the cells with 4% paraformaldehyde for 10 min.After that, we added the appropriate amount of DAPI (1 mg/mL; Molecular Probes/Invitrogen Life Technologies, Carlsbad, CA, USA) to cover the bottom of the board, after staining for 5 min in the dark.Nuclear fragments were observed at 450 nm under an inverted fluorescence microscope (Olympus IX71, Tokyo, Japan).

Annexin V-FITC/PI Staining
According to instructions, A549 cells were stained with an annexin-V FITC apoptosis detection kit (Beijing BioDee Biotech.Co., Ltd., Beijing, China).A549 cells in the logarithmic growth phase were seeded in six-well plates (4.8 × 10 4 cells/well).After incubation for 24 h at 37 • C with 5% CO 2 , various concentrations (0, 2, 5, or 10 µM) of compound 9 were added to the cultures, and the plate was incubated for a further 72 h.A549 cells were collected, washed twice with cold PBS, and centrifuged at 2400 rpm for 10 min.The resulting pellet was mixed with 200 µL of binding buffer of the Annexin V-FITC kit; then, 5 µL of FITC-labeled annexin V was added and mixed gently.After incubation at 4 • C for 10 min in the dark, 5 µL of PI was added.Then, the cells were immediately analyzed with a flow cytometer at 488 nm.

Cell Cycle
According to instructions, A549 cells were stained with the Cell Cycle and Apoptosis Analysis Kit (Beyotime Biotechnology, Shanghai, China).A549 cells in the logarithmic growth phase were seeded in six-well plates (4.8 × 10 4 cells/well).After incubation for 24 h at 37 • C with 5% CO 2 , various concentrations (0, 2, 5, or 10 µM) of compound 9 were added to the cultures, and the plate was

Figure 3 .
Figure 3. Partial least squares discriminant analysis (PLS-DA) of carbon chain length on HepG2 (human hepatocellular carcinoma).Four carbon I-a carbon chain of four carbon atoms and pyrazine were combined at the C-28 position; Four carbon II-a carbon chain of four carbon atoms was added at the C-28 position, but pyrazine was added at the C-23 position.

Figure 3 .
Figure 3. Partial least squares discriminant analysis (PLS-DA) of carbon chain length on HepG2 (human hepatocellular carcinoma).Four carbon I-a carbon chain of four carbon atoms and pyrazine were combined at the C-28 position; Four carbon II-a carbon chain of four carbon atoms was added at the C-28 position, but pyrazine was added at the C-23 position.

Figure 4 .
Figure 4. PLS-DA of combining position on HepG2.C-28-pyrazine was added at the C-28 position; C-23-pyrazine was added at the C-23 position.

Figure 4 .
Figure 4. PLS-DA of combining position on HepG2.C-28-pyrazine was added at the C-28 position; C-23-pyrazine was added at the C-23 position.

Table 2 .
The evaluation of explained variation (R 2 X) and predictive ability (Q 2 (cum) ).