Design, Synthesis, and Biological Evaluation of Novel Biotinylated Podophyllotoxin Derivatives as Potential Antitumor Agents

Podophyllotoxin has long been used as an active substance for cytotoxic activity. Fourteen novel biotinylated podophyllotoxin derivatives were designed, synthesized, and evaluated for cytotoxic activity for this study. The synthesized compounds were evaluated for cytotoxic activity in the following human cancer cell lines, SW480, MCF-7, A-549, SMMC-7721, and HL-60 by MTT assay. Most of them exhibited potent cytotoxic effects and compound 15 showed the highest cytotoxic activity among the five cancer cell lines tested, having its IC50 values in the range of 0.13 to 0.84 μM. Apoptosis analysis revealed that compound 15 caused obvious induction of cell apoptosis. Compound 15 significantly down-regulated the expression level of the marker proteins (caspase-3 and PARP) in H1299 and H1975 cells, activated the transcription of IRE1α, increased the expression of GRP78 and XBP-1s, and finally induced apoptosis of H1299 cells. In vivo studies showed that 15 at a dose of 20 mg/kg suppressed tumor growth of S180 cell xenografts in icr mice significantly. Further molecular docking studies suggested that compound 15 could bind well with the ATPase domain of Topoisomerase-II. These data suggest that compound 15 is a promising agent for cancer therapy deserving further research.


INTRODUCTION
Cancer is a kind of frequently-occurring disease that seriously threatens human health. In recent years, more attention has been focused on targeting anti-cancer drugs. Development of targeted antitumor drugs, increase of bioavailability and decrease of toxicity are the key topics which are currently being studied. Research efforts in these topics have already led to the discovery of new drug leads and molecular scaffolds important for the development of novel antitumor agents (Fulda, 2010;Qiao et al., 2011;Wen et al., 2012). Currently, targeted cancer therapy has attracted a lot of interests in cancer research and has emerged as a new treatment option for various types of cancers.
Natural compounds are valuable sources with various structures, unique biological activities, and specific selectivity. Natural products have served as important sources of lead compounds for antitumor agents which have been developed for clinical use. However, many potential drugs lack tumor selectivity and often display significant toxic side effects, which hampers their development for clinical use (Holschneider et al., 1994;Bermejo et al., 2005). In order to enhance the therapeutic specificity of anticancer drugs, various targeting strategies have been explored, including antibodies (Wu and Ojima, 2004;Schrama et al., 2006;Lambert and Berkenblit, 2018), nanocarriers (Peer et al., 2007;Bonifácio et al., 2014;Hojjat-Farsangia et al., 2014;Senthilkumar et al., 2015), peptides (Mastrobattista et al., 1999;Dharap et al., 2005), and vitamins (Sawant et al., 2008;Chen et al., 2010;Guaragna et al., 2012). In each case, molecular features overexpressed on cancer cells are being targeted.
It has been widely recognized that all living cells depend on vitamins for survival and growth and obviously cancer cells must require higher amount of vitamins to meet the need of their rapid growth. Consequently, in order to sustain their rapid cell growth and enhanced proliferation, many cancer cells over-express receptors for certain vitamins. Therefore, vitamin receptors on the surface of these cells are important biomarkers for the delivery of tumor-targeted drugs (Russell-Jonesa et al., 2004;Leamon, 2008;Lu and Low, 2012). Biotin (vitamin H) is a nutrient required for cell growth, and tumor cells need substantially higher amounts of biotin than normal cells due to their rapid growth (Russell-Jonesa et al., 2004). Recent studies have shown that many cancer cell lines express higher levels of biotin receptors (BRs) than normal cells, e.g., L1210FR (leukemia), Ov2008 (ovarian), Colo-26 (colon), P815 (mastocytoma), M109 (lung), RENCA (renal), and 4T1 (breast) cancer cell lines (Russell-Jonesa et al., 2004;Chen et al., 2010). Thus, BR has emerged as a useful biomarker for targeted delivery of anti-tumor agent, and biotin as a tumor-targeting module has been successfully employed for the construction of small molecule antitumor drug conjugates (Chen et al., 2008;Ojima, 2008;Ojima et al., 2012).
The natural lignan podophyllotoxin (PPT, 1, Figure 1) is isolated from Dysosma versipellis and shows cytotoxic activity against a variety of cancer cell lines by inhibiting microtubule assembly. However, PPT lacks tumor specificity and its high toxicity toward normal cells prevents its use in clinic for cancer treatment (Jardine, 1980;Desbene and Giorgi-Renault, 2002;Liu et al., 2007). The biological activity of PPT has led to extensive structural modification, resulting in several clinically useful compounds including etoposide (2, Figure 1), a semisynthetic glucosidic cyclic acetal of PPT. Etoposide exerts cytotoxic activity by inhibiting DNA topoisomerase II and the discovery of its novel mechanism of action led to further studies on the structure-activity relationship of PPT derivatives resulted from the structural modification at the C-4-position (Reddy et al., 2008;Zhang et al., 2014). Studies have shown that improvement in topoisomerase II inhibitory activity, water solubility, cytotoxic activity, drug resistance profile, and antitumor spectra of this class of compounds might be achieved through rational modification at C-4 position (Bromberg et al., 2003).
With the aim to improve the therapeutic efficacy and reduce the toxic side effects of podophyllotoxin in the treatment of cancer, we have designed a group of biotin-podophyllotoxin (Bio-PT) conjugates by covalently linking a biotin residue to podophyllotoxin. Such Bio-PT conjugates are anticipated to be taken up by cells through receptor-mediated endocytosis and selective delivery of these conjugates to cancer cells may be achieved due to a higher level of biotion receptors expressed on cancer cells. Here we report the synthesis of 14 biotinylated podophyllotoxin derivatives and their anticancer activity against various cancer cell lines. The compound with the highest anticancer activity was further studied to reveal the anticancer mechanisms and its antitumor effect was evaluated through in vivo studies as well.

General Information
All cancer cells were obtained from a Shanghai cell bank in China. All reagents were commercially available and were used without further purification unless otherwise indicated. Podophyllotoxin was purchased from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). Anhydrous solvents were obtained by distillation from the indicated systems immediately prior to use: dichloromethane from calcium hydride and tetrahydrofuran from sodium. Uncorrected melting points were measured on a Beijing Taike XT-4. Electrospray ionization mass spectrometry (ESI-MS) data were acquired on API Qstar Pulsar instrument; High resolution electrospray ionization mass spectrometry (HRESI-MS) data were obtained on LCMS-IT-TOF (Shimadzu, Kyoto, Japan); All NMR spectra were recorded with Bruker AV-400 or DRX-500 or Bruker AVANCE III-600 (Bruker BioSpin GmbH, Rheinstetten, Germany) instruments, with tetramethylsilane (TMS) as an internal standard: chemical shifts (δ) are given in ppm and coupling constants (J) in Hz. Column chromatography (CC) are carried out using silica gel (200-300 mesh; Qingdao Makall Group CO., LTD; Qingdao; China). All reactions were monitored by analytical thin-layer chromatography (TLC), which was visualized by ultraviolet light (254 nm) and/or 10% phosphomolybdic acid/EtOH.

Synthesis
General Procedure for the Preparation of Biotinylated Podophyllotoxin Derivatives 13-26 N,N ′ -diisopropylcarbodiimide (DIC, 0.6 mmol) and 4dimethylaminopyridine (DMAP, 0.2 mmol) were added to a solution of biotin or 6-biotinylaminocaproic acid (0.2 mmol), podophyllotoxin or its derivative (0.2 mmol) in N, N-dimethylformamide (DMF, 2.5 mL). The reaction mixture was stirred at room temperature for 24 h under N 2 . Solvents were removed under reduced pressure. The residue was purified by chromatography over silica gel (CHCl 3 /CH 3 OH = 9:1) to afford the desired product.

Cell Viability Assay
Cell viability was evaluated by 3-(4,5-dimethyl-thiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. Briefly, in each well of a 96-well cell culture plate adherent cells (100 µL) with an initial density of 1 × 10 5 cells/mL were seeded and allowed to adhere for 12 h before a test drug was added. In contrast, suspended cells with the same initial density were seeded just before drug addition. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h. After the incubation, MTT (100 µg) was added to each well, and the incubation continued at 37 • C for 4 h. The cells were lysed with SDS (200 µL) after the removal of the medium. The absorbance of the lysate was measured at 595 nm by spectrophotometry (microtiter plate reader, Bio-Rad 680). Dose response curves of cell viability were plotted and the IC 50 values of test compounds at which 50% reduction in cell growth were determined.

Cell Apoptosis Assay
The Annexin V/propidium iodide (PI) detection kit (BD Biosciences, PA, USA) was employed to quantify apoptosis using flow cytometry. H1299 and H1975 cells were seeded in each well of a 12-well plate at 2.5 × 10 5 cells/well. After incubation for 24 h, the cells were treated with compound 15 at 0.5, 1 and 2 µM or PPT (1 µM) for 24 h. Then, the cells were collected and binding buffer (100 µL), FITC annexinV (5 µL), and propidium iodide (PI, 10 µL) (eBioscience, San Diego, CA, USA) were added to the cell suspension. The cells were gently vortexed and incubated at room temperature in the dark for 15 min before measurement by flow cytometry (BD FACSCalibur TM ) within 1 h.

Western Blotting Analysis
H1299 and H1975 cell lines were treated with compound 15 at different concentrations in 6-well plates, and then the cells were collected and lysed with lysis buffer. After sonication cells were centrifuged at 14,000 rpm at 4 • C for 10 min, and total protein was extracted and detected using a bicinchoninc acid (BCA) assay kit. The samples were then separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and then the protein was transferred to nitrocellulose (NC) membranes. The membranes were probed for the following proteins with primary antibodies at 4 • C overnight: caspase-3, cleaved caspase-3, PARP, cleaved PARP, GRP78, CHOP, XBP-1, XBP-1s, and Actin. After washing the membranes with PBST (× 1), the HRP-conjugated secondary antibodies were added and incubated for 1 h at room temperature. The membranes were then washed and the HRP was detected using Luminata TM Forte Western HRP Substrate reagent. The bands of interest were visualized and imaged under chemiluminescent detection using a FluorChem E System (ProteinSimple, San Jose, CA, USA). SCHEME 2 | The synthesis of biotinylated podophyllotoxin derivatives (13-26). Reagents and reaction condition: (a) DIC, DMAP, DMF, N 2 , rt. 39-65%.

Gene Expression Assay
H1299 cells were cultured in 12-well plated at 2.5 × 10 5 cell/well in the presence of compound 15 (0.5, 1, and 2 µM) for 24 h. Total RNAs present in the cultured cells were extracted using the TransZol TM Up Reagent (TransGen Biotech, Beijing, China).
Gene expression was detected via quantitative real-time PCR (qRT-PCR) and SYBR R Premix EX Taq TM II (TaKaRa Bio, Otsu, Japan) was used to perform the analysis.

Animal Studies
All animal studies were conducted in accordance to procedures approved by the Animal Care and Use Committee at China Pharmaceutical University (Jiangsu, China). Forty icr male mice (10-20 g) were provided from the Comparative Medicine Centre of Yangzhou University (Jiangsu, China) and were housed in an SPE animal facility. S180 cancer cells were injected subcutaneously into the right axillaries of icr male mice (4.5-5.0 × 10 6 cells/spot). The mice were divided randomly into five groups: model; positive control; lowdose; medium-dose; high-dose. All mice of the therapeutic groups were injected intraperitoneally (i.p.) every day, and all mice in the positive control group were injected intravenously (i.v.) on the first day and the fourth day after inoculation. Tumor size was measured with caliper and the volume calculated using the previously reported method (Qin et al., 2015). The weight of the mice and the volume of the tumors were measured every day. At the end of the experiment, the mice were killed and the tumors were isolated and weighed.

Molecular Docking Studies
The crystal structure of Top-II (code ID: 3QX3) (Wu et al., 2011) was obtained in Protein Data Bank after eliminating the inhibitor and water molecules. The missing atoms were added by Sybyl-X 2.0 molecular modeling. The kinds of atomic charges were taken as Kollman-united-atom (Weiner et al., 1984) for the macromolecule and Gasteiger-Marsili (Gasteiger and Marsili, 1980) for the inhibitor. To find the binding mode of compound 15 to the active site of Top-II, the advanced docking program Autodock Tools v1.56 (Morris et al., 1998) was used for grid and docking. The enzyme structure was used as an input for the AutoGrid program. AutoGrid performed pre-calculated atomic affinity grid maps for each atom type in the ligand plus a separate desolvation map, and a separate desolvation map present in the substrate molecule. Docking parameters were set as the default values except docking runs was set to 100 on AutoGrid v4.01 and AutoDock v4.01.

Statistical Analysis
All data are presented as the means ± SD (n = 3). Significance was calculated using Student's t-test or one-way ANOVA. P < 0.05 was considered statistically significant. All statistical analyses were performed with the GraphPad Prism 5.0 (San Diego, CA, USA).

Chemical synthesis
Podophyllotoxin (PPT) served as the starting material for the preparation of all the derivatives. The incorporation of the azido, amino, and triazolyl groups at the 4-position of PPT followed standard procedures (Scheme 1). PPT was regioselectively demethylated with methanesulfonic acid and sodium iodide in dichloromethane (CH 2 Cl 2 ) followed by weak basic hydrolysis (water-acetone, barium carbonate) to give 4 ′ -O-demethylepipodophyllotoxin 6 by means of a previously described procedure (Kamal et al., 2000). When the reaction was carried out in acetonitrile (CH 3 CN) as a solvent, 4βepipodophyllotoxin 5 was synthesized as product. Compound 5 and 6 were converted into the corresponding 4β-azides 7 and 8 by reaction with sodium azide and trifluoroacetic acid (NaN 3 -TFA) in chloroform (CHCl 3 ) according to the known procedure (Hansen et al., 1993). The 4β-azides 7 and 8 were converted to 4β-amino substituted 9 and 10 by treatment with triphenylphosphine (Ph 3 P) and water overnight at 25 • C as previously reported (Coleman and Kong, 1998). In addition, the 4β-triazole compounds of 11 and 12 were prepared in 85-89% yield by the reaction of 7 and 8, respectively, with 2-propyn-1-ol using copper (II) acetate and sodium ascorbate as promoters in tert-butanol and water (t-BuOH-H 2 O, 1:1) at room temperature (Tae et al., 2010). Finally, biotin (3)/6-biotinylaminocaproic acid (4) and those podophyllotoxin derivatives (1, 5, 6, and 9-12) were coupled via an ester or amide bond. As shown in Scheme 2, biotin (3)/6biotinylaminocaproic acid (4) reacted with compounds 1, 5, 6, and 9-12 in the presence of diisopropylcarbodiimide (DIC) and 4-N,N-dimethylaminopyridine (DMAP) at room temperature to afford the target compounds 13-26 in 39-65% yields. All the products were structurally confirmed by 1 H and 13 C-NMR spectroscopies, as well as low resolution and high resolution mass spectrometry in electrospray ionization mode (ESI-MS and HRESI-MS). The proton and carbon-13 NMR data of these compounds were compared with those of podophyllotoxin. The configuration of C-4 in compounds 13-26 was assigned based on the coupling constant between H-3 and H-4 (J 3,4 ). Typically, compounds with C-4β-substitution have J 3,4 < 5.0 Hz as a result of H-3 and H-4 in cis relationship. The protons at C-4 of compounds 19-21 appear as a singlet. On the other hand, compounds with C-4α-substitution have J 3,4 > 6.0 Hz because H-4 is trans to H-3 (Fred Brewer et al., 1979;Belen'kiib and Schinazi, 1994). In the 13 C-NMR spectra, the C-4 of these derivatives produces a characteristic signal between 61.1 and 71.3 ppm. The triazole ting in 23-26 was readily confirmed by its C 5" -H signal (δ 7.36-7.74 ppm) in the aromatic region in the 1 H-NMR spectra, which was further supported by the characteristic carbon signals at around 123 ppm in the 13 C-NMR spectra.

Cytotoxicity and Structure-Activity Relationship
The cytotoxicity of all biotinylated podophyllotoxin derivatives 13-26 was tested with the following cancer cell lines: SW480 (colon cancer), MCF-7 (breast cancer), A-549 (lung cancer), SMMC-7721 (hepatoma), and HL-60 (leukemia), Podophyllotoxin (PPT), etoposide, and cisplatin were included for study as control drugs. The IC 50 values obtained from MTT assay are presented in Table 1. Most compounds possessed high level of cytotoxicity against all five cancer cell lines ( Table 1) and were more active than etoposide which is an antitumor agent currently in clinical use.
Biotinylated podophyllotoxin derivatives are prepared by linking a biotinylating agent, biotin (3) or 6biotinylaminocaproic acid (4), via an ester bond, an amide bond, or a trizolyl moiety. Those compounds with an ester linkage (13-18) display potent cytotoxicity with IC 50 values in sub-µM to low µM (except compound 17 against SW480 cell line). Compounds 13 and 14 are esters of podophyllotoxin while 15-18 are esters of 4-epipodophyllotoxin, and their similar potency of activity indicates that the cytotoxic activity of these compounds is not much affected by the configuration of C-4. Among the synthesized compounds, compound 15 is the most active one with IC 50 ranged from 0.13 to 0.84 µM. Compound 15 also exhibits higher activity than PPT in both  SMMC-7721 and SW480 cell lines, with PPT having IC 50 of 4.13 and 9.42 µM, respectively.
Compounds with an amide linkage (19-22) or a triazolyl moiety (23)(24)(25)(26) show weaker cytotoxicity to all tested cell lines. Most of these compounds display moderate (IC 50 > 10.36 µM) to very weak activity (IC 50 > 40 µM; except compound 20, as well as compound 24 against HL-60 cell line). The 6-aminocaproic acid linking spacer present in C-4-substituent can affect the cytotoxic potency of these compounds but not in a uniform way. For example, compounds lacking the linking spacer (15, 20, and 23) show higher activity than their counterparts bearing the linking spacer (17, 22, and 25) in all cell lines tested. In contrast, compound 13 (lacking the linking spacer) is less active than 14 (bearing the linking spacer). In most cases, the effect of 6-aminocaproic acid linking spacer on the cytotoxic potency of these compounds are relatively small except for the pair of compounds 15 and 17 in SW480 cell line (IC 50 0.56 and > 40 µM, respectively). However, it is very interesting to FIGURE 4 | Compound 15 induces apoptosis through activating the ER stress pathway: (A) H1299 cell line was treated with compound 15 (0.5, 1, and 2 µM) for 24 h, GRP78, CHOP, XBP-1, XBP-1s, ATF4, IRE-1α, and ATF6 were measured by real-time RT-PCR. The data are presented as the mean ± SD (n = 3). *p < 0.01, **p < 0.001, ***p < 0.0001 and ****p < 0.00001. (B) H1299 cell line was treated with compound 15 (0.5, 1, and 2 µM) and PPT (1 µM) for 24 h, and WB was performed to detect the expression levels of protein in the ER stress pathway. Actin was tested as a loading control.

Compound 15 Inhibits the Growth of Cancer Cell Lines
To further identify the anticancer effect and tumor selectivity of compound 15, we treated 12 more human cancer cell lines with compound 15, which included lung cancer (H460, H1975, H1299), colon cancer (LS174T, , stomach cancer (BGC-823, MGC-803), breast cancer (SKBR3, T47D), hepatoma (Bel-7402), and cervical cancer (Hela). MTT assay was employed to provide the IC 50 values of compound 15 against all these tumor cell lines as shown in Table 2. H1299 cell line was most sensitive toward compound 15 (IC 50 = 0.86 µM). For most other cancer cell lines, compound 15 showed potent anticancer activity with IC 50 values in µM range. In order to test whether compound 15 can favorably target cancer cells over normal cells, the growth inhibitory effect of 15, in comparison with PPT, on a normal human bronchial epithelial cell line (BEAS-2B) was evaluated. The IC 50 value was found to be 3.75 µM for 15 and 0.85 µM for PPT against BEAS-2B cells (see Table S1). Table 1 (0.13-0.84 µM) and Table 2 against various cancer cell lines, compound 15 does show some selectivity against certain tested cancer cell lines over the normal cells (BEAS-2B).

Compound 15 Induces Apoptosis in the H1299 and H1975 Cell Lines
Given that compound 15 exhibits broad spectrum inhibitory activity of cancer cell growth, we studied further the capacity of compound 15 in the induction of cell death through apoptosis. Lung cancer cells (H1299 and H1975) were treated with compound 15 and analyzed by flow cytometry after being stained with Annexin V/7AAD. Compound 15 at concentration of 2 µM increased significantly both H1299 and H1975 cells undergoing apoptosis when compared with the untreated control (Figure 2).

Compound 15 Regulates the Expression Levels of Apoptosis-Related Protein
It has been recognized that caspase-3 and PARP (poy ADP ribose polymerase) is a critical initiator and executioner of apoptosis (Hensley et al., 2013). H1299 and H1975 cells were treated with compound 15 at the concentration of 0.5, 1, 2 or µM for 24 h and the expression level of caspase-3, PARP, cleaved-caspase-3, and cleaved-PARP was monitored using western blot. The treatment of both H1299 and H1975 cell lines with compound 15 resulted in an increased expression level of cleaved-caspase-3 and cleaved-PARP in a dose-dependent manner (Figure 3). At the same time, the expression level of caspase-3 and PARP decreased, indicating that the treatment led to the activation of caspase-3 and the deactivation of PARP and ultimately apoptosis. These data confirmed that the compound 15 exhibits its anticancer activity through induction of apoptosis in both H1299 and H1975 cell lines.

Compound 15 Induces Apoptosis Through Activating IRE1α, a Key Mediator in the Endoplasmic Reticulum (ER) Stress Pathway
Many studies have indicated that endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), through which tumor cells can become resistant to chemotherapeutic agents (Cheng et al., 2014). PKR-like ER kinase (PERK), inostitolrequiring transmembrane kinase and endonuclease 1α (IRE-1α), and activating transcription factor 6 (ATF6) are three primary UPR sensors that lead to distinct downstream signaling pathways (Ron and Walter, 2007). Therefore, we next studied the possible involvement of compound 15 in the activation of the ER stress pathway. H1299 cell line was treated with compound 15 at the concentration of 0.5, 1, or 2 µM for 24 h and the mRNA expression level of stress related proteins (GRP78, CHOP, XBP-1, XBP-1s, ATF4, IRE-1α, and ATF6) in ER was analyzed ( Figure 4A). Interestingly, the mRNA level of all these proteins except IRE-1α was dramatically increased upon the treatment of compound 15 at 0.5 µM. The effect of 15 at other concentrations (1 or 2 µM) on the mRNA expression level of these proteins was less significant or negligible. In the case of IRE-1α, the expression level of mRNA increased by the treatment of 15 in a dose-dependent manner, suggesting that IRE-1α might play a crucial role in compound 15-induced apoptosis. We further examined the expression level of a number of these proteins (GRP78, CHOP, XBP-1, and XBP-1s) related to ER

Compound 15 Significantly Inhibits the Growth of S180 Tumor Xenografts in Icr Mice
Since compound 15 suppressed lung cancer cell proliferation in vitro, we further investigated its ability to suppress the growth of S180 tumor xenografts in icr mice ( Figure 5). As shown in Figure 5, compound 15 (5, 15, or 20 mg/kg) suppressed the growth of S180 xenografts over the course of 7 days ( Figure 5A) comparing to the Taxol R control (10 mg/kg) and the inhibition rates of compound 15 were 8.8, 15.7, and 37.7% (Figure 5D), respectively. Tumors were collected at the end of the experiment ( Figure 5B) and the tumor weights were measured. The data showed that compound 15 significantly decreased tumor weight when compared to the untreated control, indicating that 15 effectively inhibited the growth S180 tumor xenograft. In addition, compound 15 did not cause mice to die and did not affect mouse body weight significantly at a dose of up to 20 mg/kg ( Figure 5C).

Docking Studies
Based on the X-ray crystal structure of Topoisomerase-II inhibitors bound to the ATPase domain of Topo-II (PDB: 3QX3) (Wu et al., 2011), the binding mode between Topoisomerase-II and 15 or PPT was established by autodocking (Figure 6). Compound 15 binds Topo-II between the base pairs immediately flanking the two cleaved scissile phosphates ( Figure 6A). Its polycyclic podophyllotoxin core (rings A to D) sits between base pairs, while the biotin side chain and the E ring protrude toward the DNA major and minor grooves, respectively. All parts of the podophyllotoxin core contribute to drug-DNA interaction by being located between base pairs. The E ring is anchored by both interacting with GLY-478, ASP-479, and ARG-503 residues of the enzyme and being sandwiched between R503 and the deoxyribose ring of the +1 nucleotide. Compared to PPT (Figure 6B), compound 15 shows additional hydrophobic interaction with GLN-778 and ARG-820 residues through the biotin moiety. The biotin moiety in 15 provides an additional hydrophobic moiety and multiple H-bond donors/acceptor, which allows the molecule to interact more favorably with Topo-II and might lead to improved selectivity.

Chemical Stability Investigation
The chemical stability of compound 15 in aqueous phase was investigated together with podophyllotoxin (PPT, 1) for comparison. The results indicate that compound 15 degrades slowly under the physiological condition (37 ± 1 • C, pH 7.0) with 70% material remaining after 12 h (see Figures S1-S3). A similar stability profile was observed for PPT with 75% material remaining after 12 h.

CONCLUSION
In summary, a series of biotinylated podophyllotoxin derivatives (13-26) were designed, synthesized, and evaluated for cytotoxicity against five tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW480) by using MTT assay. Among them, compound 15 showed the highest anticancer activity with its IC 50 values at 0.13-0.84 µM. Preliminary structure-activity relationship (SAR) analysis indicated that derivatives bearing an amide or triazolyl linking moiety showed weaker activity than those with an ester linkage. The 6-aminocaproic acid linking spacer affected the cytotoxic potency of these compounds in an ununiform manner. Compound 15 also reduced the expression levels of caspase-3 and PARP. Importantly, the pro-apoptotic activity of compound 15 in H1299 cell line was mediated by the transcription of IRE-1α, which plays an important role in the endoplasmic reticulum stress pathway. Finally, compound 15 at a dose of 20 mg/kg suppressed the growth of S180 tumor xenografts in icr mice significantly. Molecular docking studies suggested that compound 15 could bind well with the ATPase domain of Topoisomerase-II. Continuing studies to substantiate the further development of compound 15 as an anticancer agent are underway in our laboratory and will be reported in due course.

DATA AVAILABILITY
This manuscript contains previously unpublished data. The name of the repository and accession number are not available.

AUTHOR CONTRIBUTIONS
JZ, J-MH, and Z-HJ designed and guided this study. C-TZ and F-WD conducted the chemical synthesis. LY and YL performed the cell assay. YH and F-QX participated in the cell assay. S-TY, Y-SG, and S-YF performed animal experiments. YJ performed molecular docking. LS and Z-TD performed the SPR binding assay. LS, Z-TD, and J-MH contributed reagents, materials, and analysis tools. C-TZ and Y-SG analyzed the data. C-TZ, LY, Y-SG, Z-HJ, and J-MH wrote the manuscript. All authors read and approved the final manuscript.