Synthesis and biological evaluation of 4β-benzoxazolepodophyllotoxin hybrids as DNA topoisomerase-II targeting anticancer agents

Abstract

A series of new 4β-benzoxazolepodophyllotoxin compounds (9a–j) were prepared and screened for cytotoxicity against four human tumour cell lines (HeLa, DU-145, A-159 and MCF-7). Among these compounds, 9a, 9c, 9f and 9i have shown more potent anticancer activity than etoposide with considerable IC₅₀ values. Apoptosis evaluation studies were performed using the Hoechst-33258 staining method and it was found specially that the best active compound 9i shows clear nuclear damage compared to etoposide. Molecular docking studies were also carried out to recognize the interactions against DNA topoisomerase-II and it was found that the energy calculations were in good agreement with the observed IC₅₀ value.

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Introduction

Podophyllotoxin (1) (Fig. 1) is an abundant naturally occurring cyclolignan, which is isolated from the roots and rhizomes of various species of the Podophyllum genus such as Podophyllum peltatum and some Juniperus species.¹ Podophyllotoxin exhibits important antineoplastic, antiviral and antimitotic activities.² Its prevailing cytotoxic properties have been ascribed to its binding ability to tubulin during mitosis, which inhibits microtubule assembly.³ However, its high toxicity and low bioavailability limit its anticancer applications. In this context, semisynthetic derivatives of podophyllotoxin have been widely used as anticancer agents.^4,5 The structural modifications and mechanism of action of podophyllotoxin have been studied for many decades, mainly at Sandoz Laboratories,⁶ which led to the semisynthetic etoposide (VP-16, 2) and teniposide (VM-26, 3).

Fig. 1 Schematic representing the outline of this study.

The semisynthetic derivatives of podophyllotoxin differ significantly from the parent compound podophyllotoxin in their mechanisms of action. Although podophyllotoxin inhibits the assembly of tubulin into microtubules, in contrast etoposide and teniposide analogues exhibit inhibition of DNA topoisomerase-II, which prevents the relegation of double-stranded breaks.⁷ The epipodophyllotoxin derivatives 2–5 (Fig. 1) are currently used in chemotherapy.⁸ However, their clinical use has encountered certain limitations such as poor water solubility, development of drug resistance, metabolic inactivation and certain toxic effects.⁹ To overcome the abovementioned limitations, herein, we have shown some requisite modifications to podophyllotoxin by developing the synthesis of 4β-benzoxazolepodophyllotoxins (9a–j) and their efficiency in cytotoxicity studies against four human tumor cell lines (HeLa, DU-145, A-159 and MCF-7). We also report apoptosis measurements using Hoechst-33258 staining, which represent nuclear shrinkage and molecular interactions with docking studies.

Similar to podophyllotoxin, benzoxazoles have also occupied a unique place in the field of medicinal chemistry, due to their wide range of biological activities such as anticancer activity,¹⁰ DNA topoisomerase-I, II annihilates,¹¹ antibacterial and antifungal activity.¹² It has been reported previously that the replacement of the C-4 sugar moiety of podophyllotoxin derivatives with a non-sugar substituent has improved their therapeutic value.¹³ Accordingly, we developed the following synthetic methodology to afford 4β-benzoxazolepodophyllotoxin (9a–j).

As shown in Scheme 1, the reaction of podophyllotoxin (1) with CH₃SO₃H/NaI followed by base hydrolysis using BaCO₃ gave epipodophyllotoxin (6). Compound 6 was then converted into the 4β-cyanopodophyllotoxin (7) intermediate by a simple treatment with trimethylsilyl cyanide (TMSCN) and BF₃·OEt₂/in dry CH₂Cl₂ at −15 °C for 6 hours. Thereafter, the intermediate (7) was reacted with substituted amino phenols in the presence of copper triflate in chlorobenzene under refluxing conditions to obtain 4β-benzoxazolepodophyllotoxin derivatives (9a–j) in good yields. All the synthesized compounds were characterized by ¹H, ¹³C NMR and mass spectral data.

Scheme 1 Synthesis of 4β-benzoxazolepodophyllotoxin congeners (9a–j).

In vitro cytotoxicity assay

The synthesized 4β-benzoxazolepodophyllotoxins (9a–j) were evaluated for in vitro cytotoxic ability against a panel of human cancer cell lines, including HeLa (cervical cancer), DU145 (prostate cancer), A549 (non-small cell lung cancer) and MCF-7 (breast carcinoma) selected using an MTT assay. The results are summarized in Table 1 and a well-known standard etoposide was used as a reference standard to deduce the structure–activity relationship (SAR) for the podophyllotoxin derivatives with various substitutions on the benzoxazole appendage. As shown Fig. 2, it is clear that the basic structural unit of appendage-1 is unchanged and efforts were made to deduce the SAR by modifying appendage-2. These newly synthesized 4β-benzoxazolepodophyllotoxins showed moderate to good antiproliferative potential against most of the cell lines in this investigation. Among them, compounds 9a, 9c, 9f and 9i exhibited superior activity, with IC₅₀ values ranging from 1.2 to 5.3 μM compared to that of etoposide IC₅₀, which is 2.03–5.74 μM (Table 1). The best active compound 9i with an electron donating dimethoxy substitution on appendage 2 (Fig. 2) inhibits the growth of HeLa and DU145 cells with IC₅₀ values of 1.3 μM and 1.2 μM, respectively. In addition, this compound (9i) also displayed significant growth inhibition effect in A549 (IC₅₀ = 1.8 μM) and MCF-7 (IC₅₀ = 2.0 μM) cells.

Table 1 In vitro cytotoxicity (IC₅₀ μM)^a of the 4β-benzoxazolepodophyllotoxin compounds (9a–j)

Entry	Compound	HeLa^b	DU145^c	A549^d	MCF-7^e
a Each data represents as mean ± S.D values. From three different experiments performed in triplicates.b HeLa: human cervical cancer cell line.c DU145: human prostate cancer cell line.d A549: human lung cancer epithelial cell line.e MCF-7: human breast carcinoma cell line.
1	9a	2.8 ± 0.14	3.2 ± 0.2	4.3 ± 1.3	2.6 ± 0.1
2	9b	5 ± 0.3	6.8 ± 2.4	11.7 ± 1.7	4.8 ± 2.4
3	9c	1.5 ± 0.08	1.9 ± 0.14	4.0 ± 0.2	2.5 ± 0.13
4	9d	5.6 ± 0.6	7.9 ± 0.7	8.6 ± 0.3	6.0 ± 0.4
5	9e	14.3 ± 0.9	16.7 ± 1.0	8.4 ± 0.7	10.8 ± 0.8
6	9f	2.2 ± 0.13	5.3 ± 0.7	2.6 ± 0.12	1.2 ± 0.05
7	9g	11.7 ± 0.8	14.1 ± 1.7	11.3 ± 0.8	10.3 ± 1.1
8	9h	22.8 ± 1.1	12.0 ± 1.0	23.0 ± 1.1	14.5 ± 1.5
9	9i	1.3 ± 0.03	1.2 ± 0.04	1.8 ± 0.05	2.0 ± 0.06
10	9j	14.8 ± 0.8	28.7 ± 2.2	21.8 ± 1.1	5.8 ± 0.23
11	Etoposide	5.74 ± 0.37	2.58 ± 0.25	2.03 ± 0.12	2.61 ± 0.32

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Fig. 2 Structural activity relationship (SAR).

Regarding the activity of compounds 9b and 9c, which are structural isomers possessing monomethoxy substitutions at C6 and C5 of the phenyl ring without any change at appendage 1, they exhibited varied cytotoxicity (entry 2 and 3, Table 1). When compared to compound 9b, the structural isomer 9c, which possessed C5-OCH₃, exhibited a profound activity against all four cancer cell lines used in this study with considerable IC₅₀ values. Compound 9f with a chloro (Cl) group at the C5 position of the phenyl ring also showed a considerable effect on cell growth with the highest inhibition particularly in MCF-7 (IC₅₀ = 1.2 μM). On the other hand, compound 9e with a chloro (Cl) group at C5 and an electron withdrawing nitro (–NO₂) substitution at the C6 position showed a very minimum effect on growth. The results presented in Table 1 also provide the finding that while the presence of weak electron donating substitution, such as –CH₃ (9h, 9j) and a neutral –H (9a) at appendage-2, can show moderate potency, those containing electron withdrawing –NO₂ substituents (9d and 9g) showed a diminished cytotoxic effect. Based on the structural diversity, the optimal order of substitutions on the phenyl ring of appendage-2 is methoxy > chloro > unsubstituted > methyl > nitro (Fig. 2). Overall, these results suggest that the best active compounds, which are 9a, 9c, 9f and 9i, showed excellent antiproliferative activity compared to that of the positive control etoposide.

Chromatin condensation by Hoechst-33258 staining

DNA fragmentation is the most visible marker of apoptosis and one of the major pathways of cell death. A classic characteristic of apoptosis is chromatin condensation and nuclear fragmentation/shrinkage.¹⁴ Hoechst-33258 staining is a commonly employed technique to distinguish the compact chromatin of apoptotic nuclei to identify replicating cells and to sort cells based on their DNA content. To elucidate, whether the 4β-benzoxazolepodophyllotoxin congeners (9c and 9i) induced cytotoxicity by cellular apoptosis, HeLa cells were exposed to a 3 μM concentration of the representative compounds for 24 hours. The results demonstrated that the active compounds condensed the nuclear content considerably. In particular, the best active compound 9i exhibited clear nuclear damage in comparison to the etoposide (Eto) (Fig. 3).

Fig. 3 Benzoxazolepodophyllotoxin congeners cause apoptosis in HeLa cells.

Cells were treated with 9c, 9i and etoposide (Eto) at a 3 μM concentration and control (DMSO) for 24 hours, washed with PBS, and incubated with the Hoechst-33258 stain (4 mg mL⁻¹) for 20 minutes to measure chromatin condensation. Micro photographed images were captured using a fluorescence microscope equipped with a DAPI filter.

Molecular modeling studies

Docking studies were carried out by employing the AutoDock software and the 9i, 9i + Eto (etoposide) structures were docked against the crystal structure of a human DNA topoisomerase-II receptor that was retrieved from the protein data bank with PDB: 3QX3. The best active compound 9i occupied the exact site wherein the positive control etoposide binds with the protein. The yellow coloured stick (9i) was sandwiched between and surrounded by the most important amino acid residues, such as Asp-479, Arg-503, Met-782, Gln-778, Ala-779, Gly-478, Gly-1023, Leu-852, Glu-477 and Phe-720. Strong hydrogen bonding was observed between the –O of the trimethoxy phenyl ring and –NH of Asp-479 (O⋯HN, distance: 3.0 Å). In addition, the –O of the methoxy phenyl ring showed van der Waals interactions with the –NH of Arg-503. Another hydrogen bond was noticed between the O of the lactone ring and NH of the amino acid residue Gln-778 (O⋯HN, distance: 3.0 Å). Interestingly, the modified structural unit benzoxazole also exhibited considerable interactions with topoisomerase along with the nucleotide of DNA. In comparison, the methoxy –O of the benzoxazole unit forms weak interactions with the NH of Met-782. Furthermore, some hydrophobic interactions were observed between compound 9i and amino acid residues such as Ala-779, Gly-478, Gly-1023, Leu-852, Glu-477 and Phe-720. Overall, the docking results reveal that compound 9i interacts with topoisomerase-II in a similar manner with respect to that of etoposide. A superimposition pose also demonstrates that the newly synthesized compounds induce antiproliferative potential by inhibiting DNA topoisomerase-II (Fig. 4). In addition, the docking interactions for the succeeding active compounds 9a, 9c and 9f of the same series were also evaluated (Fig. S1 see ESI†) and the calculated docking parameters are summarized in Table 2.

Fig. 4 Molecular docking poses for 9i and 9i + etoposide (Eto) in DNA topoisomerase-II.

Table 2 Estimated free energy binding and inhibition constants of the representative molecules 9a, 9c, 9f, 9i and etoposide

Entry	Compound	Free energy binding energy	Inhibition constant, Ki
1	9a	−13.24 kcal mol^−1	195.61 pM
2	9c	−13.18 kcal mol^−1	219.98 pM
3	9f	−13.42 kcal mol^−1	144.96 pM
4	9i	−12.91 kcal mol^−1	342.40 pM
5	Etoposide	−8.54 kcal mol^−1	551.55 pM

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Molecular docking poses for 9i and 9i + etoposide (Eto) in DNA topoisomerase-II. The most potent compound 9i is shown as yellow colour sticks, the interacted amino acid residues are represented as magenta sticks and the surrounding amino acids are shown as lines. The hydrogen bonding interactions with ASP-479, GLN-778 and ARG-503 are denoted as red dots. The protein DNA topoisomerase-II is shown as a pale green ribbon. The hydrophobic interactions are also shown as black dots. A pose of superimposition of the potent compound and etoposide (9i + Eto) demonstrated similar interactions with respect to the positive control. Moreover, different docking poses of compound 9i are represented in Fig S2, ESI.† The representation of yellow (9i) and magenta (Eto) sticks were proposed in the image. PyMOL was used to visualize the docking poses.

Experimental

Chemicals and reagents

All chemicals and reagents were obtained from Aldrich (Sigma-Aldrich, St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey Company, Ward Hill, MA, USA) and were used without further purification. Reactions were monitored by TLC, which were performed on silica gel glass plates containing 60 F-254 and visualization of the TLC plates was achieved by UV light or an iodine indicator. ¹H and ¹³C NMR (Nuclear Magnetic Resonance) spectra were obtained on a Gemini Varian-VXR-unity (200 and 400 MHz) or Bruker UXNMR/XWIN-NMR (300 MHz) instrument. Chemical shifts (δ) are reported in ppm downfield from the internal TMS standard. ESI spectra were obtained on a Micromass, Quattro LC using the ESI+ software with a capillary voltage of 3.98 kV and an ESI mode positive ion trap detector. Melting points were determined with an electro thermal melting point apparatus and are uncorrected.

Materials and methods

Cell culture and reagents

The cell lines used in this study were obtained from the American Type Culture Collection (ATCC). DU145 (human prostate carcinoma epithelial) cells were cultured in eagle's minimal essential medium (MEM) containing nonessential amino acids, 1 mM sodium pyruvate, and 10% FBS. HeLa (human epithelial cervical cancer), MCF-7 (human breast cancer) and A549 (human lung carcinoma epithelial) were co-cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing nonessential amino acids and 10% FBS. All cells were maintained under a humidified atmosphere of 5% CO₂ at 37 °C. Subconfluent cells were trypsinized from T75 flasks/90 mm dishes and seeded onto 96-well test plates at a density of 1 × 10⁴ cells per well in a complete medium, treated with compounds at a desired concentrations and harvested as required.¹⁵

In vitro cytotoxicity assay

Cell viability was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The pale yellow coloured tetrazolium salt (MTT) is reduced to a dark blue water-insoluble formazan by metabolically active cells, which is measured quantitatively after dissolving in DMSO (dimethyl sulfoxide). The absorbance of the soluble form of formazan is directly proportional to the number of viable cells. Cells were seeded at a density of 1 × 10⁴ cells in 200 μL of medium per well of a 96-well plate. Furthermore, the plates were incubated prior to the addition of the experimental compounds for 24 hours. Moreover, the cells were treated with the vehicle alone (0.4% DMSO in medium) or compounds (drugs were dissolved in DMSO previously) at different concentrations (1, 10 and 25 μM) for 48 hours. The assay was completed with the addition of MTT (5%, 10 μL) and incubated for 60 min at 37 °C. The supernatant was aspirated and plates were air dried and the MTT-formazan crystals were dissolved in 100 μL of DMSO. Optical density (O.D) was measured at 560 nm using a TECAN Multimode reader. The growth percentage of each treated well of the 96-well plate was calculated based on the test wells relative to the control wells. Cell growth inhibition was calculated by generating dose response curves as a plot of the percentage of surviving cells versus drug concentration. The antiproliferative activity of the cancer cells to the test compounds was expressed in terms of IC₅₀ values, which is defined as the concentration of compound that produces 50% absorbance reduction relative to the control.¹⁶

Hoechst-33258 staining

HeLa cells were incubated for a period of 24 hours in the presence or absence of test compounds 9c, 9i and etoposide (Eto) (3 μM). At the end of treatment, the medium was removed, cells were washed with medium without FBS, and Hoechst-33258 stain (invitrogen cat. no. H3570) was added to the cells for 20 minutes at 37 °C under a humidified atmosphere and the HeLa cells were washed twice with the medium. The cells were covered with the medium and observed under a fluorescence microscope equipped with a DAPI filter.¹⁷

Molecular modelling

AutoDock was used to dock the 9i derivatives in the etoposide (Eto) binding site of human DNA topoisomerase-II.¹⁸ Initial Cartesian coordinates for the protein–ligand complex structure were derived from the crystal structure of DNA topoisomerase-II (PDB ID: 3QX3). The protein targets were prepared for molecular docking simulation by removing water molecules and bound ligands. Hydrogen atoms and Kollman charges were added to each protein atom. AutoDock Tools (ADT) was used to prepare and analyze the docking simulations for the AutoDock program. The coordinates of each compound were generated using Chemdraw 11 followed by MM2 energy minimization. Grid map in AutoDock, which defines the interaction of protein and ligands in the binding pocket, was defined. The grid map was used with 60 points equally in each x, y, and z direction. AutoGrid 4 was used to produce grid maps for AutoDock calculations where the search space size utilized grid points of 0.375 Å. The Lamarckian genetic algorithm was chosen to search for the best conformers. Each docking experiment was performed 100 times, which yielded 100 docked conformations. The parameters used for the docking were as follows: population size of 150; random starting position and conformation; maximal mutation of 2 Å in translation and 50 degrees in rotations; elitism of 1; a mutation rate of 0.02 and crossover rate of 0.8; and local search rate of 0.06. Simulations were performed with a maximum of 1.5 million energy evaluations and a maximum of 50 000 generations. Final docked conformations were clustered using a tolerance of 1.0 Å root mean square deviation. The best model was picked based on the best stabilization energy. The final figures for molecular modelling were generated using PyMol.¹⁹

Conclusions

In summary, a series of 4β-benzoxazolepodophyllotoxins (9a–j) were prepared and screened for their cytotoxicity against four human tumour cell lines (HeLa, DU-145, A-549 and MCF-7) and were found to be more potent than etoposide. Some of these 4β-benzoxazolepodophyllotoxins (9a–j) have shown promising activity with considerable IC₅₀ values. Among these compounds, specifically 9a, 9c, 9f and 9i showed more potent anticancer activity than etoposide. Furthermore, compounds 9c and 9i were evaluated for Hoechst-33258 staining and clear nuclear damage was noticed in comparison to etoposide. Finally, the most active compound 9i was also investigated for molecular docking interactions against DNA topoisomerase-II and compared with etoposide. The energy calculations were in good agreement with the observed IC₅₀ values.

Acknowledgements

Dr S. Paidakula is thankful to DST-SERB, New Delhi for the award of DST-Fast Track (SB/FT/CS-015/2014) and also Dr S. Kankala thankful to the School of Physics & Chemistry, University of KwaZulu-Natal, South Africa for the facilities, NRF-South Africa and DST-India (DST/INT/SA/P-15/2011 Indo-South Africa project).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Docking poses for 9a, 9c and 9f in DNA topoisomerase-II, top 10 interaction poses of compound 9i and experimental section. See DOI: 10.1039/c5ra15366b

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