Design, synthesis , and biological and docking studies of novel epipodophyllotoxin– chalcone hybrids as potential anticancer agents

Abid Hussain Banday; Vinod V. Kulkarni; Victor J. Hruby

doi:10.1039/C4MD00325J

View PDF VersionPrevious ArticleNext Article

DOI: 10.1039/C4MD00325J (Concise Article) Med. Chem. Commun., 2015, 6, 94-104

Show CompoundsShow Chemical TermsShow Biomedical Terms

Design, synthesis, and biological and docking studies of novel epipodophyllotoxin–chalcone hybrids as potential anticancer agents†

Abid Hussain Banday *^ab, Vinod V. Kulkarni ^b and Victor J. Hruby ^b
^aDepartment of Chemistry, Islamia College of Science and Commerce, Srinagar, India-190009. E-mail: abidrrl@gmail.com; Fax: +91 520 621 8407; Tel: +91 520 248 7528
^bDepartment of Chemistry and Biochemistry, University of Arizona, Tucson, USA-85721

Received 27th July 2014 , Accepted 8th September 2014

First published on 11th September 2014

Abstract

A series of new compounds consisting of epipodophyllotoxin–chalcone hybrids was synthesized towards the development of better anticancer lead molecules. These hybrids consist of structurally different but functionally similar topoisomerase-II inhibitors which were conjugated together through click-chemistry. Their design is aimed at the synthesis of novel chemotherapeutics with a better target-protein interaction and higher bioavailability. Evaluation of the anticancer activity of these designed conjugates against a panel of six human cancer cell lines proved their potential cytotoxicity. Further, these compounds were docked against topoisomerase-II and the energy calculations were in good agreement with the observed IC₅₀ values. Compounds 6d, 6f, 7d and 7f exhibited significant in vitro cytotoxicity. Among all the compounds evaluated, compound 7f was found to be the most promising, especially selective against SW-620 and SKN-SH cell lines.

1. Introduction

Drug discovery from plant-based compounds has since long been a paradigm for the treatment of various diseases and ailments.^1,2 In this journey, a large number of plant-based products has taken a lead position in the market as active pharmaceutical agents.^3,4 Podophyllotoxin, 1, is a bioactive cyclolignan component of Podophyllum peltatum and Podophyllum hexandrum.^5,6 The C-4β epimer, epipodophyllotoxin, 2, has been a focal point for medicinal chemists in the recent past as several of its semi-synthetic derivatives, such as etoposide and teniposide had already taken a place in the market as potential anticancer drugs.^7–14 The cytotoxicity of podophyllotoxin, 1, is generally attributed to the inhibition of tubulin polymerization whereas epipodophyllotoxin, 2, and its 4β-congeners viz., etoposide and teniposide have been found to target DNA topoisomerase-II.¹⁵ Both etoposide and teneposide block the catalytic activity of topoisomerase-II by stabilizing a cleavable enzyme-DNA complex in which the DNA is cleaved and covalently linked to the enzyme which ultimately leads to cell death. Both etoposide and teneposide are active against various cancers such as lymphoma, testicular carcinoma, Kaposi's sarcoma, and small cell lung cancer.⁷ However, the therapeutic potential of these drugs is often hampered by problems of poor bioavailability and acquired drug resistance.^16,17 To address these concerns, libraries of podophyllotoxin, 1, and epipodophyllotoxin, 2, have been synthesized. As per the established structure–activity relationship (SAR),¹⁸ the 4β-position of epipodophyllotoxin is the most amenable position for modification. Most of other structural features are very essential for the cytotoxic activity and must be kept intact. Keeping this into consideration, two 4β-analogs GL-331 (ref. 11) and TOP-53 (ref. 19) were reported to exhibit potential anticancer activity.^6,20,21 Some of the derivatives such as NK-611 and NPF made it to the clinical stage,^22,23 whereas etopophos (a water-soluble prodrug), is available in the market for the treatment of a variety of malignancies.⁴⁰ SAR has revealed that bulky substituents at C-4 might be favorable for topoisomerase-II inhibition, and may enhance the cytotoxicity.²⁴ Accordingly, numerous derivatives based on modifications at the C-4 position of epipodophyllotoxin were synthesized to develop better lead molecules.

Keeping the SAR into consideration, we, in analogy with some related literature precedents,^25,26 hybridized 4β-epipodophyllotxin and chalcones both of which are proven anticancer agents. The reason behind the design lies in the similarity of their modes of action as many recent reports have revealed the topoisomerase-II inhibitory activity of chalcones,^27,28 being responsible for their cytotoxicity. Click-chemistry, which enables a modular approach to generate novel pharmacophores, was exploited to conjugate the two moieties. The rationale behind the use of click-chemistry is based on our previous work on steroidal triazoles and 4β-[(4-substituted)-1,2,3-triazol-1-yl] podophyllotoxins, wherein we established that the introduction of a triazole moiety into natural products enhances their anticancer activity.^29–32 The epipodophyllotoxin–chalcone hybrids, consisting of structurally different but functionally similar units, provide structurally compact motifs with a possible combined and/or synergistic pharmacological effect. There are chances of competition amongst the ligands when the target is the same. However, as revealed by the cytotoxicity and the docking studies, the binding sites of the two entities are likely to be different leading to better binding interactions of the hybrids with topoisomerase-II. All the designed hybrids were evaluated for their in vitro anticancer activity against a panel of six human cancer cell lines viz., SW-620 (Colon), DU-145 (Prostrate), SKN-SH (CNS), HCT-15 (Colon), Colo-205 (Colon) and HeLa (Cervix). Most of these compounds exhibited promising cytotoxicity with some having IC₅₀ values even better than etoposide. The compounds were also docked against topoisomerase-II and the binding calculations were in good agreement with the observed wet lab results (IC₅₀ values). Compounds 6d, 6f, 7d and 7f were found to be the most promising in this study especially against SW-620 and SKN-SH cell lines (Table 1).

Table 1 4-(4-Substituted)-1,2,3-triazol-1-yl epipodophyllotoxins differing in substitution at R′′


Entry	R	R′′	Entry	R	R′′
6a	CH₃		7a	H
6b	CH₃		7b	H
6c	CH₃		7c	H
6d	CH₃		7d	H
6e	CH₃		7e	H
6f	CH₃		7f	H
6g	CH₃		7g	H
6h	CH₃		7h	H
6i	CH₃		7i	H
6j	CH₃		7j	H

2. Results and discussion

Design and development of pharmacologically active chemical entities has always been the curiosity of medicinal chemists. Chemists have either used pharmacologically active natural products or relied upon small molecule designer analogs for the development of potent bioactive molecules. However, the exploitation of natural products for the synthesis of lead compounds has been the backbone of medicinal chemistry especially during the recent past.^3,4 Various efficient synthetic techniques have been used in various facets of drug discovery enabling a modular approach to generate novel pharmacophores. The present manuscript describes a rational design and synthesis of pharmacologically active compounds wherein chalcone moieties are conjugated with epipodophyllotoxin through the ‘click-chemistry’ approach. The rationality lies in the possible similarity of modes of action of the two structurally different molecules. The hybrid conjugates thus synthesized were screened for their cytotoxic activity against a panel of human cancer cell lines and were found to be very active (Fig. 1).


	Fig. 1 Structures of podophyllotoxin (1) and epipodophyllotoxin (2).

2.1. Chemistry

Podophyllotoxin was converted to C4-β-azido podophyllotoxin, 3, and C4-β-azido-4′-O-demethyl podophyllotoxin, 4, in accordance with established literature procedures.^33,34 As illustrated in Scheme 1, 4β-[(4-substituted)-1,2,3-triazol-1-yl] podophyllotoxin (6a–6j) and 4β-[(4-substituted)-1,2,3-triazol-1-yl]-4′-O-demethyl podophyllotoxin (7a–7j) derivatives were synthesized by the cycloaddition reaction of C4-β-azido podophyllotoxin, 3, and C4-β-azido-4′-O-demethyl podophyllotoxin, 4, with O-aryl-propyne ethers, 5, obtained by reacting chalcone with proporgyl bromide in the presence of potassium carbonate in acetone under reflux conditions.


	Scheme 1 Click-chemistry strategy for the synthesis of novel triazolyl epipodophyllotoxin congeners.

Azides, 3 and 4 were allowed to react with the O-propargylated phenols/chalcones, 5, as terminal alkynes in the presence of CuSO₄·5H₂O and sodium ascorbate in t-butyl alcohol and water (1 : 2) at room temperature to selectively generate a focused library of 1,2,3-triazolyl derivatives (6a–j and 7a–j) in excellent yields (Scheme 1).

All the products obtained were characterized by ¹H NMR, ¹³C NMR, IR, ESI-MS and elemental analysis. In ¹H NMR, cyclization of azides to triazoles was confirmed by the shift of 4-α H to around δ 6 and the resonance of H-5 in the triazole ring in the aromatic region. The structure was further supported by ¹³C NMR, which showed all the carbon signals corresponding to triazole derivatives. ESI-MS of all compounds showed M + H or M + Na adduct as the molecular ion.

2.2. Cytotoxicity studies

The human cancer cell lines used for the test were SW-620 (Colon), DU-145 (Prostrate), SKN-SH (CNS), HCT-15 (Colon), Colo-205 (Colon) and HeLa (Cervix). Cellular viability in the presence and absence of experimental agents was determined using the standard sulforhodamine B assay as described in Experimental section. By comparing the cytotoxic potential of compounds with different substitutions, it is evident that 4′-O-demethyl analogs (7a–j) are generally more active than the corresponding methylated analogs (6a–j). Further, the non-chalconated derivatives (6a–6c and 7a–7c) exhibited less cytotoxicity compared to the chalconated hybrids. This may be attributed to the synergistic effect which is expected from the hybridization of functionally similar molecules interacting with the same target, i.e. topoisomerase-II. Based on our previous studies,³⁰ it is more likely that the designed hybrids induced apoptosis due to topoisomerase-II inhibition. The exact mechanism of action towards the inhibition of both topoisomerase-II and topoisomerase-I, is currently under investigation. However, as indicated by the docking calculations, it is evident that the designed hybrids have much better binding affinities than etoposide towards topoisomerase-II. Most of the designed compounds exhibited better in vitro cytotoxicity than etoposide. The increased activity may be attributed either to a better target binding or to the increased solubility leading to better bioavailability. Further, the IC₅₀ values and the docking calculations of non-chalconated derivatives indicate that the presence of chalcone affects the binding of epipodophyllotoxin to the binding site of topoisomerase-II which was previously established as the ATPase domain. As the target site of chalcones on topoisomerase-II is not yet known, it is more likely that the binding site is different from that of epipodophyllotoxin. Had it been the same, the two might compete with each other resulting in lower binding-interaction (higher glide score) and higher IC₅₀ values of the hybrids. The IC₅₀ values calculated from the in vitro screening studies further revealed that the compounds 6d, 6f, 7d and 7f showed significant cytotoxic activity. Compound 7f, which contained a fluorinated chalcone, showed the highest cytotoxicity, selectively against SW-620 and SKN-SH cell lines. It is pertinent to mention here that the parent epipodophyllotoxin azide did not show any appreciable cytotoxicity on any of the cancer cell lines under scrutiny.

2.3. Docking studies

By considering the ATPase domain of topoisomerase-II (PDB ID : 1ZXM) as the binding site^30,35 observed in the co-crystallized structure of 1ZXM, all the molecules were docked as per the procedure given in Experimental section. The ATPase domain was selected for being the main binding site for epipodophyllotoxin analogs, i.e. etoposide, as reported earlier.^35,36 In agreement with the observed IC₅₀ values, the best glide scores were obtained for the potent ligands 7d (GS: −6.819) and 7f (GS: −7.409) and these were considered for interpreting the interactions. The interactions of 7d and 7f ligands with the binding domain of the receptor were found to be similar. The ligands maintained a distributed but close hydrophobic contact within the 5 Å hydrophobic pocket of Leu140, Phe142, Ile141, Pro126, Ile125, Val137 and Ala167 of the protein. The chalcone fragment of the ligands is within 4 Å from His130 and Val132. Arg98 maintains a relatively close π–π contact with the triazole group of 7d and 7f (similar contacts were observed with purine of ANP bound to topoisomerase-II of 1ZXM). H-bond strengths with the guanidinium group are 2.35 Å (for 7d), and 2.48–2.55 Å (for 7f). Ser148 has a notable H-bond interaction exhibiting a strength of 2.36 Å for 7d and 2.43 Å for 7f. Ser149 additionally offers stabilization of the lactone ring of the ligands. The phenolic moiety of the ligand is well stabilized by H-bond interactions of 2.28 Å and 2.10 Å for 7d and 7f, respectively with Ala167. The ligand interactions of the potent ligands (7d and 7f) with the receptor are shown in Fig. 2 and 3.


	Fig. 2 Interaction of compound 7d with the residues within 5 Å of the ATPase domain of topoisomerase-II.


	Fig. 3 Interaction of compound 7f with the residues within 5 Å of the ATPase domain of topoisomerase-II.

3. Conclusions

In conclusion, a series of 4β-[(4-substituted)-1,2,3-triazol-1-yl] podophyllotoxins were evaluated for the in vitro cell cytoxicity. Evaluation of the anticancer activity of these designed conjugates against a panel of six human cancer cell lines proved their cytotoxic nature. Further, the compounds were docked against topoisomerase-II and the glide scores were in good agreement with the observed IC₅₀ values. As indicated by the docking calculations, it is evident that the designed hybrids have much better binding affinities than etoposide towards topoisomerase-II. Compounds 6d, 6f, 7d and 7f exhibited significant in vitro cytotoxicity. Among all the compounds evaluated, compound 7f was found to be the most promising, especially selective against SW-620 and SKN-SH cell lines.

The exact mechanism of action of the designed hybrids towards the inhibition of both topoisomerase-II and topoisomerase-I, is currently under investigation. We, further, intend to investigate the target site of chalcones on topoisomerase-II and to study the inhibitory activity of all the active compounds against topoisomerase-II.

4. Experimental

Proton and ¹³C spectra were recorded using a Bruker DPX400 instrument in CDCl₃ as the solvent with TMS as the internal standard for protons and solvent signals as the internal standard for carbon spectra. The δ scale (ppm) has been used for reporting chemical shift values, and coupling constants are given in Hz. The Buchi MP apparatus D-545 was used for recording the melting points; IR spectra (KBr) were recorded using a Bruker Vector 22 instrument. An ESI-esquire 3000 Bruker Daltonics instrument was used for recording mass spectra. The progress of all reactions was monitored by TLC on 2 × 5 cm pre-coated silica gel 60 F254 plates of thickness 0.25 mm (Merck). The chromatograms were visualized under UV 254-366 nm and iodine.

4.1. General procedure for chemical synthesis

Compound 5 (2 mmol) was dissolved in t-BuOH/water (1 : 2, 12 mL) followed by the addition of CuSO₄·5H₂O (2.0 mmol) and sodium ascorbate (10.0 mmol). After 10 min, epipodophyllotoxin azide 3 or 4 (2 mmol) was charged into the reaction mixture which was then stirred at room temperature for 6 h. After completion, as indicated by TLC, the reaction mixture was diluted with 60 mL of water and extracted with ethyl acetate (3 × 25 mL). All the extracts were combined and dried over Na₂SO₄ followed by in vacuo evaporation. The crude products obtained after the evaporation were crystallized from diethyl ether/hexanes to yield the pure products (6a–j and 7a–j). The spectral data of all the products obtained is given as follows.

6a. 9-[4-(4-Acetyl-phenoxymethyl)-[1,2,3]triazol-1-yl]-5-(3,4,5-trimethoxy-phenyl)-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-6-one. White solid; mp: 135–137 °C; [α]²⁵_D − 51 (0.75, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 2.48 (s, 3H), 3.04 (m, 1H), 3.24 (m, 2H), 3.72 (s, 6H), 3.82 (s, 3H), 4.31 (t, J = 7.6, 1H), 4.65 (d, J = 4.8, 1H), 5.15 (s, 2H), 5.92 (d, J = 5.94, 2H), 6.07 (d, J = 3.67, 1H), 6.24 (s, 2H), 6.56 (d, J = 5.68, 2H), 6.96 (d, J =7.21, 2H), 7.37 (s, 1H), 7.84 (d, J = 7.47, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 29.4, 37.0, 41.4, 43.4, 55.9, 58.8, 60.8, 62.4, 67.5, 105.3, 111.3, 111.5, 112.0, 113.8, 113.9, 117.9, 118.0, 127.1, 134.1, 146.7, 150.4, 150.5, 151.5, 152.7, 177.3, 202.2; IR (KBr): 3346.3, 1771.6, 1731.2, 1598.5, 1516.3, 1477.1, 1338.0, 1016.6, 756.7 cm⁻¹; ESI-MS: 636.2 (M + Na). Anal. calc. for C₃₃H₃₁N₃O₉: C, 64.59; H, 5.09; N, 6.85 found: C, 64.54; H, 5.03; N, 6.90%.

6b. 5-(3,4,5-Trimethoxy-phenyl)-9-{4-[4-(3-oxo-but-1-enyl)-phenoxymethyl]-[1,2,3]triazol-1-yl}-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho[2,3-d][1,3]-6-one. White solid; mp: 129–131 °C; [α]²⁵_D − 23.3 (0.75, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 2.35 (s, 3H), 3.05 (m, 1H), 3.27 (m, 2H), 3.78 (s, 6H), 3.81 (s, 3H), 4.40 (t, J = 7.6, 1H), 4.75 (d, J = 4.8, 1H), 5.20 (s, 2H), 5.48 (s, 1H), 6.02 (d, J = 4.19, 2H), 6.11 (d, J = 4.0, 1H), 6.33 (s, 2H), 6.62 (m, 2H), 7.02 (d, J = 8.72, 2H), 7.28 (m, 2H), 7.53 (d, J = 8.78, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 25.3, 27.4, 29.7, 35.0, 41.7, 43.5, 56.5, 58.8, 60.87, 62.0, 67.4, 82.5, 84.4, 102.0, 107.8, 108.8, 110.6, 115.2, 123.1, 124.4, 125.4, 127.8, 129.6, 130.0, 133.4, 134.4, 142.9, 143.8, 146.6, 148.1, 150.5, 159.9, 172.1, 198.4; IR (KBr): 3362.9, 1771.6, 1595.5, 1505.3, 1485.2, 1238.0, 1013.6, 764.8 cm⁻¹; ESIMS: 662.2 (M + Na). Anal. calc. for C₃₅H₃₃N₃O₉: C, 65.72; H, 5.20; N, 6.57 found: C, 65.80; H, 5.24; N, 6.67%.

6c. 5-(3,4,5-Trimethoxy-phenyl)-9-{4-[2-methoxy-4-(3-oxo-but-1-enyl)-phe-noxy-methyl]-[1,2,3]triazol-1-yl}-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho [2,3-d][1,3]dioxol-6-one. White solid; mp: 124–126 °C; [α]²⁵_D − 34.6 (0.70, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 2.33 (s, 3H), 3.05 (m, 1H), 3.27 (m, 2H), 3.72 (s, 6H), 3.76 (s, 3H), 3.80 (s, 3H), 4.40 (t, J = 7.6, 1H), 4.79 (d, J = 4.8, 1H), 5.22 (s, 2H), 5.40 (s, 1H), 6.02 (d, J = 3.67, 2H), 6.25 (s, 1H), 6.53 (m, 1H), 6.69 (m, 2H), 7.01 (s, 2H), 7.28–7.48 (m, 2H), 7.53 (d, J = 8.78, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 24.3, 27.4, 29.7, 37.0, 41.6, 43.4, 55.9, 56.5, 58.8, 60.6, 62.0, 67.4, 82.5, 84.4, 102.0, 107.8, 108.9, 110.5, 115.2, 122.7, 123.4, 125.8, 127.8, 129.6, 130.0, 133.4, 134.4, 142.9, 143.8, 146.6, 148.1, 149.5, 159.9, 173.1, 196.8; IR (KBr): 3364.6, 1775.2, 1598.2, 1507.1, 1485.2, 1239.2, 1004.6, 755.7 cm⁻¹; ESI-MS: 6792.2 (M + Na). Anal. calc. for C₃₆H₃₅N₃O₁₀: C, 64.57; H, 5.27; N, 6.27 found: C, 64.54; H, 5.24; N, 6.34%.

6d. 5-(3,4,5-Trimethoxy-phenyl)-9-{4-[4-(3-oxo-3-phenyl-propenyl)-phenoxymethyl]-[1,2,3]triazol-1-yl}-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho[2,3-d] [1.3] dioxol-6-one.. White solid; mp: 145–147 °C; [α]²⁵_D − 41 (0.75, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 3.04 (m, 1H), 3.28 (m, 2H), 3.78 (s, 6H), 3.83 (s, 3H), 4.41 (t, J = 7.6, 1H), 4.76 (d, J = 4.6, 1H), 5.26 (s, 2H), 5.47 (s, 1H), 6.01 (d, J = 3.7, 2H), 6.12 (d, J = 4.0, 1H), 6.32 (s, 2H), 6.63 (d, J = 7.7, 2H), 7.05 (d, J = 8.6, 2H),7.28–7.54 (m, 3H), 7.57–7.79 (m, 4H), 8.04 (d, J = 8.78, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 29.6, 37.1, 41.7, 43.5, 56.6, 58.9, 60.8, 62.1, 67.4, 102.1, 107.9, 108.8, 110.6, 114.7, 121.8, 123.3, 124.5, 128.4, 129.0, 129.7, 130.5, 130.9, 131.8, 134.5, 135.0, 144.3, 146.7, 148.2, 149.5, 186.7; IR (KBr): 3365.7, 1778.6, 1598.5, 1504.3, 1485.2, 1233.4, 1005.3, 758.3 cm⁻¹; ESI-MS: 724.2 (M + Na). Anal. calc. for C₄₀H₃₅N₃O₉: C, 68.46; H, 5.03; N, 5.99 found: C, 68.50; H, 5.04; N, 5.93%.

6e. 5-(3,4,5-Trimethoxy-phenyl)-9-{4-[4-(3-phenyl-acryloyl)-phenoxy methyl]-[1,2,3]triazol-1-yl}-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho[2,3-d] [1,3]dioxol-6-one. White solid; mp: 137–138 °C; [α]²⁵_D − 48.8 (0.50, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 3.06 (m, 1H), 3.25 (m, 2H), 3.79 (s, 6H), 3.82 (s, 3H), 4.39 (t, J = 7.6, 1H), 4.76 (d, J = 4.5, 1H), 5.22 (s, 2H), 5.46 (s, 1H), 6.03 (d, J = 3.65, 2H), 6.14 (d, J = 3.65, 1H), 6.32 (s, 2H), 6.64 (d, J = 8.2, 2H), 7.02 (d, J = 8.6, 2H),7.24–7.32 (m, 2H), 7.34–7.54 (m, 3H), 7.56–7.77 (m, 2H), 8.04 (d, J = 8.57, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 29.5, 37.2, 41.7, 43.5, 56.6, 58.9, 60.8, 62.1, 67.4, 102.1, 107.9, 108.8, 110.6, 114.7, 121.8, 124.3, 124.5, 128.5, 129.0, 129.7, 130.5, 132.9, 131.8, 134.5, 135.0, 144.3, 146.7, 147.8, 150.4, 186.6; IR (KBr): 3342.1, 1777.7, 1587.2, 1598.3, 1485.2, 1287.0, 1001.6, 758.5 cm⁻¹; ESIMS: 724.2 (M + Na). Anal. calc. for C₄₀H₃₅N₃O₉: C, 68.46; H, 5.03; N, 5.99 found: C, 68.51; H, 5.03; N, 5.95%.

6f. 9-(4-{4-[3-(4-Fluoro-phenyl)-acryloyl]-phenoxymethyl}-[1,2,3]triazol-1-yl)-5-(3,4,5-trimethoxy-phenyl)-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho [2,3-d] [1,3] dioxol-6-one. White solid; mp: 154–156 °C; [α]²⁵_D − 33 (0.75, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 3.05 (m, 1H), 3.26 (m, 2H), 3.79 (s, 6H), 3.82 (s, 3H), 4.42 (t, J = 7.6, 1H), 4.75 (d, J = 4.7, 1H), 5.26 (s, 2H), 5.50 (s, 1H), 6.03 (d, J = 3.88, 2H), 6.12 (d, J = 4.2, 1H), 6.32 (s, 2H), 6.64 (d, J = 7.5, 2H), 7.05–7.24 (m, 5H), 7.66–7.80 (m, 3H), 8.03 (d, J = 8.77, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 35.9, 42.1, 56.9, 59.27, 60.8, 62.3, 66.2, 67.8, 67.7, 102.41, 106.8, 107.8, 109.6, 113.9, 120.7, 122.4, 124.4, 126.4, 128.3, 129.4, 129.8, 131.8, 132.0, 132.5, 133.5, 138.3, 157.6, 160.8, 162.0, 189.6; IR (KBr): 3407.1, 1776.6,1598.5, 1505.3, 1485.2, 1238.0, 1012.6, 758.8 cm⁻¹; ESI-MS: 720 (M + H). Anal. calc. for C₄₀H₃₄FN₃O₉: C, 66.75; H, 4.76; N, 5.84 found: C, 66.79; H, 4.74; N, 5.80%.

6g. 9-(4-{4-[3-(3-Fluoro-phenyl)-acryloyl]-phenoxymethyl}-[1,2,3]triazol-1-yl)-5-(3,4,5-trimethoxy-phenyl)-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho [2,3-d] [1,3] dioxol-6-one. White solid; mp: 165–167 °C; [α]²⁵_D − 58.3 (0.65, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 3.07 (m, 1H), 3.26 (m, 2H), 3.79 (s, 6H), 3.82 (s, 3H), 4.43 (t, J = 7.6, 1H), 4.76 (d, J = 4.5, 1H), 5.27 (s, 2H), 5.51 (s, 1H), 6.02 (d, J = 4.1, 2H), 6.16 (d, J = 4.1, 1H), 6.33 (s, 2H), 6.65 (d, J = 4.1, 2H), 7.08 (m, 3H), 7.22–7.35 (m, 3H), 7.54–7.73 (m, 2H), 8.06 (d, J = 8.44, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 37.3, 42.1, 43.8, 56.9, 59.3, 60.8, 62.2, 67.8, 102.4, 108.3, 109.1, 110.9, 114.5, 115.0, 117.4, 117.8, 123.3, 123.6, 124.9, 129.9, 130.8, 131.3, 131.8, 133.8, 143.2, 147.1, 148.5, 149.9, 162.3, 173.7, 190.2; IR (KBr): 3363.6, 1775.6,1598.5, 1505.3, 1485.2, 1238.0, 1002.6, 753.9 cm⁻¹; ESI-MS: 720.2 (M + H). Anal. calc. for C₄₀H₃₄FN₃O₉: C, 66.75; H, 4.76; N, 5.84 found: C, 66.78; H, 4.73; N, 5.82%.

6h. 9-(4-{4-[3-(2-Fluoro-phenyl)-acryloyl]-phenoxymethyl}-[1,2,3]triazol-1-yl)-5-(3,4,5-trimethoxy-phenyl)-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho [2,3-d] [1,3] dioxol-6-one. White solid; mp: 137––138 °C; [α]²⁵_D − 69.4 (0.75, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 3.06 (m, 1H), 3.29 (m, 2H), 3.79 (s, 6H), 3.82 (s, 3H), 4.42 (t, J = 7.5, 1H), 4.76 (d, J = 4.7, 1H), 5.26 (s, 2H), 5.49 (s, 1H), 6.03 (d, J = 3.88, 2H), 6.13 (d, J = 4.2, 1H), 6.33 (s, 2H), 6.64 (d, J = 7.5, 2H), 7.05–7.33 (m, 5H), 7.65–7.83 (m, 3H), 8.04 (d, J = 8.76, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 37.2, 42.1, 56.9, 59.27, 60.8, 66.2, 67.8, 67.7, 102.41, 106.8, 107.8, 109.6, 113.9, 120.7, 122.2, 123.4, 127.4, 128.3, 129.4, 129.8, 130.8, 132.0, 131.8, 133.5, 137.2, 156.6, 160.8, 163.0, 188.3; IR (KBr): 3415.3, 1786.5, 1587.4, 1505.1, 1485.2, 1262.0, 1062.6, 738.9 cm⁻¹; ES-IMS: 720.2 (M + H). Anal. calc. for C₄₀H₃₄FN₃O₉: C, 66.75; H, 4.76; N, 5.84 found: C, 66.77; H, 4.75; N, 5.80%.

6i. 5-(3,4,5-Trimethoxy-phenyl)-9-{4-[4-(3-m-tolyl-acryloyl)-phenoxy methyl]-[1,2,3]triazol-1-yl}-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho[2,3-d] [1,3]dioxol-6-one. White solid; mp: 122–123 °C; [α]²⁵_D − 42 (0.70, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 2.41 (s, 3H), 3.06 (m, 1H), 3.26 (m, 2H), 3.80 (s, 6H), 3.83 (s, 3H), 4.41 (t, J = 7.6, 1H), 4.75 (d, J = 4.5, 1H), 5.26 (s, 2H), 5.45 (s, 1H), 6.02 (d, J = 3.65, 2H), 6.13 (d, J = 3.65, 1H), 6.33 (s, 2H), 6.64 (d, J = 8.2, 2H), 7.08 (d, J = 8.8, 2H), 7.24–7.29 (m, 4H), 7.57–7.79 (m, 2H), 8.04 (d, J = 8.57, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 18.6, 36.0, 40.7, 42.5, 55.3, 57.9, 60.7, 61.0, 66.4, 101.0, 106.9, 107.8, 109.6, 113.6, 120.5, 122.2, 123.4, 124.8, 127.8, 128.6, 129.8, 130.3, 131.2, 132.5, 133.5, 133.9, 137.6, 143.4, 145.6, 147.1, 148.5, 160.7, 172.1, 186.7; IR (KBr): 3425, 2915, 1775, 1747, 1599, 1455, 1240, 1114, 1037, 763 cm⁻¹; ESI-MS: 716.3 (M + H). Anal. calc. for C₄₁H₃₇N₃O₉: C, 68.80; H, 5.21; N, 5.87 found: C, 68.85; H, 5.14; N, 5.89%.

6j. 5-(3,4,5-Trimethoxy-phenyl)-9-(4-{4-[3-(4-methoxy-phenyl)-acryloyl]-phenoxymethyl}-[1,2,3]triazol-1-yl)-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7] naphtho [2,3-d][1,3]dioxol-6-one. White solid; mp: 145–147 °C; [α]²⁵_D − 38.5 (0.75, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 3.08 (m, 1H), 3.26 (m, 2H), 3.79 (s, 9H), 3.82 (s, 3H), 4.41 (t, J = 7.6, 1H), 4.76 (d, J = 4.5, 1H), 5.26 (s, 2H), 5.47 (s, 1H), 6.03 (d, J = 3.83, 2H), 6.13 (d, J = 4.1, 1H), 6.33 (s, 2H), 6.64 (d, J = 7.59, 2H), 7.07 (d, J = 7.59, 2H), 7.24–7.41 (m, 4H), 7.66–7.67 (m, 2H), 8.06 (d, J = 8.78, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 36.2, 41.2, 42.5, 55.8, 58.1, 60.7, 61.0, 65.0, 66.4, 70.2, 101.0, 106.8, 106.8, 109.6, 113.9, 120.7, 122.2, 124.4, 127.4, 128.3, 129.4, 129.8, 130.8, 132.0, 132.5, 133.5, 134.0, 143.1, 145.8, 147.1, 148.2, 156.6, 160.8, 172.1, 191.7; IR (KBr): 3386.3, 1778.6, 1597.3, 1507.2, 1485.2, 1237.0, 1001.5, 733.3 cm⁻¹; ESI-MS: 732.3 (M + H). Anal. calc. for C₄₁H₃₇N₃O₁₀: C, 67.30; H, 5.10; N, 5.74 found: C, 67.35; H, 5.04; N, 5.77%.

7a. 9-[4-(4-Acetyl-phenoxymethyl)-[1,2,3]triazol-1-yl]-5-(4-hydroxy-3,5-dimethoxy-phenyl)-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-6-one. White solid; mp: 143–145 °C; [α]²⁵_D −45 (0.75, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 2.49 (s, 3H), 3.05 (m, 1H), 3.26 (m, 2H), 3.74 (s, 6H), 4.33 (t, J = 7.6, 1H), 4.65 (d, J = 4.8, 1H), 5.15 (s, 2H), 5.92 (d, J = 5.94, 2H), 6.06 (d, J = 3.67, 1H), 6.24 (s, 2H), 6.56 (d, J = 5.68, 2H), 6.96 (d, J =7.21, 2H), 7.37 (s, 1H), 7.85 (d, J = 7.47, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 29.5, 37.0, 41.4, 43.4, 55.9, 58.8, 62.2, 67.5, 105.3, 111.3, 111.5, 112.0, 113.8, 113.9, 117.9, 118.0, 127.1, 134.1, 146.7, 150.4, 150.5, 151.5, 152.8, 177.1, 201.2; IR (KBr): 3385.3, 1773.6, 1731.2, 1598.5, 1515.3, 1477.2, 1338.0, 1016.6, 759.7 cm⁻¹; ESI-MS: 622 (M + Na). Anal. calc. for C₃₂H₂₉N₃O₉: C, 64.10; H, 4.88; N, 7.01 found: C, 64.24; H, 5.01; N, 6.92%.

7b. 5-(4-Hydroxy-3,5-dimethoxy-phenyl)-9-{4-[4-(3-oxo-but-1-enyl)-phenoxymethyl]-[1,2,3]triazol-1-yl}-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho[2,3-d][1,3]-6-one. White solid; mp: 139–141 °C; [α]²⁵_D − 16.3 (0.760, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 2.36 (s, 3H), 3.06 (m, 1H), 3.26 (m, 2H), 3.80 (s, 6H), 4.40 (t, J = 7.6, 1H), 4.75 (d, J = 4.8, 1H), 5.20 (s, 2H), 5.47 (s, 1H), 6.02 (d, J = 4.19, 2H), 6.11 (d, J = 4.0, 1H), 6.33 (s, 2H), 6.62 (m, 2H), 7.00 (d, J = 8.72, 2H), 7.28 (m, 2H), 7.52 (d, J = 8.78, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 24.1, 27.4, 29.7, 37.0, 41.7, 43.5, 56.5, 58.8, 62.0, 67.4, 82.5, 84.4, 102.0, 107.8, 108.8, 110.6, 115.2, 123.1, 124.4, 125.4, 127.8, 129.6, 130.0, 133.4, 134.4, 142.9, 143.8, 146.6, 148.1, 149.5, 159.9, 173.1, 198.4; IR (KBr): 3389.9, 1778.6, 1598.5, 1505.3, 1485.2, 1238.0, 1011.6, 774.7 cm⁻¹; ESI-MS: 648 (M + Na). Anal. calc. for C₃₄H₃₁N₃O₉: C, 65.27; H, 4.99; N, 6.72 found: C, 65.10; H, 4.84; N, 6.79%.

7c. 5-(4-Hydroxy-3,5-dimethoxy-phenyl)-9-{4-[2-methoxy-4-(3-oxo-but-1-enyl)-phenoxy-methyl]-[1,2,3]triazol-1-yl}-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho [2,3-d][1,3]dioxol-6-one. White solid; mp: 136–138 °C; [α]²⁵_D − 25.6 (0.75, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 2.31 (s, 3H), 3.05 (m, 1H), 3.26 (m, 2H), 3.73 (s, 6H), 3.77 (s, 3H), 4.40 (t, J = 7.6, 1H), 4.79 (d, J = 4.8, 1H), 5.22 (s, 2H), 5.40 (s, 1H), 6.02 (d, J = 3.67, 2H), 6.25 (s, 1H), 6.53 (m, 1H), 6.69 (m, 2H), 7.01 (s, 2H), 7.28–7.48 (m, 2H), 7.52 (d, J = 8.78, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 24.1, 27.4, 29.7, 37.0, 41.6, 43.4, 55.9, 56.5, 58.8, 62.0, 67.4, 82.5, 84.4, 102.0, 107.8, 108.9, 110.5, 115.2, 122.7, 123.4, 125.8, 127.8, 129.6, 130.0, 133.4, 134.4, 142.9, 143.8, 146.6, 148.1, 149.5, 159.9, 173.1, 197.9; IR (KBr): 3388.6, 1775.2, 1598.2, 1507.1, 1485.2, 1239.2, 1002.6, 754.7 cm⁻¹; ESI-MS: 678 (M + Na). Anal. calc. for C₃₄H₃₃N₃O₉: C, 64.12; H, 5.07; N, 6.41 found: C, 64.24; H, 5.16; N, 6.53%.

7d. 5-(4-Hydroxy-3,5-dimethoxy-phenyl)-9-{4-[4-(3-oxo-3-phenyl-propenyl)-phenox-ymethyl]-[1,2,3]triazol-1-yl}-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho[2,3-d] [1,3]dioxol-6-one. White solid; mp: 151–153 °C; [α]²⁵_D − 37 (0.75, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 3.06 (m, 1H), 3.29 (m, 2H), 3.80 (s, 6H), 4.41 (t, J = 7.6, 1H), 4.76 (d, J = 4.6, 1H), 5.26 (s, 2H), 5.47 (s, 1H), 6.01 (d, J = 3.7, 2H), 6.12 (d, J = 4.0, 1H), 6.33 (s, 2H), 6.64 (d, J = 7.7, 2H), 7.05 (d, J = 8.6, 2H),7.24–7.54 (m, 3H), 7.57–7.79 (m, 4H), 8.05 (d, J = 8.78, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 29.7, 37.1, 41.7, 43.5, 56.6, 58.9, 62.1, 67.4, 102.1, 107.9, 108.8, 110.6, 114.7, 121.8, 123.3, 124.5, 128.4, 129.0, 129.7, 130.5, 130.9, 131.8, 134.5, 135.0, 144.3, 146.7, 148.2, 149.5, 186.7; IR (KBr): 3382.7, 1778.6,1598.5, 1505.3, 1485.2, 1233.4, 1005.3, 769.3 cm⁻¹; ESI-MS: 710 (M + Na). Anal. calc. for C₃₉H₃₃N₃O₉: C, 68.11; H, 4.84; N, 6.11 found: C, 68.20; H, 4.94; N, 6.02%.

7e. 5-(4-Hydroxy-3,5-dimethoxy-phenyl)-9-{4-[4-(3-phenyl-acryloyl)-phenoxy methyl]-[1,2,3]triazol-1-yl}-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho[2,3-d] [1,3]dioxol-6-one. White solid; mp: 145–148 °C; [α]²⁵_D − 44.4 (0.60, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 3.06 (m, 1H), 3.26 (m, 2H), 3.80 (s, 6H), 4.39 (t, J = 7.6, 1H), 4.75 (d, J = 4.5, 1H), 5.23 (s, 2H), 5.46 (s, 1H), 6.02 (d, J = 3.65, 2H), 6.14 (d, J = 3.65, 1H), 6.33 (s, 2H), 6.64 (d, J = 8.2, 2H), 7.02 (d, J = 8.6, 2H),7.24–7.32 (m, 2H), 7.34–7.54 (m, 3H), 7.57–7.79 (m, 2H), 8.05 (d, J = 8.57, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 29.6, 37.1, 41.7, 43.5, 56.6, 58.9, 62.1, 67.4, 101.1, 107.9, 108.8, 110.6, 114.7, 121.8, 124.3, 124.5, 128.4, 129.0, 129.7, 130.5, 132.9, 131.8, 134.5, 135.0, 144.3, 146.7, 148.2, 150.5, 186.7; IR (KBr): 3347.1, 1777.7, 1587.2, 1598.3, 1485.2, 1287.0, 1002.6, 738.6 cm⁻¹; ESI-MS: 710 (M + Na). Anal. calc. for C₃₉H₃₃N₃O₉: C, 68.11; H, 4.84; N, 6.11 found: C, 68.21; H, 4.72; N, 6.23%.

7f. 9-(4-{4-[3-(4-Fluoro-phenyl)-acryloyl]-phenoxymethyl}-[1,2,3]triazol-1-yl)-5-(4-hyd-roxy-3,5-dimethoxy-phenyl)-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho [2,3-d] [1,3] dioxol-6-one. White solid; mp: 162–164 °C; [α]²⁵_D − 28 (0.75, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 3.06 (m, 1H), 3.26 (m, 2H), 3.81 (s, 6H), 4.41 (t, J = 7.6, 1H), 4.76 (d, J = 4.7, 1H), 5.26 (s, 2H), 5.50 (s, 1H), 6.02 (d, J = 3.88, 2H), 6.12 (d, J = 4.2, 1H), 6.33 (s, 2H), 6.64 (d, J = 7.5, 2H), 7.05–7.23 (m, 5H), 7.65–7.82 (m, 3H), 8.04 (d, J = 8.77, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 36.4, 42.1, 56.9, 59.27, 60.6, 66.2, 67.8, 67.7, 102.41, 106.8, 107.8, 109.6, 113.9, 120.7, 122.2, 124.4, 126.4, 128.3, 129.4, 129.8, 131.8, 132.0, 132.5, 133.5, 138.3, 157.6, 160.8, 161.0, 189.4; IR (KBr): 3409.1, 1778.6,1598.5, 1505.3, 1485.2, 1238.0, 1012.6, 756.8 cm⁻¹; ESI-MS: 706 (M + H). Anal. calc. for C₃₉H₃₂FN₃O₉: C, 66.38; H, 4.57; N, 5.95 found: C, 66.29; H, 4.64; N, 5.83%.

7g. 9-(4-{4-[3-(3-Fluoro-phenyl)-acryloyl]-phenoxymethyl}-[1,2,3]triazol-1-yl)-5-(4-hydro-xy-3,5-dimethoxy-phenyl)-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho [2,3-d] [1,3] dioxol-6-one. White solid; mp: 173–175 °C; [α]²⁵_D − 53.3 (0.55, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 3.08 (m, 1H), 3.28 (m, 2H), 3.80 (s, 6H), 4.42 (t, J = 7.6, 1H), 4.76 (d, J = 4.5, 1H), 5.28 (s, 2H), 5.51 (s, 1H), 6.02 (d, J = 4.1, 2H), 6.15 (d, J = 4.1, 1H), 6.33 (s, 2H), 6.64 (d, J = 4.1, 2H), 7.08 (m, 3H), 7.22–7.36 (m, 3H), 7.54–7.72 (m, 2H), 8.05 (d, J = 8.44, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 37.4, 42.1, 43.8, 56.9, 59.3, 62.3, 67.8, 102.4, 108.3, 109.1, 110.9, 114.5, 115.0, 117.4, 117.8, 123.3, 123.6, 124.9, 129.9, 130.8, 131.3, 131.8, 133.8, 143.2, 147.1, 148.5, 149.9, 162.3, 173.7, 191.2; IR (KBr): 3378.6, 1778.6,1598.5, 1505.3, 1485.2, 1238.0, 1002.6, 754.9 cm⁻¹; ESI-MS: 706 (M + H). Anal. calc. for C₃₉H₃₂FN₃O₉: C, 66.38; H, 4.57; N, 5.95 found: C, 66.47; H, 4.68; N, 5.87%.

7h. 9-(4-{4-[3-(2-Fluoro-phenyl)-acryloyl]-phenoxymethyl}-[1,2,3]triazol-1-yl)-5-(4-hydro-xy-3,5-dimethoxy-phenyl)-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho [2,3-d] [1,3] dioxol-6-one. White solid; mp: 142–146 °C; [α]²⁵_D − 62.4 (0.75, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 3.06 (m, 1H), 3.29 (m, 2H), 3.80 (s, 6H), 4.41 (t, J = 7.5, 1H), 4.76 (d, J = 4.7, 1H), 5.26 (s, 2H), 5.49 (s, 1H), 6.02 (d, J = 3.88, 2H), 6.13 (d, J = 4.2, 1H), 6.33 (s, 2H), 6.64 (d, J = 7.5, 2H), 7.05–7.33 (m, 5H), 7.65–7.82 (m, 3H), 8.05 (d, J = 8.77, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 37.4, 42.1, 56.9, 59.27, 60.6, 66.2, 67.8, 67.7, 102.41, 106.8, 107.8, 109.6, 113.9, 120.7, 122.2, 123.4, 127.4, 128.3, 129.4, 129.8, 130.8, 132.0, 132.5, 133.5, 137.3, 156.6, 160.8, 163.0, 188.4; IR (KBr): 3405.3, 1789.5, 1587.4, 1505.1, 1485.2, 1262.0, 1063.6, 736.9 cm⁻¹; ESI-MS: 706 (M + H). Anal. calc. for C₃₉H₃₂FN₃O₉: C, 66.38; H, 4.69; N, 5.95 found: C, 66.29; H, 4.60; N, 5.87%.

7i. 5-(4-Hydroxy-3,5-dimethoxy-phenyl)-9-{4-[4-(3-m-tolyl-acryloyl)-phenoxy methyl]-[1,2,3]triazol-1-yl}-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7]naphtho[2,3-d] [1,3]dioxol-6-one. White solid; mp: 129–132 °C; [α]²⁵_D − 35 (0.70, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 2.40 (s, 3H), 3.06 (m, 1H), 3.28 (m, 2H), 3.80 (s, 6H), 4.41 (t, J = 7.6, 1H), 4.76 (d, J = 4.5, 1H), 5.26 (s, 2H), 5.46 (s, 1H), 6.02 (d, J = 3.65, 2H), 6.15 (d, J = 3.65, 1H), 6.33 (s, 2H), 6.64 (d, J = 8.2, 2H), 7.07 (d, J = 8.8, 2H), 7.24–7.29 (m, 4H), 7.57–7.79 (m, 2H), 8.05 (d, J = 8.57, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 18.3, 36.0, 40.7, 42.5, 55.5, 57.9, 61.0, 66.4, 101.0, 106.9, 107.8, 109.6, 113.6, 120.5, 122.2, 123.4, 124.6, 127.8, 128.6, 129.8, 130.3, 130.9, 132.5, 133.5, 133.9, 137.6, 143.4, 145.6, 147.1, 148.5, 160.7, 172.1, 187.7; IR (KBr): 3408, 2925, 1775, 1747, 1599, 1457, 1240, 1114, 1037, 760 cm⁻¹; ESI-MS:702 (M + H). Anal. calc. for C₄₀H₃₅N₃O₉: C, 68.46; H, 5.03; N, 5.99 found: C, 68.35; H, 5.14; N, 5.87%.

7j. 5-(4-Hydroxy-3,5-dimethoxy-phenyl)-9-(4-{4-[3-(4-methoxy-phenyl)-acryloyl]-phenoxymethyl}-[1,2,3]triazol-1-yl)-5,8,8a,9-tetrahydro-5aH-furo[3′,4′:6,7] naphtho [2,3-d][1,3]dioxol-6-one. White solid; mp: 152–154 °C; [α]²⁵_D − 33.2 (0.70, CHCl₃); ¹H NMR (400 MHz, CDCl₃): 3.06 (m, 1H), 3.27 (m, 2H), 3.80 (s, 9H), 4.41 (t, J = 7.6, 1H), 4.76 (d, J = 4.5, 1H), 5.26 (s, 2H), 5.48 (s, 1H), 6.02 (d, J = 3.83, 2H), 6.13 (d, J = 4.1, 1H), 6.33 (s, 2H), 6.64 (d, J = 7.59, 2H), 7.07 (d, J = 7.59, 2H), 7.22–7.42 (m, 4H), 7.64–7.66 (m, 2H), 8.05 (d, J = 8.78, 2H); ¹³C NMR (400 MHz, CDCl₃): δ 36.0, 41.2, 42.5, 55.6, 58.1, 61.0, 65.0, 66.4, 70.2, 101.0, 106.8, 107.8, 109.6, 113.9, 120.7, 122.2, 123.4, 127.4, 128.3, 129.4, 129.8, 130.8, 132.0, 132.5, 133.5, 134.0, 143.3, 145.6, 147.1, 148.2, 156.6, 160.8, 172.1, 191.9; IR (KBr): 3388.3, 1778.6, 1596.4, 1507.3, 1485.2, 1238.0, 1002.6, 732.3 cm⁻¹; ESI-MS: 718 (M + H). Anal. calc. for C₄₀H₃₅N₃O₁₀: C, 66.94; H, 4.92; N, 5.85 found: C, 66.79; H, 5.04; N, 5.97%.

4.2. Evaluation of anticancer activity

The anticancer activity of all the derivatives was screened by the inhibition of growth of various cancer cell lines. We have adopted a method recommended by the National Cancer Institute (NCI), which uses the protein-binding dye sulforhodamine B to estimate cell growth. Cellular viability in the presence and absence of experimental agents was determined by using the standard sulforhodamine B assay. The cells were harvested, counted and seeded (10⁴ cells per well in 100 μL medium) in the log phase of their growth in 96-well microtitre plates. Cultures were treated with varying concentrations of test samples (10–100 μM) which were made of 1 : 10 serial dilutions, after 24 h of incubation at 37 °C and 5% CO₂ to allow cell attachment. Six replicate wells were set up for each experimental condition. The cells were left in contact with the test samples for 48 h under the same conditions. Cold trichloroacetic acid (TCA, 50%) was used for fixing the cells which were left at 4 °C for 1 h, followed by washing and air drying. Sulforhodamine-B dye was used to stain all the cells. The adsorbed dye was dissolved in Tris–buffer and plates were gently shaken for 10 min, on a mechanical shaker. The optical density (OD) was recorded using an ELISA reader at 540 nm. Subtraction of mean OD value of the respective blank from the mean OD value of an experimental set was used for calculation of the cellular growth. Percent growth in the presence of a test material was calculated by considering the growth in the absence of any test material as 100%, and in turn percent growth inhibition in the presence of a test material was calculated in terms of IC₅₀ values. The different epipodophyllotoxin derivatives (test material) were dissolved in a mixture of DMSO : Water (1 : 1) and then introduced into the medium containing the cancer cell lines. The cells were allowed for proliferation in the presence of the test material for 48 h. With etoposide as the positive control, cell growth inhibition results are presented in terms of IC₅₀ values (Table 2).

Table 2 IC₅₀ values (μM) of in vitro cytotoxicity of 4β-[(4-substituted)-1,2,3-triazol-1-yl] podophyllotoxin congeners against a panel of human cancer cell lines^a

Entry	SW-620	DU-145	SKN-SH	HCT-15	Colo-205	HeLa
a ND = not determined; cell lines: SKN-SH (CNS), SW-620, HCT-15, COLO-205 (Colon), DU-145 (prostrate), HeLa (cervix).
6a	14.60	13.60	15.60	13.50	18.80	18.50
6b	10.21	11.32	14.40	16.30	7.60	ND
6c	3.41	ND	10.03	10.43	8.98	3.40
6d	2.02	30.20	4.13	12.80	9.12	5.34
6e	9.20	30.00	25.61	15.26	29.30	19.10
6f	2.40	6.50	3.40	12.00	13.20	ND
6g	5.21	13.30	12.40	15.60	7.32	14.23
6h	8.12	20.20	6.73	12.90	ND	17.23
6i	6.53	9.36	7.13	ND	8.23	12.20
6j	11.21	10.21	7.32	6.12	12.30	ND
7a	11.60	16.60	11.61	12.00	14.82	18.92
7b	7.50	12.02	10.32	6.82	ND	13.05
7c	5.66	6.50	5.45	12.40	18.23	5.00
7d	0.64	12.00	0.66	9.50	12.30	5.90
7e	4.40	5.45	8.30	6.75	ND	9.18
7f	0.35	12.00	0.39	12.40	12.45	4.50
7g	5.36	7.40	4.50	ND	5.15	6.40
7h	12.55	9.34	7.40	15.20	12.30	ND
7i	11.45	20.50	13.45	12.20	15.40	9.32
7j	10.35	12.45	15.60	7.45	ND	6.80
Etoposide	13.24	16.43	9.03	12.23	14.23	10.40

4.3. Molecular docking

Structure building and calculations were performed on a Mac OSX (v10.7.5) using the Schrodinger molecular modeling software (Schrödinger Release 2013-2, Schrödinger, LLC, New York, 2013). The ligands were built using Maestro (v9.5) and minimized. Conformational search application (Macromodel v10.1) was used to generate an extensive set of conformations using advanced option. OPLS-2005 (ref. 37) force field in solvent water conditions with default extended cutoff values was used. Optimizations were converged into an energy convergence or continued until the limit of 50 000 iterations was reached. For the conformational search the mixed torsional/low mode sampling method was used with 10 000 steps. The 100 lowest energy conformers were saved and were further used for docking studies (Table 3).

Table 3 Docking scores of various 4β-[(4-substituted)-1,2,3-triazol-1-yl] podophyllotoxin analogs

Entry	Glide score	Entry	Glide score
6a	−4.32	7a	−5.30
6b	−3.45	7b	−4.80
6c	−4.00	7c	−4.50
6d	−4.92	7d	−6.81
6e	−3.50	7e	−5.47
6f	−5.20	7f	−7.40
6g	−4.80	7g	−4.37
6h	−3.52	7h	−4.32
6i	−4.31	7i	−5.30
6j	−4.45	7j	−4.97
Etoposide	−3.52

The crystal structure coordinates of the human topoisomerase-II ATPase-AMP-PNP complex (PDB ID: 1ZXM from the protein data bank, http://www.rcsb.org) were imported for protein preparation using the protein preparation wizard. The protein complex was preprocessed, by replacing the missing amino acids and removing all water molecules including the ones near the co-crystallized AMP. By using the OPLS-2005 minimization method, a refined structure of the protein was obtained which was further used for grid calculations and docking studies using Glide (v60014).^38,39 The generation for the receptor-grid values was computed using OPLS-2005 force fields. The ligands were docked with the obtained conformers using extra precision settings of the docking panel with default settings.

References

T. Beghyn, R. Deprez-Poulain, N. Willand, B. Folleas and B. Deprez, Chem. Biol. Drug Des., 2008, 72, 3–15 CAS.
F. E. Koehn and G. T. Carter, Nat. Rev. Drug Discovery, 2005, 4, 206–220 CrossRef CAS PubMed.
D. J. Newman and G. M. Cragg, J. Nat. Prod., 2007, 70, 461–477 CrossRef CAS PubMed.
K. Gransalke, Drug Discovery, 2011, 11, 16–19 Search PubMed.
M. G. Kelly and J. L. Hartwell, J. Natl. Cancer Inst., 1954, 14, 967–1010 CAS.
K. H. Lee, Med. Res. Rev., 1999, 19, 569–596 CrossRef CAS.
L. Bohlin and B. Rosen, Drug Discovery Today, 1996, 1, 343–351 CrossRef CAS.
K. H. Lee, S. A. Beers, M. Mori, Z. Wang, Y. Kuo, L. Li, S. Liu, Y. Cheng, F. Han and Y. Cheng, J. Med. Chem., 1990, 33, 1364–1368 CrossRef CAS.
H. Stahelin and A. V. Wartburg, Prog. Drug Res., 1989, 33, 169–266 CAS.
T. W. Doyle, Etoposide (VP-16) Current Status and New Developments, Academic press, New York, 1984 Search PubMed.
Z. Q. Wang, Y. H. Kuo, D. Schnur, J. P. Bowen, S. Y. Liu, F. S. Han, Y. C. Cheng and K. H. Lee, J. Med. Chem., 1990, 33, 2660–2666 CrossRef CAS.
B. F. Issell, Cancer Chemother. Pharmacol., 1982, 7, 73–80 CAS.
D. R. Budman, Semin. Oncol., 1996, 23, 8–14 CAS.
F. A. Greco and J. D. Hainsworth, Semin. Oncol., 1996, 23, 45–50 CAS.
T. L. Macdonald, E. K. Lehenert, J. T. Loper, K. C. Chow and W. E. Ross, in DNA topoisomerase in cancer, ed. M. Potmesil and K. W. Kohn, Oxford University Press, New York, 1991, p. 119 Search PubMed.
P. I. Clark and M. L. Slevin, Clin. Pharmacokinet., 1987, 12, 223–252 CrossRef CAS PubMed.
A. Kamal, M. Arifuddin, B. A. Kumar and S. G. Dastidar, US Pat. 0066837, 2007.
D. Imperio, T. Pirali, U. Galli, F. Pagliai, L. Cafici, P. Luigi, G. Sorba, A. A. Genazzani and G. C. Tron, Bioorg. Med. Chem., 2007, 15, 6748–6757 CrossRef CAS PubMed.
T. Terada, K. Fujimoto, M. Nomura, J. Yamashita, K. Wierzba, R. Yamazaki, J. Shibata, Y. Sugimoto, Y. Yamada, T. Kobunai, S. Takeda, Y. Minami, K. Yoshida and H. Yamaguchi, J. Med. Chem., 1993, 36, 1689–1699 CrossRef CAS.
T. Utsugi, J. Shibata, Y. Sugimoto, K. Aoyagi, K. Wierzba, T. Kobunai, T. Terada, T. Ohhara, T. Tsuruo and Y. Yamada, Cancer Res., 1996, 56, 2809–2814 CAS.
Z. Xiao, Y. D. Xiao, A. Golbraikh, A. Tropsha and K. H. Lee, J. Med. Chem., 2002, 45, 2294–2309 CrossRef CAS PubMed.
K. Mross, A. Huttmann, K. Herbst, A. R. Hanauske, T. Schilling, C. Manegold, K. Burk and D. K. Hossfeld, Cancer Chemother. Pharmacol., 1996, 38, 217–224 CrossRef CAS.
Y. Zhang, A. Tropsha, A. T. McPhail and K. H. Lee, J. Med. Chem., 1994, 37, 1460–1464 CrossRef CAS.
Z. Xiao, K. F. Bastow, J. R. Vance and H. Lee, Bioorg. Med. Chem., 2004, 12, 3339–3344 CAS.
M. Abdel-Aziz, S. U. Park, G. E.-D. A. A. Abuo-Rahma, M. A. Sayed and Y. Kwon, Eur. J. Med. Chem., 2013, 69, 427–438 CrossRef CAS PubMed.
X. Zhang, B. Bao, X. Yu, L. Tong, Y. Luo, Q. Huang, M. Su, L. Sheng, J. Li, H. Zhu, B. Yang, X. Zhang, Y. Chen and W. Lu, Bioorg. Med. Chem., 2008, 15, 6981–6995 Search PubMed.
S. Ohira, K. Takaya, T. Mitsui, M. Kido, K. Kakumoto, K. Hayashi, A. Kuboki, H. Tani, S. Ikeda, M. Linuma, Y. Akao and H. Nozaki, Tetrahedron Lett., 2013, 54, 5052–5055 CrossRef CAS PubMed.
Y. Na and J. M. Nam, Bioorg. Med. Chem. Lett., 2011, 21, 211–214 CrossRef CAS PubMed.
A. H. Banday, S. A. Shameem, B. D. Gupta and H. M. S. Kumar, Steroids, 2010, 75, 801–804 CrossRef CAS PubMed.
D. M. Reddy, J. Srinivas, G. Chashoo, A. K. Saxena and H. M. S. Kumar, Eur. J. Med. Chem., 2011, 46, 1983–1991 CrossRef CAS PubMed.
P. B. Reddy, S. K. Agrawal, S. Singh, B. A. Bhat, A. K. Saxena, H. M. S. Kumar and G. N. Qazi, Chem. Biodiversity, 2008, 5, 1792–1802 CAS.
B. A. Bhat, P. B. Reddy, S. K. Agrawal, A. K. Saxena, H. M. S. Kumar and G. N. Qazi, Eur. J. Med. Chem., 2008, 43, 2067–2072 CrossRef CAS PubMed.
A. Kamal, B. A. Kumar and M. Arifuddin, Tetrahedron Lett., 2003, 44, 8457–8459 CrossRef CAS PubMed.
A. Kamal, N. Laxman and G. Ramesh, Bioorg. Med. Chem. Lett., 2000, 10, 2059–2062 CrossRef CAS.
D. Leroy, A. V. Kajava, C. Frei and S. M. Gasser, Biochemistry, 2001, 40, 1624–1634 CrossRef CAS PubMed.
A. K. Patel, S. Patel and P. K. Naik, Curr. Res. J. Biol. Sci., 2010, 2, 13–23 CAS.
G. A. Kaminski, R. A. Friesner, J. Tirado-Rives and W. L. Jorgensen, J. Phys. Chem. B, 2001, 105, 6474–6487 CrossRef CAS.
R. A. Friesner, R. B. Murphy, M. P. Repasky, L. L. Frye, J. R. Greenwood, T. A. Halgren, P. C. Sanschagrin and D. T. Mainz, J. Med. Chem., 2006, 49, 6177–6196 CrossRef CAS PubMed.
R. A. Friesner, J. L. Banks, R. B. Murphy, T. A. Halgren, J. J. Klicic, D. T. Mainz, M. P. Repasky, E. H. Knoll, D. E. Shaw, M. Shelley, J. K. Perry, P. Francis and P. S. Shenkin, Glide, J. Med. Chem., 2004, 47, 1739–1749 CrossRef CAS PubMed.
A. Kumar, V. Kumar, A. E. Alegria and S. V. Malhotra, Curr. Med. Chem., 2011, 18, 3853–3870 CrossRef CAS.

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4md00325j

Click here to see how this site uses Cookies. View our privacy policy here.

Design, synthesis, and biological and docking studies of novel epipodophyllotoxin–COMPOUND LINKSRead more about this on ChemSpiderDownload mol file of compoundExplore further on Open PHACTSchalcone hybrids as potential anticancer agents†