Synthesis of Saccharumoside-B analogue with potential of antiproliferative and pro-apoptotic activities

Abstract

A new series of phenolic glycoside esters, saccharumoside-B and its analogs (9b-9n, 10) have been synthesized by the Koenigs-Knorr reaction. Antiproliferative activities of the compounds (9b-9n, 10) were evaluated on various cancer cell lines including, MCF-7 breast, HL-60 leukemia, MIA PaCa-2 pancreatic, DU145 prostate, HeLa cervical and CaCo-2 colon, as well as normal human MCF10A mammary epithelial and human peripheral blood mononuclear cells (PBMC) by MTT assay. Compounds (9b-9n, 10) exhibited considerable antiproliferative effects against cancer cells with IC₅₀ range of 4.43 ± 0.35 to 49.63 ± 3.59 µM, but they are less cytotoxic on normal cells (IC₅₀ > 100 µM). Among all the compounds, 9f showed substantial antiproliferative activity against MCF-7 and HL-60 cells with IC₅₀ of 6.13 ± 0.64 and 4.43 ± 0.35, respectively. Further mechanistic studies of 9f were carried out on MCF-7 and HL-60 cell lines. 9f caused arrest of cell cycle of MCF-7 and HL-60 cells at G0/G1 phase. Apoptotic population elevation, mitochondrial membrane potential loss, increase of cytosolic cytochrome c and Bax levels, decrease of Bcl-2 levels and enhanced caspases-9 and -3 activities were observed in 9f-treated MCF-7 and HL-60 cells. These results demonstrate anticancer and apoptosis-inducing potentials of 9f in MCF-7 and HL-60 cells via intrinsic pathway.

Introduction

Cancer has become the major cause of death in the world with the changes in the living habitat of people and environment^{1, 2}. Breast cancer is the leading cause of cancer deaths in women with an estimated 1,383,500 new cases and 458,400 deaths annually^{3, 4}. Chemotherapy is one of the treatments for cancer. Hence, chemotherapeutic agents have been developed throughout the world by pharmaceutical industries and academic institutions but their usage is limited due to poor efficacy and adverse effects^5–7. Drug resistance is also another problem in cancer treatment^{8, 9}. Thus, discovery and development of safer and effective molecules are urgently required to reduce the burden of cancer.

Natural products have been isolated from natural sources and explored their use as drugs for various diseases including cancers. However, there are many challenges in discovery and development of natural products as drugs. Yields, time-consuming isolation processes and impurities are some of the major challenges in isolation and identification of natural products¹⁰. Bioactivity studies including in vitro, preclinical and clinical, require high quantities of compounds for evaluating their bioactivities¹⁰. Some of the natural products have been found with moderate bioactivities and poor pharmacokinetic properties¹¹. To overcome these problems, natural products have been synthesized chemically as in their native form and/or their analogs and explored their utility as drugs^11–13. For instance, dried leaves from almost 15 trees of Vinca rosea (Catharanthus roseus) required to obtain only 30 g of vincristine (a chemotherapeutic drug). Similarly, 27,300 kg of the bark of Taxus brevifolia required to obtain 1900 g of taxol. Later, vincristine and taxol have been synthesized in high quantities by several total and semi-synthetic methods¹⁰. Discodermolide, a marine-derived anticancer agent from marine sponge Discodermia dissoluta was not available in sufficient quantities to carry out clinical trials¹⁰. Later, discodermolide was synthesized with high yields and used in clinical trials for cancers^{14, 15}.

Phenolic glycoside esters (PG) are some of the most abundant secondary metabolites in plants with novel bioactivities¹⁶. Several PG have been isolated from the plants of the different families and reported to possess various bioactivities including anticancer activity^17–19. Previously, Tao et al.¹⁹ isolated four new PG esters including saccharumoside B from the bark of Acer saccharum (sugar maple tree) and reported cytotoxic activity of saccharumoside B on human colon cancer cell line. Mechanism of action of cytotoxic saccharumoside B in cancers remains to be elusive. The present work reports for the first time the synthesis of anticancer natural product saccharumoside B and its analogs with good yields. Further, in vitro anticancer and pro-apoptotic studies of synthesized saccharumoside-B and its analogs were performed.

Results

Chemistry

Synthesis of saccharumoside-B and its analogs (Table 1) was performed according to the pathway illustrated in Figs 1 and 2. First, α-D-glucose (1) was treated with Ac₂O/AcOH in presence of HClO₄ in Ac₂O to provide pentaacetyl-α-D-glucose (2). Subsequently, compound 2 was treated with HBr in glacial acetic acid to get α-D-acetobromo glucose (3). Different phenolic glycoside derivatives (5a-n) were prepared by the reaction of compound 3 with different 4-hydroxy benzaldehydes (4a-n) using K₂CO₃, aliquat-336 and CH₂Cl₂:H₂O (1:1). Deacylation of 5a-n was carried out in presence of NaOMe in MeOH to afford corresponding products 6a-n. Benzoylation of 6a-n using different benzoyl chlorides (7a-n) and pyridine led to the formation of a mixture of isomers, which were further separated by using silica gel column chromatography (100–200 mesh) to get pure compounds 8a-n. The aldehyde group of 8a-n was reduced by employing NaBH₄ in MeOH to give compounds 9a-n. Finally, the synthesis of naturally occurring phenolic glycoside ester, saccharumoside-B (10) (Fig. 3) was done by the debenzylation of compound 9a using Pd/CaCO₃ and TEA under H₂ atmosphere. Spectral data of synthetic sacharramoside-B was found to be identical to those of reported isolated product (Supplementary Figures 1–28)¹⁹.

Table 1.

Synthesis of saccharumoside-B and its analogs.

S.No.	Entry	R1	R2	R3	Yield (%)a	M.P (oC)
1	10	OCH3	H		40	200–202
2	9b	OCH3	H		47	187–189
3	9c	OCH3	H		45	228–230
4	9d	OCH3	H		48	199–201
5	9e	OCH3	H		50	168–170
6	9f	OCH3	H		30	209–211
7	9g	OCH3	H		45	128–130
8	9h	OCH3	H		31	194–196
9	9i	OCH3	H		30	186–188
10	9j	OCH3	OCH3		48	180–182
11	9k	OCH3	OCH3		45	201–203
12	9l	OCH3	OCH3		47	131–133
13	9m	OCH3	OCH3		30	170–172
14	9n	OCH3	OCH3		43	124–126

^aIsolated yield.

Figure 1.

Synthesis of saccharumoside-B analogs. Reagents and conditions: (a) Ac₂O/AcOH, HClO₄ in Ac₂O; (b) HBr in glacial acetic acid; (c) K₂CO₃, DCM-H₂O, aliquat-336; (d) NaOMe, MeOH; (e) Pyridine, DCM; (f) NaBH₄, MeOH.

Figure 2.

Synthesis of saccharumoside-B. Reagents and conditions: (g) Pd/CaCO₃, H₂ gas, TEA.

Figure 3.

Structures of saccharumoside-B and its analog 9f.

Antiproliferative activities of saccharumoside-B and its analogs

Anticancer activity of synthesized phenolic glycoside esters (9b-9n, 10) was tested on MCF-7 breast, MIA PaCa-2 pancreatic, DU145 prostate, CaCo-2 colon, HL-60 leukemia and HeLa cervical cancer cells as well as on normal MCF10A mammary epithelial cells and PBMCs by MTT assay. As shown in Table 2, all compounds (9b-9n, 10) showed considerable antiproliferative activity against cancer cells with IC₅₀ range of 4.43 ± 0.35 to 49.63 ± 3.59 µM. Whereas, the compounds (9b-9n, 10) showed less cytotoxicity against non-cancerous MCF-10A and PBMC cells (IC₅₀ > 100 µM) (Supplementary Figure 29). Among all compounds, 9f showed substantial antiproliferative effect on Mia PaCa-2 pancreatic, DU145 prostate, MCF-7 breast, CaCo-2 colon, HL-60 leukemia and HeLa cervical cancer cell lines with IC₅₀ of 12.87 ± 0.52, 10.8 ± 0.42, 6.13 ± 0.64, 8.66 ± 0.72, 4.43 ± 0.35 and 7.85 ± 0.57 µM, respectively, whereas camptothecin (positive control) exhibited antiproliferative activity with IC₅₀ of 4.24 ± 0.41, 6.75 ± 0.45, 5.16 ± 0.32, 3.68 ± 0.21, 4.95 ± 0.5 and 4.28 ± 0.32 µM, on respective cancer cell lines (Fig. 4). Fluoro substituent on the phenyl ring of ester group imparts enhanced anticancer activity on various cancer cells to compound 9f when compared to saccharumoside-B (Fig. 3). As significant inhibition of MCF-7 and HL-60 cells by 9f was observed, further experiments were aimed to demonstrate the mechanistic insights of 9f in MCF-7 breast and HL-60 leukemia cancer cells.

Table 2.

Antiproliferative activities of saccharumoside-B and its analogs against various cancer and normal cell lines.

S.No.	IC50 (µM)a
	Cancer cell lines						Normal cell line
	MIA PaCa-2 Pancreatic	DU145 Prostate	MCF-7 Breast	CaCo-2 Colon	HL-60 Leukemia	HeLa Cervical	MCF-10A Breast	PBMC
10	28.32 ± 0.76	33.46 ± 1.43	24.90 ± 1.67	44.67 ± 2.52	31.78 ± 1.82	39.87 ± 2.6	>100	>100
9b	39.34 ± 1.42	37.34 ± 2.54	26.43 ± 0.96	47.34 ± 1.62	22.75 ± 1.3	36.68 ± 2.31	>100	>100
9c	13.34 ± 0.25	12.36 ± 0.47	9.34 ± 0.48	13.67 ± 0.63	12.45 ± 0.81	14.22 ± 0.68	>100	>100
9d	44.56 ± 2.67	42.45 ± 1.51	28.34 ± 1.47	37.35 ± 1.65	25.21 ± 1.92	39.5 ± 2.43	>100	>100
9e	36.34 ± 1.58	49.39 ± 1.72	33.37 ± 0.76	43.45 ± 1.87	22.15 ± 1.32	41.52 ± 2.1 8	>100	>100
9 f	12.87 ± 0.52	10.8 ± 0.42	6.13 ± 0.64	8.66 ± 0.72	4.43 ± 0.35	7.85 ± 0.57	>100	>100
9 g	23.45 ± 1.53	38.32 ± 0.37	28.85 ± 0.47	43.87 ± 2.52	33.52 ± 1.45	41.92 ± 3.15	>100	>100
9 h	33.87 ± 1.41	42.11 ± 1.52	33.85 ± 0.48	49.04 ± 0.48	30.23 ± 1.32	35.36 ± 2.2	>100	>100
9i	34.78 ± 1.93	43.56 ± 1.87	34.78 ± 0.51	46.98 ± 2.58	29.62 ± 0.83	38.36 ± 1.53	>100	>100
9j	45.76 ± 0.42	36.78 ± 0.59	29.45 ± 0.79	33.56 ± 1.51	25.57 ± 0.94	30.43 ± 2.81	>100	>100
9k	23.87 ± 0.74	42.78 ± 0.59	21.97 ± 0.72	28.26 ± 1.47	18.53 ± 0.78	26.55 ± 1.37	>100	>100
9 l	43.76 ± 1.75	49.63 ± 3.59	33.84 ± 0.73	41.11 ± 1.48	31.77 ± 1.58	36.28 ± 1.76	>100	>100
9 m	47.34 ± 2.54	33.78 ± 1.27	28.43 ± 0.69	35.56 ± 1.46	21.82 ± 1.53	34.15 ± 1.83	>100	>100
9n	26.78 ± 1.25	37.89 ± 2.48	34.95 ± 1.52	45.23 ± 1.79	28.34 ± 1.72	32.11 ± 1.35	>100	>100
Camptothecin	4.24 ± 0.41	6.75 ± 0.45	5.16 ± 0.32	3.68 ± 0.21	4.95 ± 0.5	4.28 ± 0.32	>100	>100

^aData represented as mean of the three independent experiments ± S.E.M.

Figure 4.

Antiproliferative activity of 9f on cancer cell lines. Antiproliferative effect of 9f on Mia PaCa-2 pancreatic, DU145 prostate, MCF-7 breast CaCo-2 colon cancer, HL-60 leukemia and HeLa cervical cancer cell lines is presented in dose-dependent manner. IC₅₀ values of the 9f and camptothecin (positive control) are depicted in the figure. Data are presented as the mean ± SEM of three independent experiments.

Apoptosis inducing activity of 9f on MCF-7 breast cancer cell line

MCF-7 cancer cells (1 × 10⁶/ml) were exposed to compound 9f at 5 and 10 µM concentrations for 24 h and used for cell proliferation and pro-apoptotic studies (Fig. 5). Antiproliferative effect of 9f against HL-60 cells at 24 h was presented in Panel A of Fig. 5. Cell cycle distribution of MCF-7 cancer cells was analyzed by flow cytometry. The cells were stained with propidium iodide (PI) and analyzed by flow cytometry to determine the distribution of the cell population in different phases (G0/G1, S, and G2/M) of cell cycle. Camptothecin-treated cells represented as positive control. Panel B of Fig. 5 represents the percent distribution of MCF-7 cancer cells in each stage of the cell cycle following incubation with 9f for a period of 24 h. 9f-treated MCF-7 cells resulted in considerable accumulation of cells in the G0/G1 phase of cell cycle with a concomitant decrease in the number of cells in both the S and G2/M phases in a dose-dependent manner. This demonstrates the cell cycle arrest at G0/G1 phase (Supplementary Figure 30). As shown in Panel C of Fig. 5, apoptotic cell death of MCF-7 breast cancer cells by 9f was quantified by flow cytometry using Annexin V-PI staining. The results demonstrated that 9f caused a dose-dependent increase in the early apoptotic to late apoptotic population in MCF-7 cells (Panel C of Fig. 5; Supplementary Figure 31).

Figure 5.

Apoptosis-inducing activity of 9f in MCF-7 breast cancer cells. Panel A: Antiproliferative effect of 9f (5 and 10 µM) in MCF-7 at 24 h. Panel B: Effect of 9f on cell cycle distribution of MCF-7 cancer cells. Panel C: Apoptotic cell population in 9f treated MCF-7 cells. Panel D: Effect of 9f on mitochondrial membrane potential in MCF-7 cells. Panel E: Effect of 9f on Bcl-2 levels represented in percent inhibition in treated and untreated MCF-7 cancer cells. Panel F: Distribution of Bax levels represented in percent increase in 9f treated MCF-7 cancer cells. Panel G: Effect of 9f in cytosolic cytochrome c levels represented in ng/ml and percent increase in treated and untreated MCF-7 cells. Panel H: Effect of 9f on caspases 3 and 9 activities represented in fold increase in treated MCF-7 cancer cells as compared to untreated. Results of apoptosis-inducing activity of 9f are compared with camptothecin (positive control). Each bar represents mean ± SEM of three independent experiments; *P < 0.05, **P < 0.01 versus untreated control.

Mitochondrial membrane potential loss (Δψm), an indicator of mitochondria membrane disruption, is a distinctive feature of the initial stages of apoptosis. In rhodamine-123 based mitochondrial membrane potential (Δψm) assay, 9f caused 41.33 ± 1.43% and 52.67 ± 2.33% mitochondria membrane potential loss in MCF-7 cells at 5 and 10 µM concentrations, respectively. Similarly, camptothecin (positive control) caused 53.33 ± 3.06% and 62 ± 2.31% mitochondrion membrane potential loss in MCF-7 cells at 5 and 10 µM concentrations, respectively, (Fig. 5 panel D). Bcl-2 and Bax expression levels are associated with integrity of mitochondria membrane and involved in the regulation of apoptosis. Compound 9f showed dose dependent inhibition of Bcl-2 in treated MCF-7 breast cancer cells as compared to untreated cells (Fig. 5, panel E). Bax protein levels are dose dependently increased in treated MCF-7 cancer cells compared to untreated cells (Fig. 5, panel F). Overall, these results demonstrate the decreasing the ratio of Bcl-2/Bax in 9f-treated MCF-7 cancer cells (Fig. 5). This is one of the indications of apoptosis. Cytosolic cytochrome c levels elevation is a hallmark of apoptosis. To determine the release of cytochrome c from mitochondria into cytosol, cytosolic fractions were collected from the MCF-7 cells treated with compound 9f (5, 10 µM), and subjected to antibody coated ELISA based protein level estimation. Cytosolic cytochrome c levels are dose dependently increased in treated MCF-7 cancer cells (Fig. 5, panel G). Caspases activation is a key process in apoptosis. Caspases-3, -8 and -9 colorimetric assays were performed to estimate the level of caspases (3, 8 and 9) activation before and after treatment with 9f. Exposure of cancer cells to 9f enhanced caspase-3 and -9 activities (Fig. 5, panel H), but it did not show considerable effect on caspase-8 activity (data not showed). These results demonstrated the pro-apoptotic potential of 9f in MCF-7 cells via mitochondria-mediated pathway.

Apoptosis inducing activity of 9f on HL-60 leukemia cell line

Pro-apoptotic potential of 9f was also evaluated in HL-60 cells. Panel A of Fig. 6 represents the anti-proliferative effect of 9f against HL-60 cells at 24 h. 9f exhibited better antiproliferative effect than camptothecin (positive control) against HL-60 cells. In cell cycle analysis, dose-responsive accumulation of HL-60 cells in the G0/G1 phase of cell cycle was observed upon treatment with 9f for 24 h (Panel B of Fig. 6). This demonstrates the cell cycle arrest at G0/G1 phase (Supplementary Figure 30). As shown in panel C of Fig. 6, apoptotic cell death of HL-60 cancer cells by 9f was quantified by flow cytometry using Annexin V-PI staining technique. The results showed an increase in the early apoptotic to late apoptotic cells in a dose-dependent fashion in 9f-treated HL-60 cells (Panel C of Fig. 6; Supplementary Figure 31).

Figure 6.

Pro-apoptotic potential of 9f in HL-60 cells. Panel A: Antiproliferative effect of 9f in HL-60 at 24 h. Panel B: Effect of 9f on cell cycle distribution of HL-60 cells. Panel C: Apoptotic cell population in 9f-treated HL-60 cells. Panel D: Effect of 9f on mitochondrial membrane potential in HL-60 cells. Panel E: Effect of 9f on Bcl-2 levels represented in percent inhibition in treated and untreated HL-60 cells. Panel F: Distribution of Bax levels represented in percent increase in 9f treated HL-60 cells. Panel G: Effect of 9f in cytosolic cytochrome c levels represented in ng/ml and percent increase in treated and untreated HL-60 cells. Panel H: Effect of 9f on caspases 3 and 9 activities represented in fold increase in treated HL-60 cells as compared to untreated. Each bar represents mean ± S.E.M. of three independent experiments; *P < 0.05, **P < 0.01 versus untreated control.

9f caused 55.32 ± 2.14% and 65.56 ± 2.56% mitochondria membrane potential loss in HL-60 cells at 5 and 10 µM concentrations respectively compared to untreated cells (control) (Fig. 6 panel D). Compound 9f showed dose dependent inhibition of Bcl-2 and increase in Bax protein levels in treated HL-60 cells as compared to untreated cells (Fig. 6, panel E and F). Overall, these results demonstrate the decreasing the ratio of Bcl-2/Bax in 9f-treated HL-60 cancer cells (Fig. 6). This is an indication of apoptosis. Cytosolic cytochrome c levels are dose dependently increased in cytosolic fraction of 9f-treated HL-60 cells (Fig. 6, panel G). Moreover, 9f enhanced caspase-3 and -9 activities (Fig. 6, panel H), but it did not exhibit considerable effect on caspase-8 activity (data not showed) in HL-60 cells. These results confirmed that 9f induces apoptosis in HL-60 cells via activation of mitochondria-mediated pathway (Fig. 7).

Figure 7.

A diagram represents that 9f-induces apoptosis in MCF-7 breast and HL-60 cancer cells through mitochondria-mediated intrinsic pathway. 9f inhibits Bcl-2 levels and elevates Bax protein levels that may causes mitochondrial membrane potential (Δψm) loss and release of mitochondrial cytochrome c into cytosol, followed by activation of caspases 9 and 3 and DNA fragmentation.

Discussion

During last two decades, several phenolic glycoside (PG) esters have been isolated from plants and reported to possess anticancer activities^17–19. 4-O-β-D-apifuranosyl-(1 → 2)-β-D-glucopyranosyl-2-hydroxy-6-methoxyacetophenone, a new phenolic glycoside was isolated from Celosia argentea and showed cytoxicity activity against SGC-7901 gastric and BEL-7404 hepatic cancer cells²⁰. Ginnalins A–C, maple polyphenols, induced cell cycle arrest in colon and breast cancer cells by down regulating cyclins A and D1²¹. Salidroside, a phenolic glycoside from Acer tegmentosum Maxim, showed anticancer activity and induced apoptosis in human hepatocellular carcinoma Hep G2 cells²². Tao et al.¹⁹ isolated four new PG esters including saccharumoside B, from bark of the sugar maple tree and reported cytotoxic activity of saccharumoside B on human colon tumorigenic and nontumorigenic cell lines. However, the possible mechanism of anticancer natural product saccharumoside B in cancers is unknown. In present study, we have for first time synthesized saccharumoside B and its analogs (9b-9f, 10) by Koenigs-Knorr reaction and evaluated anticancer activities of these compounds on various cancer cell lines including MCF-7 breast, MIA PaCa-2 pancreatic, DU145 prostate, CaCo-2 colon, HL-60 leukemia and HeLa cervical, and non-cancerous PBMC and MCF10A cells. Compounds (9b-9f, 10) exhibited considerable cytotoxicity against cancer cells, but less cytotoxic against normal cells. Aforementioned results demonstrated that compounds are more cytotoxic towards cancerous cells than normal cells. Compound 9f showed substantial anticancer activity against HL-60 and MCF-7 breast cancer cells among other compounds. 9f showed better anticancer activity than saccharumoside B and comparable anticancer potency with camptothecin (positive control). Moreover, 9f appeared less toxic to normal cells than to cancer cells in vitro.

Further, detailed studies of 9f were carried out using MCF-7 and HL-60 cell line to establish the possible mechanism of action of anticancer compound 9f. Cell cycle analysis results demonstrated that 9f arrested MCF-7 and HL-60 cells at G0/G1. 9f caused a dose-dependent increase in the early apoptotic to late apoptotic population in MCF-7 and HL-60 cells. These observations suggest that 9f arrested cell cycle at G0/G1 phase and induced apoptosis. Apoptosis is the process of programmed cell death that may occurs through extrinsic and intrinsic pathways²³. In intrinsic pathway, anti-apoptotic Bcl-2 family proteins inhibit mitochondrial outer membrane permeabilization. Pro-apoptotic Bax increases the permeability of mitochondrial outer membrane that leads to mitochondrial membrane potential loss and form mitochondrial apoptosis-induced channels. Followed by cytochrome c and other pro-apoptotic proteins are released into cytosol through the mitochondrial membrane pores. The released cytosolic cytochrome c activates caspase-9, which in turn activates caspases cascade including caspase-3 that leads to activation of ultimate biochemical events in apoptosis^23–25. Hence, pro-apoptotic studies were carried out and results of these studies demonstrated that 9f inhibited anti-apoptotic Bcl-2 and elevated pro-apoptotic Bax protein levels that caused mitochondrial membrane potential loss in MCF-7 and HL-60 cells. Subsequently, mitochondrial cytochrome c is released into cytosol and activated caspases 9 and 3. 9f exhibited almost similar cellular effects in both MCF-7 and HL-60 cells, but it is more active against HL-60 cells. Overall, our study demonstrated that anticancer activity of saccharumoside B analog 9f against HL-60 and MCF-7 cells by arresting the cell cycle at G0/G1 and inducting apoptosis via mitochondria-mediated intrinsic pathway (Fig. 7).

Chemotherapy is the most commonly used strategy for cancer treatment. However, side effects and dug resistance are the two major problems with available chemotherapeutic drugs^5–9. Hence, discovery and development of new anticancer agents are highly desired for their clinical use. Previous studies demonstrated that new anticancer drugs with more potency have been developed from natural compounds through their structural modifications²⁶. In this scenario, the current study demonstrates that 9f (an analog of natural product saccharumoside-B) exhibited significant antiproliferative effect on cancer cells and is less toxic to normal cells in vitro. Our preliminary, but encouraging results, suggested 9f as an excellent scaffold for further study in the field of cancer chemotherapy and needed in vivo anticancer and toxicity studies to develop it as an anticancer agent.

In conclusion, we have synthesized saccharumoside-B and their analogs (9b-9n, 10) with good yields by the Koenigs-Knorr reaction. In antiproliferative studies, all synthesized compounds (9b-9n, 10) showed considerable cytotoxic effect against MCF-7 breast, MIA PaCa-2 pancreatic, DU145 prostate, CaCo-2 colon, HL-60 leukemia and HeLa cervical cancer cell lines, but they showed less cytotoxicity against non-cancerous MCF10A and PBMC cells. The results demonstrated that compounds are more cytotoxic towards cancerous cells than normal cells. Among all tested compounds, 9f showed significant anticancer activity in MCF-7 breast cancer and HL-60 leukemia cells. Moreover, cell cycle analysis in the MCF-7 and HL-60 cells has shown a significant increase in the G0/G1 and apoptotic population upon 9f treatment, which is suggestive of induction of cell cycle arrest and apoptosis. Further, mitochondrial membrane potential loss, elevation of cytosolic cytochrome c and Bax levels, decrease in Bcl-2 levels, enhanced caspase-9 and -3 activities confirm the apoptosis-inducing potential of compound 9f in MCF-7 breast cancer and HL-60 leukemia cell line via mitochondria-mediated intrinsic pathway. Thus, it is expected that 9f can be developed as therapeutic drug for cancer based on future studies.

Materials and Methods

General information

Melting points were recorded on a Mel-Temp melting point apparatus and are uncorrected. Unless otherwise stated, all the materials were obtained from the commercial suppliers and are used without further purification. Chromatography was carried on silica gel (100–200 mesh). All the reactions were monitored by thin-layer chromatography and the spots were visualized under UV light. The ¹H and ¹³C NMR spectra were recorded on Bruker FT-NMR spectrometer operating at 400 MHz for ¹H and 100 MHz for ¹³C using TMS as an internal standard. Chemical shifts are expressed in δ (ppm) and coupling constants J in Hertz (Hz). Mass spectra were recorded on an Agilent 1100 LC/MSD.

Synthesis of 4-(benzyloxy)-3-methoxybenzoyl chloride (7)

To a solution of vanillin (2.0 g, 13.15 mmol) and K₂CO₃ (5.44 g, 39.47 mmol) in acetone, benzyl chloride (2.3 g, 197.3 mmol) was added drop wise at room temperature. The reaction mixture was then stirred at room temperature for 4 h. After completion of the reaction (as indicated by TLC), the reaction mixture was filtered and concentrated under reduced pressure. The resultant residue was purified by silica gel column chromatography using hexane/ethyl acetate as eluents to afford 4-(benzyloxy)-3-methoxy benzaldehyde as off-white solid.

Aq. KOH (4.4 g, 20.2 mmol) was added to 4-(benzyloxy)-3-methoxy benzaldehyde (5.0 g, 20.06 mmol) at r.t. and the reaction mixture was heated at 180 °C for 1 h. After the completion of the reaction (as indicated by TLC), the reaction mixture was poured into crushed ice and acidified with conc. HCl up to a pH~2–3. The obtained solid was filtered, washed with distilled water and dried under reduced pressure. The obtained solid was dissolved in SOCl₂ and allowed to stir at 100 °C for 2 h under anhydrous conditions. Excess of SOCl₂ was removed under high vacuum and the obtained residue was used for the next step without further purification.

Synthesis of pentaacetyl-α-D-glucose (2)

To a solution of α-D-glucose 1 (50.0 g, 280 mmol) in glacial acetic acid and acetic anhydride (162.8 mL) and perchloric acid in acetic anhydride (10 mL) was added drop wise using an addition funnel at 0 °C and allowed to stir at room temperature for 1 h. After completion of the reaction as monitored by TLC, the reaction mixture was poured into ice-cold water, and obtained solid was filtered, dried to get pure compound 2.

Synthesis of α-D-acetobromo glucose (3)

To a solution of pentaacetyl-α-D-glucose 2 (15 g, 38.4 mmol) in CH₂Cl₂, HBr in glacial acetic acid (23.9 g, 96.0 mmol) was added slowly at 0 °C. The reaction was then allowed to stir at room temperature for 1 h. After completion of the reaction as monitored by TLC, the reaction mixture was quenched using distilled water and extracted with CHCl₃, the organic layer was dried by using anhydrous Na₂SO₄, and concentrated under vacuum to obtain compound 3.

General synthesis of tetra-acetyl phenolic glycosides (5a-n)

To a solution of 3 (2.0 g, 4.8 mmol) in CH₂Cl₂ and water (1:1), K₂CO₃ (0.88 g, 5.82 mmol), catalytic amount of aliquat-336 and corresponding 4-hydroxy benzaldehyde 4(a-n) were added and stirred at 50 °C for overnight. After completion of the reaction (as indicated by TLC), the reaction mixture was quenched with ice-cold water and extracted with ethyl acetate. The organic layer was washed with water, brine and evaporated under reduced pressure to get crude. The crude residue was purified by silica gel column chromatography to afford pure compounds 5(a-n).

General synthesis of phenolic glycosides (6a-n)

To a solution of a compound 5(a-n) (0.7 g, 1.4 mmol) in MeOH (5 mL), NaOMe (0.16 g, 2.9 mmol) was added and stirred at room temperature for 1 h. After completion of the reaction as monitored by TLC, methanol was evaporated and the residue was partitioned between water and ethyl acetate. The organic layer was evaporated, obtained crude was purified by silica gel column chromatography to get pure compound 6(a-n).

General synthesis of esters of 4-O-(β-D-glucopyranosyl) phenolic aldehydes (8a-n)

To a solution of compound 6(a-n) (0.45 g, 1.4 mmol) in DCM, pyridine (14.8 mmol) and corresponding benzoyl chloride 7(a-n) (0.59 g, 2.1 mmol) in CH₂Cl₂ were added at 0 °C. After completion of the reaction as monitored by TLC, the reaction mixture was quenched with ice-cold water, extracted with ethyl acetate. The organic layer was washed with water, brine solution. The residue was purified by silica gel column chromatography to afford pure compound 8(a-n).

General synthesis of glycosyl esters (9a-n)

To a solution of compound 8(a-n) (0.2 g, 0.36 mmol) in MeOH, NaBH₄ (0.02 g, 0.54 mmol) was added portionwise at 0 °C and the reaction was allowed to stir at room temperature for 30 min. After the completion of the reaction as monitored by TLC, methanol was evaporated and the residue was quenched with ice-cold water, acidified with 2N HCl (pH~2–3) and extracted with chloroform. The organic layer washed with water, brine and dried over anhy.Na₂SO₄ and evaporated under reduced pressure. The obtained residue was purified by using column chromatography to afford pure compound 9(a-n) (Fig. 1).

Synthesis of saccharumoside-B (10)

To a solution of compound-9a (150 mg, 2.7 mmol) in MeOH, TEA (0.07 mL, 0.54 mmol) and 5% Pd/CaCO₃ were added and the reaction mixture was stirred at room temperature under H₂ atmosphere (Fig. 2). After completion of the reaction, as determined by TLC, the reaction mass was filtered through a celite bed and the filtrate was evaporated under reduced pressure to get crude compound. The crude was purified by using column chromatography to afford pure compound 10.

Characterization data of compounds

(6-(4-(hydroxymethyl)-2-methoxyphenoxy)-tetrahydro-3,4,5-trihydroxy-2H-pyran-2-yl) methyl 4-hydroxy-3-methoxy benzoate (10)

Off- white solid, Mp: 200–202 °C; [α]^22.3 -31.8 (c 0.5, MeOH). ¹H NMR (400 MHz, MeOH-d₄): δ 7.46 (dd, J = 1.6, 8.4 Hz, 1H), 7.42 (d, J = 2.0 Hz, 1H,), 6.92 (d, J = 8.4 Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H), 6.76 (d, J = 8.4 Hz, 1H), 6.53 (d, J = 7.2 Hz, 1H), 4.78 (d, J = 7.2 Hz, 1H), 4.56 (dd, J = 1.6, 11.6 Hz, 1H), 4.39 (s, 2H), 4.28 (dd, J = 7.2, 12.0 Hz, 1H), 3.75 (s, 3H), 3.74 (s, 3H), 3.64 (ddd, J = 1.6, 7.2, 11.6 Hz, 1H), 3.44–3.40 (m, 2H), 3.34 (d, J = 9.6 Hz, 1H). ¹³C NMR (100 MHz, MeOH-d₄): δ 167.9, 153.1, 150.9, 148.8, 147.1, 137.9, 125.3, 122.6, 120.6, 118.2, 116.0, 112.8, 102.9, 77.9, 75.7, 74.9, 72.1, 65.1, 64.9, 56.8, 56.6. LC-MS (ESI, positive ion mode): m/z 489.3 [M + Na]⁺. Anal. Calcd for C₂₂H₂₆O₁₁: C, 56.65; H, 5.62; Found: C, 56.60; H, 5.66.

(6-(4-(hydroxymethyl)-2-methoxyphenoxy)-tetrahydro-3,4,5-trihydroxy-2H-pyran-2-yl)methyl benzoate (9b)

Off- white solid, Mp: 187–189 °C; [α]^22.0 -31.88 (c 0.5, MeOH). ¹H NMR (400 MHz, DMSO-d₆): δ 8.00 (d, J = 7.2 Hz, 2H), 7.74 (t, J = 7.6 Hz,1H), 7.61 (t, J = 8.0 Hz, 2H), 7.09 (d, J = 8.4 Hz, 1H), 6.98 (s,1H), 6.72 (d, J = 8.4 Hz, 1H), 4.66 (d, J = 11.2 Hz, 1H), 4.46 (d, J = 5.6 Hz, 2H), 4.30 (dd, J = 4.0, 7.6 Hz, 1H), 3.38 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 165.5, 148.7, 144.9, 136.5, 133.3, 129.7, 128.7, 118.3, 115.2, 111.1, 99.9, 76.6, 73.7, 73.2, 70.1, 64.3, 62.6, 55.6. LC-MS (ESI, positive ion mode): m/z 443.3 [M + Na]⁺. Anal. Calcd for C₂₁H₂₄O₉: C, 59.99; H, 5.75. Found: C, 59.92; H, 5.80.

(6-(4-(hydroxymethyl)-2-methoxyphenoxy)-tetrahydro-3,4,5-trihydroxy-2H-pyran-2-yl)methyl 4-methylbenzoate (9c)

Off- white solid, Mp: 228–230 °C; [α]^21.1 -31.8 (c 0.5, MeOH). ¹H NMR (400 MHz, DMSO-d₆): δ 7.89 (d, J = 7.6 Hz, 2H), 7.40 (d, J = 8.0 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.98 (s, 1H), 6.71 (d, J = 8.0 Hz, 1H), 4.99 (d, J = 6.4 Hz, 1H), 4.64 (d, J = 11.2 Hz, 1H), 4.46 (d, J = 5.6 Hz, 2H), 4.27 (dd, J = 7.6, 11.6 Hz,1H), 3.79 (s, 3H), 3.76- 3.29 (m, 4H), 2.46 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 165.5, 148.8, 145.1, 143.6, 136.5, 129.2, 129.1, 127.0, 118.3, 115.3, 111.6, 100.1, 76.7, 73.8, 73.2, 70.2, 64.1, 62.7, 55.7, 21.2. LC-MS (ESI, positive ion mode): m/z 457.3 [M + Na]⁺. Anal. Calcd for C₂₂H₂₆O₉: C, 60.82; H, 6.03; Found: C, 60.78; H, 6.10.

(6-(4-(hydroxymethyl)-2-methoxyphenoxy)-tetrahydro-3,4,5-trihydroxy-2H-pyran-2-yl)methyl 3,5-dimethoxybenzoate (9d)

Off- white solid, Mp: 199–201 °C; [α]^20.7 -32.7 (c 0.5, MeOH).¹H NMR (400 MHz, DMSO-d₆): δ 7.17 (s, 2H), 7.07 (d, J = 8.8 Hz, 1H), 7.00 (s, 1H), 6.78 (s, 1H), 6.71 (d, J = 8.0 Hz, 1H), 4.71 (d, J = 11.6 Hz, 1H), 4.57 (brs, 1H), 4.51 (brs, 2H), 4.43 (dd, J = 7.2, 8.4, 1H), 3.83 (s, 6H), 3.79 (s, 3H), 3.75 (brs, 1H), 3.58–3.44 (m, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 167.5, 162.4, 157.9, 150.8, 147.1, 147.0, 137.8, 133.2, 120.8, 120.6, 118.3, 117.9, 112.7, 108.9, 77.8, 77.7, 75.6, 74.9, 72.0, 71.9, 56.7, 56.1. LC-MS (ESI, positive ion mode): m/z 503.3 [M + Na]⁺, 519.3 [M + K]⁺. Anal. Calcd. for C₂₃H₂₈O₁₁: C, 57.50; H, 5.87; Found: C, 57.45; H, 5.90.

(6-(4-(hydroxymethyl)-2-methoxyphenoxy)-tetrahydro-3,4,5-trihydroxy-2H-pyran-2-yl)methyl 3,4,5-trimethoxybenzoate (9e)

Off- white solid, Mp: 168–170 °C; [α]^21.7 -33.7 (c 0.5, MeOH). ¹H NMR (400MHz, DMSO-d₆): δ 7.47 (d, J = 10.0 Hz, 1H), 7.23 (s, 1H), 6.98 (d, J = 8.0 Hz, 1H), 6.77 (dd, J = 2.0, 6.4 Hz, 1H), 4.66–3.8 (m, 7H), 3.79 (s, 12H). ¹³C-NMR (100 MHz, DMSO-d₆): δ 154.6, 149.2, 146.7, 138.5, 120.5, 118.7, 112.8, 109.0, 108.9, 108.4, 107.9, 103.3, 76.1, 73.4, 71.9, 66.9, 64.9, 60.6, 56.9. LC-MS (ESI, positive ion mode): m/z 533.1 [M + Na]⁺. Anal. Calcd for C₂₄H₃₀O₁₂: C, 56.47; H, 5.92. Found: C, 56.43; H, 5.96.

(6-(4-(hydroxymethyl)-2-methoxyphenoxy)-tetrahydro-3,4,5-trihydroxy-2H-pyran-2-yl)methyl 4-fluorobenzoate (9f)

Off- white solid, Mp: 209–211 °C; [α]^21.8 -30.56 (c 0.5, MeOH). ¹H NMR (400 MHz, DMSO-d₆): δ 7.98 (t, J = 6.8 Hz, 2H), 7.37 (t, J = 8.0 Hz, 2H), 7.01 (d, J = 7.6 Hz, 1H), 6.91 (s, 1H), 6.66 (d, J = 8.0 Hz, 1H), 5.32 (brs, 1H), 5.09 (d, J = 4 Hz, 1H), 4.95 (d, J = 6 Hz, 1H), 4.58 (d, J = 11.6 Hz, 1H), 4.39 (s, 2H), 4.23 (brs, 1H). ¹³C NMR (100 MHz, DMSO-d₆): δ 153.6, 137.6, 133.8, 131.6, 131.0, 129.9, 129.4, 114.9, 102.8, 77.6, 75.2, 73.6, 70.6, 66.4, 62.9. LC-MS (ESI, positive ion mode): m/z 461.3 [M + Na]⁺. Anal. Calcd for C₂₁H₂₃FO₉: C, 57.53; H, 4.33. Found: C, 57.48; H, 4.38.

(6-(4-(hydroxymethyl)-2-methoxyphenoxy)-tetrahydro-3,4,5-trihydroxy-2H-pyran-2-yl)methyl 2-iodobenzoate (9g)

Off- white solid, Mp: 128–130 °C [α]^21.3 -31.8 (c 0.5, MeOH). ¹H NMR (400 MHz, MeOH-d₄): δ 7.77 (dd, J = 1.2, 7.6 Hz, 1H), 7.72 (dd, J = 1.6, 7.6 Hz, 1H), 7.25 (dd, J = 2.0, 8.0 Hz, 1H), 7.21 (d, J = 3.6, 4.0 Hz, 1H), 7.09 (d, J = 8.0 Hz, 1H), 7.01 (d, J = 2.0 Hz,1H), 4.93 (d, J = 7.2 Hz, 1H), 4.70 (dd, J = 2.0, 12.0 Hz, 1H), 4.52 (s, 2H), 4.44 (dd, J = 5.2, 7.2 Hz, 1H), 3.86 (s, 3H), 3.78 (ddd, J = 2.0, 7.6, 9.2 Hz, 3H), 3.57–3.44 (m, 3H). ¹³C NMR (100 MHz, MeOH-d₄): δ 168.0, 151.2, 146.9, 142.1, 138.1, 136.8, 133.7, 131.2, 129.2, 120.6, 118.6, 112.7, 102.8, 77.8, 75.5, 74.9, 71.9, 65.9, 64.9, 56.7. LC-MS (ESI, positive ion mode): m/z 569.2 [M + Na]⁺. Anal. Calcd for C₂₁H₂₃IO₉: C, 46.17; H, 4.24; Found: C, 46.12; H, 4.30.

(6-(4-(hydroxymethyl)-2-methoxyphenoxy)-tetrahydro-3,4,5-trihydroxy-2H-pyran-2-yl)methyl nicotinate (9h)

Off- white solid, Mp: 194–196 °C; [α]^22.3 -27.18 (c 0.5, MeOH). ¹H NMR (400 MHz, DMSO-d₆): δ 9.07 (s, 1H), 8.78 (d, J = 7.6 Hz, 1H), 8.39 (dd, J = 1.6, 5.6 Hz, 1H), 7.59 (dd, J = 2.8, 8.0 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H,), 7.01 (s, 1H), 6.70 (d, J = 8.4 Hz, 1H), 4.94 (d, J = 7.2 Hz, 1H), 4.76 (dd, J = 2.8, 12.4 Hz, 1H), 4.49 (brs, 2H), 4.47 (d, J = 4.4 Hz, 1H), 3.86 (s, 3H), 3.80–3.48 (m, 5H). ¹³C NMR (100 MHz, DMSO-d₆): δ 166.0, 154.1, 151.2, 151.0, 146.8, 139.0, 138.0, 127.9, 125.3, 120.4, 118.2, 112.7, 102.6, 77.8, 75.4, 74.9, 72.0, 65.8, 64.9, 56.7. LC-MS (ESI, positive ion mode): m/z 422 [M + H]⁺, 444.3 [M + Na]⁺. Anal. Calcd for C₂₀H₂₃NO₉: C, 57.00; H, 5.50. Found: C, 56.93; H, 5.56.

(6-(4-(hydroxymethyl)-2-methoxyphenoxy)-tetrahydro-3,4,5-trihydroxy-2H-pyran-2-yl)methyl 3,5-dinitrobenzoate (9i)

Off- white solid, Mp: 186–188 °C; [α]^21.3 -29.16 (c 0.5, MeOH). ¹H NMR (400 MHz, DMSO-d₆): δ 8.95 (d, J = 6.4 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H), 6.80 (s, 1H), 6.53 (t, J = 8.0 Hz, 1H), 4.77 (bros, 1H), 4.43 (s, 2H), 3.74 (s, 3H), 3.65- 3.36 (m, 6H). ¹³C NMR (100MHz, DMSO-d₆): δ 169.5, 133.7, 132.4, 130.1, 129.9, 125.6, 123.5, 120.8, 118.7, 112.9, 103.2, 79.5, 77.9, 74.9, 71.5, 69.2, 65.0, 62.6, 56.8. LC-MS (ESI, negative ion mode): m/z 547.2 [M + 2H₂O-H]⁻. Anal. Calcd for C₂₁H₂₂N₂O₁₃: C, 49.42; H, 4.34; Found: C, 49.40; H, 4.38.

(6-(4-(hydroxymethyl)-2,6-dimethoxyphenoxy)-tetrahydro-3,4,5-trihydroxy-2H-pyran-2-yl)methyl benzoate (9j)

Off- white solid, Mp: 180–182 °C; [α]^21.2 -26.4 (c 0.5, MeOH). ¹H NMR (400 MHz, DMSO-d₆): δ 7.80 (d, J = 7.2 Hz, 2H); 7.65 (t, J = 7.6 Hz, 1H), 7.52 (t, J = 7.6 Hz, 2H), 6.59 (s, 1H), 4.87 (d, J = 5.6 Hz, 1H), 4.50 (d, J = 10.8 Hz, 1H), 4.41 (d, J = 5.6 Hz, 2H), 4.19 (dd, J = 6.8, 11.6 Hz, 1H), 3.66 (s, 6H), 3.27 (brs, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 165.5, 152.6, 138.5, 133.2, 132.7, 129.7, 129.0, 128.6, 104.2, 102.6, 76.3, 74.1, 73.8, 70.2, 64.0, 62.9, 56.1. LC-MS (ESI, positive ion mode): m/z 473.2 [M + Na]⁺, 489.2 [M + K]⁺. Anal. Calcd for C₂₂H₂₆O₁₀: C, 58.66; H, 5.82. Found: C, 58.62; H, 5.87.

(6-(4-(hydroxymethyl)-2,6-dimethoxyphenoxy)-tetrahydro-3,4,5-trihydroxy-2H-pyran-2-yl)methyl 4-methylbenzoate (9k)

Off- white solid, Mp: 201–203 °C; [α]^22.3 -27.16 (c 0.5, MeOH). ¹H NMR (400 MHz, MeOH-d₄): δ 7.69 (d, J = 8 Hz, 2H), 7.31 (d, J = 8 Hz, 2H), 6.59 (s, 2H), 4.86 (d, J = 6.4 Hz, 1H), 4.49 (d, J = 11.6 Hz, 1H) 4.41 (s, 2H), 4.17 (ddd, J = 2.8, 6.4, 11.6 Hz, 1H), 3.67 (s, 6H), 2.39 (s, 3H). ¹³C NMR (100 MHz, MeOH-d₄): δ 165.5, 152.6, 143.5, 138.4, 129.2, 129.1, 127.0, 104.3, 102.7, 76.4, 74.1, 73.9, 70.2, 63.9, 62.9, 56.1, 21.1. LC-MS (ESI, positive ion mode): m/z 487.3 [M + Na]⁺, 503.2 [M + K]⁺. Anal. Calcd for C₂₃H₂₈IO₁₀: C, 59.48; H, 6.08. Found: C, 59.42; H, 6.14.

(6-(4-(hydroxymethyl)-2,6-dimethoxyphenoxy)-tetrahydro-3,4,5-trihydroxy -2H-pyran-2-yl)methyl 3,5-dimethoxybenzoate (9l)

Off- white solid, Mp: 131–133 °C; [α]^22.4 -27.16 (c 0.5, MeOH). ¹H NMR (400 MHz, DMSO-d₆): δ 7.08 (dd, J = 2.8, 8.8 Hz, 1H), 6.97 (d, J = 2.0 Hz, 2H), 6.79 (s, 1H), 6.56 (s, 1H), 4.85 (d, J = 5.6 Hz, 1H), 4.47 (d, J = 6.8 Hz, 1H), 4.44 (s, 2H), 3.78 (s, 6H), 3.67 (s, 6H), 3.39–3.26 (m, 5H). ¹³C NMR (100 MHz, DMSO-d₆): δ 165.1, 160.3, 152.5, 138.4, 132.8, 131.7, 106.9, 106.8, 104.8, 104.2, 102.7, 76.2, 74.1, 73.8, 70.0, 64.1, 62.9, 56.1, 55.5, 55.4. LC-MS (ESI, positive ion mode): m/z 533.3 [M + Na]⁺. Anal. Calcd for C₂₄H₃₀O₁₂: C, 56.47; H, 5.92. Found: C, 56.40; H, 5.97.

(6-(4-(hydroxymethyl)-2,6-dimethoxyphenoxy)-tetrahydro-3,4,5-tri hydroxy -2H-pyran-2-yl)methyl 4-fluorobenzoate (9m)

Off- white solid, Mp: 170–172 °C; [α]^22.0 -18.68 (c 0.5, MeOH). ¹H NMR (400 MHz, DMSO-d₆): δ 7.89 (brs 1H), 7.39 (t, J = 8.8 Hz, 2H), 6.64 (s, 2H), 4.93 (brs, 1H), 4.55 (d, J = 11.6 Hz, 1H), 4.46 (brs, 2H), 4.24 (dd, J = 5.6, 9.2 Hz, 1H), 3.72 (m, 4H), 3.37 (m, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ 152.6, 138.4, 132.6, 132.1, 132.0, 131.9, 131.8, 115.8, 102.5, S76.3, 74.1, 73.8, 70.3, 64.2, 62.9, 56.1. LC-MS (ESI, positive ion mode): m/z 491.2 [M + Na]⁺, 507.2 [M + K]⁺. Anal. Calcd for C₂₂H₂₅FO₁₀: C, 56.41; H, 5.38. Found: C, 56.37; H, 5.40.

(6-(4-(hydroxymethyl)-2,6-dimethoxyphenoxy)-tetrahydro-3,4,5-tri hydroxy-2H-pyran-2-yl)methyl 2-iodobenzoate (9n)

Off- white solid, Mp: 124–126 °C; [α]^21.6 -0.92 (c 0.5, MeOH). ¹H NMR (400 MHz, DMSO-d₆): δ 7.98 (d, J = 8.0 Hz, 1H), 7.46 (dt, J = 2.0, 7.6 Hz, 1H), 7.42 (dd, J = 1.6, 7.6 Hz, 1H), 7.27 (ddd, J = 0.8, 1.2, 1.8 Hz, 1H), 6.59 (s, 2H), 4.88 (d, J = 6.4 Hz, 1H), 4.46 (d, J = 11.6 Hz, 1H), 4.41 (d, J = 5.6 Hz, 2H), 4.25 (dd, J = 6.8, 6.8 Hz, 1H), 3.67 (s, 6H), 3.27–3.25 (m, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 166.0, 152.6, 140.6, 138.5, 135.4, 132.9, 132.6, 130.3, 128.1, 104.1, 102.6, 94.1, 76.2, 74.0, 73.7, 70.1, 64.7, 62.9, 56.1. LC-MS (ESI, positive ion mode): m/z 599.1 [M + Na]⁺, 615.0 [M + K]⁺. Anal. Calcd for C₂₂H₂₅IO₁₀: C, 45.85; H, 4.37. Found: C, 45.81; H, 4.40.

Chemicals and reagents

RPMI-1640, minimal essential medium (MEM), Dulbecco’s modified eagle medium (DMEM), DNase-free RNase, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), camptothecin were purchased from Sigma Chemical Company (St. Louis, MO, USA). Other reagents used were of analytical grade and available locally.

Cell lines

Colon CaCo-2 pancreatic MIA PaCa-2 and promyelocytic human leukemia HL-60 cell lines were procured from European Collection of Cell Cultures (ECACC, Salisbury, Wiltshire, UK). Breast cancer cell line MCF-7 was obtained from National Cancer Institute (Frederick, MD, USA). Human prostate carcinoma DU145, human mammary epithelial MCF-10A and human cervical cancer HeLa cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). Human peripheral blood mononuclear cells (PBMC) were collected from whole blood by Ficoll Plague method²⁷. Cells were grown in RPMI-1640/DMEM/MEM medium containing 10% fetal calf serum (FCS), 100 mg of streptomycin and 100 units penicillin per ml medium. Cells were incubated in CO₂ incubator at 37 °C with 95% humidity and 5% CO₂ gas environment. Cells were treated with tested compounds dissolved in DMSO, while the untreated control cultures received only the vehicle (DMSO, <0.2%).

Cell proliferation by MTT assay

MTT assay was performed to assess the antiproliferative activities of compounds^{27, 28}. Cells were treated with compounds at various concentrations for 24 h or 48 h. 20 µL of freshly prepared MTT reagent was added to cells and incubated at 37 °C for 2 h. The supernatant growth medium was removed and replaced with DMSO (100 µL) to dissolve the formazan crystals. The optical density (absorbance) was read at 570 nm with reference wavelength of 620 nm. Three individual experiments were performed and results were expressed as percent inhibitions at various doses of each compound. IC₅₀ means the concentration of the compound, which inhibits 50% of cell growth²⁸.

Cell cycle analysis

Cancer cells (1 × 10⁶/ml) were treated with different concentrations of compound (5 and10 µM) for 24 h, fixed in cold 70% alcohol in PBS, washed, digested with DNase free RNase (400 µg/mL) at 37 °C for 45 min and stained with propidium iodide (10 µg/ml). Cells were analyzed for PI-DNA fluorescence by flow cytometerically using FACS CALIBUR (Becton Dickinson, Franklin Lakes, New Jersey, USA)^28–30. The fluorescence intensity of subG0/GI cell fraction represents the dead cell population.

Quantification of apoptosis

Apoptotic population of cancer cells was quantified by Annexin V-propidium iodide (PI) staining technique (Abcam, Cambridge, MA, USA) using a flow cytometer as per published technique³¹.

Mitochondrial Membrane Potential Changes (ΔΨm) Measurement Assay

Mitochondrial membrane potential changes (ΔΨm) were determined by flow cytometry using rhodamine-123 (green-fluorescent dye)³². MIA PaCa-2 pancreatic cancer cells (1 × 10⁶ cells/well) were exposed to the isolated compound (5 µM) for 24 h. Further, rhodamine-123 (200 nM) was added to the cells, which were kept in dark for 35 min. The cells were further centrifuged and the pellet was washed with phosphate buffered saline (1 ml). The intensity of fluorescence in the cells represented the mitochondrial membrane potential change (ΔΨm), which was measured using a flow cytometer³².

Cytosolic cytochrome c estimation

Cells were collected by centrifugation at 600 g for 5 min at 4 °C. The cell pellets were washed once with ice-cold PBS and resuspended in cytosol extraction buffer (20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol and 0.1 mM PMSF) containing 250 mM sucrose on ice for 10 min. The cells were homogenized with the grinder on ice. Further, the homogenates were centrifuged at 700 g for 10 min at 4 °C. The supernatants were centrifuged at 10,000 × g for 30 min at 4 °C. Supernatant was collected as cytosolic fraction.

Cytosolic cytochrome c levels were determined using quantitative sandwich enzyme immunoassay using microplate reader. Sample or standard (100 μL) was added into each well and incubated for 2 h at room temperature. The wells were washed several times with wash buffer. The plate was inverted and cleaned with paper toweling. Cytochrome c conjugate (200 μL) was added into each well and incubated for 2 h at room temperature. Wells were washed several times with wash buffer. Substrate solution (200 μL) was added into each well and incubated for 30 min at room temperature. Finally, stop solution (50 μL) was added into wells. The optical density (absorbance) of each well was determined using microplate reader at a 450 nm wavelength³².

Caspases assays

Caspases-3, -8 and -9 assays were measured according to manufacturer’s instructions using a caspase colorimetric protease kit (Abcam, Cambridge, MA, USA). Cells were treated with compounds for 48 h. The cells were lysed by the addition of 50 μL of chilled cell lysis buffer and incubated on ice for 10 min. The resulting cell lysate was centrifuged for 1 min at 10,000 g, and the supernatant was collected. The cell lysate containing 75 mg of protein was incubated with 4 mL of 4 mmol/L pNA-conjugated substrates (DEVD-pNA, IETD-pNA and LEHD-pNA; substrates for caspase-3, -8 and -9, respectively) at 37 °C for 3 h. The amount of pNA released was measured at 405 nm using an ELISA microplate reader (Bio rad). The caspases activity was expressed as fold difference^{32, 33}.

Quantification of Bcl-2

The intracellular content of Bcl-2 was quantified using the human Bcl-2 ELISA kit (Abcam, Cambridge, MA, USA). For the sample preparation, the cells were lysed with 1X lysis buffer. After 1 h of incubation at room temperature, the sample was spun at 1,000 g for 15 min and the supernatant was used for Bcl-2 measurement. Briefly, 80 μL of sample diluent and 20 μL of sample were added into the wells coated with monoclonal antibody to human Bcl-2, and after that the wells were washed twice with the wash buffer. Next, 50 μL of biotin-conjugate were added into the wells. After 2 h of incubation at room temperature on a microplate shaker, 100 μL of streptavidin-horseradish peroxidase were added. The sample was incubated again for 1 h at room temperature on a microplate shaker. The solution was withdrawn and the wells were washed with 3X wash buffer. Immediately, 100 μL of TMB substrate solution were added. Finally, 100 μL of stop solution were added into each well to stop the enzyme reaction. The absorbance was read using an ELISA reader at 450 and 620 nm wavelengths. Bcl-2 protein concentration was determined from the standard protein graphic using the optical densities of the samples³².

Quantification of Bax

Bax protein concentration determination was carried out using the Human Bax Enzyme Immunometric Assay kit (Assay Designs, Ann Arbor, MI, USA). The lysate (sample) preparation was carried out according to the manual. Following centrifugation (16,000 g for 15 min), the cells were resuspended in Modified Cell lysis buffer 4 [0.5 μL/mL of Sigma Protease Inhibitor Cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. Cell lysate (100 μL) (sample) was added into the wells coated with monoclonal antibody to human Bax-α in triplicate. The plate was tapped gently to mix the contents. The sample was incubated at room temperature on a plate shaker for 1 h. The wells were emptied and washed with 5X wash buffer. After the final wash, the plate was tapped gently on a lint free paper towel to remove any remaining wash buffer. The sample was incubated again for 1 h at room temperature on a plate shaker after the addition of 100 μL of specific antibody (biotinylated monoclonal antibody to Bax-α) into each well. The wells were washed again with 5X wash buffer and emptied. Next, 100 μL of blue conjugate (streptavidin conjugated to horseradish peroxidase) was added into each well. The sample was left on a plate shaker for 30 min at room temperature. The wells were washed again as in the previous step. Solution (100 μL) of 3,3′,5,5′ tetramethylbenzidine (TMB) and H₂O₂ was added into each well. Finally, 100 μL of stop solution containing hydrochloric acid in water was added to stop the enzyme reaction. The optical densities were read at 450 and 570 nm using an ELISA reader. Bax protein concentration was determined from the standard protein graphic using the optical densities of the samples³².

Statistical analysis

Data are presented as mean ± standard error of mean (S.E.M) from triplicate parallel experiments unless otherwise indicated. Statistical significance is performed using Student’s t-test. The difference of the statistical data of two groups: p ≤ 0.05 was considered as significant.

Author Contributions

Design of the experiments: S.R., N.S.Y., S.K.K., A.B., S.V., R.T., M.B., V.R.P., K.S.V.G.K.D., D.B.L., M.A.K., G.M.A., V.V.T., V.N.C., S.G.K., G.E.B., S.O.B., G.A. Execution of the experiments: S.V., N.S.Y., S.R., S.K.K., A.B., K.S.V.G.K.D., V.R.P., G.M.A., V.V.T., R.T., M.B., V.N.C., S.G.K., G.E.B., S.O.B., G.A. Analysis of the data: S.R., N.S.Y., S.K.K., A.B., S.V., R.T., M.B., V.R.P., K.S.V.G.K.D., D.B.L., M.A.K., G.M.A., V.V.T., V.N.C., S.G.K., G.E.B., S.O.B., G.A. Contribution in reagents/materials/analysis tools: S.R., N.S.Y., S.K.K., A.B., S.V., R.T., M.B., V.R.P., K.S.V.G.K.D., D.B.L., M.A.K., G.M.A., V.V.T., V.N.C., S.G.K., G.E.B., S.O.B., G.A. Writing the paper: N.S.Y., S.R., S.V., M.A.K., K.S.K., B.L.D., R.T., A.B., G.A. Research Guidance: G.A., A.B., S.V., N.S.Y.

Competing Interests

The authors declare that they have no competing interests.

Electronic supplementary material

Supplementary information accompanies this paper at doi:10.1038/s41598-017-05832-w