Sulforaphane Analogues with Heterocyclic Moieties: Syntheses and Inhibitory Activities against Cancer Cell Lines

Recent studies have shown that sulforaphane (SFN) selectively inhibits the growth of ALDH+ breast cancer stem-like cells.Herein, a series of SFN analogues were synthesized and evaluated against breast cancer cell lines MCF-7 and SUM-159, and the leukemia stem cell-like cell line KG-1a. These SFN analogues were characterized by the replacement of the methyl group with heterocyclic moieties, and the replacement of the sulfoxide group with sulfide or sulfone. A growth inhibitory assay indicated that the tetrazole analogs 3d, 8d and 9d were significantly more potent than SFN against the three cancer cell lines. Compound 14c, the water soluble derivative of tetrazole sulfide 3d, demonstrated higher potency against KG-1a cell line than 3d. SFN, 3d and 14c significantly induced the activation of caspase-3, and reduced the ALDH+ subpopulation in the SUM159 cell line, while the marketed drug doxrubicin(DOX) increased the ALDH+ subpopulation.


Introduction
The natural compound, sulforaphane (SFN), was first isolated from broccoli in 1992. Since then, SFN has been found to be an effective chemo-preventive agent, and it exhibits anti-inflammatory, antioxidant, anti-proliferative and anti-cancer activities [1][2][3][4][5][6][7]. Recently, Sun et al. reported that SFN also inhibits the growth of the ALDH + subpopulation of the breast cancer stem cell line, SUM-159, via down regulation of the Wnt/β-catenin self-renewal pathway [8]. Analogs of SFN were subsequently synthesized and their anti-cancer activities against various cancer cell lines were examined in the literature [1,4,[9][10][11][12][13][14][15][16][17][18][19], and it was found that the replacement of the methyl group yielded compounds with significant activity [1,19]. Herein, a series of SFN analogues containing a heterocyclicring were synthesized, and then were evaluated for their activities against the breast cancer cell lines MCF-7 and SUM-159, and the leukemia stem cell-like KG-1a.
As shown in Scheme 1, the phthalimide groups in compounds 1a-e were hydrolyzed to yield amines 2a-e, which were then converted to isothiocyanates 3a-e with CSCl2 under basic conditions. Sulfides 1a-1c were oxidized by tert-butyl hydroperoxide (TBHP) to provide compounds 4a-c and 5a-c (Scheme 2) [20]. These compounds were subsequently hydrolyzed to give amines 6a-c and 7a-c, respectively. Amines 6a-c and 7a-c were further converted to isothiocyanates 8a-c and 9a-c.
Following removal of the Bocgroups in compounds 11d and 11e, the free amines 12d and 12e were obtained, respectively. Treatment of 12d and 12e with CSCl2 then converted these compounds to the final isothiocyanate compounds 9d and 9e.
However, all of the tetrazole SFN analogues showed low solubility in water, and thus, were further modified to generate water soluble derivatives (Scheme 5). Sulfide 3d was converted to the water soluble derivatives, 14a-c, via treatment with thiol 13a-c under basic conditions. Unfortunately, water soluble derivatives of sulfoxide 8d and sulfone 9d could not be generated, because 8d and 9d were unstable under basic conditions, and cleavage of the heterocyclic ring occurred in each case. As shown in Scheme 1, the phthalimide groups in compounds 1a-e were hydrolyzed to yield amines 2a-e, which were then converted to isothiocyanates 3a-e with CSCl 2 under basic conditions. Sulfides 1a-1c were oxidized by tert-butyl hydroperoxide (TBHP) to provide compounds 4a-c and 5a-c (Scheme 2) [20]. These compounds were subsequently hydrolyzed to give amines 6a-c and 7a-c, respectively. Amines 6a-c and 7a-c were further converted to isothiocyanates 8a-c and 9a-c.
As shown in Scheme 4, protection of the free amines 2d and 2e with Boc groups generated compounds 10d and 10e, respectively. Sulfides 10d and 10e were then oxidized with TBHP to provide sulfones 11d and 11e.
Following removal of the Bocgroups in compounds 11d and 11e, the free amines 12d and 12e were obtained, respectively. Treatment of 12d and 12e with CSCl 2 then converted these compounds to the final isothiocyanate compounds 9d and 9e.
However, all of the tetrazole SFN analogues showed low solubility in water, and thus, were further modified to generate water soluble derivatives (Scheme 5). Sulfide 3d was converted to the water soluble derivatives, 14a-c, via treatment with thiol 13a-c under basic conditions. Unfortunately, water soluble derivatives of sulfoxide 8d and sulfone 9d could not be generated, because 8d and 9d were unstable under basic conditions, and cleavage of the heterocyclic ring occurred in each case.

Activity of Sulforaphane Analogues and Water Soluble Derivatives
These series of SFN analogues 3a-e, 8a-e, 9a-e and water soluble derivatives 14a-c were evaluated for their activities against MCF-7, SUM-159 and KG-1a cell lines, using SFN and parent molecule 3d as a positive controls. The results are summarized in Tables 1 and 2 respectively.

Activity of Sulforaphane Analogues and Water Soluble Derivatives
These series of SFN analogues 3a-e, 8a-e, 9a-e and water soluble derivatives 14a-c were evaluated for their activities against MCF-7, SUM-159 and KG-1a cell lines,using SFN and parent molecule 3d as a positive controls. The results are summarized in Tables 1 and 2, respectively. Moreover, the cytotoxicities of the water soluble compounds 14a-c were assayed, and their inhibitory effects on 293T cell line are shown in Table 3. SFN, 3d and 14c enhanced caspase-3 activity in SUM-159 cells is shown in Figure 1, and inhibitory effects on ALDH-positive cell subpopulation in SUM-159 cells is shown in Figure 2.

Activity of Sulforaphane Analogues and Water Soluble Derivatives
These series of SFN analogues 3a-e, 8a-e, 9a-e and water soluble derivatives 14a-c were evaluated for their activities against MCF-7, SUM-159 and KG-1a cell lines,using SFN and parent molecule 3d as a positive controls. The results are summarized in Tables 1 and 2, respectively. Moreover, the cytotoxicities of the water soluble compounds 14a-c were assayed, and their inhibitory effects on 293T cell line are shown in Table 3. SFN, 3d and 14c enhanced caspase-3 activity in SUM-159 cells is shown in Figure 1, and inhibitory effects on ALDH-positive cell subpopulation in SUM-159 cells is shown in Figure 2.

Activity of Sulforaphane Analogues and Water Soluble Derivatives
These series of SFN analogues 3a-e, 8a-e, 9a-e and water soluble derivatives 14a-c were evaluated for their activities against MCF-7, SUM-159 and KG-1a cell lines,using SFN and parent molecule 3d as a positive controls. The results are summarized in Tables 1 and 2, respectively. Moreover, the cytotoxicities of the water soluble compounds 14a-c were assayed, and their inhibitory effects on 293T cell line are shown in Table 3. SFN, 3d and 14c enhanced caspase-3 activity in SUM-159 cells is shown in Figure 1, and inhibitory effects on ALDH-positive cell subpopulation in SUM-159 cells is shown in Figure 2.

Activity of Sulforaphane Analogues and Water Soluble Derivatives
These series of SFN analogues 3a-e, 8a-e, 9a-e and water soluble derivatives 14a-c were evaluated for their activities against MCF-7, SUM-159 and KG-1a cell lines,using SFN and parent molecule 3d as a positive controls. The results are summarized in Tables 1 and 2, respectively. Moreover, the cytotoxicities of the water soluble compounds 14a-c were assayed, and their inhibitory effects on 293T cell line are shown in Table 3. SFN, 3d and 14c enhanced caspase-3 activity in SUM-159 cells is shown in Figure 1, and inhibitory effects on ALDH-positive cell subpopulation in SUM-159 cells is shown in Figure 2.

Activity of Sulforaphane Analogues and Water Soluble Derivatives
These series of SFN analogues 3a-e, 8a-e, 9a-e and water soluble derivatives 14a-c were evaluated for their activities against MCF-7, SUM-159 and KG-1a cell lines,using SFN and parent molecule 3d as a positive controls. The results are summarized in Tables 1 and 2, respectively. Moreover, the cytotoxicities of the water soluble compounds 14a-c were assayed, and their inhibitory effects on 293T cell line are shown in Table 3. SFN, 3d and 14c enhanced caspase-3 activity in SUM-159 cells is shown in Figure 1, and inhibitory effects on ALDH-positive cell subpopulation in SUM-159 cells is shown in Figure 2.

Activity of Sulforaphane Analogues and Water Soluble Derivatives
These series of SFN analogues 3a-e, 8a-e, 9a-e and water soluble derivatives 14a-c were evaluated for their activities against MCF-7, SUM-159 and KG-1a cell lines,using SFN and parent molecule 3d as a positive controls. The results are summarized in Tables 1 and 2, respectively. Moreover, the cytotoxicities of the water soluble compounds 14a-c were assayed, and their inhibitory effects on 293T cell line are shown in Table 3. SFN, 3d and 14c enhanced caspase-3 activity in SUM-159 cells is shown in Figure 1, and inhibitory effects on ALDH-positive cell subpopulation in SUM-159 cells is shown in Figure 2.

Activity of Sulforaphane Analogues and Water Soluble Derivatives
These series of SFN analogues 3a-e, 8a-e, 9a-e and water soluble derivatives 14a-c were evaluated for their activities against MCF-7, SUM-159 and KG-1a cell lines,using SFN and parent molecule 3d as a positive controls. The results are summarized in Tables 1 and 2, respectively. Moreover, the cytotoxicities of the water soluble compounds 14a-c were assayed, and their inhibitory effects on 293T cell line are shown in Table 3. SFN, 3d and 14c enhanced caspase-3 activity in SUM-159 cells is shown in Figure 1, and inhibitory effects on ALDH-positive cell subpopulation in SUM-159 cells is shown in Figure 2.

Activity of Sulforaphane Analogues and Water Soluble Derivatives
These series of SFN analogues 3a-e, 8a-e, 9a-e and water soluble derivatives 14a-c were evaluated for their activities against MCF-7, SUM-159 and KG-1a cell lines,using SFN and parent molecule 3d as a positive controls. The results are summarized in Tables 1 and 2, respectively. Moreover, the cytotoxicities of the water soluble compounds 14a-c were assayed, and their inhibitory effects on 293T cell line are shown in Table 3. SFN, 3d and 14c enhanced caspase-3 activity in SUM-159 cells is shown in Figure 1, and inhibitory effects on ALDH-positive cell subpopulation in SUM-159 cells is shown in Figure 2.

Activity of Sulforaphane Analogues and Water Soluble Derivatives
These series of SFN analogues 3a-e, 8a-e, 9a-e and water soluble derivatives 14a-c were evaluated for their activities against MCF-7, SUM-159 and KG-1a cell lines,using SFN and parent molecule 3d as a positive controls. The results are summarized in Tables 1 and 2, respectively. Moreover, the cytotoxicities of the water soluble compounds 14a-c were assayed, and their inhibitory effects on 293T cell line are shown in Table 3. SFN, 3d and 14c enhanced caspase-3 activity in SUM-159 cells is shown in Figure 1, and inhibitory effects on ALDH-positive cell subpopulation in SUM-159 cells is shown in Figure 2.

Activity of Sulforaphane Analogues and Water Soluble Derivatives
These series of SFN analogues 3a-e, 8a-e, 9a-e and water soluble derivatives 14a-c were evaluated for their activities against MCF-7, SUM-159 and KG-1a cell lines,using SFN and parent molecule 3d as a positive controls. The results are summarized in Tables 1 and 2, respectively. Moreover, the cytotoxicities of the water soluble compounds 14a-c were assayed, and their inhibitory effects on 293T cell line are shown in Table 3. SFN, 3d and 14c enhanced caspase-3 activity in SUM-159 cells is shown in Figure 1, and inhibitory effects on ALDH-positive cell subpopulation in SUM-159 cells is shown in Figure 2. Moreover, the cytotoxicities of the water soluble compounds 14a-c were assayed, and their inhibitory effects on 293T cell line are shown in Table 3. SFN, 3d and 14c enhanced caspase-3 activity in SUM-159 cells is shown in Figure 1, and inhibitory effects on ALDH-positive cell subpopulation in SUM-159 cells is shown in Figure 2.
The activities of water soluble compounds of 3d, i.e., 14a-c, were assayed against the MCF-7, SUM-159 and KG-1a cell lines in an independent experiment, and both SFN and 3d were applied as the positive controls.The water soluble derivative 14a, was found to be comparably active against MCF-7 cell lines, and less active against SUM-159 and KG-1a cell lines, and it was less active than the parent molecule 3d against all three of the cell lines. Compounds 14b exhibited higher activity than SFN against MCF-7, SUM-159 and KG-1a cell lines, with IC 50 values of 7.84, 5.96, and 4.73 µM, respectively. Compared with 3d, compound 14b exhibited comparable activity against all three cell lines. Compound 14c exhibited higher activities than SFN against all three cell lines, with IC 50 values of 7.30, 6.71 and 1.88 µM, respectively. As for inhibitory effect against the stem cell-like KG-1a cell line, water soluble derivative 14c was about 6.4 times more potent than SFN, and it was also more potent than its parent molecule 3d.
In the caspase-3 activity assays, SFN, 3d and 14c were found to significantly induce the activation of caspase-3 in the SUM-159 cell line (Figure 1). Based on these results, it appears that SFN, 3d and 14c potentially induce apoptosis in cancer cells by increasing caspase-3 activity.
In addition, SFN and doxorubicin (DOX) as well as 3d and 14c were assayed for their affects on the ALDH + subpopulation in the SUM-159 cell line (Figure 2). SUM-159 cells were treated with SFN (1 and 5 µM), 3d (0.5 and 2.5 µM), 14c (0.5 and 2.5 µM) and DMSO for 4 days and subject to Aldefluor assay and flow cytometry analysis.
In general, conventional drugs such as DOX are usually less active against ALDH + cells, thereby leading to an increase in this cell population. In contrast, SFN, 3d, and 14c were found to selectively inhibit the growth of ALDH + cells at concentrations that were 10-fold lower than their IC 50 values against the SUM-159 cell.

General Information
1 H-and 13 C-NMR spectra were obtained using a AV 400 spectrometer (Bruker, Madison, WI, USA) using CDCl 3 as the solvent.Chemical shifts are reported in parts per million (ppm) relative to either a tetramethylsilane internal standard or solvent signals. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br. = broad, m = multiplet), coupling constants and integration.

General Procedure for the Synthesis of Compounds 1a-e
Compounds 1a-e were prepared according to a literature methid according to the literature method [19].     2a-e, 6a-c and 7a-c To a solution of compound 1 (1 mmol) in anhydrous MeOH (10 mL) was added methylamine (10 mL, 40% water solution) at room temperature. The mixture was stirred at room temperature overnight, and the solvent was removed under reduced pressure to give crude residue, which was purified by columnchromatography to obtain 2.The synthesis of 6 and 7 were similar to that of 2.

General Procedure for the Synthesis of Sulfides 3a-e, Sulfoxides 8a-c and Sulfones 9a-c
To a solution of 2 (1 mmol) and NaOH (1 mol/L, 1.66 mmol) in anhydrous CH 2 Cl 2 (10 mL) was added CSCl 2 (2.55 mmol) at 0˝C. The mixture was stirred for 20 min, warmed to room temperature, and continued to stir for 3 h. The reaction mixture was diluted with brine (10 mL) and CH 2 Cl 2 (10 mL), and extracted with CH 2 Cl 2 (10 mLˆ2). The organic layer was dried over anhydrous Na 2 SO 4 , and solvent was removed under reduced pressure. The residue was purified by columnchromatography to obtain isothiocyanate 3. The synthesis of compounds 8 and 9 were similar to 3.

General Procedure for the Synthesis of Amides 4a-c and 5a-c
To a solution of compound 1 (1 mmol) in anhydrous CH 2 Cl 2 (20 mL) under an argon atmosphere was added Ti(O-i-Pr) 4 (1 mmol), and the reaction mixture was stirred at room temperature for 15 min. Then the reaction mixture was cooled to´20˝C for 20 min, and a solution of TBHP (2 mmol) in anhydrous CH 2 Cl 2 (3.2 mL) was added slowly. The mixture was stirred at´20˝C for 8-12 h, and water (20 mL) was added, the reaction mixture was stirred for 1 h, and the resulting gel was dissolved with ethyl acetate (30 mLˆ2). After remove the solvent under reduced pressure, and obtain crude 4 or 5, which was purified withcolumnchromatography.
(t, J = 6.8 Hz, 2H), 1.80-1.97 (m, 4H); 13  To a solution of compound 3 (1 mmol) in anhydrous CH 2 Cl 2 (20 mL) under an argon atmosphere was added MCPBA (2 mmol) at 0˝C, and the resulting reaction mixture was stirred at room temperature for overnight. Saturated sodium bicarbonate solution was added, and the aqueous layer was extracted with CH 2 Cl 2 (10 mLˆ2). The combined organic layer was dried over anhydrous Na 2 SO 4 , and the solvent was removed under reduced pressure, the residue was purified with columnchromatography to obtain 8. To a solution of compound 2 (1 mmol) in anhydrous CH 2 Cl 2 (20 mL) was added Et 3 N (2 mmol), and the reaction mixture was stirred at room temperature for 30 min. (Boc) 2 O was added, and then the mixture was stirred at room temperature for another 8 h. Water (10 mL) was added to quench the reaction, and the aqueous layer was extracted with CH 2 Cl 2 (10 mLˆ2). The combined organic layer was dried over anhydrous Na 2 SO 4 , and the solvent was removed under reduced pressure, and the crude residue was purified with columnchromatography to obtain 10.
tert-Butyl (4-((5-phenyl-1H-tetrazol-1-yl)thio)butyl)carbamate (10d): yield 63%, yellow oil; 1  To a solution of compound 10 (1 mmol) in anhydrous CH 2 Cl 2 (20 mL) under an argon atmosphere was added titanium tetraisopropoxide (1 mmol), and the reaction mixture was stirred at room temperature for 15 min, and then was cooled to´20˝C for 20 min. A solution of TBHP (2 mmol) in anhydrous CH 2 Cl 2 (3.2 mL) was added slowly, the mixture was stirred for 8 to 12 h at room temperature, and then water (20 mL) was added. The mixture was stirred for another 1 h, the resulting gel was dissolved with ethyl acetate (30 mLˆ2). After the solvent was removed under reduced pressure, the crude oil was purified by columnchromatography.

General Procedure for the Synthesis of Tetrazole Acids Compounds 14a-b
To a solution of compound 3d (1 mmol) in dried CH 2 Cl 2 (2 mL) was added DMAP (0.2 mmol) and stirred at room temperature for 5 min. Then N-acetylcysteine (0.8 mmol) or thiopronin (0.8 mmol) was added, and the reaction mixture was stirred for 4 h. Added 10% aqueous citric acid solution (10 mL) and CH 2 Cl 2 (10 mL). Then organic layer was dried with anhydrous sodium sulfate, then purified by silica gel column and got compound 14.

General Procedure for the Synthesis Tetrazole N-dimethyl Compound 14c
To a solution of 2-(dimethylamino)ethanethiol (0.71 mmol) in 95% EtOH (1 mL) was added 3d (0.71 mmol), and the reaction mixture was stirred for 10 h at 40˝C. Then organic layer was dried with anhydrous sodium sulfate, then purified by silica gel column to give compound 14c. . They were cultured in RPMI 1640 supplemented with 10% FBS at 37˝C in a 5% CO 2 incubator. Cells were seeded in 96-well plates at a density of 3000 cells per well. All cells were treated for 48 h, with increasing concentrations of different compounds.Cell viability was measured using an MTT assay which was performed following the manufacturer's protocol. The number of living cells was directly proportional to the absorbance at 490 nm of a formazan product reduced from MTT by living cells. The IC 50 value was obtained using SPSS 11.5 software. The results were derived from three independent experiments performed in triplicate.

Caspase-3 Activity Assay
SUM-159 cells were treated with SFN (12 µM), 3d (4.5 µM), 14c (9.5 µM), respectively, and collected after 48 h. The caspase-3 activity assay was determined using a caspase-3 activity kit (Beyotime, Nanjing, China). Cellular protein was extracted with the supplied lysis buffer, followed by the determination of protein concentration using BCA Protein Assay Reagents (Beyotime). The assay is based on the ability of caspase-3 to change acetyl-Asp-Glu-Val-Asp p-nitroanilide into the yellow formazan product, p-nitroaniline. The absorbance at 405 nm was determined, and the activity of caspase-3 was assessed by calculating the ratio at OD 405nm of the drug-treated cells to the untreated cells. The results were derived from three independent experiments performed in triplicate.

Aldefluor Assay
A cell population with a high aldehyde dehydrogenase (ALDH) enzyme activity was previously reported to enrich mammary stem/progenitor cells. SUM-159 cells were treated with SFN (1 and 5 µM), 3d (0.5 and 2.5 µM), 14c (0.5 and 2.5 µM) and DMSO for 4 days and subject to Aldefluor assay and flow cytometry analysis. (Stem Cell Technologies, BD, New York, NY, USA). Single cells obtained from the drug-treated cells were incubated in an Aldefluor assay buffer containing an ALDH substrate, bodipy-aminoacetaldehyde (1 µM per 1,000,000 cells), for 40 to 50 min at 37˝C. As a negative control, a fraction of cells from each sample was incubated under identical condition in the presence of the ALDH inhibitor diethylaminobenzaldehyde. Flow cytometry was used to measure ALDH-positive cell population. The results were derived from three independent experiments performed in triplicate.

Conclusions
The ALDH + population in breast cancer cell line SUM-159 was stem cell-like cells, and it was recently reported that SFN can selectively inhibit the growth of this cell population [8]. To further investigate the structure-activity relationship of SFN, a series of SFN analogues with heterocyclic moieties were synthesized in this study, and then were assayed against the breast cancer cell lines, MCF-7 and SUM-159, and the acute leukemia stem-cell like cell line KG-1a. Among the furan, methoxypyridine, methoxybenzene, tetrazole, and thiazole classes of SFN analogues, the tetrazole SFN analogues 3d, 8d, and 9d were generally the most potent. In particular, 8d exhibited up to a 15-fold greater potency than SFN against KG-1a cell line. The water soluble derivatives 14c exhibited comparable activity with the parent molecule, 3d, against MCF-7 and SUM-159, and higher potency against the KG-1a cell line. In addition, compounds 14c exhibited less effect than 3d on the human embryonic kidney cell line 293T.Caspase-3 activity data also indicated that SFN, 3d and 14c induced apoptosis in the SUM-159 cell line by increasing caspase-3 activity. Like SFN, analogues 3d and 14c also significantly reduced the ALDH + subpopulation in the SUM-159 cell line from 3.10% to 0.16% and 0.07%, respectively. In contrast, treatment with DOX increased the ALDH + population to 5.12%. Based on the results obtained for the leukemia stem-cell like cell line KG-1a, and the observed effects on the stem cell-like ALDH + subpopulation in breast cancer cells, the biological activities of 3d and 14c should be further investigated.