Glycosylation enhances the anti-migratory activities of isomalyngamide A analogs

European Journal of Medicinal Chemistry 64 (2013) 169e178

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Glycosylation enhances the anti-migratory activities of isomalyngamide A analogs Shivaji V. More a, Tzu Ting Chang a, Yu-Pin Chiao a, Shu-Chuan Jao b, Chung-Kuang Lu c, Wen-Shan Li a, d, * a

Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan Division of Herbal Drugs and Natural Products, National Research Institute of Chinese Medicine, Taipei 112, Taiwan d Doctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University, Kaohsiung 804, Taiwan b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 July 2012 Received in revised form 18 February 2013 Accepted 24 March 2013 Available online 6 April 2013

Three, new, fully synthetic glycosylated isomalyngamide A analogs 4e6 were prepared and evaluated for their anti-migratory activities in human breast cancer cells. The results of the study show that two glycosylated derivatives 4 and 5, containing mannose and galactose appendages, suppress metastatic events (e.g., migration, invasion and adhesion) in human breast adenocarcinoma MDA-MB-231 cells at “nontoxic” concentration levels. In contrast, derivative 6 that contains a lactose moiety, displays a less potent activity. The findings show that monosaccharide rather than disaccharide appendages to the isomalyngamide A backbone more greatly influence cell migration and invasive ability. Evidence has been gained for a mechanism for inhibition of metastatic activities in MDA-MB-231 cells by 4 and 5, involving inactivation of the expression of p-FAK and paxillin through the integrin-mediated antimetastatic pathway. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Cell adhesion FAK/paxillin signaling pathway Glycosylation Isomalyngamide A Invasion Metastasis

1. Introduction Cancer is one of the major diseases that both diminishes the quality of and shortens human life. Metastasis is the leading negative property of cancer [1]. The results of in vivo studies have provided direct information about the pathogenesis of metastasis, which possesses diverse and characteristic gene-expression profiles that differ from those seen in normal organs and tissues [2e5]. Metastasis is a combination of multiple biological events that results in migration of cancer cells from primary sites to secondary organs. Metastatic spread might involve a pathway in which cancer cells disseminate from a primary tumor to other sites by penetrating the walls of either lymphatic or blood vessels at initial stages of tumor growth [6e8]. Because inhibition of the multiple biological events associated with metastasis including adhesion, degradation and migration of tumor cells could impede metastasis, they are potential targets in the development of antimetastatic agents

* Corresponding author. Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan. Tel.: þ886 2 27898662; fax: þ886 2 27831237. E-mail address: [email protected] (W.-S. Li). 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.03.044

[9]. Unfortunately, only limited progress has been made in developing of antimetastatic agents [10,11]. Incorporation of sugar moieties into pharmacologically active natural products is an important and effective approach to regulate drug action, solubility, recognition, and pharmacological effects [12e19]. For example, glycorandomization profoundly influences the cytotoxic effects of colchicine on various tumors, such as Du145, HCT-116, Hep3B, SF-268, SK-OV-3, and A549 [13] and monoglycosylated vancomycins have been shown to have lower inhibitory potencies than the parent antibiotic against selected grampositive bacterial strains, including Bacillus subtilis, Staphylococcus aureus and Enterococcus faecium [17]. Like migrastatin [11], the isomalyngamide A analogs 2 and 3 (Fig. 1) have therapeutic potential in the treatment of breast cancer by acting to not only block cell proliferation but also to inhibit tumor cell migration [20]. The parent natural product, isomalyngamide A (1) (Fig. 1), is a member of a family of fatty acid amides that were isolated from the marine cyanobacterium Lyngbya majuscule [21,22]. Perhaps owing to its complicated structure, which is composed of an amide linkage between a highly functionalized amine and (
)-7(S)-methoxytetradec-4(E)-enoic acid, isomalyngamide A has not yet been synthesized [23,24].

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Fig. 1. Structures of isomalyngamide A (1), nonglycosylated analogs 2 and 3 and glycosylated analogs 4e6.

Interestingly, isomalyngamide A analogs 2 and 3 (Fig. 1), bearing fatty acid scaffolds that do not contain reactive and difficultly installed enol ether, vinyl chloride, 4E double bond and 7S methoxy functional groups, have been prepared and shown to be antimigratory agents that block tumor cell migration with respective IC50 values of 22.7 and 29.9 mM [20]. Recent advances in search of new malyngamides, the other type of fatty acid amides, isolate and discover malyngamide 2, showing anti-inflammatory activity with modest cytotoxicity to the mammalian cell line [25]. Malyngamide 2 also features a skeleton of N-substituted chiral fatty acid amides with a triol oxidized cyclohexanone ring, which could be served as bioisostere of a sugar ring [26]. Therefore, glycosylation of isomalyngamides may pave the way for the improvement of their biological activity through enhancement of either target recognition or solubility properties. Based on the observations summarized, we hypothesized that the isomalyngamide A analog 2 might be an ideal substance with which to examine the role played by glycosylation on antimigratory properties. For this purpose, we designed and synthesized three glycosylated isomalyngamide A analogs 4e6 (Fig. 1) and determined their activities against breast cancer cells. The observations made in this study show that the monoglycosylated isomalyngamide A analogs 4 and 5 suppress metastatic events (e.g., migration, invasion and adhesion) in human breast adenocarcinoma MDA-MB-231 cells at “nontoxic” concentration levels. Significantly, these substances promote a decrease of p-FAK and paxillin expression through an integrin-mediated antimetastatic pathway and, consequently, cause inhibition of cell migration/ adhesion and complete cessation of tumor cell invasion. 2. Results and discussion 2.1. Synthesis of isomalyngamide A glycosides 4e6 Isomalyngamide A analogs 4e6 (Fig. 1), bearing a fatty acid amide core structure, were prepared in adequate yields by using a convergent approach following the retrosynthetic strategy outlined in Scheme 1 involving peptide coupling of the fatty acid 17 and the peracetylated aminoethoxyl glycosides 18e20. Using this strategy, fatty acid 17 is generated by condensation of amine 15 and malonate half ester 10.

Acid 10 was synthesized in three steps starting with commercially available pentane-2,4-dione (7) (see Supplementary data (SD), Scheme S1), using the method described previously [20]. The key amine intermediate 15 was obtained by Boc-deprotection of 14, which was efficiently generated through condensation of amine 12 and fatty acid 13 using 2-(1H-benzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HBTU)/diisopropylethylamine (DIPEA) as a coupling agent (Scheme 2). Amide formation between acid 10 and amine 15, utilizing a similar coupling procedure, produced the desired ester 16, which was saponified to yield 17. Glycosylations of 2-bromoethanol, using the peracetylated mannose, galactose and lactose derivatives 21e23, were performed under Lewis acidic condition (BF3$2Et2O) to form the respective peracetylated (2-bromo) ethyl glycosides 24e26 in 65e68% yield (Scheme 3). Conversion of (2-bromo) ethyl glycosides 24e26 to the corresponding azides 27e29 was achieved by reaction with excess sodium azide (NaN3) in DMF at 60  C. Subsequent azide reduction of 27e29 with 10% Pd/C in methanol under a H2 atmosphere provided the peracetylated aminoethoxyl glycosides 18e20 in 76e92% yields. Condensation of acid 17 and the corresponding aminoethoxyl glycosides 18e20, using HBTU/DIPEA as the coupling agent, resulted in formation of the target glycosylated isomalyngamide A analogs 4e6 (Scheme 3). 2.2. Isomalyngamide A glycosides 4e6 do not affect proliferation of MDA-MB-231 cells at low concentration (30 mM) It has been reported that small molecules serve as potent antitumor [27e29] and/or antimetastatic agents [30e38], acting through complicated pathways involving inhibition of cell proliferation and blockage of cell migration. Consequently, suppression of tumor metastasis by anticancer agents may be a result of cytotoxicity activation and migration inhibition through simultaneous regulation of multifactorial and chemical processes. Thus, in order to gain a detailed understanding of the mechanism responsible for antimetastasis, cytotoxic effects of small molecules must be eradicated while sustaining their ability to inhibit cancer cell migration. The in vitro cytotoxic activities of isomalyngamide A analogs 2 and 3 and glycosides 4e6 against breast adenocarcinoma MDA-MB231 were evaluated using MTT assays. The results (Fig. 2) show that

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Scheme 1. Retrosynthetic analysis of glycosylated isomalyngamide A analogs 4e6.

4e6 do not display significant effects on the inhibition of cancer cell proliferation in the 10e30 mM concentration range, which is similar to the activity obtained from the isomalyngamide A analogs 2 and 3. This finding suggests that these glycosides can be used in “nontoxic” concentrations (10e30 mM) to study their direct effects on the metastasis pathway. 2.3. Isomalyngamide A glycosides 4e6 suppress serum-induced MDA-MB-231 cell migration in a dose-dependent manner For the purpose of evaluating the effects of the isomalyngamide A glycosides on the migration of MDA-MB-231 cells in vitro, wound-healing migration assays were performed first. As the data in Fig. 3 show, glycosides 4 and 5 have no effect on MDA-MB-231 cell proliferation at concentrations of 25 mM but at this

concentration they exhibit significant delayed wound healing activities. Unlike 4 and 5, glycoside 6, possessing a disaccharide appendage, causes a less potent reduction of cell migration at the same concentration (Fig. 3c). In comparison to their nonglycosylated analogs 2 and 3 (Fig. 3c) glycosides 4 and 5 exhibit improved potency on inhibition of wound healing toward MDAMB-231 breast cancer cells (25 mM). These observations suggest that introduction of a monosaccharide (mannose and galactose) rather than a disaccharide (lactose) moiety is superior for promoting reduction of the migratory ability of breast cancer cells. To quantify cellular migration effects, Boyden chamber assays were performed. Glycosides 4 and 5 were found to effectively suppress MDA-MB-231 cell migration (Fig. 4) in a dose-dependent manner with respective IC50 values of 11.7 and 11.8 mM (Table 1). It is interesting to note that glycosides 4 and 5 are 2e3-fold more

Scheme 2. Synthesis of the fatty acid moiety of glycosylated isomalyngamide A analog. Reagents and conditions: a) (Boc)2O, dioxane, RT, 12 h, 40%; b) HBTU, DiPEA, DMF, RT, 12 h, 77%; c) TFA, 2% dd H2O, 0  C w RT, 3 h, 90%; d) HBTU, DiPEA, DMF, RT, 12 h, 81%; e) KOH, THF/MeOH, reflux, 16 h, 93%.

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Scheme 3. Synthesis of glycosylated isomalyngamide A analogs. Reagents and conditions: a) Ac2O, pyridine, 100  C, 3 h, 93e98%; b) 2-bromoethanol, BF3$Et2O, DCM, RT, in dark, 3 h, 65e68% c) NaN3, DMF, 60  C, 12 h, 62e82%; d) Pd/C, MeOH, H2, RT, 2 h, 76e92%; e) HBTU, DiPEA, DMF, RT, 5 h, 40%.

potent than the non-glycosylated precursors 2 and 3 (IC50 ¼ 22.7 and 29.9 mM, respectively). In contrast, glycoside 6 (see Table 1 and Supplementary data (SD) Fig. S1) is at least 4-fold less potent than 4 and 5 and 2-fold less potent than 2 and 3. These results are consistent with previous observations made in wound-healing studies, which demonstrate that monosaccharides rather than

disaccharides inhibit migration of metastatic breast cancer cells. Cumulatively, wound-healing migration and Boyden chamber assays uncover glycosylation to modulate the potency of 2, and monoglycosylated compounds 4 and 5 were identified which, unlike 2 or 3, exhibited significantly delayed wound healing and led to decreased migration.

Fig. 2. Cell viability effects of glycosylated isomalyngamide A analogs 4e6. MDA-MB-231 cells were incubated with increasing concentrations (10e50 mM) of isomalyngamide A analogs 2 and 3 and glycosides 4e6 for 48 h in 96-well plates and the cell survival was measured by an MTT assay. The values plotted are representative of three independent experiments. Data are expressed as percent viability of treated cells (isomalyngamide A analogs 2e6) compared with the untreated control. Significant differences compared with untreated control: *p < 0.05; **p < 0.01; ***p < 0.001.

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Fig. 3. Effects of glycosides 4e6 on the motility of MDA-MB-231 cells. In control experiments, cells were grown in tissue culture plates until confluence, followed by a scratch wound applied with a sterile plastic pipette tip from time of injury (a) to 24 h (b). Cells were treated with isomalyngamide A analogs 2 and 3 and glycosides 4e6 (25 mM) from time of injury (a) to 24 h (c).

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Fig. 4. Chamber assay of the effects of glycosides 4 and 5 on the serum-induced migration of MDA-MB-231 cells. (a) and (b) The images from Boyden chamber assay (top) and the quantitative results (bottom) are shown. IC50 values represent mean  SD of three independent experiments. Data are expressed as percent migration of treated cells (isomalyngamide A analogs 4 and 5) compared with the untreated control. Significant differences compared with untreated control: *p < 0.05; **p < 0.01; ***p < 0.001.

2.4. Isomalyngamide A glycosides 4e6 reduce the invasive ability of MDA-MB-231 cells The potential for isomalyngamide A analogs 2 and 3 and glycosides 4e6 to invade MDA-MB-231 cells was determined using Table 1 Inhibition of cancer cell migration by isomalyngamide A glycosides.a Cpds #

Inhibition of migration IC50 (mM)b

2 3 4 5 6

22.7 29.9 11.7 11.8 >50

a

   

1.320 0.620 0.2 0.3

Results from chamber cell migration assay. IC50 values are reported as the mean of three independent determinations. b

standard Transwell invasion assays, in which cancer cells are allowed to transfer across a reconstituted basement membrane (Matrigel) for 24 h. As the results in Fig. 5 show, after treatment with glycosides 4 and 5 at 20 mM, significant suppression of MDAMB-231 cell invasion takes place. Furthermore, the observations demonstrate that treatment of cells with 30 mM of glycosides 4 and 5 results in complete inhibition of tumor cell invasion. Unlike glycosides 4 and 5, which exhibit more potent effects against MDA-MB-231, glycoside 6, possessing the lactose structural unit, displays a low Matrigel invasion-suppressive response (Fig. 5e). In contrast, the parent 2 and analog 3, no sugar-bearing scaffolds, displayed a significant reduction in potency compared to those of glycosides 4 and 5 toward breast cancer cell line (Fig. 5a and b). These results match those obtained in earlier cell migration studies (Figs. 3 and 4), which show that the presence of a monosaccharide (mannose and galactose) rather than disaccharide

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Fig. 5. Glycosides 4e6 inhibit the cancer cell invasive capacity through Matrigel-coated transwell filters. Cells were seeded onto precoated transwell inserts with serum-free medium containing at a density of 8  104 cells/mL and then treated with different doses of isomalyngamide A analogs 2 and 3 and glycosylated isomalyngamide A analogs 4e6 or DMSO for 24 h. Data are expressed as percent invasion ability of treated cells (isomalyngamide A analogs 2e6) compared with the untreated control. Significant differences compared with untreated control: *p < 0.05; **p < 0.01; ***p < 0.001.

(lactose) leads to significantly enhanced efficacy against tumor cell invasion. 2.5. Isomalyngamide A glycosides 4e6 inhibit fibronectin- and collagen type IV-mediated cell adhesion Integrin-mediated cancer cell adhesion to the ECM proteins fibronectin, collagen IV, vitronectin and laminin, plays multiple roles in mediating the diverse processes involving cell adhesion, migration, and invasion that cause the spread of metastatic tumors [39e45]. Given the fact that 4e6 have a broad range of antimetastatic activities (migration and invasion) in MDA-MB-231 cells, inhibition of cancer cell adhesion by these glycosides was

evaluated. As the data in Fig. 6 demonstrate, treatment of MDA-MB231 cells with glycosides 4e6 inhibits their adhesion to fibronectin and collagen IV in a concentration-dependent manner. For example, the percent of cells that adhere to fibronectin decrease from 100% (control) to 79%, 69% and 58%, when they are pretreated for 48 h with 5 at respective concentrations of 10, 20, and 30 mM (Fig. 6a). In contrast, isomalyngamide A analogs 2 and 3 displayed low potency in inhibition of cancer cell adhesion (Fig. 6). This finding raises an interesting question about whether or not an antiproliferative/cytotoxic effect is directly involved. As the data in Fig. 2 show, glycosides 4e6 exhibit negligible effects on cell growth inhibition in the concentration range of 0e30 mM, suggesting that the impact of cytotoxicity on the cell adhesion can be ignored.

Fig. 6. Glycosides 4e6 inhibit the adhesive property of human MDA-MB-231 cells. Cancer cells were incubated with indicated concentrations of isomalyngamide A analogs 2 and 3 and glycosides 4e6 for 48 h then followed by adhesion assays using fibronectin- and collagen type IV-coated 96-well plates as described in Experimental section. Data are expressed as percent adhesive ability of treated cells (isomalyngamide A analogs 2e6) compared with the untreated control. Significant differences compared with untreated control: *p < 0.05; **p < 0.01; ***p < 0.001.

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Fig. 7. Glycoside 4- and 5-induced anti-migratory activities in MDA-MB-231 cells. Western blot analysis of p-FAK (Y397), FAK, paxillin, and Rac1 in MDA-MB-231 cells treated with indicated concentrations of 4 and 5. Actin was probed as an internal control.

2.6. Isomalyngamide A glycosides 4e5 decrease cell migration ability through inhibition of the FAK/paxillin signaling pathway Integrins, a heterodimeric transmembrane receptor family, have been shown to be intracellular signaling receptors that are crucial for cellular adhesion and motility to extracellular matrix (ECM) proteins during the cell migration process [46,47]. Activation of FAK by phosphorylation of tyrosine-397, the major autophosphorylation site upon integrin ligation, is commonly thought to be involved in initial obliteration of focal adhesion following promotion of cancer cell migration [48]. It has been shown that association of FAK with the focal adhesion targeting region of the adaptor protein paxillin further regulates cell motility and survival [49]. Because the interaction of FAK and paxillin is one of the most intensely studied systems in integrin-regulated signaling cascades, the effects of glycosides 4 and 5 on these focal adhesion-associated proteins were investigated using western blot analysis (Fig. 7). The results show that treatment of MDA-MB-231 cells with glycosides 4 and 5 leads to dose-dependent decreases in the phospho-signals of FAK (pY397). The findings indicate that the isomalyngamide A glycosides reduce FAK-Y397 phosphorylation, which subsequently triggers the induction of downstream effects related to cell motility. However, expression levels of FAK are either unchanged or less affected when cells are treated with 4 or 5 for 24 h. Whether or not the level of paxillin that is associated with increased cancer cell adhesion to collagen is also altered by treatment with glycoside 4 or 5 (Fig. 7) was explored next. The findings show that the amount of paxillin decreases significantly in a dosedependent manner when the cells are treated with glycosides 4 or 5 for 24 h. This result is consistent with previous observations, which show that treatment of MDA-MB-231 cells with glycoside 4 or 5 induces inhibition of collagen type IV-mediated cell adhesion (Fig. 6b). Furthermore, the data arising from the western blot analyses also demonstrate that glycosides 4 and 5 do not effect expression levels of Rac1, a member of the Rho family of GTPases that is activated by integrin signaling (Fig. 7) [50]. Taken together, the results suggest that isomalyngamide A glycoside-induced antimigratory activities are perhaps associated with modulation of the pathway involving FAK-paxillin, a subset of integrin signaling molecules.

that isomalyngamide A monosaccharides 4 and 5 rather than the disaccharide derivative 6 are potent inhibitors of tumor metastasis that act by efficiently blocking cell migration, adhesion and invasive ability. Observations made in the investigation suggest that glycosylation of isomalyngamides improves biological activity through, perhaps, enhancement of target recognition, pharmacokinetic profile or their sensitivity toward certain cancer cell lines rather than solubility properties. Also, the current findings are pertinent to our previous studies, which have demonstrated that nonglycosylated isomalyngamide A analogs 2 and 3 activate synchronous suppression of integrin b1 (CD29) and p-FAK in MDA-MB-231 cells, indicating that these substances modulate the progression of integrin signaling. This action is supported by the current findings, which show that treatment of MDA-MB-231 cells with glycosylated isomalyngamide A analogs 4 and 5 induces a decrease of p-FAK and paxillin expression and subsequently results in inhibition of cell migration/adhesion and complete cessation of tumor cell invasion. Also, the results of the study described herein demonstrate how the impact of glycosylation upon 2 affects the ability of 4e6 to modulate biological properties involving motility, migration, invasive capacity and adhesive property. This effort should pave the way for the development of other glycosylated isomalyngamide A analogs as effective antimetastatic agents. 4. Experimental section 4.1. Materials All materials were obtained from commercial sources and used as purchased. 1H NMR and 13C NMR spectra were recorded with Bruker AMX400 or 500 MHz. Proton chemical shifts are reported in parts per million (ppm) relative to the singlet at 7.24 ppm for residual CHCl3 in deuteriochloroform or the quintet at 3.30 ppm for residual CHD2OD in the methanol-d4. Carbon chemical shifts are reported in parts per million (ppm) relative to the internal 13C signals in CDCl3 (77.0 ppm) and CD3OD-d4 (49.0 ppm). Mass spectra were obtained with a FAB JMS-700 double focusing mass spectrometer (JEOL, Tokyo, Japan). Analytical thin layer chromatography (TLC) was performed on silica gel 60 F254 (Merck). Flash column chromatography was performed with silica gel 60 (230e400 mesh) (Merck). 4.2. Synthesis and characterization of isomalyngamide A analogs 4e6 4.2.1. General procedure for the preparation of isomalyngamide A analogs 4e6 To independent solutions of 17 (0.260 mmol) in DMF (5 mL) was added HBTU (0.312 mmol) followed by DIPEA (0.520 mmol) slowly and the resulting solution was stirred for 5 min. The respective peracetylated aminoethoxyl glycosides 18, 19 or 20 (0.260 mmol) were added to these solutions and resulting mixtures were stirred at room temperature for 12 h, concentrated, dissolved in ethyl acetate (10 mL) and washed with 6% aq. HCl (3  10 mL). In each case, the combined organic layers were dried over MgSO4 and concentrated in vacuo giving a residue that was subjected to silica gel column chromatography (hexane/ethyl acetate, 90:10 volume) to afford the corresponding glycoside (4 (40%), 5 (40%) or 6 (40%)).

3. Conclusions The studies described above have led to an effective strategy for the development of new glycosylated isomalyngamide A analogs, which have potential medical applications as antimetastatic agents for the treatment of human breast cancer. The results demonstrate

4.2.1.1. (2R,3R,4S,5S,6S)-2-(Acetoxymethyl)-6-(2-(2,2-dimethyl-3oxo-3-((2-tetradecanamidoethyl)amino)propanamido)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (4). Yield 40%; 1H NMR (CDCl3, 400 MHz) 0.77 (t, J ¼ 7.0 Hz, 3H), 1.15 (br s, 20H), 1.34 (br s, 6H), 1.48e1.51 (m, 2H), 1.89 (s, 3H), 1.94 (s, 3H), 2.00 (s, 3H), 2.05 (s,

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3H), 2.07e2.09 (m, 2H), 3.28 (br s, 4H), 3.35e3.40 (m, 2H), 3.45e 3.50 (m, 1H), 3.65e3.70 (m, 1H), 3.89e3.92 (m, 1H), 3.99e4.03 (m, 1H), 4.15e4.20 (m, 1H), 4.74 (s, 1H), 5.14e5.19 (m, 3H), 6.58 (br s, 1H), 7.23e7.25 (m, 2H); 13C NMR (100 MHz) 13.90, 20.47, 20.51, 20.52, 20.65, 22.46, 23.75, 23.79, 25.56, 29.12, 29.14, 29.16, 29.30, 29.42, 31.69, 36.34, 38.94, 39.12, 40.60, 49.18, 62.24, 65.83, 66.56, 68.48, 68.98, 69.18, 97.35, 169.47, 169.86, 169.88, 170.51, 173.44, 174.60, 174.92; HRMS: calcd. for C37H63N3O13Na (M þ Na)þ 780.4259, found 780.4264. 4.2.1.2. (2R,3S,4S,5R,6R)-2-(Acetoxymethyl)-6-(2-(2,2-dimethyl-3oxo-3-((2-tetradecanamidoethyl)amino)propanamido)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (5). Yield 40%; 1H NMR (CDCl3, 500 MHz) 0.83 (t, J ¼ 7.0 Hz, 3H), 1.20 (br s, 20H), 1.40 (br s, 6H), 1.53e1.57 (m, 2H), 1.95 (s, 3H), 2.01 (s, 3H), 2.06 (s, 3H), 2.12 (s, 3H), 2.10e2.14 (m, 2H), 3.31e3.35 (m, 5H), 3.46e3.51 (m, 1H), 3.62e 3.66 (m, 1H), 3.84e3.90 (m, 2H), 4.07e4.15 (m, 2H), 4.47 (d, J ¼ 8.0 Hz, 1H), 4.99 (dd, J ¼ 3.5 Hz, 10.5 Hz, 1H), 5.14 (dd, J ¼ 8.0.Hz, 10.5 Hz, 1H), 5.35 (t, J ¼ 3.0 Hz, 1H), 6.24 (d, J ¼ 4.9 Hz, 1H), 6.94 (t, J ¼ 5.4 Hz, 1H), 7.28 (d, J ¼ 4.6 Hz, 1H); 13C NMR (125 MHz) 14.04, 20.48, 20.56, 20.59, 20.75, 22.62, 23.71, 24.08, 25.66, 29.29, 29.45, 29.58, 31.85, 36.60, 39.55, 39.57, 40.58, 49.31, 61.23, 66.99, 68.03, 68.80, 70.79, 70.82, 101.04, 169.63, 170.02, 170.11, 170.34, 173.77, 174.43, 174.67; HRMS: calcd. for C37H63N3O13Na (M þ Na)þ 780.4259, found 780.4260. 4.2.1.3. (2R,3S,4S,5R,6S)-2-(Acetoxymethyl)-6-(((2R,3R,4S,5R,6R)-4,5diacetoxy-2-(acetoxymethyl)-6-(2-(2,2-dimethyl-3-oxo-3-((2tetradecanamidoethyl)amino)propanamido)ethoxy)tetrahydro-2H-pyran-3-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (6). Yield 40%; 1 H NMR (CDCl3, 400 MHz) 0.82 (t, J ¼ 6.8 Hz, 3H), 1.19 (br s, 20H), 1.37 (s, 6H), 1.53e1.54 (m, 2H), 1.91 (s, 3H), 1.98e2.02 (m, 12H), 2.06e209 (m, 8H), 2.83 (s, 1H), 3.31e3.33 (m, 5H), 3.41e3.45 (m, 1H), 3.55e3.60 (m, 2H), 3.73e3.77 (m, 2H), 3.82e3.85 (m, 1H), 4.00e4.08 (m, 3H), 4.41e4.46 (m, 2H), 4.80e4.84 (m, 1H), 4.89e4.93 (m, 1H), 5.02e5.07 (m, 1H), 5.12 (t, J ¼ 9.2 Hz, 1H), 5.30 (d, J ¼ 3.2 Hz, 1H), 6.28 (br s, 1H), 6.50 (br s, 1H), 6.93e6.97 (m, 1H); 13C NMR (100 MHz) 14.02, 20.41, 20.53, 20.70, 22.58, 23.64, 23.99, 25.63, 29.25, 29.41, 29.54, 31.81, 36.50, 39.39, 39.54, 40.63, 49.23, 60.67, 61.83, 66.53, 68.28, 69.03, 70.60, 70.87, 71.50, 72.72, 76.07, 100.41, 100.99, 169.02, 169.67, 169.75, 169.96, 170.04, 170.25, 170.33, 173.58, 174.52, 174.69; HRMS: calcd. for C49H79N3O21Na (M þ Na)þ 1068.5104, found 1068.5111. 4.3. Biology Cell growth inhibition assays [51,52] and western blot analyses [53] were performed by using the modified procedures previously described. 4.3.1. Cell culture Human breast carcinoma MDA-MB-231 was purchased from Bioresource Collection and Research Center (BCRC 60425) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; HyClone) with 10% fetal bovine serum (FBS; GIBCO), 2 mM L-glutamine and antibiotics (containing 100 mg/L Streptomycin, 100 U/mL Penicillin G, and 0.25 mg/L Amphotericin B) at 37  C in a humidified atmosphere containing 5% CO2 in air. 4.3.2. Cell growth inhibition assay Cancer cells were seeded at a density of 1.5  104 per well in triplicates in 96-well culture dishes. After 24 h incubation, cells were treated with different concentrations of glycosylated isomalyngamide A analogs or DMSO as control for 48 h. After treating with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 1 mg/mL, Sigma) at 37  C for 4 h, cell viability was

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determined by measuring the spectrophotometric absorbance at 540 nm (Molecular Devices SPECTRAmax PLUS384). 4.3.3. Wound healing assay MDA-MB-231 cells were seeded in duplicates on 6-well plates in a monolayer of 100% confluence at 37  C for overnight and a scratch wound was applied with a sterile 100 mL plastic pipette tip. Debris was removed by washing and glycosylated isomalyngamide A analog was added in the presence of 10% serum. MDA-MB-231 cells were allowed to migrate into the scratch for 24 h and visualized using a microscope. Photographs were taken immediately at 0 h and 24 h at a 100 magnification. 4.3.4. Cell migration assay Boyden chamber assay was used to determine the migration ability of breast cancer cells. The 8 mm pore size polycarbon membrane (coated with 30 mg/well Matrigel) was used to allow cancer cells to migrate from the top chamber to the bottom chamber (Neuro Probe AP-48). A total of 2.5  105 cells/mL trypsinized in serum free medium containing various concentrations of glycosylated isomalyngamide A analogs were added into the upper chamber. The lower chamber was filled with 500 mL of appropriate medium with 5% FBS as chemoattractant. The chamber was incubated for overnight at 37  C and cells on the upper surface of the membrane were completely wiped out with a cotton swab. The cells that penetrated to the lower chamber were fixed with 100% methanol and stained with 10% Giemsa for nucleus. Photographs of three random fields were taken and the cells were counted to calculate the average number of transmigrated cells from three independent experiments. 4.3.5. Transwell invasion assay Matrigel invasion assay was performed using transwell chambers with a filter insert (8-mm pore; Falcon, BD Biosciences), precoated with Matrigel (BD Bioscience) in DMEM medium. Cells were seeded onto precoated transwell insert with serum-free medium containing at a density of 8  104 cells/mL and then treated with different doses of glycosylated isomalyngamide A analogs or DMSO for 24 h. Conditioned medium obtained from 5% fetal bovine serum in tissue culture medium was added into the lower wells as chemoattractant. Thereafter, invading cells were fixed and stained after 24 h. These experiments were carried out in triplicate. Three random views were photographed and the mean number of invasive cells was counted under the microscope. 4.3.6. Adhesion assay Fibronectin and Collagen IV, the ECM proteins, were diluted to 10 mg/mL in 1  phosphate buffered saline (PBS) with sterile water. The 96-well tissue culture plate was coated with 100 mL of each protein overnight at 4  C. Next, the wells were washed with 1  PBS for three times before the binding of cells to the matrix. Then, nonspecific cell adhesions were blocked by the addition of 0.1% bovine serum albumin (BSA) in 100 mL DMEM per well for 60 min. Cells were treated with different concentrations of glycosylated isomalyngamide A analogs for 48 h, harvested by trypsinization, and adjusted to 2  105 cells/mL in DMEM containing 0.1% BSA. Each well was added 100 mL cell suspension. The plate was incubated at 37  C in a humidified incubator with 5% CO2 atmosphere for 60 min. Adherent cells were fixed with 10% formaldehyde in PBS for 10 min and then stained with 0.05% crystal violet in deionized water for 10 min. The dye was extracted with 100 mL of 0.1% acetic acid and 50% ethanol solution was used to dissolve the crystal violet, which simultaneously quantified by measuring the optical absorbance at 595 nm on a spectrophotometer.

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4.3.7. Western blot analysis Samples containing equal amounts of protein (20 mg) were separated on a 10% SDS-PAGE. After electrophoresis, proteins were electroblotted onto a polyvinylidene fluoride transfer membrane (PVDF, Pall Corporation) in a transfer buffer. Immunoblotting was performed using antibodies against total FAK (BD Biosciences), phospho-FAK (Y397) (Cell Signaling), total paxillin (BD Biosciences), and Rac1 (Millipore). b-Actin (Chemicon) served as an internal control. Antibody reaction was visualized using enhanced western blot chemiluminescence reagent (PerkinElmer). 4.3.8. Statistical analysis Cell growth inhibition, migration, invasion and adhesion studies were performed in triplicate. The results were expressed as mean  standard deviation (mean  SD) and analyzed with t-test. A probability, P, of less than 0.05 is considered significant in this study. Acknowledgments We are grateful for financial support of this work provided by the Academia Sinica and the National Science Council. Instrumentation support was provided by the NMR and Mass Spectrometry facilities of the Institute of Chemistry at Academia Sinica, Taiwan. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.ejmech.2013. 03.044. References [1] A. Jemal, R. Siegel, E. Ward, Y. Hao, J. Xu, M.J. Thun, CA Cancer J. Clin. 59 (2009) 225e249. [2] M. Gerlinger, A.J. Rowan, S. Horswell, J. Larkin, D. Endesfelder, E. Gronroos, P. Martinez, N. Matthews, A. Stewart, P. Tarpey, I. Varela, B. Phillimore, S. Begum, N.Q. McDonald, A. Butler, D. Jones, K. Raine, C. Latimer, C.R. Santos, M. Nohadani, A.C. Eklund, B. Spencer-Dene, G. Clark, L. Pickering, G. Stamp, M. Gore, Z. Szallasi, J. Downward, P.A. Futreal, C. Swantonm, N. Engl. J. Med. 366 (2012) 883e892. [3] S. Casimiro, I. Luis, A. Fernandes, R. Pires, A. Pinto, A.G. Gouveia, A.F. Francisco, J. Portela, L. Correia, L. Costa, Clin. Exp. Metastasis 29 (2012) 155e164. [4] Z. Shaikhibrahim, A. Lindstrot, B. Langer, R. Buettner, N. Wernert, Int. J. Mol. Med. 27 (2011) 811e819. [5] A.J. Minn, G.P. Gupta, P.M. Siegel, P.D. Bos, W. Shu, D.D. Giri, A. Viale, A.B. Olshen, W.L. Gerald, J. Massagué, Nature 436 (2005) 518e524. [6] I. Malanchi, A. Santamaria-Martínez, E. Susanto, H. Peng, H.A. Lehr, J.F. Delaloye, J. Huelsken, Nature 481 (2011) 85e89. [7] G.P. Gupta, D.X. Nguyen, A.C. Chiang, P.D. Bos, J.Y. Kim, C. Nadal, R.R. Gomis, K. Manova-Todorova, J. Massagué, Nature 446 (2007) 765e770. [8] P.S. Steeg, Nat. Med. 12 (2006) 895e904. [9] P.S. Steeg, D. Theodorescu, Nat. Clin. Pract. Oncol. 5 (2008) 206e219. [10] R. Gupte, R. Patil, J. Liu, Y. Wang, S.C. Lee, Y. Fujiwara, J. Fells, A.L. Bolen, K. Emmons-Thompson, C.R. Yates, A. Siddam, N. Panupinthu, T.C. Pham, D.L. Baker, A.L. Parrill, G.B. Mills, G. Tigyi, D.D. Miller, ChemMedChem 6 (2011) 922e935. [11] L. Chen, S. Yang, J. Jakoncic, J.J. Zhang, X.Y. Huang, Nature 464 (2010) 1062e1066. [12] C.J. Thibodeaux, C.E. Melançon, H.W. Liu, Nature 446 (2007) 1008e1016. [13] A. Ahmed, N.R. Peters, M.K. Fitzgerald, J.A. Watson Jr., F.M. Hoffmann, J.S. Thorson, J. Am. Chem. Soc. 128 (2006) 14224e14225.

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