Usnic Acid Benzylidene Analogues as Potent ... - ACS Publications

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Usnic Acid Benzylidene Analogues as Potent Mechanistic Target of Rapamycin Inhibitors for the Control of Breast Malignancies Hassan Y. Ebrahim, Mohamed R. Akl, Heba E. Elsayed, Ronald A. Hill, and Khalid A. El Sayed* Department of Basic Pharmaceutical Sciences, School of Pharmacy, University of Louisiana at Monroe, Monroe, Louisiana 71209, United States S Supporting Information *

ABSTRACT: (+)-Usnic acid (1) is a common bioactive lichen-derived secondary metabolite with a characteristic dibenzofuran scaffold. It displayed low micromolar antiproliferative activity levels and, notably, induced autophagy in a panel of diverse breast cancer cell lines, suggesting the mechanistic (formerly “mammalian”) target of rapamycin (mTOR) as a potential macromolecular target. The cellular autophagic markers were significantly upregulated due to the inhibition of mTOR downstream effectors. Additionally, 1 showed an optimal binding pose at the mTOR kinase pocket aided by multiple interactions to critical amino acids. Rationally designed benzylidene analogues of 1 displayed excellent fitting into a targeted deep hydrophobic pocket at the core of the kinase cleft, through stacking with the phenolic side chain of the Tyr2225 residue. Several potent analogues were generated, including 52, that exhibited potent (nM concentrations) antiproliferative, antimigratory, and anti-invasive activities against cells from multiple breast cancer clonal lines, without affecting the nontumorigenic MCF-10A mammary epithelial cells. Analogue 52 also exhibited potent mTOR inhibition and autophagy induction. Furthermore, 52 showed potent in vivo antitumor activity in two athymic nude mice breast cancer xenograft models. Collectively, usnic acid and analogues are potential lead mTOR inhibitors appropriate for future use to control breast malignancies.

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inhibitors, including INK128, AZD8055, and AZD2014, have been developed and progressed to various phases of clinical trials.9 Autophagy is a lysosomal-mediated degradation and recycling process of proteins, organelles, and other cellular components. It involves the formation of double-membrane vesicles (autophagosomes) that engulf proteins and organelles and then fuse with lysosomes to form autolysosomes, where hydrolases degrade the cargo of the vacuoles to their small-molecule constituents.10 Autophagy machinery is tightly controlled by intricate signaling networks, most of which connect with the mTOR pathway.11 In the past two decades, numerous studies indicated a strong association of autophagy with cancer development, progression, and therapy. Yet, the existence of any clear roles played by autophagy in conjunction with cancer initiation or progression remains controversial.12 Some studies suggested autophagy as a protective mechanism exploited by cancerous cells and, as such, can be considered a potential chemoresistance mechanism.13 Other researchers have adopted an opposing view, hypothesizing autophagy as obligate for antitumor effects to be exerted by some chemotherapeutics.14 Extreme autophagic degradation of cellular components will ultimately inhibit cancerous cell growth and ultimately induce apoptosis.

he mechanistic target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine protein kinase that belongs to the family of phosphatidylinositol 3-kinase-related kinases (PIKKs). In mammals, including humans, cellular mTOR exists in two distinct multiprotein complexes, mTORC1 and mTORC2.1 mTORC1 integrates diverse signals from the extracellular environment with intracellular metabolic changes to coordinate regulation of cell growth, proliferation, survival, and metabolism.2 In particular, mTORC1 regulation of protein translation is its best-characterized action in relation to oncogenesis. These regulatory effects are mediated by phosphorylation (activation) of two well-known downstream effectors: the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and the p70 ribosomal S6 kinase (S6K).3 The other mTOR complex, mTORC2, promotes cell motility and invasion through stimulation of cytoskeleton F-actin stress fibers, Rac1, and paxillin.4 In addition, mTORC2 phosphorylates Akt/ PKB at Ser473, a required step for Akt/PKB full activation, and hence regulates cell survival, apoptosis, and metabolism.5 Rapamycin, a microbial-derived macrolide and a prototype mTOR inhibitor, has been extensively characterized as an mTORC1 allosteric inhibitor. Rapalogs are rapamycin analogues with improved pharmacokinetic profiles, approved by the FDA for treatment of multiple malignancies.6,7 However, the feedback activation of Akt/PKB limited the clinical use of rapalogs as monotherapy and triggered the need to develop additional ATP-competitive inhibitors (second-generation mTOR inhibitors) that target the kinase domain of both mTORC1 and mTORC2. 8 Numerous small-molecule © 2017 American Chemical Society and American Society of Pharmacognosy

Received: October 7, 2016 Published: February 28, 2017 932

DOI: 10.1021/acs.jnatprod.6b00917 J. Nat. Prod. 2017, 80, 932−952

Journal of Natural Products

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Figure 1. Effect of usnic acid (1) on proliferation of multiple human breast cancer cell lines. (A) Mean percent proliferation for each cell line at different concentrations of 1 after 72 h incubation. (B) Bar graphs showing mean IC50 ± SEM of 1 against each breast cancer cell line.

Breast cancer remains the most common malignancy diagnosed among the female population and the second leading cause of cancer-related deaths in women.15 Numerous therapeutic approaches have been designed to rationally interfere with different oncogenic signaling pathways. Estrogen receptor modulators, tyrosine kinase inhibitors, and HER2 monoclonal antibodies are the most accepted targeted therapies for breast cancers. However, these drugs quickly develop acquired resistance and cause significant side effects. Therefore, continuous discovery of novel therapeutic entities is a dire need. The aberrant activation of the oncogenic mTOR signaling axis appears to be central for breast cancer growth, survival, motility, as well as constituting a mechanism of resistance to many chemotherapeutics.16 Consequently, interference with mTOR oncogenic functions by smallmolecule inhibitors is a valid strategy for the control of breast malignancies, either as monotherapy or in combination with chemotherapeutics to potentiate and reverse acquired resistance to their effects.16 (+)-Usnic acid (1) is a lichen-derived secondary metabolite with a unique dibenzofuran skeleton and is commonly found in lichenized fungi of the genera Usnea, Ramalina, and Cladonia. Usnic acid has been incorporated for years in cosmetics, perfumery, and traditional medicines.17,18 It has a wide range of bioactivities including antimicrobial, antiviral, anti-inflammatory, and analgesic.19 The sodium salt of usnic acid has been marketed in the United States as an active component of a dietary supplement for weight control.19 This formula was later withdrawn due to acute liver toxicities reported with chronic consumption. Consequently, numerous studies were carried out and published describing 1-induced hepatotoxicity in animal models, especially at relatively higher doses.19,20 Moreover, metabolomic characterization of 1-induced liver injury in rats was reported for doses of 100, 200, and 240 mg/kg.20 Regardless of this toxicity, much attention has been devoted to usnic acid as an anticancer lead during the past 10 years. In 2013, a study showed the moderate antiproliferative and apoptotic effects of usnic acid against the

lung cancer A549 cell line through the induction of mitochondrial membrane depolarization.21 Recently, the in vitro and in vivo growth-inhibitory effects of 1 against the estrogendependent human breast cancer MCF-7 cells in a mouse model were reported.22 The mechanism of activity of 1 in this study was proposed to be exerted via the generation of reactive oxygen species.22 The promising and diverse biological activities of 1 encouraged multiple semisynthesis modification studies to enhance its bioactivities. Usnic acid−polyamine conjugates displayed enhanced cytotoxicity against multiple murine and human cancer cell lines.23 Moreover, the enamine, pyrazole, and chalcone analogues of usnic acid have been reported with significant antiviral activity against influenza A virus.24 The current study describes the discovery of usnic acid as a novel mTOR inhibitor with activities against a panel of human breast cancer cell lines. A rational semisynthesis extension tactic, accomplished through C−C coupling, was adopted to improve usnic acid interactions with the mTOR kinase to enhance its cellular and in vivo potency.

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RESULTS AND DISCUSSION

Usnic Acid Effects on the Growth and Viability of Human Breast Cancer Cells. The lichen-derived (+)-usnic acid (1) was screened against a panel of human breast cancer cell lines in an MTT proliferation assay. The panel comprises an array of cell lines with diverse molecular and phenotypic characteristics. For instance, MDA-MB-231 and MDA-MB-468 cells are triple-negative for receptors of estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER2), while overexpressing the proto-oncogene receptor tyrosine kinase c-Met. BT-474 and SK-BR-3 are HER2-overepressing cells, while MCF-7 and T-47D are ER-dependent cell lines. Thus, selective potency of 1 toward a particular cell line should help in identifying the potential macromolecular target(s). Compound 1 exhibited a moderate, albeit panantiproliferative activity against all tested breast cancer cell lines at low micromolar concentrations (Figure 1). Therefore, 1 was 933



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hypothesized to target (a) common pathway(s) involved in the growth, proliferation, and survival of multiple breast cancer cells. Usnic Acid Autophagy Induction in Human Breast Cancer Cell Lines. Microscopic examination of the breast cancer cells treated with 1 revealed the presence of distinct autophagic vacuoles (Figure 2A). It is well established that formation of autophagosomes depends on the activity of class-III phosphatidylinositol 3-kinase (PI3K) as a vacuolar sorting protein to induce formation of a complex with Beclin-1 (Atg6),25 whereas the elongation stage requires cleavage of the microtubule-associated protein 1 light chain-3 (Atg8/LC3) by Atg4 protease, resulting in the formation of cytosolic LC3-I protein, which in turn conjugates with phosphatidylethanolamine to form membrane-bound LC3-II.25 Thus, the levels of these characteristic autophagic markers were assessed by Western blot analysis of lysates from 1-treated MCF-7 and MDA-MB-231 cells. Immunoblots (Figure 2B) revealed a significant dose-dependent upregulation of the autophagic marker LC3, which is required for the formation of autophagosomes. Moreover, the important regulator of autophagic protein localization to the preautophagosomal structure (PAS), Beclin-1, showed a significant dose-dependent elevation upon treatment with various doses of 1. These results supported the preliminary observation of autophagy and directed the study focus to the master regulator of protein synthesis and autophagy, namely, the mechanistic target of rapamycin. Usnic Acid Docking Results at the mTOR Kinase Domain. To validate the hypothesis that 1 targets mTOR, it was docked at the ATP-binding pocket of an mTOR crystal structure to explore docking poses and binding interactions. The dibenzofuran scaffold binds at the adenine site, as do most of the small-molecule kinase inhibitors, and interacts with the kinase hinge through a hydrogen bonding of its C-12 carbonyl oxygen with the Val2240 backbone amide hydrogen (Figure 3). In addition, a characteristic edge-to-face (T-shaped) π−π stacking of the dibenzofuran scaffold of 1 with the hinge Trp2239 side-chain indole moiety was observed. Interestingly, previous studies attributed the nanomolar-range potency of selective mTOR inhibitors, such as Torin2, to the π−π stacking with the hinge Trp2239.26 In addition, 1 was anchored to the kinase N-lobe through a hydrogen bonding of its C-3 enolate oxygen and C-10 carbonyl oxygen with the side-chain polar hydrogens of Lys2187 and Ser2165, respectively. Collectively, these virtual results suggested the mTOR kinase domain as a potential molecular target of 1, which required further cellular level validation. Oncogenic mTOR Axis as a Valid Molecular Target for Usnic Acid in Breast Cancer Cells. Breast cancer MCF-7 and MDA-MB-231 cells were the most sensitive to treatment with 1 in an MTT proliferation assay (Figure 1B). Interestingly, these two cell lines are best known for their dysregulated mTOR signaling pathway, through PI3K catalytic subunit activating mutation (MCF-7 cells) or overexpression of upstream oncogenic receptor tyrosine kinase c-Met (MDA-MB-231 cells).27,28 Lysates pooled from MCF-7 and MDA-MB-231 cells treated with different concentrations of 1 were analyzed by Western blot for evaluating mTOR-linked critical biomarkers. Immunoblots (Figure 4) revealed a significant phosphorylation inhibition of the principle mTOR downstream effectors: Compound 1 induced a significant reduction of p-S6K, which regulates the phosphorylation of the critical component of the translation machinery ribosomal protein S6. The phosphorylated level of the key translation repressor, eukaryotic

Figure 2. Autophagy induction by usnic acid (1) in multiple breast cancer cell lines. (A) Microscopic photographs of MDA-MB-231, MCF-7, SK-BR-3, T-47D, MDA-MB-468, and BT-474 cells treated with 1 at 15 μM showing characteristic autophagic vacuoles compared to control cells. (B) Western blot analysis of main cellular autophagic markers in the vehicle-treated control and MCF-7 and MDA-MB-231 cells treated with 1. Data show a concentration−response upregulation of Beclin-1 and LC3 in cells treated with 1 compared to control. 934



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translation initiation factor 4E-binding protein 1 (4E-BP1), was significantly reduced in 1-treated cells as compared to untreated cells. The level of p-Ser473 Akt/PKB, a common regulatory site for the full function of Akt/PKB that is known to be regulated by catalytic activity of mTORC2, was assessed. The Ser473 phosphorylation of Akt/PKB was significantly suppressed in 1-treated cells, indicative of mTORC2 inhibition (Figure 4). Ultimately, this inhibition mechanism should offer the advantage of mitigating the compensatory Akt/PKB activation associated with the mTORC1 inhibitors, rapamycin and rapalogs. Collectively, these results strongly support the ability of 1 to inhibit mTOR catalytic activity and thereby interfere with mTORC1/2 cellular functions. Usnic Acid as Breast Cancer Migration and Invasion Inhibitor. Numerous proteins and signaling cascades have been implicated in cancer cell motility and invasion, including the PI3K/mTOR signaling pathway.4 The wound healing scratch assay was implemented to assess the antimigratory effect of 1 against metastatic human MDA-MB-231 breast cancer cells. Treatment with 1 resulted in a dose-dependent inhibition of cell migration and wound closure after 23 h of incubation (Figure 5), with a calculated IC50 value of 13.7 μM. The anti-invasive activity of 1 against MDA-MB-231 cells was also assessed using the CultreCoat cell invasion platform. Cells treated with 1 displayed a significantly lower invasion capability, indicated by higher cell density at the upper surface of invasion chambers, compared to control wells, which showed lower cell density and thus greater cell invasion (Figure 6). The IC50 value of 1 in this assay was 14.2 μM. Therefore, 1 proved to be a proliferation, migration, and invasion inhibitor of the triplenegative and metastatic breast cancer MDA-MB-231 cells; however, the potency level of bioactivity still needed improvement. Rational Design of Usnic Acid Semisynthesis Analogues. The surface representation of the usnic acid binding

Figure 4. Western blot analysis of the usnic acid (1) effects on the mTOR downstream effectors in MFC-7 and MDA-MB-231 breast cancer cells. Concentration-dependent downregulation of the mTOR downstream effectors p-4EBP1, p-S6K, and p-Akt in cells treated with 1 was observed versus vehicle-treated control.

mode in the mTOR kinase pocket suggested the molecular basis for the structural extension toward a deep pocket at the core of the kinase cleft (Figure 7A). The chemical environment of the targeted pocket was examined to prioritize the chemical nature of the proposed structural extensions. The pocket comprises the hydrophobic amino acids Leu2185, Tyr2225, Ile2237, Ile2356, and Phe2358. In particular, the nearby Tyr2225 residue with its phenolic side chain was observed to be in close proximity of the usnic acid C-6 acetyl group (Figure 7B). Therefore, molecular extension of the parent scaffold with a phenyl moiety that is capable of π−π stacking with the targeted Tyr2225 phenolic side chain would be

Figure 3. Binding mode and key interactions of usnic acid (1) at the mTOR kinase domain. 1 binds to the kinase hinge through a hydrogen bonding of its C-12 carbonyl oxygen to the backbone amide hydrogen of Val2240 and T-shaped π−π stacking of its dibenzofuran moiety to the side-chain indole of Trp2239. Compound 1 anchors to the N-lobe of the kinase domain through hydrogen bonding of its C-3 enolate oxygen and C-10 carbonyl oxygen to side-chain moieties of Lys2187 and Ser2165, respectively. 935



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Figure 5. Antimigratory activity of usnic acid (1) against MDA-MB-231 breast cancer cells using the wound healing assay. (A) Representative microscopic photographs of wounds at zero time and 23 h postincubation with either vehicle or 15 μM with usnic acid. (B) Mean (±SEM) percent cell migration at different concentrations of 1 after 23 h incubation. The lichen-derived norstictic acid was used as a positive control antimigratory compound.38 The symbol * indicates a statistical significance at P < 0.05.

phenolic side chain (Figure 7C). To further validate the rational design based on the molecular extension of usnic acid, 2 was evaluated against breast cancer cells in an MTT proliferation assay. MCF-7 and MDA-MB-231 cells were chosen to monitor the biological activity of new analogues, since they in each case harbor a dysregulated mTOR signaling pathway (vide supra). Analogue 2 exerted antiproliferative activity more than 3-fold potency of 1 against both tested cell lines (Table 1). These results strongly supported the rational design hypothesis and motivated further optimization of additional substituted benzylidene moieties. The side-product 3 was identified as a tetracyclic epimeric mixture of a cyclized intermediate obtained during the preparation of 2 (Scheme 1). This analogue offered the opportunity to compare the effect of coplanarity of the extended conjugated benzylidene system in 2 versus the nonplanar structure of 3. Interestingly, the epimeric mixture 3 did not show the same antiproliferative activity improvement in cellular assays when compared to 2 (Table 1), which offers evidence supporting the importance of the coplanarity of the conjugated enone of 2 for properly and optimally orienting the benzylidene moiety for better stacking with the Tyr2225 phenolic side chain of mTOR. Moreover, docking of the two possible C-1′ epimers of 3, in which the phenyl moiety is either α- or β-oriented, into the mTOR kinase domain suggested that both isomers enabled orientation of their benzylidene moieties for T-shaped stacking with the Tyr2225 phenolic side chain in similar fashion to 2, but with the dibenzofuran core shifted away from the hinge region. This resulted in the loss (in silico, at least) of the critical stacking with the hinge Trp2239 indole moiety, which may well account for the observed significant activity reduction for 3. In addition, both hydrogen bonds mediating the anchorage of analogue 2 to the

expected to improve target interactions and enhance the overall cellular potency. Claisen−Schmidt condensation chemistry was implemented to carbon−carbon couple the C-6 acetyl group with diverse aromatic aldehydes, providing for the planned molecular extension (Scheme 1). Previous structural modifications of 1 suggested the reactivity preference of the C-2 acetyl group.24 The exact condensation site, at either the C-2 or C-6 acetyl moiety, was ascertained by 1D and 2D NMR spectroscopy. The chemical shifts of individual proton and carbon atoms of the parent 1 were assigned based on NMR data (Figure S1, Supporting Information) and comparing with literature.29 The 3J HMBC cross-peaks of the aromatic proton H-4 singlet (δ 5.59) and the 11-methyl singlet (δ 2.64) with C-2 (δ 105.3) confirmed their identity in 1. Similarly, 3J HMBC cross-peak correlation of the 13-methyl singlet (δ 2.65) with the quaternary C-6 (δ 101.6) permitted their unambiguous assignment in 1. The 13C NMR chemical shifts of the C-2 acetyl group (C-10 and C-11) of 2 were identical to those of the parent 1. The 2J and 3J HMBC correlations of the H-13 and H-1′ doublets, respectively, with the C-12 carbonyl confirmed this assignment. The C-12 carbonyl carbon was also upfield-shifted by 10.2 ppm, due to the extended conjugation with the newly introduced benzylidene moiety, thus confirming coupling of the aldehyde at the 13-methyl group (Figure S54, Supporting Information). This first proposed semisynthesis analogue 2, the expected coupling product of 1 with benzaldehyde, was docked into the mTOR kinase cleft. The binding mode suggested a molecular superposition of the dibenzofuran scaffold of 2 with that of the parent 1, in addition to an edge-to-face stacking (T-shaped stacking) of the benzylidene moiety with the targeted Tyr2225 936



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Figure 6. Effect of usnic acid (1) on MDA-MB-231 cell invasion. (A) Microscopic photographs of upper surface of invasion wells after 24 h of incubation with DMSO or 10 μM treatment of 1. The lower cell density indicates a higher cell invasion, and the higher cell density indicates a lower cell invasion. (B) Mean (±SEM) percent cell invasion at different concentrations of 1 after 24 h incubation. Norstictic acid was used as a positive control anti-invasive compound.38 The symbol * indicates a statistical significance at P < 0.05.

N-lobe Val2240 and Lys2187 residues were lost, providing possible sourcing for activity reduction, at least in part, from unrecovered desolvation penalty (Figure S55, Supporting Information). Optimizations then aimed at exploring the biological space of various benzylidene moiety substituents having different electronic and hydrophobic/hydrophilic properties. Initially, the effects of nature, number, and position of halogen atoms on the benzylidene moiety were explored. Thus, analogues 4−19 (Table 2) were considered, synthesized, purified, and tested in an MTT proliferation assay. All halogenated analogues were relatively more potent than the nonsubstituted 2, however with variable cellular potencies. The p-chlorobenzylidene analogue 4 was more potent than its m-chlorinated counterpart 5, per their IC50 values against MCF-7 and MDA-MB-231 cells (Table 1). The o,p-dichlorinated analogue 7 was relatively more active than the o,o-dichlorinated analogue 9, which was in turn more potent than m,m-dichlorobenzylidene analogue 11 (Table 1). This clearly indicated the activity preference for the paraposition of chloro substituents on the benzylidene moiety and the superiority of the dichlorinated over the monochlorinated analogues. The intermediate aldol 12 was obtained as an epimeric mixture, rather than the tetracyclic epimeric mixture, when m,m-dichlorobenzaldehyde was the reactant. All side-products

(6, 8, 10, and 12) were relatively less potent than their benzylidene counterparts (Table 1). This strongly emphasized the importance of benzylidene and dibenzofuran moieties in maintaining proper mTOR interactions and cellular potency. Similarly, different fluorinated benzylidene analogues 13−18 were prepared and evaluated in proliferation assays. The difluorinated benzylidene analogues, 17 (o,o-difluoro) and 18 (m,m-difluoro), were relatively more potent than the monofluorinated analogues: 13 (p-fluoro), 15 (o-fluoro), and 16 (m-fluoro). However, no differences in cellular potencies were observed regarding the positioning of the fluorine atom among both mono- and difluoro analogues (Table 1). The fluorinated analogues were relatively less potent than their chlorinated counterparts. The electron-withdrawing property of the substituent should play a role in enhancing the cellular potency based on the proposed ligand−target interaction; however, increased hydrophobicity can also be expected to impart better bioactivity in this situation, given that cell membrane penetration is necessary to reach the targeted cytoplasmic-domain kinase. Hydrophobicity may account for the greater activity of chlorinated analogues, which, as noted above, were generally more potent than their fluorinated analogues. This conclusion is further supported by the enhanced bioactivity displayed by the 937



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Figure 7. continued

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Figure 7. Rational design of usnic acid (1) semisynthesized analogues. (A) Surface representation of usnic acid (1) binding mode at the mTOR kinase domain disclosing the nearby pocket for prospective molecular extension indicated by the arrow. (B) Binding mode of 1 into the mTOR kinase domain showing the nearby Tyr2225 phenolic side chain targeted for the molecular extension. (C) Binding pose and overlay of 1 and its benzylidene analogue 2 at the mTOR kinase domain revealing the superposition of the main dibenzofuran scaffold and T-shaped stacking of the benzylidene moiety with the phenolic side chain of Tyr2225. (D) Binding mode of the most active analogue 52 at the mTOR kinase domain. The extended benzylidene moiety is oriented for stacking with the Tyr2225 phenolic side chain in the targeted pocket, and the p-OCF3 substituent forms hydrophobic interactions with side chains of Val2227 and Ile2237, at a small hydrophobic subpocket.

Scheme 1. General Synthesis Route Towards Analogues 2−53

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Table 1. Antiproliferative Activities of (+)-Usnic Acid (1) and Semisynthesized Analogues 2−53 against MCF-7 and MDA-MB231 Breast Cancer Cell Lines breast cancer cell line (IC50 ± SEM, μM) compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

MCF-7 11.20 4.50 7.26 2.10 2.70 6.32 0.93 3.64 1.53 4.92 2.44 5.81 2.98 6.22 3.74 3.63 1.96 1.82 1.13 9.38 12.82 1.33 4.21 0.84 3.43 0.97 3.73

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.25 0.42 1.02 0.36 0.43 0.63 0.19 0.43 0.25 0.63 0.28 0.33 0.29 0.45 0.41 0.44 0.30 0.30 0.20 1.04 0.62 0.24 0.23 0.16 0.34 0.18 0.37

MDA-MB-231

compound

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53

13.10 5.10 8.04 2.63 3.92 6.41 1.36 4.01 5.69 4.31 2.95 4.44 3.31 6.89 3.54 4.79 2.38 2.09 1.53 10.57 14.41 1.65 5.99 1.03 3.65 0.96 2.94

0.76 0.80 078 0.34 0.49 0.91 0.28 0.63 0.62 0.63 0.45 0.41 0.49 0.36 0.43 0.67 0.59 0.43 0.29 1.18 0.92 0.32 0.64 0.17 0.49 0.16 0.43

p-bromobenzylidene analogue 19 (Table 1). The finding that electron-withdrawing substituents with hydrophobic character potentiate the activity was further corroborated by the dramatic activity decrease observed for the p-carboxylate analogue 20, having a hydrophilic and extensively ionizable (at physiological pH) electron-withdrawing group, as well as for its tetracyclic side-product analogue 21 (Table 1). This was furthermore in accord with the unfavorable impact of polar para-position benzylidene moiety substituents on the activity. The effect of electron-donating substituents was explored by preparing and evaluating the biological activity of benzylidene analogues 22 (p-methyl) and 24 (p-tert-butyl) in proliferation assays. Both analogues exhibited significant improvement of the antiproliferative activities, when compared to the parent 2 (Table 1), indicating the tolerability of the hydrophobic electron-donating groups. Moreover, 24 was more potent than 22, supporting the earlier conclusion that increasing the hydrophobicity of benzylidene moiety substituents would enhance antiproliferative activity. However, analogues with longer aliphatic chains were not considered for further synthesis and testing due to the expected metabolic considerations as discussed later. A number of alkyloxy- and alkylthiobenzylidene analogues (26−36) were thence considered, synthesized, and tested. The p-ethoxybenzylidene analogue 30 was more potent than its p-methoxy counterpart 26. This potentially indicated that increasing the length of the p-alkyloxy side chain would, in general, improve the antiproliferative activity. Accordingly, the p-isopentyloxy analogue 33 was the most potent alkyloxy-substituted analogue, indicating that long and branched substituents would impart better bioactivity. In contrast, the

MCF-7 1.22 0.93 0.71 3.36 2.18 5.92 0.53 2.91 0.39 0.72 0.54 0.74 0.52 1.14 6.53 0.75 4.32 0.87 3.09 1.23 0.73 4.77 0.91 5.24 0.28 1.51

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.13 0.20 0.13 0.41 0.28 0.57 0.09 0.24 0.05 0.09 0.06 0.06 0.09 0.16 0.46 0.11 0.42 0.11 0.32 0.23 0.13 0.63 0.14 0.52 0.04 0.17

MDA-MB-231 1.63 1.15 0.84 3.68 2.03 6.13 0.62 2.23 0.58 0.89 0.73 0.72 0.68 0.95 5.71 0.91 5.58 0.93 4.42 1.16 0.71 3.93 0.72 1.56 0.32 1.79

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.25 0.49 0.16 0.46 0.38 0.78 0.07 0.28 0.07 0.13 0.13 0.11 0.11 0.17 0.79 0.18 0.60 0.17 0.50 0.26 0.16 0.50 0.10 0.17 0.03 0.26

corresponding o-ethoxy analogue 32 exhibited a significantly lower antiproliferative activity (Table 1). This further substantiated the hypothesis of preferential para over ortho positioning of the substituents attached to the benzylidene moiety, regardless of their electronic nature. The alkylthiobenzylidene analogues 37 and 38 were in each case more potent than their alkyloxy counterparts 26 and 30. The enhanced activity of the thio-analogues could be attributed, at last in part, to the greater hydrophobicity imparted by the sulfur atom as compared with the oxygen atom. However, further alkylthio or alkyloxy analogues were not considered for subsequent synthesis and evaluation, due to the plausible in vivo toxicity and metabolic considerations as discussed later. Benzylidene moiety extension with another phenyl group was considered, in order to assess whether or not the pocket would accommodate another aromatic ring, a possibility suggested by our in silico studies. Thus, the p-phenylbenzylidene analogue 39 was synthesized and tested. It exhibited more potent antiproliferative activities than the parent 2 (Table 1). The p-fluorophenylbenzylidene analogue 40 was then considered, synthesized, and tested. Gratifyingly, 40 showed enhanced potencies versus 39 (Table 1). Additional extended structures, with p-benzyloxybenzylidene moieties, including 41−46, were synthesized. Unexpectedly, all of them were relatively less potent than the p-phenylbenzylidenes 39 and 40 (Table 1). The reduced potencies may be attributed, in part, to the entropic penalty resulting from free rotation of the introduced methyleneoxy linker.30 Multiobjective optimization plays a significant role in successful early stage drug creation pipelines. Frequently, biological 940



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Table 2. Chemical Structures of (+)-Usnic Acid (1) and Semisynthesized Analogues 2−53

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Table 2. continued

potency is the only driving force during the in vitro hit-to-lead optimization, whereas if expected in vivo toxicities or metabolic instabilities of proposed analogues to be synthesized are concurrently taken into account, later attrition during preclinical evolution can reasonably be expected to decrease. Thus, such considerations were contemplated during the semisynthesis optimization of the benzylidene analogues of 1, which led to

avoiding synthesizing some analogues in particular directions (vide supra), hence saving time, effort, and resources. For instance, the in vitro results indicated enhanced potency of the p-alkylbenzylidene analogues 22 and 24; however, further analogues with longer aliphatic chains were not considered for subsequent synthesis. These analogues would reasonably be predicted to undergo facile ω or ω-1 hydroxylation in vivo, 942



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Figure 8. Antiproliferative effect of analogue 52 against multiple human breast cancer cell lines. (A) Mean percent proliferation (±SEM) for each cell line treated with different concentrations of 52 after 72 h of incubation. (B) Bar graphs showing the IC50 ± SEM of 52 against the tested breast cancer cell lines.

potency against cells of both cancer lines, when comparing to the mono-CF3 counterpart 47 (m-CF3-benzylidene), but less enhancement than analogue 48 (p-CF3-benzylidene). It had been observed earlier that the p-OCH3-benzylidene analogue 26 showed enhanced activities in cell-based assays, but this compound would be predicted to undergo rapid in vivo metabolic deactivation through CYP-catalyzed O-demethylation. Therefore, functional hybridization between OCH3 and CF3 groups was considered, and 52 (p-OCF3-benzylidene analogue) was thus considered and synthesized. The p-OCF3 substituent was hypothesized to be a hydrophobic group beneficial to activity according to the accrued SAR at this stage and to also be metabolically stable (i.e., not liable to O-dealkylation). When tested, 52 exhibited the most potent antiproliferative activity among all synthesized analogues against cells of the mTOR dysregulated MCF-7 and MDAMB-231 cell lines (Table 1). Moreover, 52 inhibited the proliferation of other breast cancer cell lines at nanomolar concentrations. A docking study was launched to better understand the hypothesized binding mode of 52 at the mTOR kinase pocket. When bound in the energetically most favorable pose, the dibenzofuran moiety of analogue 52 was positioned (Figure 7D) almost identical to 1 (Figure 7B), maintaining the critical stacking with Trp2239 at the mTOR kinase pocket hinge. The extended benzylidene moiety was oriented for efficient stacking with the Tyr2225 phenolic side chain at the targeted hydrophobic pocket, and the p-OCF3 was oriented toward a small hydrophobic subpocket created by the side chains of Val2227 and Ile2237 (Figure 7D). Moreover, an additional hydrogenbonding interaction of the C-7 phenolic hydrogen with the C-lobe Cys2243 backbone amide carbonyl was observed. Collectively, these critical interactions correlate with the improved mTOR inhibitory activity of 52, which may account for its nanomolar-level potency against cells of all tested breast cancer lines (Figure 8).

rapidly producing polar metabolites having unfavorably reduced antiproliferative activity (i.e., unfavorable pharmacokinetic profiles). Moreover, although the p-alkyloxybenzylidene analogues exhibited a remarkable activity improvement, such analogues are expected to be efficiently biotransformed, through CYP450-mediated O-dealkylation, to their corresponding phenols, which will create polar phenolic groups with generally expected lesser activity and significant potential for subsequent production of toxicologically deleterious electrophilic metabolites. Additionally, the enhanced bioactivity displayed by the alkylthiobenzylidene analogues over their corresponding alkyloxy counterparts was attributed, at least in part, to the hydrophobicity imparted by the sulfur atom. However, additional thio-analogues were not considered as candidates in further optimization studies, in order to avoid the anticipated toxicities associated with the arylthioethers.31,32 In parallel, the extended p-biphenylbenzylidene analogue 39 showed enhanced activity compared to its parent 2; however, analogues without a p-substituted phenyl moiety are expected to undergo CYPmediated aromatic hydroxylation, which, as noted above for the p-alkyloxybenzylidene analogues, could likewise be accompanied by the formation of highly toxigenic electrophilic areneoxide intermediate(s).33 It was partly on this basis that the p-fluorobiphenylbenzylidene analogue 40 was considered and synthesized as a possibly less toxic counterpart to 39. Altogether, adaptive design modifications during the hit-to-lead optimizations likely improved the lead-like characteristics, furthermore saving time and resources while aiming to avert plausible future preclinical challenges and impediments. Benzylidene analogues with hydrophobic and more electronwithdrawing substituents, represented herein by compounds 47, 48, and 50, were subsequently designed, synthesized, and tested. The trifluoromethyl group was proposed to be optimal for improved bioactivity and expected metabolic stability, when compared to a methyl group. The m,m-di-CF3-benzylidene analogue 50 showed relatively enhanced antiproliferative 943



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Analogue 52 as Oncogenic mTOR Axis Inhibitor in Breast Cancer Cells. Semisynthesis efforts resulted in the most active hit 52, exhibiting nanomolar activity levels in cellular assays against cells of multiple breast cancer lines. Western blot analyses were performed on lysates from cells treated with 52 and vehicle-treated control cells to confirm the mTOR kinase inhibitory activity in MCF-7 and MDA-MB-231 cells. The phosphorylation levels of mTORC1 downstream effectors, including ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), were significantly reduced in cells from both cancer lines compared to control cells (Figure 9). Modulation of

Figure 9. Western blot analysis showing the effects of analogue 52 on mTOR downstream effectors in MCF-7 and MDA-MB-231 breast cancer cells. Data show a concentration-dependent downregulation of p-Akt, p-4E-BP1, and p-S6K in cells treated with 52 compared to vehicle-treated control cells.

mTORC1 downstream effectors invariably attenuated the protein synthesis machinery and thus reduced cancer cell growth and proliferation capacities. In addition, the downregulation of p-Ser473 Akt/PKB in both cell lines further indicated mTORC2 inhibition. Accordingly, proliferation, survival, motility, and invasion of breast cancer cells were significantly hampered upon treatment with 52. Also in this regard, it is worth noting once again the differing nature of the cancer-associated defect, though both convergent on mTORassociated pathways, in the two different cancer cell lines. Collectively, immunoblotting data strongly support the hypothesis that the antiproliferative activity of 52 is mediated, at least in part, through the inhibition of mTOR catalytic activity in breast cancer cells. Analogue 52 as Autophagy Inducer in Breast Cancer Cell Lines. Treatment of breast cancer cell lines with 52 at effective antiproliferative concentrations resulted in a massive autophagy response (Figure 10), as previously observed for 1. The human breast cancer MDA-MB-231 and MCF-7 cells were treated with 52, then lysed, and analyzed for various autophagyrelated protein markers by Western blot to characterize its effects on the autophagic cascade at the molecular level. The immunoblots (Figure 10) showed a significant upregulation of the major constituent of the autophagosomes, LC3, in addition to elevated levels of Beclin-1 that are critically involved

Figure 10. Autophagy induction by analogue 52 in different breast cancer cell lines. (A) Microscopic photographs of MDA-MB-231, MCF-7, SK-BR-3, T-47D, MDA-MB-468, and BT-474 cells treated with 52 showing characteristic autophagic vacuoles compared to control cells (B) Western blot analysis of main cellular autophagic markers in MCF-7 and MDA-MB-231 cells treated with vehicle and 52. Data show a concentration-dependent upregulation of Beclin-1 and LC3 I/II in cells treated with 52 as compared to vehicle control. 944



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Figure 11. Effect of analogue 52 on MDA-MB-231 cell migration in the wound healing assay. (A) Representative microscopic photographs of wounds at zero time and 23 h postincubation and treatment with either vehicle or 1 μM 52. (B) Mean (±SEM) percent cell migration at different concentrations of 52 after 23 h of incubation. Norstictic acid was used as a positive control.38 The symbol * indicates a statistical significance at P < 0.05.

effect of 52 on the clonogenic growth of human breast cancer cells and further motivated in vivo efficacy studies of 52 in various experimental animal models of breast cancer, but selectivity for cancerous versus normal cells remained an open question to first be addressed. Selective Toxicity of Anlogue 52. Toxicities exerted on normal cells constitute one of the major challenges impeding the success of many chemotherapeutic clinical candidates. Thus, discovery of novel bioactive hit entities that display selective in vitro growth and proliferation inhibition of breast cancer cells without a significant toxicity to normal cells strongly supports the promise of success in future in vivo studies. In this regard, the cytotoxic effect of 52 against nontumorigenic MCF-10A mammary epithelial cells was assessed. Treatment with 52, at achievably efficacious antiproliferative concentrations, resulted in minimal MCF-10A cell death (Figure S56, Supporting Information). Promisingly, 52 did not result in significant cytotoxicity even at a concentration up to 6-fold its IC50 value against different malignant cell lines. This clearly suggests the tolerability of the nontumorigenic cells to treatment with 52 at effective antiproliferative concentrations, further paving the way for in vivo studies. In Vivo Activity of Analogue 52 against Breast Cancer in Nude Mice Xenograft Models. The antitumor efficacy of 52 was assessed using mTOR-dysregulated orthotopic xenograft breast cancer models, generated by inoculating nude mice with MCF-7 or MDA-MB-231/GFP cells. Intraperitoneal administration of 52 at a dose regimen of 10 mg/kg, 3×/week, significantly attenuated tumor growth in both models by the end of the study (Figure 14). In the MDA-MB-231/GFP model, the mean tumor volume at the sacrifice time (34 days post-inoculation) in vehicle-treated control group animals was 1835 ± 251 mm3 (±SE). Meanwhile, the mean tumor volume

in autophagic vesicle nucleation. Therefore, large autophagy induction and inhibition of protein synthesis by 52 can well account for the ultimate inhibition of cell proliferation. Analogue 52 as Breast Cancer Migration and Invasion Inhibitor. The potent mTOR inhibitory activity of 52 in cells of two disparate breast cancer lines encouraged the evaluation of its ability to inhibit migratory and invasive phenotypes of the human metastatic MDA-MB-231 breast cancer cells. Analogue 52 exhibited a dose-dependent inhibition of the 2D cell migration and wound closure, with an observed IC50 value of 0.51 μM in the wound healing assay (Figure 11). Analogue 52 significantly inhibited the invasion of MDA-MB-231 cells through the extracellular matrix in the Cultrcoat cell invasion assay, with an IC50 value of 0.42 μM (Figure 12). Overall, these results imparted additional bioactivity importance to 52 and thus encouraged additional in vivo assessment of antitumor efficacy against breast cancer cells having aggressive and invasive phenotypes. Analogue 52 as Inhibitor of MCF-7 Breast Cancer Cell Clonogenic Growth. The ability of transformed cells to grow independently on a solid surface (anchorage-independent growth) is a carcinogenesis hallmark.34 The soft agar colony formation assay was implemented to characterize the suppressive effect of 52 on the clonogenic survival of the human breast cancer MCF-7 cells that readily form 3D colonies in vitro. Untreated MCF-7 cells retained the capacity to proliferate and formed notable colonies within approximately 8 days postplating, with an average diameter of 500 μm (Figure 13). In contrast, in the presence of 0.2 and 0.6 μM concentrations of 52, a significant colony growth inhibition was observed with average colony diameters of 370 and 122 μm, respectively, corresponding to 22.9% and 75.3% colony forming inhibition, respectively. These results provided solid evidence for the significant inhibitory 945



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Figure 12. Effect of analogue 52 on MDA-MB-231 cell invasion. (A) Microscopic photographs of upper surfaces of invasion wells after a 24 h incubation period with DMSO or treatment with 0.5 μM of 52. The low cell density in control wells indicates a higher invasion, and the high cell density in treatment wells indicates a lower invasion. (B) Mean (±SEM) percent cell invasion at different concentrations of 52 after 23 h of incubation. Norstictic acid was used as a positive anti-invasive control compound.38 The symbol * indicates a statistical significance at P < 0.05.

Figure 13. Effect of analogue 52 on the clonogenic growth of MCF-7 cells. Representative microscopic photographs of colonies after 8 days of incubation comparing cells treated with the vehicle and 52 at indicated concentrations.

in treated mice reached 692 ± 146 mm3, representing a nearly 62.3% tumor growth inhibition in the treatment group (Figure 14A). In the MCF-7 model, vehicle-treated control group mice showed mean tumor volumes of 1093 ± 185 mm3 by the end of the study (75 days postinoculation). On the other hand, mice receiving treatment with 52 harbored tumors with a mean volume of only 385 ± 96 mm3 (Figure 14), reflecting a 64.8% mean tumor growth inhibition. Collectively, these results clearly demonstrated the robust efficacy of 52 in attenuating MCF-7 and MDA-MB-231/GFP breast tumor growth, cells of both lines having dysregulated function convergent on mTORgoverned pathways. Moreover, the body weights of the animals were monitored for the duration of the study and results taken

as a general indicator of toxicity. Analogue 52 did not induce any gross toxicity signs or significant reduction of the mean body weights of animals in treatment groups as compared to the vehicle-treated control groups (Figure S57, Supporting Information). Tumor samples from control and treatment groups were used to validate mTOR inhibition as a mechanistic basis for the observed efficacy. Thus, excised malignant tissues from each group were homogenized, lysed, and analyzed by Western blot to evaluate the phosphorylation levels of critical mTOR downstream effectors. The p-4E-BP1 levels in the tumors excised from treatment group animals (both models) were significantly downregulated, compared to their levels in vehicle-treated 946



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Figure 14. In vivo antitumor effect of analogue 52 in different breast cancer models. (A) MDA-MB-231/GFP breast cancer model. (i) Effect of 52 on the mean tumor volume throughout the study. (ii) Representative pictures of control mice and 52-treated groups showing the difference in tumor volume (indicated by arrows) at the study end. (B) MCF-7 breast cancer model. (i) Effects of 52 on the mean tumor volume throughout the study. (ii) Representative pictures of mice treated with the vehicle and 52 showing the difference in tumor volume (indicated by arrows) at the study end. (C) Western blot analyses of tumor tissue homogenates from control and treatment groups from both models. Data show downregulation of mTOR downstream effectors, p-S6K and p-4E-BP1, in both treatment groups and in controls for comparison. The symbol * indicates a statistical significance at P < 0.05.

control group tumors (Figure 14C). Nonphosphorylated 4E-BP1 tightly binds to the cap-binding protein eIF4E and

represses cap-dependent mRNA translation, protein synthesis, and, ultimately, tumor growth.35 In addition, phosphorylation 947



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(6bS)-8-Acetyl-6,9-dihydroxy-5,6b-dimethyl-3-phenyl-2,3-dihydro-1H-benzofurano[2,3-f ]chromene-1,7(6bH)-dione (3): yield (22.6 mg, 34.9%); 1H and 13C NMR data (Supporting Information, Figure S3); ESIMS m/z 431.2 [M − H]− (calcd for C25H20O7). (S,E)-2-Acetyl-6-[3-(4-chlorophenyl)acryloyl]-3,7,9-trihydroxy8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (4): yield (13.5 mg, 19.3%); 1 H and 13 C NMR data (Supporting Information, Figure S4); ESIMS m/z 465.2 [M − H]− (calcd for C25H19ClO7). (S,E)-2-Acetyl-6-[3-(3-chlorophenyl)acryloyl]-3,7,9-trihydroxy8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (5): yield (11.8 mg, 16.9%); 1H and 13C NMR data (Supporting Information, Figure S5); ESIMS m/z 465.2 [M − H]− (calcd for C25H19ClO7). (6bS)-8-Acetyl-3-(4-chlorophenyl)-6,9-dihydroxy-5,6b-dimethyl2,3-dihydro-1H-benzofurano[2,3-f ]chromene-1,7(6bH)-dione (6): yield (9.7 mg, 13.9%); 1H and 13C NMR data (Supporting Information, Figure S6); ESIMS m/z 465.2 [M − H]− (calcd for C25H19ClO7). (S,E)-2-Acetyl-6-[3-(2,4-dichlorophenyl)acryloyl]-3,7,9-trihydroxy8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (7): yield (6.8 mg, 9.1%); 1H and 13C NMR data (Supporting Information, Figure S7); ESIMS m/z 499.3 [M − H]− (calcd for C25H18Cl2O7). (6bS)-8-Acetyl-3-(2,4-dichlorophenyl)-6,9-dihydroxy-5,6b-dimethyl-2,3-dihydro-1H-benzofurano[2,3-f ]chromene-1,7(6bH)dione (8): yield (7.2 mg, 9.6%); 1H and 13C NMR data (Supporting Information, Figure S8); ESIMS m/z 499.3 [M − H]− (calcd for C25H18Cl2O7). (S,E)-2-Acetyl-6-[3-(2,6-dichlorophenyl)acryloyl]-3,7,9-trihydroxy8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (9): yield (10.8 mg, 14.4%); 1H and 13C NMR data (Supporting Information, Figure S9); ESIMS m/z 499.3 [M − H]− (calcd for C25H18Cl2O7). (6bS)-8-Acetyl-3-(2,6-dichlorophenyl)-6,9-dihydroxy-5,6b-dimethyl-2,3-dihydro-1H-benzofuro[2,3-f ]chromene-1,7(6bH)-dione (10): yield (21.3 mg, 28.3%); 1H and 13C NMR data (Supporting Information, Figure S10); ESIMS m/z 499.2 [M − H]− (calcd for C25H18Cl2O7). (S,E)-2-Acetyl-6-[3-(3,5-dichlorophenyl)acryloyl]-3,7,9-trihydroxy8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (11): yield (8.4 mg, 11.2%); 1H and 13C NMR data (Supporting Information, Figure S11); ESIMS m/z 499.2 [M − H]− (calcd for C25H18Cl2O7). (9bS)-2-Acetyl-6-[3-(3,5-dichlorophenyl)-3-hydroxypropanoyl]3,7,9-trihydroxy-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (12): yield (17.9 mg, 23.1%); 1H and 13C NMR data (Supporting Information, Figure S12); ESIMS m/z 517.4 [M − H]− (calcd for C25H20Cl2O8). (S,E)-2-Acetyl-6-[3-(4-fluorophenyl)acryloyl]-3,7,9-trihydroxy8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (13): yield (8.1 mg, 12.0%); 1H and 13C NMR data (Supporting Information, Figure S13); ESIMS m/z 449.2 [M − H]− (calcd for C25H19FO7). (6bS)-8-Acetyl-3-(4-fluorophenyl)-6,9-dihydroxy-5,6b-dimethyl2,3-dihydro-1H-benzofurano[2,3-f ]chromene-1,7(6bH)-dione (14): yield (20.6 mg, 30.5%); 1H and 13C NMR data (Supporting Information, Figure S14); ESIMS m/z 449.2 [M − H]− (calcd for C25H19FO7). (S,E)-2-Acetyl-6-[3-(2-fluorophenyl)acryloyl]-3,7,9-trihydroxy8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (15): yield (16.9 mg, 25.0%); 1H and 13C NMR data (Supporting Information, Figure S15); ESIMS m/z 449.2 [M − H]− (calcd for C25H19FO7). (S,E)-2-Acetyl-6-[3-(3-fluorophenyl)acryloyl]-3,7,9-trihydroxy8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (16): yield (13.4 mg, 19.9%); 1H and 13C NMR data (Supporting Information, Figure S16); ESIMS m/z 449.2 [M − H]− (calcd for C25H19FO7). (S,E)-2-Acetyl-6-[3-(2,6-difluorophenyl)acryloyl]-3,7,9-trihydroxy8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (17): yield (17.1 mg, 24.4%); 1H and 13C NMR data (Supporting Information, Figure S17); ESIMS m/z 467.2 [M − H]− (calcd for C25H18F2O7). (S,E)-2-Acetyl-6-[3-(3,5-difluorophenyl)acryloyl]-3,7,9-trihydroxy8,9b-dimethyldibenzo-[b,d]furan-1(9bH)-one (18): yield (19.2 mg, 27.4%); 1H and 13C NMR data (Supporting Information, Figure S18); ESIMS m/z 467.2 [M − H]− (calcd for C25H18F2O7). (S,E)-2-Acetyl-6-[3-(4-bromophenyl)acryloyl]-3,7,9-trihydroxy8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (19): yield (12.5 mg, 16.3%); 1H and 13C NMR data (Supporting Information, Figure S19); ESIMS m/z 509.1 [M − H] and 511.2 [M + 2 − H]− (calcd for C25H19BrO7).

levels of ribosomal protein S6 kinase (S6K) were significantly lower in tumors from animals in treatment groups of both models, compared to their levels in vehicle-treated control group tumors. Nonphosphorylated ribosomal protein S6 loosely binds to the cap-binding complex of the 40S ribosomal subunit and, hence, represses protein translation initiation.36 In conclusion, this study strongly supports mTOR inhibition by 52 as at least a prominent source of the significant tumor growth attenuation observed in nude mouse breast cancer models. Rational design and testing of natural-product-based semisynthesis analogues is a versatile strategy for hit-to-lead optimizations starting with natural bioactive hits, enabling improvement of lead-likeness, facilitating the reasoned exploration of structure-activity correlates, and providing robust lead compounds and guidances for successful optimization and ultimate generation of viable, mechanistically novel anticancer clinical candidates

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EXPERIMENTAL SECTION

General Experimental Procedures. 1H and 13C NMR spectra were recorded at 400 and 100 MHz, respectively, in CDCl3 (CDCl3− methanol-d4, 9:1, mixture was used to enhance the solubility of analogues 20 and 21), using the residual solvent peaks (δH = 7.26 and δC = 77.1) as references, on a JEOL Eclipse ECS-400 NMR spectrometer (JEOL Inc., Peabody, MA, USA). Carbon multiplicities were determined using PENDANT experiments. The ESI-MS experiments were conducted using a 3200 Q-trap LC/MS/MS system (Applied Biosystems, Foster City, CA, USA) using Analyst version 1.4.1 software (MDS Sciex, Toronto, Canada). A purity of ≥95% (unless otherwise stated) for each compound used in the biological assays was confirmed by analytical HPLC. Chromatograms were acquired using a Shimadzu semipreparative HPLC system with LC-20AP analytical/preparative pump systems and an SPD-20A variable-wavelength UV detector under the following conditions: column, Cosmosil 5C18-AR-II packed (4.6 i.d. × 250 mm); mobile phase, isocratic CH3CN−H2O (9:1); flow rate, 1.0 mL/min, UV detection at λ = 254 nm. (+)-Usnic acid (1) was purchased from Sigma-Aldrich (St. Louis, MO, USA), and the different aldehydes were purchased from either Sigma-Aldrich or Alfa Aesar (Ward Hill, MA, USA). All chemicals were used directly without prior purification. Si gel 60 (230−400 mesh, Natland International Corporation, Reserach Triangle Park, NC, USA) was used for column chromatography and analytical TLC was carried out on precoated Si gel 60 F254 TLC plates (EMD Millipore, Billerica, MA, USA), using appropriate developing systems. TLC spots were detected under UV light at 254 and 365 nm and then chemically visualized by spraying with a solution of freshly prepared p-anisaldehyde−EtOH−HOAc−H2SO4 (2:170:20:10 v/v) and heating at 105 °C until a maximal spot color developed. General Method for Semisynthesis of (+)-Usnic Acid Benzylidene Analogues 2−53. A stirring suspension of 1 (50 mg, 0.15 mmol) in 10 mL of MeOH was treated with 300 μL of 10% KOH dropwise until 1 was completely dissolved, and the mixture was left to stir for 15 min at rt. Different aldehydes (0.15 mmol) were added to each stirring mixture over approximately 10 min. Reaction mixtures were left for 1−3 days at room temperature (rt) and periodically monitored by TLC. Solvents were evaporated under vacuum and the residues partitioned between 2% aqueous HCl and EtOAc (10 mL each). Organic layers were pooled, washed with brine, and dried over anhydous Na2SO4. Concentrated organic layers were adsorbed on Celite and purified over Si gel 60 columns using mixtures of n-hexane and EtOAc to yield pure benzylidene analogues and other side products. (S)-2-Acetyl-6-cinnamoyl-3,7,9-trihydroxy-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (2): yield (11.4 mg, 17.6%); 1H and 13C NMR data (Supporting Information, Figure S2); ESIMS m/z 431.2 [M − H]− (calcd for C25H20O7). 948



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(17.9 mg, 23.0%); 1H and 13C NMR data (Supporting Information, Figure S36); ESIMS m/z 517.4 [M − H]− (calcd for C30H30O8). (S,E)-2-Acetyl-3,7,9-trihydroxy-8,9b-dimethyl-6-[3-(4(methylthio)phenyl)acryloyl]dibenzo[b,d]furan-1(9bH)-one (37): yield (8.1 mg, 11.3%); 1H and 13C NMR data (Supporting Information, Figure S37); ESIMS m/z 477.1 [M − H]− (calcd for C26H22O7S). (S,E)-2-Acetyl-6-[3-(4-(ethylthio)phenyl)acryloyl]-3,7,9-trihydroxy-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (38): yield (6.3 mg, 8.5%); 1H and 13C NMR data (Supporting Information, Figure S38); ESIMS m/z 491.2 [M − H]− (calcd for C27H24O7S). (S,E)-2-Acetyl-6-[3-(biphenyl-4-yl)acryloyl]-3,7,9-trihydroxy-8,9bdimethyldibenzo[b,d]furan-1(9bH)-one (39): yield (8.4 mg, 11.0%); 1 H and 13C NMR data (Supporting Information, Figure S39); ESIMS m/z 507.2 [M − H]− (calcd for C31H24O7). (S,E)-2-Acetyl-6-[3-(4′-fluorobiphenyl-4-yl)acryloyl]-3,7,9-trihydroxy-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (40): yield (15.7 mg, 19.9%); 1H and 13C NMR data (Supporting Information, Figure S40); ESIMS m/z 525.2 [M − H]− (calcd for C31H23FO7). (S,E)-2-Acetyl-6-[3-(4-(benzyloxy)phenyl)acryloyl]-3,7,9-trihydroxy-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (41): yield (14.3 mg, 17.7%); 1H and 13C NMR data (Supporting Information, Figure S41); ESIMS m/z 537.4 [M − H]− (calcd for C32H26O8). (6bS)-8-Acetyl-3-[4-(benzyloxy)phenyl]-6,9-dihydroxy-5,6b-dimethyl-2,3-dihydro-1H-benzofurano[2,3-f ]chromene-1,7(6bH)dione (42): yield (22.1 mg, 27.4%); 1H and 13C NMR data (Supporting Information, Figure S42); ESIMS m/z 537.4 [M − H]− (calcd for C32H26O8). (S,E)-2-Acetyl-6-{3-[4-(4-fluorobenzyloxy)phenyl]acryloyl}-3,7,9trihydroxy-8,9b-dimethyldibenzo-[b,d]furan-1(9bH)-one (43): yield (14.6 mg, 17.5%); 1H and 13C NMR data (Supporting Information, Figure S43); ESIMS m/z 555.2 [M − H]− (calcd for C32H25FO8). (6bS)-8-Acetyl-3-[4-(4-fluorobenzyloxy)phenyl]-6,9-dihydroxy5,6b-dimethyl-2,3-dihydro-1H-benzofuro[2,3-f ]chromene-1,7(6bH)dione (44): yield (24.2 mg, 29.0%); 1H and 13C NMR data (Supporting Information, Figure S44); ESIMS m/z 555.2 [M − H]− (calcd for C32H26O8). (S,E)-2-Acetyl-6-{3-[4-(2-chloro-6-fluorobenzyloxy)phenyl]acryloyl}-3,7,9-trihydroxy-8,9b-dimethyldibenzo[b,d]furan-1(9bH)one (45): yield (18.8 mg, 21.2%); 1H and 13C NMR data (Supporting Information, Figure S45); ESIMS m/z 589.3 [M − H]− (calcd for C32H24ClFO8). (6bS)-8-Acetyl-3-[4-(2-chloro-6-fluorobenzyloxy)phenyl]-6,9-dihydroxy-5,6b-dimethyl-2,3-dihydro-1H-benzofurano[2,3-f ]chromene-1,7(6bH)-dione (46): yield (23.1 mg, 19.3%); 1H and 13C NMR data (Supporting Information, Figure S46); ESIMS m/z 589.4 [M − H]− (calcd for C32H24ClFO8). (S,E)-2-Acetyl-3,7,9-trihydroxy-8,9b-dimethyl-6-[3-(3(trifluoromethyl)phenyl)acryloyl]dibenzo[b,d]furan-1(9bH)-one (47): yield (12.8 mg, 17.1%); 1H and 13C NMR data (Supporting Information, Figure S47); ESIMS m/z 499.2 [M − H]− (calcd for C26H19F3O7). (S,E)-2-Acetyl-3,7,9-trihydroxy-8,9b-dimethyl-6-{3-[4(trifluoromethyl)phenyl]acryloyl}dibenzo[b,d]furan-1(9bH)-one (48): yield (8.7 mg, 11.6%); 1H and 13C NMR data (Supporting Information, Figure S48); ESIMS m/z 449.3 [M − H]− (calcd for C26H19F3O7). (6bS)-8-Acetyl-6,9-dihydroxy-5,6b-dimethyl-3-[4-(trifluoromethyl)phenyl]-2,3-dihydro-1H-benzofuro[2,3-f ]chromene-1,7(6bH)-dione (49): yield (11.6 mg, 15.5%); 1H and 13C NMR data (Supporting Information, Figure S49); ESIMS m/z 449.3 [M − H]− (calcd for C26H19F3O7). (S,E)-2-Acetyl-6-{3-[3,5-bis(trifluoromethyl)phenyl]acryloyl}-3,7,9trihydroxy-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (50): yield (19.7 mg, 23.1%); 1H and 13C NMR data (Supporting Information, Figure S50); ESIMS m/z 567.1 [M − H]− (calcd for C27H18F6O7). (9bS)-2-Acetyl-6-{3-[3,5-bis(trifluoromethyl)phenyl]-3-hydroxypropanoyl}-3,7,9-trihydroxy-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (51): yield (22.1 mg, 25.1%); 1H and 13C NMR data (Supporting Information, Figure S51); ESIMS m/z 585.3 [M − H]− (calcd for C27H20F6O8). (S,E)-2-Acetyl-3,7,9-trihydroxy-8,9b-dimethyl-6-{3-[4-(trifluoromethoxy)phenyl]acryloyl}dibenzo[b,d]furan-1(9bH)-one (52): yield

(S,E)-4-[3-(8-Acetyl-1,3,7-trihydroxy-2,9a-dimethyl-9-oxo-9,9adihydrodibenzo[b,d]furan-4-yl)-3-oxoprop-1-enyl]benzoic acid (20): yield (13.6 mg, 19.1%); 1H and 13C NMR data (Supporting Information, Figure S20); ESIMS m/z 475.1 [M − H]− (calcd for C25H20O9). 4-[(6bS)-8-Acetyl-6,9-dihydroxy-5,6b-dimethyl-1,7-dioxo2,3,6b,7-tetrahydro-1H-benzofurano[2,3-f ]chromen-3-yl]benzoic acid (21): yield (13.3 mg, 18.6%); 1H and 13C NMR data (Supporting Information, Figure S21); ESIMS m/z 475.1 [M − H]− (calcd for C25H20O9). 4-[(6bS)-8-Acetyl-6,9-dihydroxy-5,6b-dimethyl-1,7-dioxo2,3,6b,7-tetrahydro-1H-benzofurano[2,3-f ]chromen-3-yl]benzoic acid (22): yield (7.8 mg, 11.7%); 1H and 13C NMR data (Supporting Information, Figure S22); ESIMS m/z 445.2 [M − H]− (calcd for C26H22O7). (6bS)-8-Acetyl-6,9-dihydroxy-5,6b-dimethyl-3-p-tolyl-2,3-dihydro-1H-benzofuro[2,3-f ]chromene-1,7(6bH)-dione (23): yield (14.5 mg, 21.7%); 1H and 13C NMR data (Supporting Information, Figure S23); ESIMS m/z 445.3 [M − H]− (calcd for C26H22O7). (S,E)-2-Acetyl-6-[3-(4-tert-butylphenyl)acryloyl]-3,7,9-trihydroxy8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (24): yield (13.4 mg, 18.3%); 1H and 13C NMR data (Supporting Information, Figure S24); ESIMS m/z 487.4 [M − H]− (calcd for C29H28O7). (6bS)-8-Acetyl-3-(4-tert-butylphenyl)-6,9-dihydroxy-5,6b-dimethyl-2,3-dihydro-1H-benzofurano[2,3-f ]chromene-1,7(6bH)-dione (25): yield (26.1 mg, 35.6%); 1H and 13C NMR data (Supporting Information, Figure S25); ESIMS m/z 487.4 [M − H]− (calcd for C29H28O7). (S,E)-2-Acetyl-3,7,9-trihydroxy-6-[3-(4-methoxyphenyl)acryloyl]8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (26): yield (11.5 mg, 16.6%); 1H and 13C NMR data (Supporting Information, Figure S26); ESIMS m/z 461.2 [M − H]− (calcd for C26H22O8). (6bS)-8-Acetyl-6,9-dihydroxy-3-(3-methoxyphenyl)-5,6b-dimethyl-2,3-dihydro-1H-benzofuro[2,3-f ]chromene-1,7(6bH)-dione (27): yield (28.3 mg, 40.8%); 1H and 13C NMR data (Supporting Information, Figure S27); ESIMS m/z 461.3 [M − H]− (calcd for C26H22O8). (S,E)-2-Acetyl-3,7,9-trihydroxy-8,9b-dimethyl-6-[3-(3,4,5trimethoxyphenyl)acryloyl]dibenzo[b,d]-furan-1(9bH)-one (28): yield (13.2 mg, 16.9%); 1H and 13C NMR data (Supporting Information, Figure S28); ESIMS m/z 512.2 [M − H]− (calcd for C28H26O10). (S,E)-2-Acetyl-6-[3-(2-fluoro-4,5-dimethoxyphenyl)acryloyl]-3,7,9trihydroxy-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (29): yield (17.6 mg, 23.0%); 1H and 13C NMR data (Supporting Information, Figure S29); ESIMS m/z 509.1 [M − H]− (calcd for C27H23FO9). (S,E)-2-Acetyl-6-[3-(4-ethoxyphenyl)acryloyl]-3,7,9-trihydroxy8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (30): yield (10.2 mg, 14.3%); 1H and 13C NMR data (Supporting Information, Figure S30); ESIMS m/z 475.1 [M − H]− (calcd for C27H24O8). (6bS)-8-Acetyl-3-(4-ethoxyphenyl)-6,9-dihydroxy-5,6b-dimethyl2,3-dihydro-1H-benzofurano[2,3-f ]chromene-1,7(6bH)-dione (31): yield (22.7 mg, 31.8%); 1H and 13C NMR data (Supporting Information, Figure S31); ESIMS m/z 475.2 [M − H]− (calcd for C27H24O8). (S,E)-2-Acetyl-3,7,9-trihydroxy-6-[3-(2-methoxyphenyl)acryloyl]8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (32): yield (7.3 mg, 10.2%); 1H and 13C NMR data (Supporting Information, Figure S32); ESIMS m/z 475.3 [M − H]− (calcd for C27H24O8). (6bS)-8-Acetyl-6,9-dihydroxy-3-(2-methoxyphenyl)-5,6b-dimethyl-2,3-dihydro-1H-benzofurano[2,3-f ]chromene-1,7(6bH)-dione (33): yield (16.8 mg, 23.5%); 1H and 13C NMR data (Supporting Information, Figure S33); ESIMS m/z 475.2 [M − H]− (calcd for C27H24O8). (S,E)-2-Acetyl-6-[3-(4-butoxyphenyl)acryloyl]-3,7,9-trihydroxy8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (34): yield (10.6 mg, 14.0%); 1H and 13C NMR data (Supporting Information, Figure S34); ESIMS m/z 503.3 [M − H]− (calcd for C29H28O8). (6bS)-8-Acetyl-3-(4-butoxyphenyl)-6,9-dihydroxy-5,6b-dimethyl2,3-dihydro-1H-benzofurano[2,3-f ]chromene-1,7(6bH)-dione (35): yield (24.2 mg, 32.0%); 1H and 13C NMR data (Supporting Information, Figure S35); ESIMS m/z 503.4 [M − H]− (calcd for C29H28O8). (S,E)-2-Acetyl-3,7,9-trihydroxy-6-[3-(4-(isopentyloxy)phenyl)acryloyl]-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one (36): yield 949



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(13.3 mg, 17.2%); 1H and 13C NMR data (Supporting Information, Figure S52); ESIMS m/z 515.2 [M − H]− (calcd for C26H19F3O8). (6bS)-8-Acetyl-6,9-dihydroxy-5,6b-dimethyl-3-[4-(trifluoromethoxy)phenyl]-2,3-dihydro-1H-benzofuro[2,3-f ]chromene-1,7(6bH)-dione (53): yield (19.5 mg, 25.2%); 1H and 13C NMR data (Supporting Information, Figure S53); ESIMS m/z 515.3 [M − H]− (calcd for C26H19F3O8). Molecular Modeling Studies. The in silico experiments were carried out using the Schrödinger molecular modeling software package installed on an iMac 27-in. Z0PG workstation with a 3.5 GHz Quad-core Intel Core i7, Turbo Boost up to 3.9 GHz, processor, and 16 GB RAM (Apple, Cupertino, CA, USA). The X-ray crystal structure of mTOR (PDB code: 4JT5) was retrieved from the Protein Data Bank.37 The Protein Preparation Wizard of the Schrödinger suite was used to prepare the kinase domain. The protein was reprocessed by assigning bond orders, adding hydrogens, creating disulfide bonds, and optimizing H-bonding networks using PROPKA (Jensen Research Group, Copenhagen, Denmark). Finally, energy minimization with a root-mean-square deviation value of 0.30 Å was applied using an Optimized Potentials for Liquid Simulation (OPLS_2005, Schrö dinger, New York, NY, USA) force field. For ligand structure preparation, the 2D structures of usnic acid and semisynthesized analogues were sketched in the Maestro 9.3 panel (Maestro, version 9.3, 2012, Schrödinger). The LigPrep 2.3 module (LigPrep, version 2.3) of the Schrödinger suite was utilized to generate 3D structures and to search for different conformers. The Optimized Potentials for Liquid Simulation (OPLS_2005) force field was applied to geometrically optimize the ligands and to compute partial atomic charges. Finally, at most 32 poses per ligand were generated with different spatial features for subsequent docking studies. The prepared mTOR kinase domain was employed to generate energy grids using the default value of the protein atomic scale (1.0 Å) within the cubic box centered on experimental cocrystallized ligand. After receptor grid generation, structures were docked using the Glide 5.8 module (Glide, version 5.8, 2012, Schrödinger). Cell Lines and Culture Conditions. Human breast cancer cell lines MDA-MB-231, MDA-MB-468, MCF-7, T-47D, BT-474, and SK-BR-3 and the human nontumorigenic mammary epithelial MCF-10A cell line were all obtained from the American Type Culture Collection (Manassas, VA, USA). The breast cancer MDA-MB-231/ green florescent protein-tagged (MDA-MB-231/GFP) cell line was purchased from Cell Biolabs (San Diego, CA, USA). Cells were cultured and maintained in RPMI-1640 medium (Corning, Manassas, VA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Valley Biomedical, Winchester, VA, USA) in a 5% CO2 humidified incubator at 37 °C. MCF-7 cells were cultured and maintained in DME medium (Gibco by Life Technologies, Grand Island, NY, USA) supplemented with 10% hyclone fetal bovine serum (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The immortalized mammary epithelial MCF-10A cells were maintained in DME/high glucose medium (Life Technologies) supplemented with 10% horse serum (Gibco by Life Technologies), 20 ng/mL EGF (Peprotech, Rocky Hill, NJ, USA), 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, and 10 μg/mL insulin. Cell Proliferation Assay. Cells were seeded into 96-well plates at a density of 5 × 103 cells/well in 100 μL of culture medium, and the plates were incubated overnight at 37 °C in a 5% CO2 humidified incubator so that the cells could recover and attach. Media were removed and cells were washed with PBS. Test compounds were prepared as stock solutions (10 mM) in DMSO and immediately added to culture media (supplemented with 100 ng/mL HGF, for c-Met expressing cells) to prepare the final working concentrations. About 100 μL of treatment media was added, in triplicates, and cells were incubated at 37 °C for 72 h. The media were gently aspirated and cells were rinsed with PBS at the end of the incubation period. About 100 μL of fresh media and 50 μL of MTT solution were added to each well and cells were incubated for an additional 4 h. Supernatants were carefully removed and formazan crystals were dissolved in 100 μL of DMSO. The plates were incubated for 5 min with gentle

shaking before measuring the absorbance at 570 nm using a Synergy 2 microplate reader (BioTek, Winooski, VT, USA). Cell numbers were derived from a standard curve executed at the beginning of each experiment. IC50 values were calculated using GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA, USA). Cell Migration Assay. MDA-MB-231 cells were seeded into a 24-well plate at a density of 1 × 105 cells/well and incubated overnight to recover and attach at 37 °C in a 5% CO2 humidified incubator. Wounds were inflicted in confluent monolayers using sterile 200 μL pipet tips. Cells were washed with PBS and reincubated in serum-free media for 5 h, after which the media were replaced with fresh ones supplemented with HGF (100 ng/mL) and containing different concentrations of test compounds or DMSO as vehicle control. The lichen-derived norstictic acid (15 μM) was used as positive control antimigratory compound.38 Wounds were photographed at zero time and monitored for closing up to 24 h. When wounds were about to close, media were gently aspirated and cells were rinsed with PBS and fixed with cold MeOH for 15 min at 4 °C. Finally, wounds were photographed for treatments, including the vehicle-treated control wells for comparison. Percentages of cell migration were calculated using the following formula: Percent cell migration = [T0 − Tt − Tdmso]/[T0 − Tdmso] × 100, where T0 is wound thickness at zero time, Tdmso is wound thickness in DMSO-treated control wells and Tt is wound thickness in treatment wells. IC50 values were calculated using GraphPad Prism version 5.01 (GraphPad Software). Cell Invasion Assay. The experiment was performed according to the manufacturer’s (Cultrecoat invasion kit) procedures with optimization regarding the number of MDA-MB-231 cells per well. The 96-well invasion chamber was equilibrated at rt for 1 h prior to use. Inserts were rehydrated by adding 25 μL of warm RPMI-1640 medium and incubation at 37 °C for 1 h. MDA-MB-231 cells in culture plates were serum-starved for 16 h in serum-free medium prior to the assay. Cells were harvested, resuspended, and counted to prepare a working concentration of 1 × 106 cells/mL in RPMI-1640 culture medium. Cells (25 μL of 2.5 × 104 cells) were added to the top hydrated inserts, and 150 μL of serum-free media, supplemented with HGF (100 ng/mL) and containing either test compounds or DMSO, were added to the bottom chamber. The lichen-derived norstictic acid (15 μM) was used as a positive control anti-invasive natural product.38 Plates were assembled and incubated at 37 °C in a 5% CO2 humidified incubator for 24 h. After incubation, the top chamber was inverted to remove the media and transferred to a black receiver plate. Wells of the top chamber were washed with 100 μL of warm washing buffer, 100 μL of cell dissociation solution/calcein AM was added to each well of the lower chamber, and plates were incubated for an additional 1 h. Finally, the top chamber was removed and the fluorescence in lower plate wells was measured at 485 nm excitation, 520 nm emission using a Synergy 2 microplate reader (BioTek). Relative fluorescence units (RFU) were used to calculate the number of invaded cells in control and treatment wells, using a standard curve constructed prior to the start of the experiment. Mean percent invasion values were calculated relative to the vehicle-treated control wells. IC50 values were calculated using GraphPad Prism version 5.01 (GraphPad Software). Clonogenic Colony Formation Assay. The colony-forming capability of MCF-7 breast cancer cells was assessed utilizing the CytoSelect 96-well in vitro tumor sensitivity assay kit (Cell Biolabs, Inc., San Diego, CA, USA) according to the manufacturer’s protocol. Briefly, 50 μL of base agar matrix layer was dispensed into a 96-well microplate, and the plate was gently tapped to ensure even coverage of the wells. The plate was kept at 4 °C for 30 min to allow the agar matrix layer to solidify. MCF-7 cells (5 × 103) in 75 μL of cell suspension/agar matrix layer were dispensed into each well. The plate was transferred back to 4 °C for 20 min to allow the cell suspension/ agar matrix layer to solidify. Cells were treated with 50 μL of 10% serum media containing 0.2 and 0.6 μM of compound 52 or DMSO. The plate was monitored daily for cellular growth and colony formation. After 8 days, colonies were photographed under an inverted phase-contrast microscope. Matrix solubilization buffer (125 μL) was added to each well and pipetted several times to ensure solubilization. About 100 μL of the mixture was transferred to a 96-well microplate, 950



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and 10 μL of MTT solution was added to each well. The plate was incubated for 4 h at 37 °C. About 100 μL of detergent solution was added to each well, and the plate was incubated for an additional 4 h at rt. Finally, the absorbance was measured at 570 nm using a Synergy 2 microplate reader (BioTek). Percent cell growth was normalized to the DMSO-treated control cells. Cytotoxicity Assay. The human nontumorigenic MCF-10A breast epithelial cells were seeded into a 96-well plate at a density of 3 × 104 cells/well. Cells were incubated overnight at 37 °C in a 5% CO2 humidified incubator to allow cell recovery and attachment. Media were carefully removed and cells were washed with PBS. Various concentrations of analogue 52 in serum-free media were added in triplicates, while vehicle control wells were treated with media containing the maximum amount of DMSO added in treatment sets. Doxorubicin (10 μM) was used as a positive cytotoxic control drug.38 Cells were incubated for 24 h. At the end of the incubation period, media were removed and cells were washed with PBS. About 100 μL of fresh culture media and 50 μL of MTT solution were added to each well, and the cells were reincubated for 4 h and checked periodically for the formation of formazan crystals. Supernatants were carefully removed, and formazan crystals were dissolved in 100 μL of DMSO after crystals were fully grown. The plate was incubated in the dark for 5 min and gently swirled before measuring the absorbance at 570 nm in a Synergy 2 microplate reader (BioTek). Average values from triplicate readings were calculated and subtracted from the mean value obtained for blank wells. Cell numbers were deduced from a standard curve executed at the beginning of the experiment. Percent cell viability was calculated by comparing numbers of cells in treatment wells to the mean for DMSO-treated control wells. Western Blot Analysis. The human breast cancer MCF-7 and MDA-MB-231 cells were seeded at a density of 5 × 105 cells/100 mm culture dish and incubated overnight to recover and attach. Cells were treated with either test compound or DMSO as vehicle control for 72 h in culture media (supplemented with 100 ng/mL HGF for the c-Met-dependent MDA-MB-231 cells). Total cellular protein contents were obtained using RIPA lysis and extraction buffer (Thermo Fisher Scientific, Madison, WI, USA) supplemented with mammalian protease arrest (G-Biosciences, St. Louis, MO, USA). Samples were diluted in Laemmli buffer (BIO-RAD, Hercules, CA, USA) containing 5% β-mercaptoethanol (Sigma-Aldrich) prior to loading on gels. Cell lysates (30 μg) were electrophoresed on Mini-PROTEAN TGX precast polyacrylamide gels (BIO-RAD) using Tris/glycine/SDS running buffer and transferred to Immuno-Blot PVDF membranes (BIO-RAD). Blotted membranes were blocked with 5% BSA (Cell Signaling Technology, Beverly, MA, USA) in TBST (10 mM TrisHCl, 150 mM NaCl, 0.1% Tween-20) for 2 h with gentle agitation at rt. Immunoblots were incubated overnight at 4 °C with appropriate primary antibodies (Cell Signaling Technology). After incubation, membranes were washed three times with TBST and incubated with HRP-labeled secondary antibodies (Cell Signaling Technology) for 1 h with agitation at rt. Chemiluminescence detection was performed using the Supersignal West Pico kit (Thermo Fisher Scientific, Madison, WI, USA) and G. BOX imaging system with a highresolution 100m pixel camera (Syngene, Fredrick, MD, USA). In Vivo Nude Mice Xenograft Models. Female athymic nude mice (5−6 weeks old) were purchased from Harlan Laboratories (Cumberland, IN, USA). Animals were housed at the Animal Facility (School of Pharmacy, University of Louisiana at Monroe) and maintained under clean conditions in sterile filter-top cages, at a temperature of 24 ± 2 °C, 50 ± 10% relative humidity, and 12:12 h artificial light−dark cycle. Mice received mouse chow and water ad libitum. All procedures were conducted in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and with the approval by the Institutional Animal Care and Use Committee (IACUC). MDA-MB-231 green florescent protein-tagged (MDA-MB-231/ GFP) cells were harvested, washed with PBS, and resuspended in RPMI-1640 medium. Cells (2 × 106 cells/25 μL) were injected into the mammary fat pad of each nude mouse, using a 29G hypodermic needle. Animals were observed daily for the growth of palpable tumors

at the site of injection. Ten days postimplantation, tumors became visible with an approximate average volume of 50 mm3. Mice were randomized and allocated to control and treatment groups (5 mice/ group). In the MCF-7 xenografted animals, a 17β-estradiol pellet delivering controlled release over 90 days (Innovative Research of America, Sarasota, FL, USA) was implanted beneath the skin of each nude mouse. After recovery from surgery (5 days), animals were injected with 2 × 106 cells suspended in 25 μL of serum-free media into the mammary fat pad of each nude mouse. It took approximately 20 days for generated tumors to reach the average volume of 50 mm3. Mice were randomized and allocated to control and treatment groups (5 mice/group). Analogue 52 was prepared as a stock solution in sterile DMSO (1 mg/20 μL), diluted with sterile PBS containing 0.1% Tween 80, and injected intraperitoneally at a dose regimen of 10 mg/kg body weight, three times per week, for the indicated times in each set of experiments. Animals in the control groups received the same volume of vehicle, following the same treatment protocol. Tumor dimensions were measured using a digital caliper (VWR, Radnor, PA, USA). Tumor volume was calculated using the wellestablished formula: tumor volume (mm3) = [(length × width2)/2]. Animals were monitored daily for any signs of treatment- or vehicleassociated toxicity. Animals were sacrificed at the indicated times, unless they appeared to be moribund or a tumor showed signs of necrosis. At termination, tumors were excised from the connective tissues and snap-frozen for subsequent analysis. Statistical Analyses. All in vitro experiments were performed in triplicate. Pooled data were subjected to statistical analyses using GraphPad Prism version 5.01 (GraphPad Software). Differences between means from two different groups were subjected to Student’s “t” test, whereas one-way analysis of variance (ANOVA) was used to test for significant differences between three or more groups. The in vivo tumor growth data were subjected to two-tailed Student’s “t” tests. Results were considered to be significantly different when P < 0.05, indicated by * symbol.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00917. 1 H and 13C NMR data and spectra of (+)-usnic acid (1) and its semisynthesized analogues 2−53 (Figures S1−S53); 1D and 2D NMR expansions of analogue 3 (Figure S54); binding modes of possible epimeric structures of analogue 3 (α- or β-phenyl) at the mTOR kinase domain (Figure S55); effect of analogue 52 on the viability of the human nontumorigenic MCF-10A mammary epithelial cells (Figure S56); mean body weight of the nude mice in control and treatment groups of both breast cancer models throughout the study (Figure S57) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 318-342-1725. Fax: 318-342-1737. E-mail: elsayed@ulm. edu. ORCID

Khalid A. El Sayed: 0000-0002-1456-4064 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Research reported in this manuscript was supported in part by the National Cancer Institute of the National Institutes of Health under Award Number R15CA167475. 951



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