- Email: [email protected]
(+)-Dehydroabietylamine derivatives target triple-negative breast cancer
European Journal of Medicinal Chemistry 102 (2015) 9e13
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
(þ)-Dehydroabietylamine derivatives target triple-negative breast cancer lez b, Lekh Nath Gautam a, Michele Connelly a, Taotao Ling a, My Tran a, Miguel A. Gonza Rachael K. Wood c, Iram Fatima c, Gustavo Miranda-Carboni c, Fatima Rivas a, * a b c
Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN 38105-3678, USA nica, Universidad de Valencia, 46100 Burjassot, Valencia, Spain Departamento de Química Orga Department of Medicine, The University of Tennessee Health Science Center, Memphis, TN 38163, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 June 2015 Received in revised form 15 July 2015 Accepted 17 July 2015 Available online 23 July 2015
Breast cancer remains the leading cause of cancer-related death among women. The invasive triplenegative subtype is unresponsive to estrogen therapy, and few effective treatments are available. In search of new chemical scaffolds to target this disease, we conducted a phenotypic screen against the human breast carcinoma cell lines MDA-MB-231, MA11, and MCF-7 using terrestrial natural products. Natural products that preferentially inhibited proliferation of triple-negative MDA-MB-231 cells over estrogen receptorepositive cells were further studied; herein we focused on the abietanes. The activity of the abietane carnosol prompted us to generate a focus library from the readily available (þ)-dehydroabietylamine. The lead compound 61 displayed a promising EC50 of 9.0 mM against MDA-MB-231 and our mechanistic studies indicate it induced apoptosis, which was associated with activation of caspase-9 and -3 and the cleavage of PARP. Here we describe our current progress towards this promising therapeutic candidate. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: (þ)-Dehydroabietylamine Abietane Apoptosis Breast cancer Triple-negative
1. Introduction Natural products (NPs) are an integral component of drug discovery programs and have had their greatest success in the oncology field. Approximately 60% of clinically approved anticancer drugs are based on secondary metabolites found in nature [1]. The natural world is a vast, rich, and relatively untapped source of molecular scaffolds for reagent compounds or therapeutic agents. As more sensitive screening assays are developed, promising scaffolds from NPs will be identified as potential leads. Breast cancer is a leading cause of premature death worldwide; in the United States, it was estimated to cause more than 40,000 deaths in 2014 [2]. Breast cancer is classified according to its expression of estrogen receptor (ER), progesterone receptor (PR), and/or human epidermal growth factor receptor (Her2/neu) [3], and current therapies target these receptors. Triple-negative breast carcinoma (TNBC) is a heterogeneous subset of tumors
* Corresponding author. Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place Memphis, TN 381053678, USA. E-mail address: [email protected] (F. Rivas). http://dx.doi.org/10.1016/j.ejmech.2015.07.034 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
defined only by their aggressive nature and the absence of the mentioned receptors. Its poor overall prognosis reflects not only its inherent aggressiveness but also the lack of targeted therapies [4]. As approximately one million breast cancers are diagnosed each year and about 15% are TNBC [4], there is a clear and urgent need for new therapies. The main goal of our drug discovery program is to identify novel NP molecular scaffolds for research and treatment of TNBC. We reasoned that a broad phenotypic screen against breast cancer cell lines using pure NPs would lay the foundation for a medicinal chemistry program. Here we present our lead findings. 2. Results and discussion We used the triple-negative MDA-MB-231 cell line and the ERpositive MA11 and MCF7 cell lines as cellular models of breast carcinoma. The MDA-MB-231 line has been extensively characterized and provides a robust in vitro model of TNBC. Given the rise of questionable cell lines, we opted for only validated models [5]. We screened a library of pure NPs (2300 compounds) primarily from terrestrial sources for their antiproliferative effect on the three cell lines using CellTiterGlo [12] (see SI).
10
T. Ling et al. / European Journal of Medicinal Chemistry 102 (2015) 9e13
Several hits were observed in the ER-positive cell lines, while few were selective for TNBC. A few representatives of the abietane family members showed promising results. However, the non-aromatic abietanes, abietic acid 1 and primaric acid 2 (Fig. 1) showed no activity against the TNBC cell line. Compounds 3e7 displayed anti-proliferative activity against MDA-MB-231 cells at 50 mM, with no detectable cytotoxicity observed in Hek293 and HepG2 cell lines. Of particular interest were compounds 6 and 7, showing activity against MDAMB-231 cells at 22 and 29 mM, respectively (see SI). The abietanes are diterpenoids possessing an ABC ring system; dehydroabietic acid is a classic example (Fig. 1) [6]. They are found in the resin of various terrestrial plants of the Podocarpaceae and Cupressaceae families and in other angiosperms [7]. The abietanes have demonstrated a broad range of biological properties, particularly anticancer activities. For example, carnosic acid has been established as a BCL-9 inhibitor and has shown potency against colon cancer in vitro and in vivo [8,9], while related NPs have shown activity against human pancreatic tumor cell lines [9]. Because these compounds are abundant in nature, semi-syntheses and derivatization programs are sustainable and can generate powerful structure-function studies to characterize their medicinal properties. Rosin Amine D, an inexpensive oil consisting of ~65% DHAA plus a mixture of the dihydro- and tetrahydro-abietylamines as well as other amines. These compounds are the product of basetreated Rosin acid nitriles, which are naturally abundant in resins [10]. DHAA (4, Fig. 1, featuring a chemical handle at C18) serves as a resolving reagent for racemic carboxylic acids and as a chiral synthon for pharmaceutical preparations [10]. Conformational evaluation of these abietane compounds suggested the hypothesis that the aromatic C ring might be required for the observed anti-proliferative activity. Fig. 2 shows the superposition of model compounds 1, 2 and 4 at the lowest energy conformation (DFT). While they possess a closely related pharmacophore, the A and C rings show clear differences (no hydrogens are shown for clarity). In search of compounds with greater anti-TNBC potency, we chose compound 4 (DHAA) as the starting point for our synthetic plan. We focused our attention on the C18 of the abietane core. Given the obvious versatility of the primary amine, this approach would probe a broad range of chemical space near the A ring while maintain the B and C rings intact. Our findings support the further use of this unique chemical scaffold for development of therapeutic agents against TNBC. Being affordable, abietanes can provide inexpensive lead candidates. After column purification of the Rosin D mixture, DHAA was obtained as 50 g batches of light yellow oil in roughly 64% yield. As the DHAA supply was sufficient, we converted it to both amide and corresponding sulfonamide derivatives (Fig. 3). We first synthesized the DHAA amides by peptide coupling of DHAA with a broad range of carboxylic chlorides to evaluate electronic properties. DHAA was treated with 1.1 equivalent of the corresponding
Fig. 2. Representative western blots of MDA-MB231 cells treated with compounds 7, 43, and 61. Panel A. After 6 hrs of treatment. Panel B. After 12 hrs of treatment. Cells near 70% confluence were treated with the compounds, and the indicated cellular proteins were probed in 3 independent experiments. Loading control, b-actin; positive control, 2.5 mM staurosporine; vehicle (28 mM DMSO).
acid chloride and 3.0 equivalents of triethylamine in dichloromethane (DCM) at room temperature, stirring for 1e12 h (Fig. 3). The products were purified by column chromatography to afford the neat compounds 8e29 in 82%e95% chemical yield. The focused amide library included aliphatic and aromatic functional groups for a thorough evaluation of chemical space and electronic properties [11]. The amide library included electrondonating groups (8e17), those with electron-withdrawing groups (18e24), and electronically neutral heterocycles (25e29) (Fig. 4). The sulfonamide functional groups have been essential motifs in medicinal chemistry since the early discovery of sulfonamidecontaining antibacterial drugs [11]. We made the conscious decision to ensure a comprehensive range of sulfonamide derivatives were synthesized. DHAA was dissolved in anhydrous DCM, and 1.1 equivalent of the sulfonyl chloride and 3.0 equivalents of triethylamine were added at 0 C. The mixture was then stirred at room temperature for 1e12 h, generating sulfonamide derivatives with aliphatic and aromatic functional groups (Fig. 5). The products were purified by column chromatography to afford the neat compounds 30e62 (Fig. 5) in 78e98% chemical yield. The compound library was evaluated against the three breast cancer cell lines mentioned above by using an established CellTiterGlo proliferation assay [12]. Their general cytotoxicity was also evaluated in human embryonic kidney (Hek293) and hepatocellular carcinoma (HepG2) cell lines at concentrations <100 mM. A representative heat map of our findings is shown (Fig. 6). Compounds 8e29 showed no antiproliferative activity and no cytotoxicity at 52e65 mM (data not shown) in all the tested cell lines. The electron-donating vs. -withdrawing nature of the amides did not affect the bioactivity properties of these compounds. The sulfonamides 30e62 also provided few bioactive compounds. Gratifyingly, compounds 43 and 61 showed promising activity against MDA-MB-231 cells at 35 mM and 9 mM, respectively (Fig. 6). At the tested concentrations, compound 61 showed no antiproliferative activity against MCF7, indicating this derivative targets a specific pathway (yet to be identified) in the triple-negative
Fig. 1. Constituents of dehydroabietylamine (DHAA, Rosin Amine D) and related abietanes.
T. Ling et al. / European Journal of Medicinal Chemistry 102 (2015) 9e13
Fig. 3. Synthesis of DHAA derivatives.
Fig. 4. DHAA amide library, compounds 8e29.
Fig. 5. DHAA sulfonamide library, compounds 30e62.
Fig. 6. Representative heat map of selected compounds.
11
12
T. Ling et al. / European Journal of Medicinal Chemistry 102 (2015) 9e13
phenotype. Compound 43 showed weak activity against the MA11 cell line at 48 mM and no activity against MCF7 (Fig. 6). Compounds 52e60 may have activity in MDA-MB-231 at higher concentrations (>52.3 mM) as suggested by their activity curves. However, we opted not to pursue those compounds and further evaluate compound 61. Our results demonstrated no obvious structure-activity relationship (SAR) between the amide versus the sulfonamide compounds, indicating that molecular subtleties such as electronic cloud densities in the sulfonamide might be responsible for the biological activity. While structural similarities in the A and B ring of these compounds exist, molecular modeling indicates significant differences in total surface charge densities (see SI). Further biologic target identification will be required to guide the SAR studies for this scaffold. We next conducted in vitro ADME studies of compound 61 (Table 1, SI), using various known drugs as controls [13,14]. The compound showed a good pharmacokinetic profile, with excellent stability in human and mouse microsomes (t1/2 ¼ 0.93 h and t1/ 2 ¼ 0.51 h, respectively) and in human and mouse plasma (t1/ 2 ¼ 6.76 h and t1/2 ¼ 37.69 h, respectively). Its membrane permeability was acceptable (62.5 106 cm/s by PAMPA assay), as was its efflux ratio (0.8 in CACO cells). Its plasma binding affinity was slightly higher (94.8% in human plasma, 99.3% in mouse plasma) than that of the verapamil control (87.6% in human plasma, 88.0% in mouse plasma). Ongoing formulation studies will improve the compound's physicochemical properties (solubility and permeability at different pH levels) and suitability for specific delivery method (s). To identify the potential mechanisms of cell death, compounds 7, 43 and 61 were further evaluated by cell cycle arrest/apoptosis studies (see SI for detailed procedures and results) and immunoblot analysis (Fig. 7). Arrest of the cell cycle is regarded as an effective strategy to inhibit tumor cell proliferation [13]. Our results show that all three compounds (7, 43 and 61) caused cell cycle arrest at G0/G1 (50.76%, 63.41%, and 61.26%, respectively; positive control 43%, negative control 48.36%). The compounds (7, 43 and 61) also showed inhibition of cell cycle progression at G2/M phase (19%, 16.9% and 18.3%, respectively (see SI). Furthermore, compounds 43 and 61 were observed to cause DNA fragmentation in a time dependent manner (12 h point treatment shown in SI). Induction of apoptosis by compounds 7, 43 and 61 was analyzed by flow cytometry with Annexin V and propidium iodide (PI) double staining after 12 h incubation. Three cell populations were evaluated by using established protocols [15], viable
(negative for Annexin V-FITC and PI staining), early apoptotic (Annexin V-FITCepositive, PI-negative), and late apoptotic or dead (positive for Annexin V-FITC and PI) cells. Mean Percentages of apoptotic cells after treatment were 4.4%, 3.4% and 21.1%, respectively (positive control, 25%; negative control, 3.1%, see SI for details). We next conducted experiments to probe key enzymes involved in programmed cell death [16], specifically poly-(ADP-ribose) polymerase (PARP), DR5 and caspase 9, 8, 7, and 3 by western blot analysis at two time points (6 and 12 h) that had shown cellular stress by cell cycle. All three compounds (7, 43 and 61) showed a time dependent PARP increase. Significant amount of cleaved-PARP for compound 43 and 61 at the 12 h time point was observed. DR5 increase was recorded at the 12 h time point for all three compounds. DR5 is a proapoptotic receptor activated by TRAIL and it signals through an intracellular death domain, which includes the Fas-associated death domain and pro-caspase-8 or pro-caspase-10. Death-inducing signaling complex-activated autocleavage of procaspase 8 to active caspase-8 allows downstream cleavage of caspase 9, 7, and 3 among others, leading to the execution of apoptosis [16]. Only traces of cleaved caspase-8 were captured, and a significant amount of procaspase-8 was observed from the 6 to the 12 h time point for compounds 43 and 61 (Fig. 7). Cleaved caspase-9 (or cleaved caspase-3) was present at the 6 h point with diminished expression of full length caspase-9 (or caspase-3) at the 12 h point for compound 43 and 61. However, no cleaved caspase-9 at either time point was observed for compound 7. Full length caspase-7 expression was diminished over time from 6 to 12 h for 7, 43 and 61. The pilot data suggest these compound series may lead to apoptosis in a time dependent manner. Further mechanistic studies, in particular BAX/BAK evaluation, are warranted to gain more knowledge on the detailed mechanism of cell death. 3. Conclusions This study showcases the potential of NP screens in drug discovery. We have identified promising compounds active against the TNBC model (MDA-MB-231 cell line). Our findings indicate that DHAA derivatives (compound 43 and 61) inhibit cell proliferation through G0/G1 phase arrest. Furthermore, compounds 43 and 61 induce apoptosis through activation of caspase-9, caspase-3 and the cleavage of PARP in the above TNBC model. Our current objective is to study the pharmacological effects of compound 61 on critical biological processes involved in breast cancer such as cell migration and invasion. Future studies will be focused on
Fig. 7. Representative western blots of MDA-MB231 cells treated with compounds 7, 43, and 61.
T. Ling et al. / European Journal of Medicinal Chemistry 102 (2015) 9e13
improving the physicochemical properties of these lead compounds for in vivo evaluation. Competing interests The authors declare that they have no competing interests. Acknowledgments This study was supported by the American Lebanese Syrian Associated Charities (ALSAC). The authors thank the HighThroughput Analytical Chemistry core Facility for analytical support, the Cell Cycle Facility for technical assistance and the scientific editing facility at St. Jude Children's Research Hospital. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2015.07.034. References [1] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs over the 30 years from 1981 to 2010, J. Nat. Prod. 75 (2012) 311e335. [2] a) R. Siegel, J. Ma, Z. Zou, A. Jemal, Cancer statistics, 2014, CA Cancer J. Clin. 64 (2014) 9e29; b) M.R. Clemens, O.A. Gladkov, E. Gartner, V. Vladimirov, J. Crown, J. Steinberg, F. Jie, A. Keating, Phase II, multicenter, open-label, randomized study of YM155 plus docetaxel as first-line treatment in patients with HER2-negative metastatic breast cancer, Breast Cancer Res. Treat. 149 (2015) 171e179. [3] Cancer Genome Atlas Network, Comprehensive molecular portraits of human breast tumours, Nature 490 (2012) 61e70. [4] a) A. Papa, D. Caruso, S. Tomao, L. Rossi, E. Zaccarelli, F. Tomao, Triple-negative breast cancer: investigating potential molecular therapeutic target, Expert Opin. Ther. Targets 19 (2015) 55e75; b) M.K. Muellner, B. Mair, Y. Ibrahim, C. Kerzendorfer, H. Lechtermann, C. Trefzer, F. Klepsch, A. Muller, E. Leitner, S.M. Maschler, G.S. Furga, K.L. Bennett, J. Baselga, U. Rix, S. Kubicek, J. Colinge, V. Serra, S. Nijman, Targeting a cell state common to triple-negative breast cancer, Mol. Sys. Biol. 11 (2015) 789; c) J. Crown, J. O’shaughnessy, G. Gullo, Emerging targeted therapies in triplenegative breast cancer, Ann. Oncol. 23 (Suppl. 6) (2012) 56e65; d) K. Santos, M. Laranjo, M. Abrantes, A.F. Brito, C. Goncalves, A.B.S. Ribeiro, M.F. Botelho, M.I.L. Soares, A.S.R. Oliverira, T.M.V.D.P. Melo, Targeting triplenegative breast cancer cells with 6,7-bis(hydroxymethyl)-1H,3h-pyrrolo[1,2c]thiazoles, Eur. J. Med. Chem. 79 (2014) 273e281; e) P. Morales, S.B. Benito, C. Andradas, M.G. Canas, J.M. Flores, P. Goya, J.F. Ruiz, C. Sanchez, N. Jagerovic, Selective, nontoxic CB2 cannabinoid o-quinone with in vivo activity against triple-negative breast cancer, J. Med. Chem. 58 (2015) 2256e2264; f) S. Abdelhamed, S. Yokoyama, A. Refaat, K. Ogura, H. Yagita, S. Awale, I. Saiki, Piperine enhances the efficacy of trail-based therapy for triple-negative breast cancer, Anticancer Res. 34 (2014) 1893e1900. [5] K.J. Chavez, V. Sireesha, S.V. Garimella, S. Lipkowitz, Triple negative breast cancer cell lines: one tool in the search for better treatment of triple negative breast cancer, Breast Dis. 32 (2010) 35e48. [6] a) C.W. Brandt, L.G. Neubauer, Miro resin. Part I. Ferruginol, J. Chem. Soc. (1939) 1031e1037; b) G.C. Harris, Resin acids. V. The composition of the gum oleoresin acids of Pinus Palustris, J. Am. Chem. Soc. 70 (1948) 3671e3674; lez, Aromatic abietane diterpenoids: total syntheses and sync) M.A. Gonza thetic studies, Tetrahedron 71 (2015) 1883e1908; lez, Aromatic abietane diterpenoids: their biological activity and d) M.A. Gonza synthesis, Nat. Prod. Rep. 32 (2015) 684e704.
13
[7] A.M. Gonz alez, J. Clark, M. Connelly, F. Rivas, Antimalarial activity of abietane ferruginol analogues possessing a phthalimide group, Bioorg. Med. Chem. Lett. 24 (2014) 5234e5237. [8] M. Bispo de Jesus, W.F. Zambuzzi, , et al.R.R. Ruela de Sousa, C. Areche, A.C. Santos de Souza, H. Aoyama, G. Guillermo Schmeda-Hirschmann, J.A. Rodriguez, A.R. Monteiro de Souza Brito, M.P. Peppelenbosch, J.D. Hertog, E. De Paula, C.V. Ferreira, Ferruginol suppresses survival signaling pathways in androgen-independent human prostate cancer, Biochimie 90 (2008) 843e858. [9] a) M. Fronza, E. Lamy, S. Gunther, B. Heinzmann, S. Laufer, I. Merfort, Abietane diterpenes induce cytotoxic effects in human pancreatic cancer cell line MIA PaCa2 through different mode of action, Phytochemistry 78 (2012) 107e119; b) M.S. Kang, S. Hirai, T. Goto, K. Kuroyanagi, J.Y. Lee, T. Uemura, Y. Ezaki, N. Takahashi, T. Kawada, Dehydroabietic acid, a phytochemical, acts as ligand for PPARs in macrophages and adipocytes to regulate inflammation, Biochem. Biophys. Res. Comm. 369 (2008) 333e338; c) A. Fallarero, M. Skogman, J. Kujala, M. Rajaratnam, V.M. Moreira, J.Y. Kauhaluoma, P. Vuorela, (þ)-Dehydroabietic acid, an abietane-type diterpene, inhibits staphyloccus aureus biofilms in vitro, Int. J. Mol. Sci. 14 (2013) 12054e12072; d) I.C. Guerrero, L. Andre, L.G. Leon, R.P. machine, J.M. Padron, J.G. Lius, J. Delgadillo, Abietane diterpenoids from Salvia pachyphylla and S. clevelandii with cytotoxic activity against human cancer cell lines, J. Nat. Prod. 69 (2006) 1803e1805; e) M. Roche, A.E.K. Ibrahim, J. Mieszczanek, M. Bienz, LEF1 and B9L shield bcatenin from inactivation by axin, desensitizing colorectal cancer cells to tankyrase inhibitors, Cancer Res. 74 (2014) 1495e1505; f) M. Roche, T.J. Rutherford, D. Gupta, D.B. Veprintsev, B. Saxty, S.M. Freund, M. Bienz, An intrinsically labile a-helix abutting the BCL9-binding site of bcatenin is required for its inhibition by carnosic acid, Nat. Comm. 3 (680) (2012) 1e10. [10] a) Amine D, formerly Rosin Amine D, is a trade-name of the Hercules. b) J.N. Borglin, Soap Sand. Chem. 23 (1947) 147. Chem. Abstr., 43, 9397a, 1949; c) United States Patent, US 7,825,162 B2, Nov 2, 2010. [11] a) A. Kalgutkar, R. Jones, A. Sawant, RSC Drug Discovery Series No. 1, in: D.A. Smith (Ed.), In Metabolism Pharmacokinetics and Toxicity of Functional Groups, RSC, Cambridge, UK, 2010 (Chapter 5); b) C. Hansch, P.G. Sammes, J.B. Taylor, Comprehensive Medicinal, Chemistry, vol. 2, Pergamon Press, Oxford, UK, 1990 (Chapter 7).1; c) N.A. Meanwell, Synopsis of some recent tactical application of bioisosteres in drug design, J. Med. Chem. 54 (2011) 2529e2591. [12] a) Y. Tu, C. Jeffries, H. Ruan, C. Nelson, D. Smithson, A.A. Shelat, K.M. Brown, X.C. Li, J.P. Hester, T. Smillie, I.A. Khan, L. Walker, K. Guy, B. Yan, An automated high-throughput system to fractionate plant natural products for drug discovery, J. Nat. Prod. 73 (2010) 751e754; b) V. Hadi, M. Hotard, T. Ling, Y.G. Salinas, G. Palacios, M. Connelly, F. Rivas, Structure activity relationship of Jatropha isabelli natural products and their synthetic analogs as potential antimalarial therapeutic agents, Eur. J. Med. Chem. 65 (2013) 376e380. [13] L. Di, E.H. Kerns, X.J. Ma, Y. Huang, G.T. Carter, Applications of high throughput microsomal stability assay in drug discovery, Comb. Chem. High. Throughput Screen 11 (2008) 469e476. [14] A.M. Marino, M. Yarde, H. Patel, S. Chong, P.V. Balimane, Validation of the 96 well Caco-2 cell culture model for high throughput permeability assessment of discovery compounds, Int. J. Pharm. 297 (2005) 235e241. [15] a) C. Liu, Z. Liu, M. Li, X. Li, Y.-S. Wong, S.-M. Ngai, W. Zheng, Y. Zhag, T. Chen, Enhancement of auranofin-induced apoptosis in MCF-7 human breast cells by selenocystine, a synergistic inhibitor of thioredoxin reductase, PLOS One 8 (2013) e53945; b) G.H. Williams, K. Stoeber, The cell cycle and cancer, J. Pathol. 226 (2011) 352e364. [16] a) L. Galluzzi, I. Vitale, J.M. Abrams, E.S. Alnemri, E.H. Baehrecke, G. Kroemer, et al., Molecular definitions of cell death subroutines recommendations of the nomenclature committee on cell death 2012, Cell Death Differ. 19 (2012) 107e120; b) Y. Fuchs, H. Stellar, Programmed cell death in animal development and diseases, Cell 147 (2011) 742e758; c) N. Ozoren, W.S. El-Deiry, Cell surface death receptor signaling in normal and cancer cells, Semin. Cancer Biol. 13 (2003) 135e147.
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
(þ)-Dehydroabietylamine derivatives target triple-negative breast cancer lez b, Lekh Nath Gautam a, Michele Connelly a, Taotao Ling a, My Tran a, Miguel A. Gonza Rachael K. Wood c, Iram Fatima c, Gustavo Miranda-Carboni c, Fatima Rivas a, * a b c
Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN 38105-3678, USA nica, Universidad de Valencia, 46100 Burjassot, Valencia, Spain Departamento de Química Orga Department of Medicine, The University of Tennessee Health Science Center, Memphis, TN 38163, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 June 2015 Received in revised form 15 July 2015 Accepted 17 July 2015 Available online 23 July 2015
Breast cancer remains the leading cause of cancer-related death among women. The invasive triplenegative subtype is unresponsive to estrogen therapy, and few effective treatments are available. In search of new chemical scaffolds to target this disease, we conducted a phenotypic screen against the human breast carcinoma cell lines MDA-MB-231, MA11, and MCF-7 using terrestrial natural products. Natural products that preferentially inhibited proliferation of triple-negative MDA-MB-231 cells over estrogen receptorepositive cells were further studied; herein we focused on the abietanes. The activity of the abietane carnosol prompted us to generate a focus library from the readily available (þ)-dehydroabietylamine. The lead compound 61 displayed a promising EC50 of 9.0 mM against MDA-MB-231 and our mechanistic studies indicate it induced apoptosis, which was associated with activation of caspase-9 and -3 and the cleavage of PARP. Here we describe our current progress towards this promising therapeutic candidate. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: (þ)-Dehydroabietylamine Abietane Apoptosis Breast cancer Triple-negative
1. Introduction Natural products (NPs) are an integral component of drug discovery programs and have had their greatest success in the oncology field. Approximately 60% of clinically approved anticancer drugs are based on secondary metabolites found in nature [1]. The natural world is a vast, rich, and relatively untapped source of molecular scaffolds for reagent compounds or therapeutic agents. As more sensitive screening assays are developed, promising scaffolds from NPs will be identified as potential leads. Breast cancer is a leading cause of premature death worldwide; in the United States, it was estimated to cause more than 40,000 deaths in 2014 [2]. Breast cancer is classified according to its expression of estrogen receptor (ER), progesterone receptor (PR), and/or human epidermal growth factor receptor (Her2/neu) [3], and current therapies target these receptors. Triple-negative breast carcinoma (TNBC) is a heterogeneous subset of tumors
* Corresponding author. Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place Memphis, TN 381053678, USA. E-mail address: [email protected] (F. Rivas). http://dx.doi.org/10.1016/j.ejmech.2015.07.034 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
defined only by their aggressive nature and the absence of the mentioned receptors. Its poor overall prognosis reflects not only its inherent aggressiveness but also the lack of targeted therapies [4]. As approximately one million breast cancers are diagnosed each year and about 15% are TNBC [4], there is a clear and urgent need for new therapies. The main goal of our drug discovery program is to identify novel NP molecular scaffolds for research and treatment of TNBC. We reasoned that a broad phenotypic screen against breast cancer cell lines using pure NPs would lay the foundation for a medicinal chemistry program. Here we present our lead findings. 2. Results and discussion We used the triple-negative MDA-MB-231 cell line and the ERpositive MA11 and MCF7 cell lines as cellular models of breast carcinoma. The MDA-MB-231 line has been extensively characterized and provides a robust in vitro model of TNBC. Given the rise of questionable cell lines, we opted for only validated models [5]. We screened a library of pure NPs (2300 compounds) primarily from terrestrial sources for their antiproliferative effect on the three cell lines using CellTiterGlo [12] (see SI).
10
T. Ling et al. / European Journal of Medicinal Chemistry 102 (2015) 9e13
Several hits were observed in the ER-positive cell lines, while few were selective for TNBC. A few representatives of the abietane family members showed promising results. However, the non-aromatic abietanes, abietic acid 1 and primaric acid 2 (Fig. 1) showed no activity against the TNBC cell line. Compounds 3e7 displayed anti-proliferative activity against MDA-MB-231 cells at 50 mM, with no detectable cytotoxicity observed in Hek293 and HepG2 cell lines. Of particular interest were compounds 6 and 7, showing activity against MDAMB-231 cells at 22 and 29 mM, respectively (see SI). The abietanes are diterpenoids possessing an ABC ring system; dehydroabietic acid is a classic example (Fig. 1) [6]. They are found in the resin of various terrestrial plants of the Podocarpaceae and Cupressaceae families and in other angiosperms [7]. The abietanes have demonstrated a broad range of biological properties, particularly anticancer activities. For example, carnosic acid has been established as a BCL-9 inhibitor and has shown potency against colon cancer in vitro and in vivo [8,9], while related NPs have shown activity against human pancreatic tumor cell lines [9]. Because these compounds are abundant in nature, semi-syntheses and derivatization programs are sustainable and can generate powerful structure-function studies to characterize their medicinal properties. Rosin Amine D, an inexpensive oil consisting of ~65% DHAA plus a mixture of the dihydro- and tetrahydro-abietylamines as well as other amines. These compounds are the product of basetreated Rosin acid nitriles, which are naturally abundant in resins [10]. DHAA (4, Fig. 1, featuring a chemical handle at C18) serves as a resolving reagent for racemic carboxylic acids and as a chiral synthon for pharmaceutical preparations [10]. Conformational evaluation of these abietane compounds suggested the hypothesis that the aromatic C ring might be required for the observed anti-proliferative activity. Fig. 2 shows the superposition of model compounds 1, 2 and 4 at the lowest energy conformation (DFT). While they possess a closely related pharmacophore, the A and C rings show clear differences (no hydrogens are shown for clarity). In search of compounds with greater anti-TNBC potency, we chose compound 4 (DHAA) as the starting point for our synthetic plan. We focused our attention on the C18 of the abietane core. Given the obvious versatility of the primary amine, this approach would probe a broad range of chemical space near the A ring while maintain the B and C rings intact. Our findings support the further use of this unique chemical scaffold for development of therapeutic agents against TNBC. Being affordable, abietanes can provide inexpensive lead candidates. After column purification of the Rosin D mixture, DHAA was obtained as 50 g batches of light yellow oil in roughly 64% yield. As the DHAA supply was sufficient, we converted it to both amide and corresponding sulfonamide derivatives (Fig. 3). We first synthesized the DHAA amides by peptide coupling of DHAA with a broad range of carboxylic chlorides to evaluate electronic properties. DHAA was treated with 1.1 equivalent of the corresponding
Fig. 2. Representative western blots of MDA-MB231 cells treated with compounds 7, 43, and 61. Panel A. After 6 hrs of treatment. Panel B. After 12 hrs of treatment. Cells near 70% confluence were treated with the compounds, and the indicated cellular proteins were probed in 3 independent experiments. Loading control, b-actin; positive control, 2.5 mM staurosporine; vehicle (28 mM DMSO).
acid chloride and 3.0 equivalents of triethylamine in dichloromethane (DCM) at room temperature, stirring for 1e12 h (Fig. 3). The products were purified by column chromatography to afford the neat compounds 8e29 in 82%e95% chemical yield. The focused amide library included aliphatic and aromatic functional groups for a thorough evaluation of chemical space and electronic properties [11]. The amide library included electrondonating groups (8e17), those with electron-withdrawing groups (18e24), and electronically neutral heterocycles (25e29) (Fig. 4). The sulfonamide functional groups have been essential motifs in medicinal chemistry since the early discovery of sulfonamidecontaining antibacterial drugs [11]. We made the conscious decision to ensure a comprehensive range of sulfonamide derivatives were synthesized. DHAA was dissolved in anhydrous DCM, and 1.1 equivalent of the sulfonyl chloride and 3.0 equivalents of triethylamine were added at 0 C. The mixture was then stirred at room temperature for 1e12 h, generating sulfonamide derivatives with aliphatic and aromatic functional groups (Fig. 5). The products were purified by column chromatography to afford the neat compounds 30e62 (Fig. 5) in 78e98% chemical yield. The compound library was evaluated against the three breast cancer cell lines mentioned above by using an established CellTiterGlo proliferation assay [12]. Their general cytotoxicity was also evaluated in human embryonic kidney (Hek293) and hepatocellular carcinoma (HepG2) cell lines at concentrations <100 mM. A representative heat map of our findings is shown (Fig. 6). Compounds 8e29 showed no antiproliferative activity and no cytotoxicity at 52e65 mM (data not shown) in all the tested cell lines. The electron-donating vs. -withdrawing nature of the amides did not affect the bioactivity properties of these compounds. The sulfonamides 30e62 also provided few bioactive compounds. Gratifyingly, compounds 43 and 61 showed promising activity against MDA-MB-231 cells at 35 mM and 9 mM, respectively (Fig. 6). At the tested concentrations, compound 61 showed no antiproliferative activity against MCF7, indicating this derivative targets a specific pathway (yet to be identified) in the triple-negative
Fig. 1. Constituents of dehydroabietylamine (DHAA, Rosin Amine D) and related abietanes.
T. Ling et al. / European Journal of Medicinal Chemistry 102 (2015) 9e13
Fig. 3. Synthesis of DHAA derivatives.
Fig. 4. DHAA amide library, compounds 8e29.
Fig. 5. DHAA sulfonamide library, compounds 30e62.
Fig. 6. Representative heat map of selected compounds.
11
12
T. Ling et al. / European Journal of Medicinal Chemistry 102 (2015) 9e13
phenotype. Compound 43 showed weak activity against the MA11 cell line at 48 mM and no activity against MCF7 (Fig. 6). Compounds 52e60 may have activity in MDA-MB-231 at higher concentrations (>52.3 mM) as suggested by their activity curves. However, we opted not to pursue those compounds and further evaluate compound 61. Our results demonstrated no obvious structure-activity relationship (SAR) between the amide versus the sulfonamide compounds, indicating that molecular subtleties such as electronic cloud densities in the sulfonamide might be responsible for the biological activity. While structural similarities in the A and B ring of these compounds exist, molecular modeling indicates significant differences in total surface charge densities (see SI). Further biologic target identification will be required to guide the SAR studies for this scaffold. We next conducted in vitro ADME studies of compound 61 (Table 1, SI), using various known drugs as controls [13,14]. The compound showed a good pharmacokinetic profile, with excellent stability in human and mouse microsomes (t1/2 ¼ 0.93 h and t1/ 2 ¼ 0.51 h, respectively) and in human and mouse plasma (t1/ 2 ¼ 6.76 h and t1/2 ¼ 37.69 h, respectively). Its membrane permeability was acceptable (62.5 106 cm/s by PAMPA assay), as was its efflux ratio (0.8 in CACO cells). Its plasma binding affinity was slightly higher (94.8% in human plasma, 99.3% in mouse plasma) than that of the verapamil control (87.6% in human plasma, 88.0% in mouse plasma). Ongoing formulation studies will improve the compound's physicochemical properties (solubility and permeability at different pH levels) and suitability for specific delivery method (s). To identify the potential mechanisms of cell death, compounds 7, 43 and 61 were further evaluated by cell cycle arrest/apoptosis studies (see SI for detailed procedures and results) and immunoblot analysis (Fig. 7). Arrest of the cell cycle is regarded as an effective strategy to inhibit tumor cell proliferation [13]. Our results show that all three compounds (7, 43 and 61) caused cell cycle arrest at G0/G1 (50.76%, 63.41%, and 61.26%, respectively; positive control 43%, negative control 48.36%). The compounds (7, 43 and 61) also showed inhibition of cell cycle progression at G2/M phase (19%, 16.9% and 18.3%, respectively (see SI). Furthermore, compounds 43 and 61 were observed to cause DNA fragmentation in a time dependent manner (12 h point treatment shown in SI). Induction of apoptosis by compounds 7, 43 and 61 was analyzed by flow cytometry with Annexin V and propidium iodide (PI) double staining after 12 h incubation. Three cell populations were evaluated by using established protocols [15], viable
(negative for Annexin V-FITC and PI staining), early apoptotic (Annexin V-FITCepositive, PI-negative), and late apoptotic or dead (positive for Annexin V-FITC and PI) cells. Mean Percentages of apoptotic cells after treatment were 4.4%, 3.4% and 21.1%, respectively (positive control, 25%; negative control, 3.1%, see SI for details). We next conducted experiments to probe key enzymes involved in programmed cell death [16], specifically poly-(ADP-ribose) polymerase (PARP), DR5 and caspase 9, 8, 7, and 3 by western blot analysis at two time points (6 and 12 h) that had shown cellular stress by cell cycle. All three compounds (7, 43 and 61) showed a time dependent PARP increase. Significant amount of cleaved-PARP for compound 43 and 61 at the 12 h time point was observed. DR5 increase was recorded at the 12 h time point for all three compounds. DR5 is a proapoptotic receptor activated by TRAIL and it signals through an intracellular death domain, which includes the Fas-associated death domain and pro-caspase-8 or pro-caspase-10. Death-inducing signaling complex-activated autocleavage of procaspase 8 to active caspase-8 allows downstream cleavage of caspase 9, 7, and 3 among others, leading to the execution of apoptosis [16]. Only traces of cleaved caspase-8 were captured, and a significant amount of procaspase-8 was observed from the 6 to the 12 h time point for compounds 43 and 61 (Fig. 7). Cleaved caspase-9 (or cleaved caspase-3) was present at the 6 h point with diminished expression of full length caspase-9 (or caspase-3) at the 12 h point for compound 43 and 61. However, no cleaved caspase-9 at either time point was observed for compound 7. Full length caspase-7 expression was diminished over time from 6 to 12 h for 7, 43 and 61. The pilot data suggest these compound series may lead to apoptosis in a time dependent manner. Further mechanistic studies, in particular BAX/BAK evaluation, are warranted to gain more knowledge on the detailed mechanism of cell death. 3. Conclusions This study showcases the potential of NP screens in drug discovery. We have identified promising compounds active against the TNBC model (MDA-MB-231 cell line). Our findings indicate that DHAA derivatives (compound 43 and 61) inhibit cell proliferation through G0/G1 phase arrest. Furthermore, compounds 43 and 61 induce apoptosis through activation of caspase-9, caspase-3 and the cleavage of PARP in the above TNBC model. Our current objective is to study the pharmacological effects of compound 61 on critical biological processes involved in breast cancer such as cell migration and invasion. Future studies will be focused on
Fig. 7. Representative western blots of MDA-MB231 cells treated with compounds 7, 43, and 61.
T. Ling et al. / European Journal of Medicinal Chemistry 102 (2015) 9e13
improving the physicochemical properties of these lead compounds for in vivo evaluation. Competing interests The authors declare that they have no competing interests. Acknowledgments This study was supported by the American Lebanese Syrian Associated Charities (ALSAC). The authors thank the HighThroughput Analytical Chemistry core Facility for analytical support, the Cell Cycle Facility for technical assistance and the scientific editing facility at St. Jude Children's Research Hospital. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2015.07.034. References [1] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs over the 30 years from 1981 to 2010, J. Nat. Prod. 75 (2012) 311e335. [2] a) R. Siegel, J. Ma, Z. Zou, A. Jemal, Cancer statistics, 2014, CA Cancer J. Clin. 64 (2014) 9e29; b) M.R. Clemens, O.A. Gladkov, E. Gartner, V. Vladimirov, J. Crown, J. Steinberg, F. Jie, A. Keating, Phase II, multicenter, open-label, randomized study of YM155 plus docetaxel as first-line treatment in patients with HER2-negative metastatic breast cancer, Breast Cancer Res. Treat. 149 (2015) 171e179. [3] Cancer Genome Atlas Network, Comprehensive molecular portraits of human breast tumours, Nature 490 (2012) 61e70. [4] a) A. Papa, D. Caruso, S. Tomao, L. Rossi, E. Zaccarelli, F. Tomao, Triple-negative breast cancer: investigating potential molecular therapeutic target, Expert Opin. Ther. Targets 19 (2015) 55e75; b) M.K. Muellner, B. Mair, Y. Ibrahim, C. Kerzendorfer, H. Lechtermann, C. Trefzer, F. Klepsch, A. Muller, E. Leitner, S.M. Maschler, G.S. Furga, K.L. Bennett, J. Baselga, U. Rix, S. Kubicek, J. Colinge, V. Serra, S. Nijman, Targeting a cell state common to triple-negative breast cancer, Mol. Sys. Biol. 11 (2015) 789; c) J. Crown, J. O’shaughnessy, G. Gullo, Emerging targeted therapies in triplenegative breast cancer, Ann. Oncol. 23 (Suppl. 6) (2012) 56e65; d) K. Santos, M. Laranjo, M. Abrantes, A.F. Brito, C. Goncalves, A.B.S. Ribeiro, M.F. Botelho, M.I.L. Soares, A.S.R. Oliverira, T.M.V.D.P. Melo, Targeting triplenegative breast cancer cells with 6,7-bis(hydroxymethyl)-1H,3h-pyrrolo[1,2c]thiazoles, Eur. J. Med. Chem. 79 (2014) 273e281; e) P. Morales, S.B. Benito, C. Andradas, M.G. Canas, J.M. Flores, P. Goya, J.F. Ruiz, C. Sanchez, N. Jagerovic, Selective, nontoxic CB2 cannabinoid o-quinone with in vivo activity against triple-negative breast cancer, J. Med. Chem. 58 (2015) 2256e2264; f) S. Abdelhamed, S. Yokoyama, A. Refaat, K. Ogura, H. Yagita, S. Awale, I. Saiki, Piperine enhances the efficacy of trail-based therapy for triple-negative breast cancer, Anticancer Res. 34 (2014) 1893e1900. [5] K.J. Chavez, V. Sireesha, S.V. Garimella, S. Lipkowitz, Triple negative breast cancer cell lines: one tool in the search for better treatment of triple negative breast cancer, Breast Dis. 32 (2010) 35e48. [6] a) C.W. Brandt, L.G. Neubauer, Miro resin. Part I. Ferruginol, J. Chem. Soc. (1939) 1031e1037; b) G.C. Harris, Resin acids. V. The composition of the gum oleoresin acids of Pinus Palustris, J. Am. Chem. Soc. 70 (1948) 3671e3674; lez, Aromatic abietane diterpenoids: total syntheses and sync) M.A. Gonza thetic studies, Tetrahedron 71 (2015) 1883e1908; lez, Aromatic abietane diterpenoids: their biological activity and d) M.A. Gonza synthesis, Nat. Prod. Rep. 32 (2015) 684e704.
13
[7] A.M. Gonz alez, J. Clark, M. Connelly, F. Rivas, Antimalarial activity of abietane ferruginol analogues possessing a phthalimide group, Bioorg. Med. Chem. Lett. 24 (2014) 5234e5237. [8] M. Bispo de Jesus, W.F. Zambuzzi, , et al.R.R. Ruela de Sousa, C. Areche, A.C. Santos de Souza, H. Aoyama, G. Guillermo Schmeda-Hirschmann, J.A. Rodriguez, A.R. Monteiro de Souza Brito, M.P. Peppelenbosch, J.D. Hertog, E. De Paula, C.V. Ferreira, Ferruginol suppresses survival signaling pathways in androgen-independent human prostate cancer, Biochimie 90 (2008) 843e858. [9] a) M. Fronza, E. Lamy, S. Gunther, B. Heinzmann, S. Laufer, I. Merfort, Abietane diterpenes induce cytotoxic effects in human pancreatic cancer cell line MIA PaCa2 through different mode of action, Phytochemistry 78 (2012) 107e119; b) M.S. Kang, S. Hirai, T. Goto, K. Kuroyanagi, J.Y. Lee, T. Uemura, Y. Ezaki, N. Takahashi, T. Kawada, Dehydroabietic acid, a phytochemical, acts as ligand for PPARs in macrophages and adipocytes to regulate inflammation, Biochem. Biophys. Res. Comm. 369 (2008) 333e338; c) A. Fallarero, M. Skogman, J. Kujala, M. Rajaratnam, V.M. Moreira, J.Y. Kauhaluoma, P. Vuorela, (þ)-Dehydroabietic acid, an abietane-type diterpene, inhibits staphyloccus aureus biofilms in vitro, Int. J. Mol. Sci. 14 (2013) 12054e12072; d) I.C. Guerrero, L. Andre, L.G. Leon, R.P. machine, J.M. Padron, J.G. Lius, J. Delgadillo, Abietane diterpenoids from Salvia pachyphylla and S. clevelandii with cytotoxic activity against human cancer cell lines, J. Nat. Prod. 69 (2006) 1803e1805; e) M. Roche, A.E.K. Ibrahim, J. Mieszczanek, M. Bienz, LEF1 and B9L shield bcatenin from inactivation by axin, desensitizing colorectal cancer cells to tankyrase inhibitors, Cancer Res. 74 (2014) 1495e1505; f) M. Roche, T.J. Rutherford, D. Gupta, D.B. Veprintsev, B. Saxty, S.M. Freund, M. Bienz, An intrinsically labile a-helix abutting the BCL9-binding site of bcatenin is required for its inhibition by carnosic acid, Nat. Comm. 3 (680) (2012) 1e10. [10] a) Amine D, formerly Rosin Amine D, is a trade-name of the Hercules. b) J.N. Borglin, Soap Sand. Chem. 23 (1947) 147. Chem. Abstr., 43, 9397a, 1949; c) United States Patent, US 7,825,162 B2, Nov 2, 2010. [11] a) A. Kalgutkar, R. Jones, A. Sawant, RSC Drug Discovery Series No. 1, in: D.A. Smith (Ed.), In Metabolism Pharmacokinetics and Toxicity of Functional Groups, RSC, Cambridge, UK, 2010 (Chapter 5); b) C. Hansch, P.G. Sammes, J.B. Taylor, Comprehensive Medicinal, Chemistry, vol. 2, Pergamon Press, Oxford, UK, 1990 (Chapter 7).1; c) N.A. Meanwell, Synopsis of some recent tactical application of bioisosteres in drug design, J. Med. Chem. 54 (2011) 2529e2591. [12] a) Y. Tu, C. Jeffries, H. Ruan, C. Nelson, D. Smithson, A.A. Shelat, K.M. Brown, X.C. Li, J.P. Hester, T. Smillie, I.A. Khan, L. Walker, K. Guy, B. Yan, An automated high-throughput system to fractionate plant natural products for drug discovery, J. Nat. Prod. 73 (2010) 751e754; b) V. Hadi, M. Hotard, T. Ling, Y.G. Salinas, G. Palacios, M. Connelly, F. Rivas, Structure activity relationship of Jatropha isabelli natural products and their synthetic analogs as potential antimalarial therapeutic agents, Eur. J. Med. Chem. 65 (2013) 376e380. [13] L. Di, E.H. Kerns, X.J. Ma, Y. Huang, G.T. Carter, Applications of high throughput microsomal stability assay in drug discovery, Comb. Chem. High. Throughput Screen 11 (2008) 469e476. [14] A.M. Marino, M. Yarde, H. Patel, S. Chong, P.V. Balimane, Validation of the 96 well Caco-2 cell culture model for high throughput permeability assessment of discovery compounds, Int. J. Pharm. 297 (2005) 235e241. [15] a) C. Liu, Z. Liu, M. Li, X. Li, Y.-S. Wong, S.-M. Ngai, W. Zheng, Y. Zhag, T. Chen, Enhancement of auranofin-induced apoptosis in MCF-7 human breast cells by selenocystine, a synergistic inhibitor of thioredoxin reductase, PLOS One 8 (2013) e53945; b) G.H. Williams, K. Stoeber, The cell cycle and cancer, J. Pathol. 226 (2011) 352e364. [16] a) L. Galluzzi, I. Vitale, J.M. Abrams, E.S. Alnemri, E.H. Baehrecke, G. Kroemer, et al., Molecular definitions of cell death subroutines recommendations of the nomenclature committee on cell death 2012, Cell Death Differ. 19 (2012) 107e120; b) Y. Fuchs, H. Stellar, Programmed cell death in animal development and diseases, Cell 147 (2011) 742e758; c) N. Ozoren, W.S. El-Deiry, Cell surface death receptor signaling in normal and cancer cells, Semin. Cancer Biol. 13 (2003) 135e147.