• ISSN 16748301
  • CN 32-1810/R
Volume 33 Issue 2
Mar.  2019
Article Contents

Citation:

Design, synthesis and biological evaluation of selective survivin inhibitors

  • Corresponding author: Wei Li, wli@uthsc.edu
  • Received Date: 2016-12-24
    Accepted Date: 2017-01-04
  • The differential distribution between cancer cells and normal adult tissues makes survivin a very attractive cancer drug target. We have previously reported a series of novel selective survivin inhibitors with the most potent compound MX106 reaching nanomolar activity in several cancer cell lines. Further optimization of the MX106 scaffold leads to the discovery of more potent and more selective survivin inhibitors. Various structural modifications were synthesized and their anticancer activities were evaluated to determine the structure activity relationships for this MX106 scaffold. In vitro anti-proliferative assays using two human melanoma cell lines showed that several new analogs have improved potency compared to MX106. Very interestingly, these new analogs generally showed significantly higher potency against P-glycoprotein overexpressed cells compared with the corresponding parental cells, suggesting that these compounds may strongly sensitize tumors that have high expressions of the Pglycoprotein drug efflux pumps. Western blotting analysis confirmed that the new MX106 analogs maintained their mechanism of actions by selectively suppressing survivin expression level among major inhibitors of apoptotic proteins and induced strong apoptosis in melanoma tumor cells.
  • 加载中
  • [1] Athanasoula KCh, Gogas H, Polonifi K, et al. Survivin beyond physiology:orchestration of multistep carcinogenesis and therapeutic potentials[J]. Cancer Lett, 2014, 347(2):175-182. doi: 10.1016/j.canlet.2014.02.014
    [2] Garg H, Suri P, Gupta JC, et al. Survivin:a unique target for tumor therapy[J]. Cancer Cell Int, 2016, 16:49. doi: 10.1186/s12935-016-0326-1
    [3] Andersen MH, Svane IM, Becker JC, et al. The universal character of the tumor-associated antigen survivin[J]. Clin Cancer Res, 2007, 13(20):5991-5994. doi: 10.1158/1078-0432.CCR-07-0686
    [4] Hartman ML, Czyz M. Anti-apoptotic proteins on guard of melanoma cell survival[J]. Cancer Lett, 2013, 331(1):24-34. doi: 10.1016/j.canlet.2013.01.010
    [5] Mita AC, Mita MM, Nawrocki ST, et al. Survivin:key regulator of mitosis and apoptosis and novel target for cancer therapeutics[J]. Clin Cancer Res, 2008, 14(16):5000-5005. doi: 10.1158/1078-0432.CCR-08-0746
    [6] Carmena M, Wheelock M, Funabiki H, et al. The chromosomal passenger complex (CPC):from easy rider to the godfather of mitosis[J]. Nat Rev Mol Cell Biol, 2012, 13(12):789-803. doi: 10.1038/nrm3474
    [7] Knauer SK, Mann W, Stauber RH. Survivin's dual role:an export's view[J]. Cell Cycle, 2007, 6(5):518-521. doi: 10.4161/cc.6.5.3902
    [8] Altieri DC. Targeting survivin in cancer[J]. Cancer Lett, 2013, 332(2):225-228. doi: 10.1016/j.canlet.2012.03.005
    [9] Chen X, Duan N, Zhang C, et al. Survivin and tumorigenesis:molecular mechanisms and therapeutic strategies[J]. J Cancer, 2016, 7(3):314-323. doi: 10.7150/jca.13332
    [10] O'Connor DS, Wall NR, Porter AC, et al. A p34(cdc2) survival checkpoint in cancer[J]. Cancer Cell, 2002, 2(1):43-54. doi: 10.1016/S1535-6108(02)00084-3
    [11] Jha K, Shukla M, Pandey M. Survivin expression and targeting in breast cancer[J]. Surg Oncol, 2012, 21(2):125-131. doi: 10.1016/j.suronc.2011.01.001
    [12] Kanwar JR, Kamalapuram SK, Kanwar RK. Targeting survivin in cancer:patent review[J]. Expert Opin Ther Pat, 2010, 20(12):1723-1737. doi: 10.1517/13543776.2010.533657
    [13] Cho HJ, Kim HR, Park YS, et al. Prognostic value of survivin expression in stage Ⅲ non-small cell lung cancer patients treated with platinum-based therapy[J]. Surg Oncol, 2015, 24(4):329-334. doi: 10.1016/j.suronc.2015.09.001
    [14] Huang YJ, Qi WX, He AN, et al. The prognostic value of survivin expression in patients with colorectal carcinoma:a meta-analysis[J]. Jpn J Clin Oncol, 2013, 43(10):988-995. doi: 10.1093/jjco/hyt103
    [15] Singh N, Krishnakumar S, Kanwar RK, et al. Clinical aspects for survivin:a crucial molecule for targeting drug-resistant cancers[J]. Drug Discov Today, 2015, 20(5):578-587. doi: 10.1016/j.drudis.2014.11.013
    [16] Tonini G, Vincenzi B, Santini D, et al. Nuclear and cytoplasmic expression of survivin in 67 surgically resected pancreatic cancer patients[J]. Br J Cancer, 2005, 92(12):2225-2232. doi: 10.1038/sj.bjc.6602632
    [17] Wang J, Lu Z, Yeung BZ, et al. Tumor priming enhances siRNA delivery and transfection in intraperitoneal tumors[J]. J Control Release, 2014, 178:79-85. doi: 10.1016/j.jconrel.2014.01.012
    [18] Groner B., Weiss A.Targeting survivin in cancer:novel drug development approaches[J]. BioDrugs:clinical immunotherapeutics, biopharmaceuticals and gene therapy, 2014, 28(1):27-39. doi: 10.1007/s40259-013-0058-x
    [19] Xiao M, Li W. Recent advances on small-molecule survivin inhibitors[J]. Curr Med Chem, 2015, 22(9):1136-1146. doi: 10.2174/0929867322666150114102146
    [20] Ling X, Cao S, Cheng Q, et al. A novel small molecule FL118 that selectively inhibits survivin, Mcl-1, XIAP and cIAP2 in a p53-independent manner, shows superior antitumor activity[J]. PLoS One, 2012, 7(9):e45571. doi: 10.1371/journal.pone.0045571
    [21] Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer:role of ATP-dependent transporters[J]. Nat Rev Cancer, 2002, 2(1):48-58. doi: 10.1038/nrc706
    [22] Li W, Zhang H, Assaraf YG, et al. Overcoming ABC transporter-mediated multidrug resistance:Molecular mechanisms and novel therapeutic drug strategies[J]. Drug Resist Updat, 2016, 27:14-29. doi: 10.1016/j.drup.2016.05.001
    [23] Kita A., Nakahara T., Takeuchi M., Kinoyama I., Yamanaka K., Minematsu T., Mitsuoka K., Fushiki H., Miyoshi S., Sasamata M., Miyata K.Survivin supressant:a promising target for cancer therapy and pharmacological profiles of YM155[J]. Nihon yakurigaku zasshi. Folia pharmacologica Japonica, 2010, 136(4):198-203. doi: 10.1254/fpj.136.198
    [24] Rauch A, Hennig D, Schäfer C, et al. Survivin and YM155:how faithful is the liaison[J]? Biochim Biophys Acta, 2014, 1845(2):202-220.
    [25] Wang J, Li W. Discovery of novel second mitochondria-derived activator of caspase mimetics as selective inhibitor of apoptosis protein inhibitors[J]. J Pharmacol Exp Ther, 2014, 349(2):319-329. doi: 10.1124/jpet.113.212019
    [26] Xiao M, Wang J, Lin Z, et al. Design, synthesis and structureactivity relationship studies of novel survivin inhibitors with potent anti-proliferative properties. PLoS One, 2015, 10(6):e0129807. doi: 10.1371/journal.pone.0129807
    [27] Wang J, Chen J, Miller DD, et al. Synergistic combination of novel tubulin inhibitor ABI-274 and vemurafenib overcome vemurafenib acquired resistance in BRAFV600E melanoma[J]. Mol Cancer Ther, 2014, 13(1):16-26. doi: 10.1158/1535-7163.MCT-13-0212
    [28] Hwang DJ, Wang J, Li W, et al. Structural optimization of indole derivatives acting at colchicine binding site as potential anticancer agents[J]. ACS Med Chem Lett, 2015, 6(9):993- 997. doi: 10.1021/acsmedchemlett.5b00208
    [29] Akiyama S, Fojo A, Hanover JA, et al. Isolation and genetic characterization of human KB cell lines resistant to multiple drugs[J]. Somat Cell Mol Genet, 1985, 11(2):117-126. doi: 10.1007/BF01534700
    [30] Robey RW, Shukla S, Finley EM, et al. Inhibition of Pglycoprotein (ABCB1)- and multidrug resistance-associated protein 1(ABCC1)-mediated transport by the orally administered inhibitor, CBT-1((R))[J]. Biochem Pharmacol, 2008, 75(6):1302-1312. doi: 10.1016/j.bcp.2007.12.001
    [31] Carmichael J, DeGraff WG, Gazdar AF, et al. Evaluation of a tetrazolium-based semiautomated colorimetric assay:assessment of chemosensitivity testing. Cancer Res, 1987, 47(4):936-942.
    [32] Verniest G, Wang X, De Kimpe N, et al. Heteroaryl crosscoupling as an entry toward the synthesis of lavendamycin analogues:a model study. J Org Chem, 2010, 75(2):424-433. doi: 10.1021/jo902287t
    [33] Chouhan M, Kumar K, Sharma R, et al. NiCl2 center dot 6H(2)O/NaBH4 in methanol:a mild and efficient strategy for chemoselective deallylation/debenzylation of aryl ethers[J]. Tetrahedron Lett, 2013, 54(34):4540-4543. doi: 10.1016/j.tetlet.2013.06.072
    [34] Ludwig JA, Szakács G, Martin SE, et al. Selective toxicity of NSC73306 in MDR1-positive cells as a new strategy to circumvent multidrug resistance in cancer[J]. Cancer Res, 2006, 66(9):4808-4815. doi: 10.1158/0008-5472.CAN-05-3322
    [35] Liu F, Xie ZH, Cai GP, et al. The effect of survivin on multidrug resistance mediated by P-glycoprotein in MCF-7 and its adriamycin resistant cells[J]. Biol Pharm Bull, 2007, 30(12):2279-2283. doi: 10.1248/bpb.30.2279
    [36] Shi Z, Liang YJ, Chen ZS, et al. Overexpression of Survivin and XIAP in MDR cancer cells unrelated to P-glycoprotein[J]. Oncol Rep, 2007, 17(4):969-976.
    [37] Liu F, Liu S, He S, et al. Survivin transcription is associated with P-glycoprotein/MDR1 overexpression in the multidrug resistance of MCF-7 breast cancer cells[J]. Oncol Rep, 2010, 23(5):1469-1475.
  • JBR-2016-0173-supplementary.pdf
  • 加载中

Figures(12) / Tables(3)

Article Metrics

Article views(159) PDF downloads(16) Cited by()

Related
Proportional views
通讯作者: 陈斌, bchen63@163.com
  • 1. 

    沈阳化工大学材料科学与工程学院 沈阳 110142

  1. 本站搜索
  2. 百度学术搜索
  3. 万方数据库搜索
  4. CNKI搜索

Design, synthesis and biological evaluation of selective survivin inhibitors

    Corresponding author: Wei Li, wli@uthsc.edu
  • 1. Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN, 38163, USA
  • 2. Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University, Queens, NY 11439, USA

Abstract: The differential distribution between cancer cells and normal adult tissues makes survivin a very attractive cancer drug target. We have previously reported a series of novel selective survivin inhibitors with the most potent compound MX106 reaching nanomolar activity in several cancer cell lines. Further optimization of the MX106 scaffold leads to the discovery of more potent and more selective survivin inhibitors. Various structural modifications were synthesized and their anticancer activities were evaluated to determine the structure activity relationships for this MX106 scaffold. In vitro anti-proliferative assays using two human melanoma cell lines showed that several new analogs have improved potency compared to MX106. Very interestingly, these new analogs generally showed significantly higher potency against P-glycoprotein overexpressed cells compared with the corresponding parental cells, suggesting that these compounds may strongly sensitize tumors that have high expressions of the Pglycoprotein drug efflux pumps. Western blotting analysis confirmed that the new MX106 analogs maintained their mechanism of actions by selectively suppressing survivin expression level among major inhibitors of apoptotic proteins and induced strong apoptosis in melanoma tumor cells.

    HTML

Introduction
  • Survivin is the smallest (molecular weight 16.5 kDa) member of the IAP (Inhibitor of Apoptosis Protein) family[1-2]. It is a unique anti-tumor therapy target because: (a) it acts as an essential anti-apoptosis guard which protects cells from the apoptotic cascade by binding to the activated caspases and neutralizing proapoptotic receptor[3-5]; (b) it is a key regulator of mitosis as a component of the chromosomal passenger complex (CPC)[6-7]; (c) it widely participates in tumorigenesis signaling pathways such as Akt, p53, Wnt-2 and MDM2[8-10]; (d) it is highly expressed in most types of human cancer cells and embryonic tissues where rapid cell growth is needed, but has very low expression in normal adult differentiated tissues[11-12]; (e) its expression level is closely correlated with tumor metastasis, poor disease prognosis, and high risk of chemo/radio-resistance[8, 11, 13-17].

    However, due to the nature of survivin function (protein–protein interaction) and its cell cycle dependent expression, the existing armory of survivin inhibitors is still very limited[8, 18]. Most current small molecular survivin inhibitors do not directly bind to the survivin protein, but rather interfere with survivin production (gene transcription, post-translation modification) or accelerate its degradation[8, 12, 18-20].

    Multidrug resistance (MDR) remains a major obstacle in cancer chemotherapy, accounting for more than 90% failure in clinical treatment to cancer patients. A family of ATP binding cassette (ABC) transporters, which is a class of drug efflux pumps, plays a key role in developing MDR[21]. At least 15 human ABC transporters, such as P-glycoprotein (P-gp), multidrug resistant protein1 (MRP1) and breast cancer resistant protein (BCRP) have been found to mediate MDR by inducing drug efflux[22]. Among them, P-gp has been the most extensively identified MDR in cancer cells and a broad range of chemotherapy drugs are known to be substrates transported by P-gp. As the most potent small molecule survivin inhibitor reported so far, YM155 is a survivin gene promotor inhibitor and is well known to be highly susceptible to clinically relevant MDR, including but not limited to P-gp overexpression[18-19, 23-24]. It also shows potential toxicities, which calls for special dose administration arrangements. Therefore, it is important to develop novel selective survivin inhibitors that can overcome clinically relevant MDR.

    Utilizing high throughput virtual screening and biological test oriented strategy, our laboratory has previously developed a platform of cell permeable small molecular survivin inhibitors, which selectively and effectively reduced survivin expression level in human melanoma and prostate cancer cells, with potent antitumor growth efficacy as validated by a human melanoma xenograft model[25-26]. UC112 is the lead compound we identified through virtual screening and in vitro biological studies. Preliminary optimization of UC112 leads to the discovery of a potent and selective survivin inhibitor, MX106. The structure of UC112 and MX106 are shown in Fig. 1. In this report, we described our efforts to further optimize the MX106 scaffolds which lead to the discovery of more potent MX106 analogs for selective survivin inhibition in tumor cells. We report that these new analogs show strong antiproliferative potency against human melanoma and colorectal cancer cells, effectively overcome P-gp mediated drug resistance, and maintain their mechanisms of action by selectively inhibiting survivin expression and inducing cancer cell apoptosis. Structure-activity relationships (SAR) are determined for these new analogs to support further development of this unique scaffold as a potential anticancer agent.

    Figure 1.  Structures of our previously reported compounds UC112 and MX106

Materials and methods

    General

  • All reagents were purchased from Sigma-Aldrich Chemical Co., Alfa Aesar (Ward Hill, MA), and AK Scientific (Mountain View, CA) and were used without further purification. Routine thin layer chromatography (TLC) was performed on aluminum-backed Uniplates (Analtech, Newark, DE). NMR spectra were obtained on a Varian Inova-500 spectrometer (Agilent Technologies, Santa Clara, CA) or a Bruker Ascend 400 (Billerica, MA) spectrometer. Chemical shifts are reported as parts per million (ppm) relative to TMS in CDCl3. High resolution mass spectra were collected in positive detection mode on a Waters Xevo G2-S Tof instrument equipped with an electron-spray ionization (ESI) source (Milford, MA).

  • Synthesis

    Preparation of 5-chloromethyl-8-quinolinol hydrochloride (2)
  • A mixture of 5.84 g (40.0 mmol) of 8-quinolinol, 50 ml of concentrated hydrochloric acid, and 6.4 ml of 37% formaldehyde was treated with 0.6 g of zinc chloride and stirred for 12 hours. The mixture was filtered, washed with copious acetone and dried to give compound 2 as a yellow solid (7.2 g, 78%). 1H NMR (400 MHz, deuterium oxide) δ 9.12 (dd, J = 8.7, 1.4 Hz, 1H), 8.88 (dd, J = 5.5, 1.4 Hz, 1H), 7.97 (dd, J = 8.7, 5.4 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.24 (d, J = 7.9 Hz, 1H), 4.93 (s, 2H).

  • General procedure for the synthesis of compounds (3a-c)
  • To a solution of substituted benzyl alcohol 3 (6 mmol) in anhydrous THF (30 mL) was added sodium hydride (60% dispersion in mineral oil, 0.72 g, 18 mmol) at 0℃. The suspension was stirred at 0℃ for 30 minutes. Salt 2 (1.15 g, 5mmol) was added to the suspension. The mixture was stirred at r.t for 3 hours. Water was added to the suspension, and the mixture became homogeneous. The mixture was extracted by ethyl acetate and washed with brine, dried over anhydrous sodium sulfate and concentrated to get the crude. The crude compound was purified by flash chromatography (ethyl acetate: hexane 1:3)

  • 5-(((4-azidobenzyl)oxy)methyl)quinolin-8-ol (3a)
  • 1HNMR (400 MHz, CDCl3) δ 8.78 (d, J = 2.8 Hz, 1H), 8.41 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 7.2 Hz, 1H), 7.24 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 7.2 Hz, 1H), 4.84 (s, 2H), 4.52 (s, 2H).

  • 5-(((4-ethynylbenzyl)oxy)methyl)quinolin-8-ol (3b)
  • 1HNMR (400 MHz, CDCl3) δ 8.77 (d, J = 2.8 Hz, 1H), 8.44 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 7.2 Hz, 1H), 7.25 (d, J = 8.0 Hz, 2H), 7.10 (d, J = 7.2 Hz, 1H), 4.84 (s, 2H), 4.50 (s, 2H), 3.07 (s, 3H).

  • 5-(((2-bromo-4-methylbenzyl)oxy)methyl)quinolin-8-ol (3c)
  • 1H NMR (400 MHz, Chloroform-d) δ 8.85 (dd, J = 4.3, 1.6 Hz, 1H), 8.54 (dd, J = 8.5, 1.5 Hz, 1H), 7.60 – 7.51 (m, 2H), 7.46 (d, J = 7.8 Hz, 1H), 7.23 (d, J = 7.7 Hz, 1H), 7.17 (td, J = 5.0, 2.5 Hz, 2H), 4.89 (s, 2H), 4.50 (s, 2H), 2.41 (s, 3H).

  • General procedure for the synthesis of compounds (4a-c)
  • An equimolar mixture of the substrates 3, paraformaldehyde, and the pyrrolidine in anhydrous ethanol (30 mL) was refluxed for 4 hours under argon. After cooling, the solvent was evaporated under reduced pressure. The crude compound was purified by flash chromatography (Dichloromethane: methanol 20: 1).

  • 5-(((4-azidobenzyl)oxy)methyl)-7- (pyrrolidin-1-ylmethyl) quinolin-8-ol (4a)
  • 1HNMR (400 MHz, CDCl3) δ 8.89 (dd, J = 4, 1.6 Hz, 1H), 8.37 (dd, J = 8.4, 1.6 Hz, 1H), 7.41 (dd, J = 8.4, 4.0 Hz, 1H), 7.31 (d, J = 8.4 Hz, 2H), 7.21 (s, 1H), 7.00 (d, J = 8.4 Hz, 2H), 4.84 (s, 2H), 4.53 (s, 2H), 3.98 (S, 2H), 2.70 (m, 4H), 1.88 (m, 4H). HRMS (ESI): m/z calculated for C22H23N5O2 + H+ [M + H]+: 390.1930; Found: 390.1942.

  • 5-(((4-ethynylbenzyl)oxy)methyl)-7- (pyrrolidin-1- ylmethyl)quinolin-8-ol (4b)
  • 1HNMR (400 MHz, CDCl3) δ 8.88 (dd, J = 8.4, 1.6 Hz, 1H), 8.37 (dd, J = 8.4, 1.6 Hz, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.41 (dd, J = 8.4, 4.0 Hz, 1H), 7.28 (d, J = 8.4 Hz, 2H), 7.21 (s, 1H), 4.84 (s, 2H), 4.55 (s, 2H), 3.99 (s, 2H), 3.07 (s, 1H), 2.72-2.69 (m, 4H), 1.89-1.86 (m, 4H). HRMS (ESI): m/z calculated for C24H24N2O2 + H+ [M + H]+: 373.1916; Found: 373.1924.

  • 5-(((2-bromo-4-methylbenzyl)oxy)methyl)-7- (pyrrolidin- 1-ylmethyl)quinolin-8-ol (4c)
  • 1H NMR (400 MHz, Chloroform-d) δ 8.79 (d, J = 4.1 Hz, 1H), 8.43 – 8.22 (m, 1H), 7.47 – 7.29 (m, 3H), 7.16 – 7.04 (m, 2H), 4.78 (s, 2H), 4.43 (s, 2H), 4.08 (s, 2H), 2.96 – 2.70 (m, 4H), 2.31 (s, 3H), 1.97 – 1.76 (m, 4H). Exact mass for C23H25BrN2O2: 442.1079; HRMS: [M + H]+: 443.1170

  • Preparation of 5-((naphthalen-2-ylmethoxy)methyl)quinolin-8-ol (5)
  • To a solution of the alcohol in anhydrous DMF (10 mL) was added sodium hydride (60% dispersion in mineral oil, 0.72 g, 18 mmol) at 0℃. The suspension was stirred at 0℃ for 30 minutes. Salt 2 (1.15 g, 5 mmol) was added to the suspension. The mixture was stirred at r.t for 3 hours. Water was added to the suspension, the mixture became homogeneous. The mixture was extracted by ethyl acetate and washed with brine, dried over anhydrous sodium sulfate and concentrated to get the crude. The crude compound was purified by flash chromatography (ethyl acetate: hexane 1:3) 1H NMR (400 MHz, Chloroform-d) δ 8.77 (dd, J = 4.2, 1.6 Hz, 1H), 8.38 (dd, J = 8.5, 1.6 Hz, 1H), 8.07 – 8.00 (m, 1H), 7.92 – 7.77 (m, 2H), 7.52 – 7.40 (m, 5H), 7.36 (dd, J = 8.5, 4.2 Hz, 1H), 7.11 (d, J = 7.7 Hz, 1H), 4.99 (s, 2H), 4.90 (s, 2H).

  • Preparation of 5-((naphthalen-2-ylmethoxy)methyl)-7- (pyrrolidin-1-ylmethyl)quinolin-8-ol (6)
  • An equimolar mixture of the substrates 5, paraformaldehyde, and the pyrrolidine in anhydrous ethanol (30 mL) was refluxed for 4 hours under argon. After cooling, the solvent was evaporated under reduced pressure. The crude compound was purified by flash chromatography (Dichloromethane: methanol 20: 1). 1H NMR (400 MHz, Chloroform-d) δ 8.85 (dd, J = 4.2, 1.7 Hz, 1H), 8.30 (dd, J = 8.5, 1.6 Hz, 1H), 8.12 – 7.98 (m, 1H), 7.92 – 7.77 (m, 2H), 7.53 – 7.40 (m, 5H), 7.35 – 7.28 (m, 2H), 5.02 (s, 2H), 4.90 (s, 2H), 4.04 (s, 2H), 2.87 – 2.67 (m, 4H), 1.90 (p, J = 3.2 Hz, 4H). Exact mass for C26H26N2O2: 398.1994; HRMS: [M + H]+: 399.2071

  • Preparation of 5- (morpholinomethyl)quinolin-8-ol (7)
  • To a solution of morpholine (6 mmol) in THF was added salt 2 (2 mmol). The mixture was refluxed for 3hr. The white precipitate was filtered, the filtrate was evaporated. The crude was purified by flash chromatography to produce 7. 1H NMR (400 MHz, chloroform-d) δ 8.79 (dd, J = 4.2, 1.6 Hz, 1H), 8.67 (dd, J = 8.5, 1.6 Hz, 1H), 7.47 (dd, J = 8.5, 4.2 Hz, 1H), 7.33 (d, J = 7.7 Hz, 1H), 7.07 (d, J = 7.7 Hz, 1H), 3.79 (s, 2H), 3.70 – 3.61 (m, 4H), 2.45 (dd, J = 5.6, 3.7 Hz, 4H).

  • Preparation of 5- (morpholinomethyl)-7- (pyrrolidin-1- ylmethyl)quinolin-8-ol (8)
  • An equimolar mixture of the substrates 7, paraformaldehyde, and the pyrrolidine in anhydrous ethanol (30 mL) was refluxed for 4 hours under argon. After cooling, the solvent was evaporated under reduced pressure. The crude compound was purified by flash chromatography (Dichloromethane: methanol 20: 1). 1H NMR (400 MHz, Chloroform-d) δ 8.86 (ddd, J = 5.9, 4.1, 1.6 Hz, 1H), 8.60 (td, J = 8.5, 1.7 Hz, 1H), 7.40 (dt, J = 8.4, 4.2 Hz, 1H), 7.19 (d, J = 29.0 Hz, 1H), 3.95 (d, J = 31.1 Hz, 2H), 3.86 (s, 1H), 3.81 – 3.74 (m, 3H), 3.66 (t, J = 4.7 Hz, 2H), 2.76 – 2.58 (m, 4H), 2.57 – 2.40 (m, 4H), 1.95 – 1.69 (m, 4H). Exact mass for C19H25N3O2: 327.1947; HRMS: [M + H]+: 328.2026

  • Preparation of 2-chloroquinolin-8-ol (10)
  • Quinoline-2, 8-diol (5.30 g, 32.89 mmol) was suspended in anhydrous DMF (30 mL). Thionyl chloride (9.45 mL, 131.50 mmol) was added dropwise via a syringe at 0℃ under argon. The resulted solution was stirred and heated to 50℃ for 4 hours. Then, the reaction was quenched by pouring the solution into ice water followed by extraction with ethyl acetate (3×50 mL), washed with brine (2×50 mL), dried over anhydrous MgSO4 and concentrated under reduced pressure and purified by silica gel column chromatography (eluting with CH2Cl2) to give a pale yellow solid product, 5.0 g, 84.7% yield.

  • Preparation of 2-chloro-5- (chloromethyl)quinolin-8-ol hydrochloride (11)
  • 2-Chloroquinolin-8-ol (3.42 g, 19.04 mmol) was dissolved in 12N concentrated HCl (10 mL). 37% Formal aldehyde solution (8 mL) and ZnCl2 (1.10 g) were added. The reaction mixture was stirred at 40℃ overnight. The precipitate was filtered and washed with copious acetone and dried under vacuum to give a yellow solid product, 3.25 g, 74.9% yield.

  • 2-chloro-5-(((4-isopropylbenzyl)oxy)methyl)quinolin-8-ol (12)
  • 2-Chloro-5- (chloromethyl)quinolin-8-ol hydrochloride (0.55 g, 2.08 mmol) was suspended in 2 mL of 4- isopropylbenzyl alcohol. The reaction mixture was stirred and heated to 90℃ under argon for 3 hours. The resulted solution was stirred in 10 mL of saturated NaHCO3 solution, extracted with ethyl acetate and washed with water. The extract was dried over anhydrous MgSO4 followed by filtration and concentration under reduced pressure. The residue was purified by silica gel column chromatography (eluting with CH2Cl2) to give a colorless oil product, 0.70 g, 98.5% yield. 1HNMR (400 MHz, CDCl3) δ 8.42 (d, J = 8.8 Hz, 1H), 7.77 (s, 1H), 7.41 (d, J = 8.8 Hz, 2H), 7.26 (s, 1H), 7.20 (d, J = 8.8 Hz, 2H), 7.13 (d, J = 8.0 Hz, 1H), 4.83 (s, 2H), 4.49 (s, 2H), 2.94-2.87 (m, 1H), 1.24 (d, J = 6.8 Hz, 6H).

  • Preparation of 2-chloro-5-(((4-isopropylbenzyl)oxy) methyl)-7- (pyrrolidin-1-ylmethyl)quinolin-8-ol (13)
  • 2-chloro-5-(((4-isopropylbenzyl)oxy)methyl)quinolin-8-ol (0.50 g, 1.46 mmol), paraformaldehyde (44 mg, 1.46 mmol) and pyrrolidine (0.10 g, 1.46 mmol) were mixed together in 10 mL of ethanol. The reaction mixture was stirred and heated to reflux for 4 hours under argon. Then, the volatile was removed under reduced pressure. The residue was purified by silica gel column chromatography (eluting with CH2Cl2/MeOH = 9/1 v/v) to give a yellow solid product, 0.56 g, 90.3% yield. 1HNMR (400 MHz, CDCl3) δ 8.29 (d, J = 8.4 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8.4 Hz, 2H), 7.20 (s, 1H), 4.80 (s, 2H), 4.53 (s, 2H), 3.99 (s, 2H), 2.94-2.88 (m, 1H), 2.75-2.71 (m, 4H), 1.90-1.87 (m, 4H), 1.25 (d, J = 6.8 Hz, 6H). HRMS (ESI): m/z calculated for C25H29ClN2O2 + H+ [M + H]+: 425.1996; Found: 425.1997.

  • Preparation of 5-(((4-Isopropylbenzyl)oxy)methyl)-2- (4- methylpiperazin-1-yl)quinolin-8-ol (14)
  • 2-Chloro-5-(((4-isopropylbenzyl)oxy)methyl)quinolin-8-ol (0.20 g, 0.58 mmol) and (0.59 g, 5.85 mmol) were mixed together in 5 mL of pyridine. The reaction mixture was stirred and heated to reflux for 8 hours under argon. Then, the volatile was removed under reduced pressure. The residue was purified by silica gel column chromatography (eluting with CH2Cl2/MeOH = 9/1 v/v) to give a yellow solid product, 0.21 g, 87.5% yield.. 1HNMR (400 MHz, CDCl3) δ 8.21 (d, J = 9.2 Hz, 1H), 8.12 (s, 1H), 7.13 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 7.06 (s, 1H), 6.97 (d, J = 9.2 Hz, 1H), 4.69 (s, 2H), 4.36 (s, 2H), 4.74 (t, J = 4.8 Hz, 4H), 2.94- 2.87 (m, 1H), 2.56 (t, J = 4.8 Hz, 4H), 2.37 (s, 3H), 2.07-2.05 (m, 4H), 1.20 (d, J = 6.8 Hz, 6H). HRMS (ESI): m/z calculated for C25H31N3O2 + H+ [M + H]+: 406.2495; Found: 406.2485.

  • Preparation of 5-(((4-isopropylbenzyl)oxy)methyl)-2- (isopropylthio)-7- (pyrrolidin-1-ylmethyl)quinolin-8-ol (15)
  • Isopropyl thiol (0.21 g, 2.94 mmol) was dissolved in anhydrous THF (40 mL). NaH (60%wt in mineral oil, 0.14 g, 3.54 mmol) was added in portions at 0℃ under argon. After stirred at room temperature for 2 hours, 2- Chloro-5-(((4-isopropylbenzyl)oxy)methyl)-7- (pyrrolidin-1-ylmethyl)quinolin-8-ol (13) (0.50 g, 1.18 mmol) was added. The resulted mixture was stirred at room temperature for 6 hours. Then, the reaction was quenched by addition of 50 mL of water. The mixture was extracted with CH2Cl2 (3×50 mL). The extracts was dried over anhydrous MgSO4, filtered and concentrated to dryness. The residue was purified by silica gel column chromatography (eluting with CH2Cl2/MeOH = 9/1 v/v) to give a yellow solid product, 0.46 mg, 83.9% yield. 1HNMR (400 MHz, CDCl3) δ 8.15 (d, J = 8.8 Hz, 1H), 7.29 (s, 1H), 7.23 (d, J = 8.8 Hz, 2H), 7.20 (d, J = 8.8 Hz, 2H), 7.19 (d, J = 8.0 Hz, 2H), 4.80 (s, 2H), 4.50 (s, 2H), 4.26-4.19 (m, 1H), 4.00 (s, 2H), 2.94-2.85 (m, 1H), 2.78-2.76 (m, 4H), 1.90-1.87 (m, 4H), 1.49 (d, J = 6.4 Hz, 6H), 1.24 (d, J = 6.8 Hz, 6H). HRMS (ESI): m/z calculated for C28H36N2O2S + H+ [M + H]+: 465.2576; Found: 465.2580.

  • Preparation of 5-(((4-isopropylbenzyl)oxy)methyl)-2- (4- methylpiperazin-1-yl)-7- (pyrrolidin-1-ylmethyl)quinolin- 8-ol (16)
  • 2-Chloro-5-(((4-isopropylbenzyl)oxy)methyl)-7- (pyridine-1-ylmethyl)quinolin-8-ol (13) (0.26 g, 0.76 mmol) and 4-methylpiperazine (0.76 g, 7.62 mmol) were mixed together in 5 mL of pyridine. The reaction mixture was stirred and heated to reflux for 2 hours under argon. Then, the volatile was removed under reduced pressure. The residue was purified by silica gel column chromatography (eluting with CH2Cl2/MeOH = 9/1 v/v) to give a yellow solid product, 0.17 g, 45.9% yield. 1HNMR (400 MHz, CDCl3) δ 8.15 (d, J = 9.2 Hz, 1H), 7.25 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H), 7.03 (s, 1H), 6.74 (d, J = 9.2 Hz, 1H), 4.77 (s, 2H), 4.47 (s, 2H), 3.83 (s, 2H), 3.60 (t, J = 6.4 Hz, 4H), 2.94-2.87 (m, 1H), 2.75-2.72 (m, 4H), 2.67-2.64 (m, 4H), 2.38 (s, 3H), 2.07-2.05 (m, 4H), 1.24 (d, J = 6.8 Hz, 6H). HRMS (ESI): m/z calculated for C30H40N4O2 + H+ [M + H]+: 489.3230; Found: 489.3236.

  • Preparation of 7((1H-imidazol-1-yl)methyl-2-chloro-5- (((4-isopropylbenzyl)oxy)methyl)quinolin-8-ol (17)
  • 2-Chloro-5-(((4-isopropylbenzyl)oxy)methyl)-7- (pyrrolidin-1-ylmethyl)quinolin-8-ol (13) (0.12 g, 0.28 mmol) and imidazole (0.19 g, 2.82 mmol) were mixed together in anhydrous pyridine (5 mL) under argon. The reaction mixture was stirred and heated to reflux for 6 hours under argon. Then, the volatile was removed under reduced pressure. The residue was purified by silica gel column chromatography (eluting with CH2Cl2/MeOH = 19/1 v/v) to give a yellow solid product, 65 mg, 54.6% yield. 1HNMR (400 MHz, CDCl3) δ 8.56 (s, H), 8.41 (d, J = 9.2 Hz, 1H), 7.48 (d, J = 8.8 Hz, 1H), 7.25-7.23 (m, 6H), 5.51 (s, 2H), 4.82 (s, 2H), 2.96-2.89 (m, 1H), 2.16 (s, 2H), 1.26 (d, J = 7.2 Hz, 6H). HRMS (ESI): m/z calculated for C24H24ClN3O2 + H+ [M + H]+: 422.1635; Found: 422.1648.

  • Procedure for the preparation of compound 22

    2-Hydroxyquinolin-8-yl acetate (18)
  • Quinoline-2, 8-diol (5.00 g, 31.00 mmol) was dissolved in anhydrous THF (30 mL) at room temperature under argon. Acetic anhydride (10 mL) was added via a syringe. The reaction solution was stirred and heated to reflux overnight. Then, the volatile was removed under reduced pressure. The residue was purified by silica gel column chromatography (eluting with CH2Cl2/MeOH = 19/1 v/v) to give a white solid product, 5.80 g, 92.1% yield.

  • 2-Bromoquinolin-8-yl acetate (19)
  • 2-Hydroxyquinolin-8-yl acetate (2.50 g, 12.30 mmol) was dissolved in chloroform (10 mL). POBr3 (8.82 g, 30.75 mmol) was added at room temperature. The reaction mixture was stirred and heated to reflux for 6 hours under argon. The reaction solution was cooled to room temperature and poured into ice water followed by extraction with CH2Cl2, dried over anhydrous MgSO4/ K2CO3, filtration and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with CH2Cl2) to give a white solid product, 2.80 g, 86.0% yield. 1HNMR (400 MHz, CDCl3) δ 8.97 (d, J = 8.4 Hz, 1H), 7.69 (dd, J = 8.4, 1.2 Hz, 1H), 7.57-7.52 (m, 2H), 7.46 (dd, J = 8.4, 1.2 Hz, 1H), 2.50 (s, 3H).

  • 2-Bromo-5- (chloromethyl)quinolin-8-ol hydrochloride (20)
  • 2-Bromoquinolin-8-yl acetate (1.20 g, 4.51 mmol) was suspended in concentrated HCl (10 mL) at room temperature. ZnCl2 (0.40 g) was added. The reaction mixture was stirred and heated to 40℃ for 8 hours. The yellow precipitate was separated, washed with acetone and dried under vacuum to give a yellow solid product, 0.92 g, 66.0% yield. 1HNMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 7.2 Hz, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.11 (d, J = 8.0 Hz, 1H), 5.19 (s, 2H).

  • 2-Bromo-5-(((4-isopropylbenzyl)oxy)methyl)quinolin-8-ol (21)
  • 2-Bromo-5- (chloromethyl)quinolin-8-ol hydrochloride (0.50 g, 1.62 mmol) was suspended in 5 mL of 4- isopropylbenzyl alcohol. The reaction mixture was stirred and heated to 90℃ under argon for 4 hours. The resulted solution was stirred in 10 mL of saturated NaHCO3 solution, extracted with ethyl acetate and washed with water. The extract was dried over anhydrous MgSO4 followed by filtration and concentration under reduced pressure. The residue was purified by silica gel column chromatography (eluting with CH2Cl2) to give a colorless oil product, 0.58 g, 92.7% yield. 1HNMR (400 MHz, CDCl3) δ 8.41 (d, J = 8.8 Hz, 1H), 7.77 (s, 1H), 7.41 (d, J = 8.8 Hz, 2H), 7.24 (d, J = 8.8 Hz, 1H), 7.20 (d, J = 8.8 Hz, 2H), 4.84 (s, 2H), 4.40 (s, 2H), 2.94-2.87 (m, 1H), 1.24 (d, J = 7.2 Hz, 6H).

  • 2-Bromo-5-(((4-isopropylbenzyl)oxy)methyl)-7- (pyrrolidin-1-ylmethyl)quinolin-8-ol (22)
  • 2-Bromo-5-(((4-isopropylbenzyl)oxy)methyl)quinolin-8-ol (0.84 g, 2.17 mmol), paraformaldehyde (65 mg, 2.17 mmol) and pyrrolidine (0.16 g, 2.17 mmol) were mixed together in 10 mL of ethanol. The reaction mixture was stirred and heated to reflux for 4 hours under argon. Then, the volatile was removed under reduced pressure. The residue was purified by silica gel column chromatography (eluting with CH2Cl2/MeOH = 9/1 v/v) to give a yellow solid product, 0.74 g, 72.6% yield. 1HNMR (400 MHz, CDCl3) δ 8.30 (d, J = 8.8 Hz, 1H), 7.36 (d, J = 8.8 Hz, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8.4 Hz, 2H), 7.23 (s, 1H), 4.80 (s, 2H), 4.53 (s, 2H), 4.01 (s, 2H), 2.94-2.87 (m, 1H), 2.74-2.72 (m, 4H), 1.91-1.88 (m, 4H), 1.24 (d, J = 6.8 Hz, 6H). HRMS (ESI): m/z calculated for C25H29BrN2O2 + H+ [M + H]+: 469.1491; Found: 469.1490.

  • Synthesis of 4-((tert-butyldimethylsilyl)oxy)-1- naphthaldehyde (24)

  • To a solution of aldehyde 15 (2 mmol) in DCM was added TBDMSCl (3 mmol) and imidazole (4 mmol). The mixture was stirred overnight. Water was added and the mixture was extracted with DCM. The organic layer was dried over anhydrous sodium sulfate. The crude was purified by flash chromatography to generate 24. 1H NMR (400 MHz, Chloroform-d) δ 10.12 (s, 1H), 9.21 (dt, J = 8.7, 1.0 Hz, 1H), 8.18 (ddd, J = 8.5, 1.5, 0.7 Hz, 1H), 7.77 (d, J = 7.9 Hz, 1H), 7.59 (ddd, J = 8.5, 6.9, 1.5 Hz, 1H), 7.48 (ddd, J = 8.3, 6.9, 1.2 Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H), 1.01 (s, 9H), 0.27 (s, 6H).

  • Synthesis of (4-((tert-butyldimethylsilyl)oxy) naphthalen-1-yl)methanol (25)

  • To the protected aldehyde 16 (1 mmol) in methanol was added NaBH4 (3 mmol). The mixture was stirred at room temperature for 2 hours. The solvent was evaporated and extracted with ethyl acetate. The crude was subject to flash chromatography to produce alcohol 25. 1H NMR (400 MHz, Chloroform-d) δ 8.24 (ddd, J = 8.1, 1.5, 0.7 Hz, 1H), 8.16 – 8.03 (m, 1H), 7.52 (dddd, J = 22.0, 8.1, 6.8, 1.4 Hz, 2H), 7.34 (d, J = 7.6 Hz, 1H), 6.81 (d, J = 7.7 Hz, 1H), 5.06 (s, 2H), 1.10 (s, 9H), 0.29 (s, 6H).

  • Synthesis of((4-((benzyloxy)methyl)naphthalen-1-yl) oxy) (tert-butyl)dimethylsilane (26)

  • To a solution of alcohol 25 in anhydrous THF (10ml) was added sodium hydride (60% dispersion in mineral oil) at 0℃. The suspension was stirred at 0℃ for 30 minutes. Benzyl bromide was added to the suspension. The mixture was stirred at r.t for 3 hours. Water was added to the suspension, the mixture became homogeneous. The mixture was extracted by ethyl acetate and washed with brine, dried over anhydrous sodium sulfate and concentrated to get the crude. The crude compound was purified by flash chromatography (ethyl acetate: hexane 1:3). 1H NMR (400 MHz, Chloroform-d) δ 8.31 – 8.24 (m, 1H), 7.92 – 7.82 (m, 1H), 7.45 – 7.34 (m, 3H), 7.34 – 7.27 (m, 3H), 7.26 – 7.19 (m, 2H), 6.73 (d, J = 7.9 Hz, 1H), 5.13 (s, 2H), 5.00 (s, 2H), 0.83 (s, 9H), 0.00 (s, 6H).

  • Synthesis of 4-((benzyloxy)methyl)naphthalen-1-ol (27)

  • To a solution of alcohol 18 in THF was added TBAF (1.5 eq) and stirred for 1 hour. The mixture was extracted by ethyl acetate. The crude was subject to flash chromatography to produce phenol 27. 1H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 8.18 (ddd, J = 8.2, 1.6, 0.7 Hz, 1H), 8.02 (ddd, J = 8.4, 1.3, 0.7 Hz, 1H), 7.58 – 7.38 (m, 3H), 7.36 – 7.25 (m, 5H), 6.82 (d, J = 7.6 Hz, 1H), 4.84 (s, 2H), 4.54 (s, 2H).

  • Synthesis of 4-((benzyloxy)methyl)-2- (pyrrolidin-1- ylmethyl)naphthalen-1-ol (28)

  • An equimolar mixture of the substrates 27, paraformaldehyde, and the pyrrolidine in anhydrous ethanol (30 mL) was refluxed for 4 hours under argon. After cooling, the solvent was evaporated under reduced pressure. The crude compound was purified by flash chromatography (Dichloromethane: methanol 20: 1). 1H NMR (400 MHz, Chloroform-d) δ 8.31 – 8.23 (m, 1H), 8.05 – 7.94 (m, 1H), 7.57 – 7.44 (m, 2H), 7.42 – 7.26 (m, 5H), 7.10 (s, 1H), 4.88 (s, 2H), 4.61 (s, 2H), 3.99 (s, 2H), 2.89 – 2.52 (m, 4H), 2.00 – 1.77 (m, 4H). Exact mass for C23H25NO2: 347.1885; HRMS: [M + H]+: 348.1959

  • Synthesis of 1- (benzyloxy)-4-((benzyloxy)methyl) benzene (30)

  • To a solution of alcohol 29 (3 mmol) in anhydrous THF (30ml) was added sodium hydride (60% dispersion in mineral oil, 6 mmol) at 0℃. The suspension was stirred at 0℃ for 30 minutes. Benzyl bromide (4 mmol) was added to the suspension. The mixture was stirred at r.t for 3 hours. Water was added to the suspension, the mixture became homogeneous. The mixture was extracted by ethyl acetate and washed with brine, dried over anhydrous sodium sulfate and concentrated to get the crude. The crude compound was purified by flash chromatography (ethyl acetate: hexane 1:3). 1H NMR (400 MHz, Chloroform-d) δ 7.52 – 7.18 (m, 12H), 7.03 – 6.83 (m, 2H), 5.07 (s, 2H), 4.53 (s, 2H), 4.49 (s, 2H).

  • Synthesis of 4-((benzyloxy)methyl)phenol (31)

  • To a solution of intermediate 12 (1.0 equiv.) and NiCl2.6H2O (1.5 equiv.) in methanol (5 mL) at 0℃, NaBH4 (3.0 equiv.) was added and stirred until complete consumption of the starting material. The progress of the reaction was monitored by TLC. After completion, the reaction mixture was quenched with methanol and stirred for another 20 minutes. It was then filtered through celite pad and the filtrate was concentrated under reduced pressure. The crude product, thus obtained, was purified by column chromatography on activated silica gel using ethyl acetate-hexane mixture as eluent to afford the pure phenol 13. 1H NMR (400 MHz, Chloroform-d) δ 7.39 – 7.33 (m, 4H), 7.32 – 7.27 (m, 1H), 7.26 – 7.21 (m, 2H), 6.82 – 6.75 (m, 2H), 4.54 (s, 2H), 4.47 (s, 2H).

  • Synthesis for 4-((benzyloxy)methyl)-2- (pyrrolidin-1- ylmethyl)phenol (32)

  • An equimolar mixture of the substrates 13, paraformaldehyde, and the pyrrolidine in anhydrous ethanol (30 mL) was refluxed for 4 hours under argon. After cooling, the solvent was evaporated under reduced pressure. The crude compound was purified by flash chromatography (Dichloromethane: methanol 20: 1). 1H NMR (400 MHz, Chloroform-d) δ 7.39 – 7.33 (m, 4H), 7.31 – 7.27 (m, 1H), 7.13 (dd, J = 8.2, 2.2 Hz, 1H), 6.99 (d, J = 2.2 Hz, 1H), 6.79 (d, J = 8.2 Hz, 1H), 4.54 (s, 2H), 4.43 (s, 2H), 3.82 (s, 2H), 2.71 – 2.53 (m, 4H), 1.91 – 1.80 (m, 4H). Exact mass for C19H23NO2: 297.1729; HRMS: [M + H]+: 298.1812

  • Synthesis of 5-(((4-isopropylbenzyl)oxy)methyl)-7- (pyrrolidin-1-ylmethyl)quinolin-8-yl trifluoromethanesulfonate (33)

  • To a solution of MX106 in anhydrous THF was added sodium hydride at 0℃. The mixture was added at 0℃ for 30 minutes. N-Phenyl-bis (trifluoromethanesulfonimide) was added. The mixture was refluxed for 4hr. The mixture was extracted by ethyl acetate. The crude was subject to flash chromatography to generate 33. 1H NMR (400 MHz, Chloroform-d) δ 9.00 (dd, J = 4.2, 1.6 Hz, 1H), 8.47 (dd, J = 8.6, 1.6 Hz, 1H), 7.84 (s, 1H), 7.51 (dd, J = 8.5, 4.2 Hz, 1H), 7.29 (d, J = 8.2 Hz, 2H), 7.24 (d, J = 8.2 Hz, 2H), 4.95 (s, 2H), 4.59 (s, 2H), 3.95 (s, 2H), 2.93 (p, J = 6.9 Hz, 1H), 2.74 – 2.44 (m, 4H), 1.96 – 1.71 (m, 4H), 1.26 (d, J = 6.9 Hz, 6H). Exact mass for C26H29F3N2O4S: 522.1800; HRMS: [M + H]+: 523.1884

  • Synthesis of 5-(((4-isopropylbenzyl)oxy)methyl)-7- (pyrrolidin-1-ylmethyl)quinoline (34)

  • To a solution of compound 33 in methanol was added Pd/c (10%, 0.05eq), Mg (0.1 eq) and ammonium acetate (2 eq). The mixture was stirred at room temperate for 5 hours. The mixture was filtered by celite. The crude was purified to generate compound 25. 1H NMR (400 MHz, Chloroform-d) δ 8.93 – 8.77 (m, 1H), 8.59 – 8.45 (m, 1H), 8.07 (s, 1H), 7.82 (d, J = 1.7 Hz, 1H), 7.46 (dd, J = 8.6, 4.3 Hz, 1H), 7.20 (d, J = 7.5 Hz, 3H), 7.13 (d, J = 8.2 Hz, 2H), 4.91 (s, 2H), 4.52 (s, 2H), 4.41 (s, 2H), 3.57 (s, 2H), 3.06 (s, 2H), 2.82 (p, J = 6.9 Hz, 1H), 2.08 (s, 5H), 1.16 (d, J = 6.9 Hz, 6H). Exact mass for C25H30N2O: 374.2358; HRMS: [M + H]+: 375.2439

  • Synthesis for 5-(((4-isopropylbenzyl)oxy)methyl)-7- (pyrrolidin-1-ylmethyl)-1, 2, 3, 4-tetrahydroquinoline (35)

  • To a solution of compound 33 in methanol was added Pd/C (10%, 0.1eq), Mg (0.2 eq) and ammonium acetate (2 eq). The mixture was stirred at room temperate overnight. The mixture was filtered by celite. The crude was purified to generate compound 35. 1H NMR (400 MHz, Chloroform-d) δ 9.95 (s, 1H), 7.21 (d, J = 8.2 Hz, 2H), 7.17 – 7.13 (m, 2H), 6.67 (d, J = 1.9 Hz, 1H), 6.52 (d, J = 1.9 Hz, 1H), 4.46 (s, 2H), 4.37 (s, 2H), 3.96 (s, 2H), 3.56 (s, 3H), 3.24 – 3.12 (m, 2H), 2.94 – 2.74 (m, 2H), 2.60 (t, J = 6.5 Hz, 2H), 2.11 (d, J = 8.5 Hz, 2H), 1.96 (d, J = 7.9 Hz, 2H), 1.84 (td, J = 6.4, 4.6 Hz, 2H), 1.18 (d, J = 6.9 Hz, 6H). Exact mass for C25H34N2O: 378.2671; HRMS: [M + H]+: 379.2750

  • General procedure for the synthesis of compounds (36a-b)

  • To a solution of alcohol (6 mmol) in anhydrous THF (30ml) was added sodium hydride (60% dispersion in mineral oil, 0.72 g, 18 mmol) at 0℃. The suspension was stirred at 0℃ for 30 minutes. Salt 2 (1.15 g, 5mmol) was added to the suspension. The mixture was stirred at r.t for 3 hours. Water was added to the suspension, the mixture became homogeneous. The mixture was extracted by ethyl acetate and washed with brine, dried over anhydrous sodium sulfate and concentrated to get the crude. The crude compound was purified by flash chromatography (ethyl acetate: hexane 1:3)

  • (R)-5-((1- (2, 6-dichloro-3-fluorophenyl)ethoxy)methyl) quinolin-8-ol (36a)
  • 1H NMR (400 MHz, Chloroform-d) δ 8.83 (dd, J = 4.3, 1.6 Hz, 1H), 8.59 (dd, J = 8.6, 1.6 Hz, 1H), 7.54 (dd, J = 8.5, 4.3 Hz, 1H), 7.35 (d, J = 7.7 Hz, 1H), 7.28 – 7.25 (m, 1H), 7.12 (d, J = 7.7 Hz, 1H), 7.05 (ddd, J = 8.9, 8.0, 4.6 Hz, 1H), 5.34 (q, J = 6.8 Hz, 1H), 4.85 (d, J = 11.8 Hz, 1H), 4.66 (d, J = 11.8 Hz, 1H), 1.60 (d, J = 6.8 Hz, 3H).

  • (S)-5-((1- (2, 6-dichloro-3-fluorophenyl)ethoxy)methyl) quinolin-8-ol (36b)
  • 1H NMR (400 MHz, Chloroform-d) δ 8.78 (dd, J = 4.2, 1.6 Hz, 1H), 8.52 (dd, J = 8.5, 1.5 Hz, 1H), 7.49 (dd, J = 8.5, 4.2 Hz, 1H), 7.33 – 7.27 (m, 1H), 7.25 – 7.22 (m, 1H), 7.07 – 7.04 (m, 1H), 7.03 – 6.99 (m, 1H), 5.58 (s, 1H), 5.31 (q, J = 6.8 Hz, 1H), 4.82 (dd, J = 11.7, 0.6 Hz, 1H), 4.62 (d, J = 11.8 Hz, 1H), 1.65 (d, J = 6.9 Hz, 3H).

  • General procedure for the synthesis of compounds (37a-b)

  • An equimolar mixture of the substrates 36, paraformaldehyde, and the pyrrolidine in anhydrous ethanol (30 mL) was refluxed for 4 hours under argon. After cooling, the solvent was evaporated under reduced pressure. The crude compound was purified by flash chromatography (Dichloromethane: methanol 20: 1).

  • (R)-5-((1- (2, 6-dichloro-3-fluorophenyl)ethoxy)methyl)-7- (pyrrolidin-1-ylmethyl)quinolin-8-ol (37a)
  • 1H NMR (400 MHz, Chloroform-d) δ 8.79 (dd, J = 4.2, 1.7 Hz, 1H), 8.38 (dd, J = 8.5, 1.7 Hz, 1H), 7.35 (dd, J = 8.5, 4.2 Hz, 1H), 7.13 (dd, J = 8.9, 4.9 Hz, 1H), 7.05 (s, 1H), 6.92 (dd, J = 8.9, 8.0 Hz, 1H), 5.24 – 5.16 (m, 2H), 4.73 (d, J = 11.8 Hz, 1H), 4.56 (d, J = 11.8 Hz, 1H), 3.92 (d, J = 1.6 Hz, 2H), 2.76 – 2.55 (m, 4H), 1.88 – 1.73 (m, 4H), 1.50 (d, J = 6.8 Hz, 3H). Exact mass for C23H23Cl2FN2O2: 448.1121; HRMS: [M + H]+: 449.1191

  • (S)-5-((1- (2, 6-dichloro-3-fluorophenyl)ethoxy)methyl)-7- (pyrrolidin-1-ylmethyl)quinolin-8-ol (37b)
  • 1H NMR (400 MHz, Chloroform-d) δ 8.87 (dd, J = 4.1, 1.6 Hz, 1H), 8.45 (dd, J = 8.5, 1.7 Hz, 1H), 7.41 (dd, J = 8.5, 4.2 Hz, 1H), 7.20 (dd, J = 8.8, 4.9 Hz, 1H), 7.09 (s, 1H), 6.99 (dd, J = 8.9, 8.0 Hz, 1H), 5.35 – 5.22 (m, 2H), 4.80 (d, J = 11.8 Hz, 1H), 4.62 (d, J = 11.8 Hz, 1H), 3.97 (s, 2H), 2.81 – 2.59 (m, 4H), 1.95 – 1.77 (m, 4H), 1.57 (d, J = 6.8 Hz, 3H). Exact mass for C23H23Cl2FN2O2: 448.1121; HRMS: [M + H]+: 449.1205

  • Synthesis of 5-(((4-trifluoromethylbenzyl)amino) methyl)quinolin-8-ol (38)

  • 5- (Chloromethyl)quinolin-8-ol hydrochloride (0.50 g, 2.17 mmol) and 4-trifluorobenzylamine (1.14 g, 6.52 mmol) were mixed together in 40 mL of ethyl acetate/ DMF (1/3). The reaction mixture was stirred and heated to 60℃ for 24 hours. The reaction was quenched by addition of 50 mL of saturated sodium bicarbonate solution and extracted with CH2Cl2 (3×50 mL). The extracts was dried over anhydrous MgSO4 followed by filtration and concentration under reduced pressure. The residue was purified by silica gel column chromatography (eluting with CH2Cl2/MeOH 19/1 v/v) to give a gray solid, 0.26 g, 36.1% yield. 1HNMR (400 MHz, CDCl3) δ 8.81 (d, J = 3.2 Hz, 1H), 8.52 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H), 7.41 (d, J = 8.0 Hz, 1H), 7.11 (d, J = 8.0 Hz, 1H), 4.16 (s, 2H), 3.95 (s, 2H).

  • Synthesis of 2-bromo-5-(((4-isopropylbenzyl)oxy) methyl)-7- (pyrrolidin-1-ylmethyl)quinolin-8-ol. (39)

  • 5-(((4-trifluoromethylbenzyl)amino)methyl)quinolin- 8-ol (0.16 g, 0.48 mmol), para-formaldehyde (14 mg, 0.48 mmol) and pyrrolidine (34 mg, 0.48 mmol) were mixed together in 10 mL of ethanol. The reaction mixture was stirred and heated to reflux for 4 hours under argon. Then, the volatile was removed under reduced pressure. The residue was purified by silica gel column chromatography (eluting with CH2Cl2/MeOH = 9/1 v/v) to give a yellow solid product, 0.14 g, 70.4% yield. 1HNMR (400 MHz, CDCl3) δ 8.79 (d, J = 3.6 Hz, 1H), 8.01 (dd, J = 8.4, 1.6 Hz, 1H), 7.50 (d, J = 7.2 Hz, 1H), 7.31 (d, J = 8.4 Hz, 2H), 7.27 (d, J = 8.4 Hz, 2H), 7.15 (s, 1H), 3.96 (s, 2H), 3.57 (s, 2H), 3.28 (s, 2H), 2.68-2.65 (m, 4H), 1.82-1.80 (m, 4H). HRMS (ESI): m/ z calculated for C23H24F3N3O + H+ [M + H]+: 416.1950; Found: 416.1965.

  • Cell culture and in vitro antiproliferative assays in human melanoma cells

  • Human melanoma A375 and M14 cell lines were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA), and cultured in DMEM media (Mediatech, Inc., Manassas, VA) at 37℃ in a humidified atmosphere containing 5% CO2. The culture media were supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and 1% antibiotic-antimycotic mixture (Sigma-Aldrich, St. Louis, MO). Compounds were dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich) to make a stock solution of 10 mmol/L. Compound solutions were freshly prepared by diluting stocks with cell culture medium before use (final solution contained less than 0.5% DMSO). Five thousand cells in logarithm growing phase were seeded overnight into each well of a 96-well plate. Then the cells were continuously incubated for 48 hours with sequential diluted compound solution (3 nmol/L to 100 mmol/L, 100 mL per well) in cell culture medium. The cell viability was determined in MTS assay and IC50 was calculated (n = 4), following similar procedures as described previously[25-28].

  • Cell culture and cytotoxicity assays in human epidermoid carcinoma and colorectal cancer cells

  • Dulbecco's modified Eagle's Medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin and trypsin 0.25% were purchased from Hyclone (GE Healthcare Life Science, Pittsburgh, PA). Phosphate buffered saline (PBS) was purchased from Invitrogen GIBCO (Grand Island, NY). Dimethyl sulfoxide (DMSO) and 3- (4, 5-dimethylthiazole-2-yl)-2, 5-biphenyl tetrazolium bromide (MTT) were purchased from Sigma Chemical Co (St. Louis, MO).

    The P-glycoprotein (P-gp) overexpressing KB-C2 cell line was established from a parental human epidermoid carcinoma cell line KB-3-1, by a stepwise selection of KB-3-1 in increasing concentrations of colchicine up to 2 mg/mL[29]. SW620/Ad300, which is also a P-gp overexpressing drug resistant cell line, was established by stepwise exposure of the parental human colon cancer cell line SW620 to increasing concentrations of doxorubicin up to 300 ng/mL[30]. The SW620 and SW620/Ad300 cell lines were kindly provided by Dr. Susan E. Bates and Dr. Robert W. Robey (NCI, NIH, Bethesda, MD, USA). All the cell lines were grown in DMEM supplemented with 10% FBS and 100 unit/mL penicillin/streptomycin in a humidified incubator containing 5% CO2 at 37℃.

    The MTT colorimetric assay was used to measure the sensitivity of the cells against the synthesized compounds. The assay detects the formazan product formed from the reduction of MTT in active cells thus assesses the cell viability[31]. Cells were seeded in 96-well plates at 5, 000 cells/well (KB-3-1 or KB-C2 cells) or at 7000 cells/well (SW620 or SW620/Ad300 cells) in 180 mL completed medium and cultured overnight. Then various concentrations of the compounds (20 mL) were added to the designated wells. After 72 hours continuous drug incubation, 20 mL of MTT reagent (4 mg/mL) was added to each well and the plates were incubated at 37℃ for 4 hours. Subsequently, the medium was removed and 100 µL of DMSO were added to dissolve the formazan crystals in each well. The absorbance was determined at 570 nm by the accuSkanTM GO UV/Vis Microplate Spectrophotometer (Fisher Sci., Fair Lawn, NJ). The IC50 values of each compound on each cell line were calculated from the survival curves to represent the cytotoxicity of the compounds. The fold of drug resistance was calculated by dividing the IC50 of the P-gp overexpressing cells by that of the parental cells. Two known P-gp substrates, YM155 and paclitaxel, were used as positive controls for P-gp overexpressing cell lines. On the other hand, cisplatin, which is not a substrate of P-gp, was used as negative control drug.

  • Western blotting

  • To determine the change of protein levels of survivin and closely related IAPs, lysates of A375 or M14 melanoma cells treated by the compound solution for 24 hours were used for Western blotting analysis. Primary rabbit antibodies against IAP proteins including survivin (#2808), XIAP (#2045), cIAP1 (#7065), Livin (#5471), Cleaved PARP (#9185) and the loading control protein GAPDH (HRP Conjuga