Synthesis of Î±, Î²-Unsaturated Carbonyl-Based Compounds, Oxime

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Synthesis of #, #-unsaturated carbonyl-based compounds, oxime and oxime ether analogs as potential anticancer agents for overcoming cancer multidrug resistance by modulation of efflux pumps in tumor cells Huali Qin, Jing Leng, Cheng-Pan Zhang, Ibrahim Jantan, Muhammad Wahab Amjad, Muhammad Sher, Muhammad Naeem ul Hassan, Muhammad Ajaz Hussain, and Syed Nasir Abbas Bukhari J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00276 • Publication Date (Web): 24 Mar 2016 Downloaded from http://pubs.acs.org on March 24, 2016

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Journal of Medicinal Chemistry

Synthesis of α, β-unsaturated carbonyl-based compounds, oxime and oxime ether analogs as potential anticancer agents for overcoming cancer multidrug resistance by modulation of efflux pumps in tumor cells Hua-Li Qin*a‡, Jing Leng a, Cheng-Pan Zhang a, Ibrahim Jantan b, Muhammad Wahab Amjad b, Muhammad Sher c, Muhammad Naeem-ul-Hassan c, Muhammad Ajaz Hussain c, Syed Nasir Abbas Bukhari*b‡ a

Department of Pharmaceutical Engineering, School of chemistry, chemical engineering and

life science, Wuhan University of Technology, 205 Luoshi Road, Wuhan,430070, P.R.China. b

Drug and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia. c

‡

Department of Chemistry, University of Sargodha, Sargodha 40100, Pakistan.

These authors contributed equally

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ABSTRACT Sixty nine novel α, β-unsaturated carbonyl based compounds, including cyclohexanone, tetralone, oxime and oxime ether analogs were synthesized. The antiproliferative activity determined by using seven different human cancer cell lines provided a structure-activity relationship. Compound 8ag exhibited high antiproliferative activity against Panc-1, PaCa-2, A549 and PC-3 cell lines, with IC50 value of 0.02 µM, comparable to the positive control Erlotinib. Ten most active antiproliferative compounds were assessed for mechanistic effects on BRAFV600E, EGFR TK kinases and tubulin polymerization, and were investigated in vitro to reverse efflux-mediated resistance developed by cancer cells. Compound 8af exhibited the most potent BRAFV600E inhibitory activity with an IC50 value of 0.9µM. Oxime analog 7o displayed the most potent EGFR TK inhibitory activity with an IC50 of 0.07 µM, which was analogous to the positive control. Some analogs including 7f, 8af and 8ag showed a dual role as anticancer and MDR reversal agents. Keywords: cyclohexanone; tetralone; tubulin polymerization; epidermal growth factor receptor (EGFR); chemotherapy.


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INTRODUCTION A sophisticated system of signaling pathways in multicellular organisms regulates cellular behavior and ensures that cells multiply only when they are needed by the body as a whole throughout healing or development. A defect in these signaling mechanisms leads to the breakdown of normal growth regulation, resulting in cancer. Multiple pathways and processes by which cancer arises from normal cells and tissues make it very challenging to develop anticancer agents possessing effects on all receptors, pathways and signaling mechanisms. Moreover, the tumor cells adapt themselves enhancing their resistance against chemotherapy to escape programmed death by activating anti-apoptotic pathways, after recurrent treatment with anticancer chemotherapeutic agents.1-6 In spite of combination of chemotherapeutic agents used to prevent drug resistance, the cancer cells adapt and develop one or more drug resistance pathways eventually leading to failure in the treatment of cancer.7,

8

Some key targets for

treatment of cancer and multidrug resistance (MDR) in cancer cells are described below. A transmembrane glycoprotein, epidermal growth factor receptor (EGFR), belongs to the erbB family of closely linked cell membrane receptors that includes EGFR (erbB-1 or HER1), erbB-2 (HER2), erbB-3 (HER3), and erbB-4 (HER4).9-13 Many human solid tumors, including head and neck, colorectal, non-small cell lung (NSCLC), ovarian and breast cancers exhibit the expression, over expression, or dysregulation of EGFR.14-18 The activation of EGFR might support the growth of a tumor by increasing motility, cell proliferation19, invasive capacity and adhesion20, and by inhibiting apoptosis.21 The importance of controlling EGFR signaling as a cancer treatment strategy has been demonstrated recently by the wide-ranging collection of molecular inhibitors that have been developed and are undergoing clinical trials.



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The RAS-RAF-MEK-ERK is a rapidly activated downstream pathway of receptor tyrosine kinases (RTK) which controls a number of cell responses to extracellular signals.22 The pathway constitutively activates in cancer (particularly melanoma) and triggers propagation, survival, and tumor development. A total of 3 RAF (ARAF, BRAF, and CRAF) and 3 RAS (NRAS, KRAS and HRAS) genes are found in humans, and critically, BRAF, KRAS and NRAS are mutated in 45%, 2% and 20% of the melanomas, respectively.23 The cancer-cell proliferation and survival are stimulated by BRAF

V600E

via signaling through the mitogen-activated protein kinase (MAPK)

pathway24. The development of oncogenic BRAF inhibitors, specifically type I BRAF inhibitors, that block the active conformation of BRAF kinase, has resulted in a great degree of objective tumor responses and enhancement in overall survival, in contrast to standard chemotherapy25, 26. Chemotherapy is amongst the most vital approaches for the treatment of cancers. However, successful cancer chemotherapy is hindered by MDR in cancer cells27. The mechanism behind the development of resistance to anticancer drugs by cancer cells is complex. Up untill now, the best categorized mechanism of MDR comprises the overexpression of ATP-binding cassette (ABC) transporter Pgp (also referred to as ABCB1, MDR1) on cell membranes of tumor cells. A wide range of structurally different anticancer drugs can be effluxed out of the cell by Pgp, thus lowering their intracellular levels and therapeutic efficacy28. MDR can be reversed by the inhibition of this efflux mechanism. The complications in chemotherapy can be overcome by the development of pharmacological modulators or agents of MDR efflux pumps that reverse multidrug resistance. Several natural and synthetic α,β-unsaturated carbonyl based compounds including chalcones, curcumin and their synthetic analogs are recognized to show antitumor activities 29, 30. The ketone functional group of these compounds has been postulated to work in cancer


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chemotherapy by thiol alkylation without any reactivity towards cellular hydroxyl or amino groups, and thus, due to this reactivity, enones may have potential benefit over conventional alkylators as these compounds are predicted to have less genotoxic effects, related with many anticancer drugs

31, 32

. Furthermore, the antitumor activity of enone containing materials is

associated with numerous effects such as inhibition of NF-kappaB (NF-kB)33 and mitochondrial mediated pathways34, triggering of death receptors (DRS) of tumor necrosis factor (TFN)35, inhibition of cyclin-dependent kinases36 or DNA topoisomerase II37 and so on. Therefore, the synthesis of novel α,β-unsaturated ketones is presently of high importance. Most of the research on α, β-unsaturated carbonyl based compounds as chalcones and curcumin analogs is focused on the evaluation against cancer and comparatively less is known of their capability to reverse MDR. More information is required to explain whether the lack of a β-diketone group affects the capability of these compounds to inhibit ABC transporters and if these compounds can be better chemotherapeutic agents possessing dual properties as anticancer and efficient MDR reversal agents. In the current study, we extended our formerly reported work30, 38 by synthesizing 54 new α, β-unsaturated carbonyl based compounds and evaluating their anticancer potential on human cancer cell lines. Among these compounds, the 10 most active were designated as precursors for further synthesis to their oxime derivatives. The anticancer activity of all new oxime derivatives was evaluated and 5 most active oxime analogs were then selected for additional synthesis as oxime ether analogs. From all 69 new compounds, the 10 most potent were selected for anticancer mechanistic studies, for their effects on BRAFV600E, EGFR TK kinases, and tubulin polymerization and were tested in vitro to reverse efflux mediated resistance developed by cancer cells.



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RESULTS Chemistry Sixty nine novel compounds of four different types including, cyclohexanone, tetralone, oxime and oxime ether backbone were synthesized in the current study as reported previously47 (Table 1-3). To synthesize the desired α, β-unsaturated carbonyl based compounds, Claisene Schmidt condensation was used between different ketones and suitable aryl aldehydes at a molar ratio 1:2 for cyclohexanone derivatives (4a-u) and at a molar ratio 1:1 for tetralone derivatives (5a-ag) (Scheme 1). All synthesized compounds were characterized by using various analytical techniques and characterization data of compounds including, melting point (MP), 1H NMR, 13C NMR, HRMS, microanalysis (CHNS) is provided in experimental section. All compounds have a purity grade ≥95%. Cell viability assay Human mammary gland epithelial cell line (MCF-10A) was used to perform the cell viability assay. The MCF-10A cells were treated with the synthesized compounds for 4 days and a 3-(4,5Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay was used to measure the viability of cells. The findings are presented according to the toxicity percentage (Table 4, 5 & 6). All compounds were discovered to be nontoxic with most of them exhibiting more than 90% cell viability.

Effects on human cancer cell lines The antiproliferative activity of all synthetic compounds on human prostate cancer cells (PC-3), colon cancer cells (HT-29), breast cancer cells (MCF-7), lung cancer cells (H-460), epithelial


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cancer cells (A-549), pancreatic carcinoma cells (PaCa-2) and pancreas cancer cell line (Panc-1) was investigated using the propidium iodide (PI) fluorescence assay. Generally, investigated compounds showed almost similar antiproliferative activity against different cancer cells and the average variance in their inhibitory activities was less than 8% against all types of cancer cells. GraphPad Prism software (GraphPad Software, San Diego, CA, USA) was used to calculate the median inhibition concentration (IC50) for all compounds. On the basis of IC50, a significant association was witnessed between the analogs in terms of their structural features. The three most active compounds (4c, 4f, 4o) among cyclohexanone derivatives or α, βunsaturated carbonyl-based compounds exhibited potent inhibition of cancer cells growth (Table 4). Compounds 4f and 4o showed nearly comparable and maximum anticancer activity against all cancer cell lines with IC50 ranging from 1.1±0.2 to 1.9±0.8 µM. Moreover, compound 4c also displayed strong activity with an IC50 of 2.0±1.5 µM for the HT-29 cell line. The findings of these compounds (4c, 4f, 4o) were similar against all seven cancer cell lines. Seven compounds such as 5(f, i, o, u, x, af, and ag) from new tetralone derivatives (5a-5ag) showed potent growth inhibition of all cell lines (Table 5). The compound 5ag displayed the highest anticancer potential with IC50 1.2±0.9 µM for Panc-1. After 5ag, the compound 5o exhibited significant anticancer activity with IC50 1.4±0.5 µM. The derivatives 5i , 5af, 5f, 5u, 5x also exhibited noteworthy inhibition of cancer cells These findings of the anticancer assay motivated us to extend our synthesis work. In doing so, ten new oxime analogs were synthesized by taking the three most active cyclohexanone derivatives 4(c, f, o) and seven most active tetralone derivatives 5(f, i, o, u, x, af, and ag) as bases. The newly synthesized oxime analogs were then subjected to the antiproliferative assay using seven cancer cell lines. In contrast to



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parent α, β-unsaturated carbonyl-based compounds, all analogs showed potent antiproliferative activity against seven cell lines with IC50 ranging from 0.02±0.08 to 2.9±0.2 µM (Table 6). The most potent oxime analogs were screened for subsequent synthesis as oxime ether analogs. And the resultant analogs were also assessed for antiproliferative activity (Table 6). In contrast to the parent oximes, all oxime ether analogs exhibited less inhibition of cancer cells. Following the antiproliferative assay, all four series including tetralones (33 compounds), cyclohexanones (21 compounds), oximes (10 compounds), and oxime ethers (5 compounds) were subjected to an overall comparison. The ten most potent compounds among the above mentioned series were selected for anticancer mechanistic experiments including their effect on tubulin polymerization, EGFR-TK and BRAFV600E, and were also assessed in vitro to reverse multidrug resistance developed by cancer cells. Effects on tubulin polymerization The effect of all ten selected synthetic compounds on tubulin polymerization has been summarized in Figure 1. A significant influence on tubulin assembly was exhibited by most compounds; where compounds 7f and 8f inhibited tubulin assembly strongly. According to the propidium iodide fluorescence assay, just two compounds (7o and 10o) did not exhibit tubulin assembly inhibition which suggests a different process for the witnessed cytotoxicity than inhibition of tubulin. No synthetic compound exhibited microtubule-stabilizing action comparable to recognized antimitotic chemotherapeutic drug docetaxel39. EGFR inhibitory activity An EGFR-TK assay was performed to assess the EGFR inhibitory strength of new compounds and the results are presented in Table 7. The results from this assay complement the findings of the cancer cell-based assays. All investigated compounds exhibited strong inhibition of EGFR


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with IC50 ranging from 0.07±0.03 to 8.4±0.2 µM. According to Table 7, compounds 7o and 10o were found to be most potent (IC50 = 0.07±0.03 µM and 0.1±0.05 µM for EGFR), and their EGFR inhibitory activities were similar to the positive control erlotinib (IC50 = 0.05±0.02 µM). This experiment shows that α, β-unsaturated carbonyl based oxime compounds and their ether analogs are potent EGFR inhibitors and can possibly be used as anticancer agents. BRAFV600E inhibitory activity As compared to standard chemotherapy, the development of oncogenic BRAF inhibitors has caused a high percentage of objective tumor responses and improvement in general survival. Therefore, small molecule inhibitors which target V600E mutant BRAF protein kinase are being developed for the treatment of cancer. An in vitro assay was performed to investigate the BRAFV600E inhibitory potential of the ten most active synthesized compounds. According to Table 7, all investigated compounds showed IC50 in the range of 0.9±0.4 to 6.3±0.4 µM. All investigated synthetic α, β-unsaturated carbonyl based oxime compounds were discovered to be strong BRAFV600E inhibitors. Interestingly, two of them (8af and 8ag) displayed nearly the same BRAF inhibitory potential and were also found to be potent against the proliferation of cancer cells. These findings show that the investigated compounds are prospective anticancer agents and also effectively inhibit BRAF enzyme. MDR reversal activity A Rhodamine accumulation test was performed to study the effect of the synthesized compounds (at non-toxic concentrations) on the drug accumulation of MDR cancer cells (Table 7). A Trypan blue experiment was used to investigate the toxic effects of these compounds. During a short incubation period, the compounds (at 50 µg/mL concentration) exhibited no apparent toxicity on



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cells. At 5 µg/ml concentration, most of compounds were ineffective on R123 accumulation. Nonetheless, the multidrug resistance on the mouse lypmhoma cells was reversed by the compounds at 50 µg/mL concentration and an incubation time of 30 min. Verapamil served as a positive control in the experiment. Table 7 shows the findings related to MDR. The oxime analogs based on tetralone series 8 (f, i, o, u, x, af, and ag) were discovered to be most effective whereas compounds 7f, 7o and 10o were almost ineffective. No compound was found to be inactive against human mdr1 gene-transfected mouse lymphoma cells. A combination of traditional chemotherapeutics and resistance modifiers can be used to treat MDR cancer cells. So, compounds possessing dual characteristics as strong anticancer agents as well as MDR modulators can be prospective contenders for cancer treatment. DISCUSSION A broad range of structurally correlated α, β-unsaturated carbonyl-based compounds such as tetralone, cyclohexanone and their oxime analogs were synthesized and investigated for anticancer activity against numerous cancer cell lines. Some of the synthesized compounds exhibited anticancer activity against certain cancer cell lines. The most active of these compounds were screened for mechanistic experiments and assessed for their effects on BRAFV600E, EGFR-TK and tubulin polymerization, in addition to an in vitro investigation of the reversal of efflux-mediated resistance developed by cancer cells. The most complex challenge encountered by drug candidates from the design to the development phase is the rule of three (activity-exposure-toxicity). To regulate the balance of characteristics vital to convert lead compounds into drugs that are both safe and effective, absorption, distribution, metabolism and excretion (ADME) studies are widely used in drug discovery40. Therefore, human mammary gland epithelial cell line (MCF-10A) was used to perform an in


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vitro cell viability assay. All compounds were discovered to be safe with most of them exhibiting cell viability of more than 90%. Hence, the synthesized compounds were discovered to be biologically safe and might be used as potential therapeutic agents for future drug discovery experiments. Based on the assessments among the antiproliferative activities of tetralone and cyclohexanone derivatives, the subsequent structure-activity relationships can be witnessed: (1) On assessment, it was found that oxime analogs (series 7 & 8) exhibited maximum antiproliferative activity against cancer cell lines in comparison to tetralone, cyclohexanone and oxime ether analogs. As compared to cyclohexanone, tetralone derivatives were discovered to be more promising. Nonetheless, some cyclohexanone derivatives (4c, 4f, and 4o) displayed potent antiproliferative activity. In comparison to their parent oxime analogs, oxime ether analogs were found to be less active. (2) A remarkable structure-activity relationship was witnessed among cyclohexanone derivatives. R1 position substitution in the cyclohexanone ring played a vital part in the activities of these types of compounds. A similar phenomenon was also observed for these types of compounds in our previously reported experiments for anti-inflammatory activities41. The compounds 4(a, b and c) showed good activity without R1 substitution, However, the presence of a N-CH3 at R1 exhibited the greatest activity. Methyl substitution also showed significant effects on antiproliferative activity and it was also found that the methoxy group’s occurrence improved the antiproliferative activity. The methoxy group was present at position 5 in all compounds. However substitution patterns at R3, R4, and R6 differed between the compounds and the occurrence of methoxy group at position 4 improved the activities. Cyclohexanone derivatives possessing three



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methoxy groups at positions 3, 4 and 5 were discovered to be more active in comparison to those having two methoxy groups at positions 4 and 5. All compounds contained halogens (Cl or Br) at position 6 on the benzene ring. The influence of halogens on the activity of compounds could not be recognized as no compound was halogen-free. The most potent compounds in the cyclohexanone series (4c, 4f, and 4o) possessed Br at R6 and three methoxy groups at positions 3, 4 and 5, respectively. (3) The same substitution pattern as the cyclohexanone derivatives was present for all tetralone derivatives as similar aldehydes were used to synthesize tetralone derivatives. Remarkably, our structure-activity relationship as mentioned above for cyclohexanone derivatives was verified and maintained by the antiproliferative activity of tetralone derivatives. In comparison to those bearing two methoxy groups at positions Rʹ4 and Rʹ5, all compounds possessing three methoxy groups at Rʹ3, Rʹ4 and Rʹ5 were discovered to be more potent. As compared to cyclohexanone derivatives, another substitution configuration on the linked phenyl ring of the tetralone moiety is present in tetralone derivatives designated as R2, R3, R4 and R5. Compounds (5af and 5ag) having nitro group substitution at R3 were found to be most potent. As compared to 5af, tetralone derivative 5ag proved to be a stronger antiproliferative agent, therefore verifying that three methoxy groups at positions Rʹ3, Rʹ4 and Rʹ5 added more antiproliferative activity compared with two methoxy groups at Rʹ4 and Rʹ5. The three most active tetralone derivatives 5(o, u and x) possessed similar functional groups on the phenyl contributed by the aldehyde, however in comparison to 5u and 5x, 5o was found to be more active due to a different substitution arrangement on the phenyl group of the tetralone moiety. Hence, the activities of these three compounds show that the methoxy group substitution at position


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R4 is more effective in comparison to methoxy groups at R2 and R5 or at position R3 and R4 of the tetralone moiety. The polymerization activity of the ten most active antiproliferative compounds was investigated. The findings showed that compounds 7f, 8i, 8o, 8u, 8x, and 8af exhibited slight to moderate inhibition of microtubule assembly, while the microtubules were best destabilized by compounds 7f and 8f in comparison to both positive control drugs (docetaxel as microtubule stabilizing agent and vincristine as microtubule destabilizing agent)42. The R1 position in the cyclohexanone of the tetralone moiety of the most potent oxime analogs (7f and 8f) contained methyl substitutions. Tubulin assembly has been previously reported to be inhibited by the methyl substitutions at the R1 position in the cyclohexanone ring43. The two most inactive compounds (7o and 10o) possessed N-CH3 substitution at R1 in the cyclohexanone of the tetralone moiety. An important target for the development of melanoma drugs is BRAF. The persistent MAPK activity is promoted by BRAFV600E resulting in an enhanced survival and proliferation. Cell cycle arrest can occur due to the acute inhibition of BRAFV600E by kinase inhibitors or genetic depletion and in some cases, apoptosis in melanomas addicted to this oncogene44. Among ten selected compounds, two (8af and 8ag) having oxime moiety and nitro substitution at position R3 were discovered to be potent BRAF inhibitors. According to Li et al., para-nitro substitutions on phenyl ring showed better inhibition of BRAFV600E activity45. The understanding of the significance of EGFR signaling pathway in cancer has resulted in the development of numerous anti-EGFR drugs, such as reversible first generation tyrosine kinase inhibitors (TKIs) including erlotinib and gefitinib, together with monoclonal antibodies such as panitumumab and cetuximab. The EGFR inhibitory activities of the ten most potent antiproliferative oxime and oxime ether analogs have been presented in Table 7. The N-CH3



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substitution at the R1 position in the cyclohexanone of tetralone moiety enhanced the potency of the compounds (7o and 10o). These compounds did not affect tubulin polymerization but were discovered to be strong EGFR inhibitors. Methoxy group substitution at position R3 of thetetralone moiety (compound 8u) considerably decreased the potency of EGFR (IC50 = 8.4 µM). The cell efflux pump can be considered as an enzyme with exceptionally broad substrate specificity, and it involves ABC transporters. To overcome MDR, the ABC transporter inhibition of tumor cells can be a capable prospect. The ten most active anticancer compounds were selected and assessed as MDR modulators using multidrug-resistant mouse lymphoma cells. All investigated compounds were discovered to be potent MDR reversing agents. The oxime ether analog (10o) did not exhibit significant activity. The compounds with a fluorescence activity ratio (FAR) of 1 or more indicated the reversal of MDR. The modulators of MDR attach to the transmembrane domains of Pgp, thus bringing a structural change in Pgp, which in turn prevents the ABC transporters activity46. Compounds (8af and 8ag) possessing nitro substitution exhibited same and potent potency of MDR reversal. Among two oxime analogs (8o and 8u) with identical aldehyde substitutions on the phenyl ring, 8u showed maximum MDR reversal activity while the activity of 8o was 15% less. The enhanced MDR reversal activity of 8u may be attributed to the additional methoxy group at position R3 of the tetralone moiety. In summary, a series of α, β-unsaturated tetralone and cyclohexanone derivatives with various substitutions were synthesized, and assessed in vitro for their antiproliferative activities using human cancer cell lines. The compounds which exhibited the most potent antiproliferative activities were selected for subsequent synthesis as oxime and oxime ether analogs followed by assessment through mechanistic experiments. This study has revealed some new oxime analogs


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as EGFR-TK, BRAFV600E, and tubulin polymerization inhibitors. Most of these compounds also exhibited MDR reversal activity. The compounds 8af and 8ag were found to be extremely active in all experiments. Hence, we have recognized active anticancer agents exhibiting a dual-role as anticancer and as MDR reversal agents, and have compiled some valuable SAR information that might prove useful in synthesizing more MDR reversal agents with intrinsic antitumor activity. EXPERIMENTAL SECTION Chemistry General Information: All chemicals and reagents used in this project were procured from Sigma-Aldrich, Merck and Acros Organics. The reagents were of analytical grade and were used as supplied. A typical experiment consisted of brine washing and desiccating the organic layer with magnesium sulfate prior to concentration in vacuum. JEOL ECP spectrometer was used to record the 1H and

13

C

NMR spectra operating at 500 MHz, with Me4Si as internal standard and dimethyl sulfoxide (DMSO-d6) and CDCl3 as the solvents. MicroTOF-Q mass spectrometer (Bruker) was used to record the high resolution mass spectra (HRMS). The microanalyses data were obtained using Fison EA 1108 elemental analyzer. Pre-coated silica plates (kiesel gel 60 F254, BDH) were used to perform thin layer chromatography (TLC) while flash column chromatography was carried out using silica gel 60 (230-400mesh) (Merck). Electrothermal instrument was used to determine the melting points of the compounds. The synthesized compounds were observed under ultraviolet (UV) light (254 nm) or by using vanillin stain successively burning on a hotplate. Analytical HPLC were recorded for purity determination of new compounds with Beckmann system Gold HPLC using a linear gradient from 0% to 100% solvent B (60% acetonitrile/40% water/0.1% TFA) and a Kinetex C18 Coloumn (250mmX mm, 5µm) was used for this system. Purity values for all tested compounds were found to be above 95%.



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Synthesis of α, β-unsaturated carbonyl based compounds New α, β-unsaturated carbonyl based compounds (Table 1) were synthesized by using a direct coupling method47(Scheme 1). The reaction was performed via base-catalyzed Claisen-Schmidt condensation, by reacting various kinds of ten ketones with a suitable aromatic aldehyde at molar feed ratio of 2:1 for the synthesis of 21 compounds (4a-4u), and at 1:1 for synthesizing 33 compounds (5a-5ag). A synthesis route for α, β-unsaturated carbonyl based compounds is shown in scheme 1. Concisely, ketone (10 mmol, 1 equivalent) and particular aromatic aldehyde (20 mmol, 2 equivalant) were mixed and dissolved in 15 mL ethanol in a round bottom flask, and stirred for few min at 5°C. For several minutes, a 40% NaOH solution (in ethanol) was added drop wise into the above solution. The resultant mixture was stirred at 27 °C for 1-24 h. Change in color of the reaction mixture and appearance of precipitate indicated the formation of product. The reaction was monitored using TLC and acidified ice was added to the mixture for stopping the reaction upon completion. The compounds were isolated by recrystallization and/or column chromatography. 2,6-Bis-(2-bromo-3,4,5-trimethoxy-benzylidene)-cyclohexanone (4c) Yield: 74%; mp: 102-103 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.82 (s, 2H), 6.89 (s, 2H), 3.75 (s, 18H), 2.32 (t, J = 8.0 Hz, 4H), 1.82 (m, 2H); 13C NMR (500 MHz, CDCl3) δ: 185.4, 151.2, 146.5, 144.2, 144.9, 135.9, 130.5, 108.4, 98.6, 56.5, 56.1, 55.5, 28.5, 27.3; HRMS (ESI) m/z: 613.32 [M+H]+, Microanalysis calculated for C26H28Br2O7 (612.30), C: 51.00%, H: 4.61%. Found C: 51.22%, H: 4.59%.


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2,6-Bis-(2-bromo-3,4,5-trimethoxy-benzylidene)-4-methyl-cyclohexanone (4f ) Yield: 69%; Mp: 105-106 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.84 (s, 2H), 6.87 (s, 2H), 3.79 (s, 18H), 2.02 (d, J = 8.5 Hz, 4H), 1.72 (m, H); 1.16 (d, J=8.5 Hz, 3H); 13C NMR (500 MHz, CDCl3) δ: 185.6, 150.8, 146.9, 146.4, 145.9, 134.8, 130.6, 107.5, 99.8, 56.8, 56.5, 56.0, 31.5, 29.3, 22.5; HRMS (ESI) m/z: 627.65 [M+H]+, Microanalysis calculated for C27H30Br2O7 (626.33), C: 51.78%, H: 4.83%. Found C: 51.92%, H: 4.99%. 3,5-Bis-(2-bromo-3,4,5-trimethoxy-benzylidene)-1-methyl-piperidin-4-one (4o) Yield: 55%; Mp: 140-141 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.81 (s, 2H), 6.84 (s, 2H), 3.81 (s, 18H), 2.72 (s, 4H), 2.16 (s, 3H);

13

C NMR (500 MHz, CDCl3) δ: 184.9, 150.6, 147.5, 145.2,

144.9, 135.3, 131.8, 106.2, 99.5, 56.7, 56.1, 55.9, 41.5, 16.5; HRMS (ESI) m/z: 628.32 [M+H]+, Microanalysis calculated for C26H29Br2NO7 (627.32), C: 49.78%, H: 4.66%, N: 2.23%. Found C: 49.91%, H: 4.86%, N: 2.12%. 2-(2-Bromo-3,4,5-trimethoxy-benzylidene)-4-methyl-tetralone (5f) Yield: 69%; Mp: 90-91 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.69 (s, H), 7.27 (d, J = 7.5 Hz, H), 7.19 (d, J = 7.5 Hz, H), 7.07 (t, J = 7.5 Hz, H), 6.91 (t, J = 7.0 Hz, H), 6.47 (s, H), 3.56 (s, 9H), 2.19 (d, J = 8.5 Hz, 2H), 1.79 (m, H); 1.19 (d, J = 8.5 Hz, 3H); 13C NMR (500 MHz, CDCl3) δ: 184.5, 150.7, 149.1, 147.1, 146.8, 145.2, 137.3, 134.5, 134.0, 130.2, 127.3, 125.5, 124.8, 106.9, 98.7, 56.9, 56.1, 55.8, 30.4, 29.7, 21.8; HRMS (ESI) m/z: 418.32 [M+H]+, Microanalysis calculated for C21H21BrO4 (417.29), C: 60.44%, H: 5.07%. Found C: 60.52%, H: 5.15%.



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Page 18 of 48

2-(2-Bromo-3,4,5-trimethoxy-benzylidene)-5-hydroxy-tetralone (5i) Yield: 72%; Mp: 94-95 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.68 (s, H), 7.15 (d, J = 7.5 Hz, H), 7.02 (t, J = 7.0 Hz, H), 6.90 (d, J = 7.5 Hz, H), 6.42 (s, H), 5.81 (s, H), 3.52 (s, 9H), 2.52 (t, J = 8.0 Hz, 2H), 2.13 (t, J = 8.0 Hz, 2H); 13C NMR (500 MHz, CDCl3) δ: 184.9, 159.6, 149.2, 147.2, 146.1, 140.2, 137.8, 134.4, 134.0, 130.5, 126.2, 122.4, 120.2, 105.5, 96.1, 56.8, 56.0, 55.8, 31.2, 27.8; HRMS (ESI) m/z: 420.46 [M+H]+, Microanalysis calculated for C20H19BrO5 (419.27), C: 57.29%, H: 4.57%. Found C: 57.52%, H: 4.65%. 2-(2-Bromo-3,4,5-trimethoxy-benzylidene)-7-methoxy-tetralone (5o) Yield: 76%; Mp: 92-93 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.80 (s, H), 7.21 (d, J = 8.0 Hz, H), 7.02 (s, H), 6.85 (d, J = 8.0 Hz, H), 6.55 (s, H), 3.59 (s, 3H), 3.50 (s, 9H), 2.50 (t, J = 8.0 Hz, 2H), 2.18 (t, J = 8.0 Hz, 2H); 13C NMR (500 MHz, CDCl3) δ: 185.3, 159.9, 148.9, 147.4, 146.3, 141.0, 137.2, 134.5, 134.2, 130.5, 126.5, 123.0, 119.1, 102.7, 99.4, 56.8, 56.2, 56.0, 55.8, 30.6, 25.9; HRMS (ESI) m/z: 456.62 [M+Na]+, Microanalysis calculated for C21H21BrO5 (433.29), C: 58.21%, H: 4.89%. Found C: 58.51%, H: 4.91%. 2-(2-Bromo-3,4,5-trimethoxy-benzylidene)-7,8-dimethoxy-tetralone (5u) Yield: 72%; Mp: 98-99 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.89 (s, H), 7.12 (s, H), 6.75 (s, H), 6.25 (s, H), 3.58 (s, 6H), 3.64 (s, 9H), 2.54 (t, J = 8.0 Hz, 2H), 2.18 (t, J = 8.5 Hz, 2H);

13

C

NMR (500 MHz, CDCl3) δ: 186.5, 152.7, 148.3, 147.2, 146.0, 140.6, 135.9, 134.9, 134.4, 131.2, 125.2, 122.6, 119.6, 102.2, 93.2, 56.1, 55.9, 55.5, 55.3, 55.0, 29.4, 21.2; HRMS (ESI) m/z: 464.72 [M+H]+, Microanalysis calculated for C22H23BrO6 (463.32), C: 57.03%, H: 5.00%. Found C: 57.25%, H: 5.15%.


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2-(2-Bromo-3,4,5-trimethoxy-benzylidene)-6,9-dimethoxy-tetralone (5x) Yield: 69%; Mp: 98-99 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.85 (s, H), 7.05 (d, J = 7.5 Hz, H), 6.82 (d, J = 7.5 Hz, H), 6.24 (s, H), 3.72 (s, 15H), 2.52 (t, J = 8.0 Hz, 2H), 2.10 (t, J = 8.0 Hz, 2H);

13

C NMR (500 MHz, CDCl3) δ: 186.2, 151.9, 148.5, 147.4, 146.0, 140.4, 136.2, 134.6,

134.3, 131.0, 125.4, 123.1, 119.5, 102.5, 94.0, 56.2, 55.9, 55.4, 55.2, 55.0, 29.8, 21.6; HRMS (ESI) m/z: 464.36 [M+H]+, Microanalysis calculated for C22H23BrO6 (463.32), C: 57.03%, H: 5.00%. Found C: 57.24%, H: 5.12%. 2-(2-Chloro-3,4-dimethoxy-benzylidene)-8-nitro-tetralone (5af) Yield: 62%; Mp: 94-95 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.96 (s, H), 7.64 (d, J = 8 Hz, H), 7.32 (d, J = 8 Hz, H), 7.12 (d, J = 7.5 Hz, H), 6.92 (d, J = 7.5 Hz, H), 6.52 (s, H), 3.89 (s, 6H), 2.82 (t, J = 8.0 Hz, 2H), 2.24 (t, J = 8.0 Hz, 2H); 13C NMR (500 MHz, CDCl3) δ: 190.2, 154.5, 149.5, 147.1, 146.6, 138.5, 132.1, 130.5, 129.3, 127.5, 125.2, 124.6, 120.8, 100.6, 94.4, 56.8, 56.6, 27.3, 19.4; HRMS (ESI) m/z: 374.80 [M+H]+, Microanalysis calculated for C19H16ClNO5 (373.79), C: 61.05%, H: 4.31%, N: 3.75%. Found C: 61.46%, H: 4.49%, N: 3.54%. 2-(2-Bromo-3,4,5-trimethoxy-benzylidene)-8-nitro-tetralone (5ag) Yield: 54%; Mp: 98-99 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.92 (s, H), 7.66 (d, J = 8 Hz, H), 7.14 (s, H), 6.89 (d, J = 7.5 Hz, H), 6.58 (s, H), 3.81 (s, 9H), 2.88 (t, J = 8.0 Hz, 2H), 2.19 (t, J=8.0 Hz, 2H); 13C NMR (500 MHz, CDCl3) δ: 191.0, 154.2, 148.6, 147.4, 146.2, 137.2, 132.6, 130.1, 128.8, 127.1, 125.3, 124.6, 121.2, 99.4, 95.5, 56.9, 56.6, 55.8, 25.4, 20.2; HRMS (ESI) m/z: 449.50 [M+H]+, Microanalysis calculated for C20H18BrNO6 (448.26), C: 53.59%, H: 4.05%, N: 3.12%. Found C: 53.64%, H: 4.22%, N: 3.06%.



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Synthesis of oxime analogs Particular α, β-unsaturated carbonyl based compounds acted as precursors for the synthesis of oxime analogs. Hydroxylamine hydrochloride (2 mmol) was reacted with each selected compound (1 mmol) in 10 mL ethanol to yield the respective oxime (7c, 7f, 7o, 8f, 8i, 8o, 8u, 8x, 8af and 8ag). The average completion time of a reaction was around 6-8 h, as determined by TLC. The mixture was filtered and the solvent was evaporated using rotary evaporator after the completion of reaction. To extract organic compounds, dichloromethane (15 mL x 3) and distilled water (15 mL) were added to the mixture. Anhydrous MgSO4 was used to dry the organic extracts, followed by their filtration and concentration under reduced pressure. The purity of the product was determined by using TLC. The product was recrystallized from ethylacetate to yield solid powder. Column chromatography using ethylacetate: hexane (70:30 v/v) as eluent was used to purify certain products. 2,6-Bis-(2-bromo-3,4,5-trimethoxy-benzylidene)-cyclohexanone oxime (7c) Yield: 55%; Mp: 105-106 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.39 (s, H), 7.14 (s, 2H), 6.21 (s, 2H), 3.75 (s, 18H), 2.10 (d, J=8.5 Hz, 4H), 1.81 (m, 2H); 13C NMR (500 MHz, CDCl3) δ: 164.5, 148.6, 146.5, 145.4, 142.2, 134.5, 130.1, 105.2, 98.8, 56.9, 56.5, 56.0, 29.2, 28.1; HRMS (ESI) m/z: 628.35 [M+H]+, Microanalysis calculated for C26H29Br2NO7 (627.32), C: 49.78%, H: 4.66%, N: 2.23%. Found C: 49.92%, H: 4.72%, N: 2.19%. 2,6-Bis-(2-bromo-3,4,5-trimethoxy-benzylidene)-4-methyl-cyclohexanone oxime (7f) Yield: 49%; Mp: 108-109 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.42 (s, H), 7.12 (s, 2H), 6.27 (s, 2H), 3.72 (s, 18H), 2.12 (d, J = 8.5 Hz, 4H), 1.75 (m, H); 1.24 (d, J = 8.5 Hz, 3H);

13

C NMR

(500 MHz, CDCl3) δ: 162.2, 148.8, 146.4, 145.4, 141.6, 134.6, 130.7, 107.4, 98.2, 56.4, 56.2,


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56.0, 30.2, 28.4, 22.2; HRMS (ESI) m/z: 642.37 [M+H]+, Microanalysis calculated for C27H31Br2NO7 (641.35), C: 50.56%, H: 4.87%, N: 2.18%. Found C: 50.92%, H: 4.92%, N: 2.10%. 3,5-Bis-(2-bromo-3,4,5-trimethoxy-benzylidene)-1-methyl-piperidin-4-one oxime (7o) Yield: 42%; Mp: 132-133 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.38 (s, H), 7.14 (s, 2H), 6.29 (s, 2H), 3.75 (s, 18H), 2.61 (s, 4H), 2.14 (s, 3H);

13

C NMR (500 MHz, CDCl3) δ: 164.1, 150.2,

147.3, 145.5, 142.2, 135.5, 131.6, 105.9, 99.1, 56.7, 56.3, 56.1, 42.0, 16.2; HRMS (ESI) m/z: 643.14 [M+H]+, Microanalysis calculated for C26H30Br2N2O7 (642.33), C: 48.62%, H: 4.71%, N: 4.36%. Found C: 48.85%, H: 4.79%, N: 4.25%. 2-(2-Bromo-3,4,5-trimethoxy-benzylidene)-4-methyl-tetralone oxime (8f) Yield: 50%; Mp: 114-115 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.29 (s, H), 7.42 (s, H), 6.85 (t, J=7Hz, H), 6.79 (t, J=7Hz, H), 6.44 (d, J=7.5Hz, H), 6.35 (d, J=7.5Hz, H), 6.28 (s, H), 3.71 (s, 9H), 2.51 (d, J=7 Hz, 2H), 2.0 (m, H); 1.25 (d, J=8.5 Hz, 3H);

13

C NMR (500 MHz, CDCl3) δ:

167.2, 151.9, 148.6, 146.5, 144.5, 139.5, 135.7, 134.1, 132.5, 131.2, 125.5, 122.2, 118.5, 99.8, 92.5, 56.8, 56.2, 55.5, 29.6, 24.5, 22.5; HRMS (ESI) m/z: 433.31 [M+H]+, Microanalysis calculated for C21H22BrNO4 (432.31), C: 58.34%, H: 5.13%, N: 3.24%. Found C: 58.55%, H: 5.29%, N: 3.12%. 2-(2-Bromo-3,4,5-trimethoxy-benzylidene)-6-hydroxy-tetralone oxime (8i) Yield: 57%; Mp: 119-120 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.32 (s, H), 7.46 (s, H), 6.82 (t, J=7Hz, H), 6.70 (d, J=7.5Hz, H), 6.38 (d, J=7.5Hz, H), 6.22 (s, H), 4.09 (s, H), 3.75 (s, 9H), 2.57 (t, J=7 Hz, 2H), 1.98 (t, J=7.5 Hz, 2H); 13C NMR (500 MHz, CDCl3) δ: 167.5, 152.2, 148.4,



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Page 22 of 48

146.1, 143.9, 139.7, 134.9, 133.8, 132.5, 131.1, 124.9, 121.6, 118.7, 99.2, 92.7, 56.9, 56.1, 55.8, 29.2, 24.1; HRMS (ESI) m/z: 435.29 [M+H]+, Microanalysis calculated for C20H20BrNO5 (434.28), C: 55.31%, H: 4.64%, N: 3.23%. Found C: 55.52%, H: 4.68%, N: 3.20%. 2-(2-Bromo-3,4,5-trimethoxy-benzylidene)-7-methoxy-tetralone oxime (8o) Yield: 55%; Mp: 117-118 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.28 (s, H), 7.42 (s, H), 6.81 (d, J=7Hz, H), 6.76 (d, J=7.5Hz, H), 6.42 (s, H), 6.25 (s, H), 3.78 (s, 12H), 2.55 (t, J=7 Hz, 2H), 1.99 (t, J=7.5 Hz, 2H); 13C NMR (500 MHz, CDCl3) δ: 168.2, 152.8, 148.1, 146.5, 142.1, 139.6, 134.2, 133.9, 132.4, 130.2, 124.6, 120.8, 118.2, 99.1, 92.5, 57.5, 56.8, 56.2, 55.7, 29.5, 25.1; HRMS (ESI) m/z: 449.35 [M+H]+, Microanalysis calculated for C21H22BrNO5 (448.31), C: 56.26%, H: 4.95%, N: 3.12%. Found C: 56.29%, H: 4.99%, N: 3.09%. 2-(2-Bromo-3,4,5-trimethoxy-benzylidene)-7,8-dimethoxy-tetralone oxime (8u) Yield: 59%; Mp: 111-112 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.29 (s, H), 7.45 (s, H), 6.75 (s, H), 6.42 (s, H), 6.25 (s, H), 3.78 (s, 15H), 2.52 (t, J=7 Hz, 2H), 1.97 (t, J=7.5 Hz, 2H);

13

C NMR

(500 MHz, CDCl3) δ: 168.9, 152.4, 147.6, 146.2, 141.9, 139.5, 134.3, 132.4, 131.9, 130.0, 124.2, 120.9, 118.7, 98.8, 92.4, 57.5, 56.9, 56.2, 55.7, 55.2, 29.4, 25.8; HRMS (ESI) m/z: 479.35 [M+H]+, Microanalysis calculated for C22H24BrNO6 (478.33), C: 55.24%, H: 5.06%, N: 2.93%. Found C: 55.29%, H: 5.10%, N: 2.88%. 2-(2-Bromo-3,4,5-trimethoxy-benzylidene)-6,9-dimethoxy-tetralone oxime (8x) Yield: 52%; Mp: 112-114 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.39 (s, H), 7.15 (s, H), 6.43 (d, J = 7.2 Hz, H), 6.38 (d, J = 7.4 Hz, H), 6.29 (s, H), 3.68 (s, 15H), 2.51 (t, J = 6.8 Hz, 2H), 1.99 (t, J = 7.5 Hz, 2H); 13C NMR (500 MHz, CDCl3) δ: 166.5, 151.3, 148.1, 146.2, 145.0, 139.2, 136.7,


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134.2, 133.9, 131.5, 124.8, 123.5, 117.2, 100.1, 94.6, 56.7, 56.4, 56.2, 55.9, 55.5, 29.6, 21.5; HRMS (ESI) m/z: 479.45 [M+H]+, Microanalysis calculated for C22H24BrNO6 (478.33), C: 55.24%, H: 5.06%, N: 2.93%. Found C: 55.29%, H: 5.18%, N: 2.91%. 2-(2-Chloro-3,4-dimethoxy-benzylidene)-8-nitro-tetralone oxime (8af) Yield: 57%; Mp: 124-125 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.47 (s, H), 7.26 (s, H), 7.14 (d, J = 8.4Hz, H), 7.04 (d, J = 7.4Hz, H), 6.87 (d, J = 7.2Hz, H), 6.65 (d, J = 7.5Hz, H), 6.50 (s, H), 3.74 (s, 6H), 2.75 (t, J = 8.2 Hz, 2H), 2.14 (t, J = 8.2 Hz, 2H); 13C NMR (500 MHz, CDCl3) δ: 164.5, 152.3, 149.2, 147.8, 145.2, 138.6, 132.9, 130.1, 128.7, 127.1, 125.2, 124.1, 121.3, 99.9, 93.7, 57.1, 56.8, 28.8, 19.1; HRMS (ESI) m/z: 389.80 [M+H]+, Microanalysis calculated for C19H17ClN2O5 (388.80), C: 58.69%, H: 4.41%, N: 7.21%. Found C: 58.72%, H: 4.49%, N: 7.25%. 2-(2-Bromo-3,4,5-trimethoxy-benzylidene)-8-nitro-tetralone oxime (8ag) Yield: 54%; Mp: 98-99 ˚C; 1H NMR (500 MHz, CDCl3) δ: 8.45 (s, H), 7.32 (s, H), 7.22 (d, J = 8.2 Hz, H), 7.10 (s, H), 6.75 (d, J = 8.2 Hz, H), 6.48 (s, H), 3.72 (s, 9H), 2.81 (t, J = 8.0 Hz, 2H), 2.09 (t, J = 8.0 Hz, 2H); 13C NMR (500 MHz, CDCl3) δ: 167.9, 154.6, 148.4, 146.5, 145.1, 137.6, 131.9, 130.0, 128.5, 126.9, 125.1, 124.7, 120.8, 98.8, 95.2, 56.8, 56.5, 56.0, 25.2, 19.7; HRMS (ESI) m/z: 449.50 [M+H]+, Microanalysis calculated for C20H19BrN2O6 (463.28), C: 51.85%, H: 4.13%, N: 6.05%. Found C: 51.59%, H: 4.29%, N: 6.14%. Synthesis of oxime ether analogs Dry acetone (10.0 mL), baked K2CO3 (5 mmol), haloalkylamine hydrochloride (1.2 mmol) and respective oxime analog (1 mmol) were taken in a round bottom flask and refluxed for 8 h under



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Page 24 of 48

anhydrous conditions. TLC was used to monitor the reaction (silica gel plate as stationary phase while 5% methanol in chloroform as mobile phase). Once the reaction was completed, K2CO3 was filtered off from the mixture followed by washing with acetone (3x10 mL). The filtrate was concentrated and purification of crude product was carried out using distilled hexane-based basic alumina column chromatography. 3,5-Bis-(2-bromo-3,4,5-trimethoxy-benzylidene)-1-methyl-cyclohexanone-O-(2diethylamino-ethyl)-oxime (10f) Yield: 62%; Mp: 106-107 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.12 (s, 2H), 6.87 (s, 2H), 4.51 (m, 2H), 3.81 (s, 18H), 2.60 (m, 4H), 2.51 (m, 2H), 2.14 (d, J=8.5 Hz, 4H), 1.76 (m, H); 1.70 (t, J=7.0Hz, 6H); 1.25 (d, J=8.5 Hz, 3H);13C NMR (500 MHz, CDCl3) δ: 164.5, 152.5, 147.5, 145.1, 140.8, 135.9, 132.4, 104.1, 98.9, 72.1, 56.9, 56.2, 55.8, 52.1, 48.2, 42.5,30.9, 17.5, 14.9; HRMS (ESI) m/z: 741.52 [M+H]+, Microanalysis calculated for C32H44Br2N2O7 (740.52), C: 53.52%, H: 5.99%, N: 3.78%. Found C: 53.54%, H: 6.12%, N: 3.62%. 3,5-Bis-(2-bromo-3,4,5-trimethoxy-benzylidene)-1-methyl-piperidin-4-one-O-(2diethylamino-ethyl)-oxime (10o) Yield: 50%; Mp: 111-113 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.10 (s, 2H), 6.89 (s, 2H), 4.52 (m, 2H), 3.78 (s, 18H), 2.62 (s, 4H), 2.55 (m, 2H), 2.42 (m, 4H), 2.19 (s, 3H), 1.75 (t, J = 7.0 Hz, 6H); 13C NMR (500 MHz, CDCl3) δ: 164.7, 151.3, 147.8, 145.5, 141.4, 135.9, 132.0, 104.7, 99.6, 72.2, 56.8, 56.6, 56.2, 52.0, 48.4, 42.1, 17.7, 14.4; HRMS (ESI) m/z: 742.55 [M+H]+, Microanalysis calculated for C32H43Br2N3O7 (741.51), C: 51.83%, H: 5.85%, N: 5.67%. Found C: 51.89%, H: 5.92%, N: 5.52%.


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2-(2-Bromo-3,4,5-trimethoxy-benzylidene)-8-nitro-tetralone-O-(2-diethylamino-ethyl)oxime (11x) Yield: 49%; Mp: 121-122 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.25 (s, H), 7.02 (d, J=8.0Hz, H), 6.91 (s, H), 6.65 (d, J=8.0Hz, H), 4.50 (m, 2H), 3.68 (s, 15H), 2.87 (t, J=8.0 Hz, 2H), 2.55 (m, 2H), 2.40 (m, 4H), 2.08 (t, J=8.0 Hz, 2H); 1.70 (t, J=7.0Hz, 6H); 13C NMR (500 MHz, CDCl3) δ: 170.2, 153.5, 148.7, 146.1, 145.2, 135.6, 131.0, 129.5, 127.2, 126.2, 125.0, 124.1, 118.4, 98.2, 95.1, 72.5, 56.8, 56.2, 56.0, 55.5, 55.2, 52.4, 48.8, 25.1, 19.9, 14.2; HRMS (ESI) m/z: 578.55 [M+H]+, Microanalysis calculated for C28H37BrN2O6 (577.51), C: 58.23%, H: 6.46%, N: 4.85%. Found C: 58.25%, H: 6.72%, N: 4.75%. 2-(2-Chloro-,4,5-dimethoxy-benzylidene)-8-nitro-tetralone-O-(2-diethylamino-ethyl)oxime (11af) Yield: 47%; Mp: 117-118 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.29 (s, H), 6.99 (d, J=8.0Hz, H), 6.89 (d, J=8.0Hz, H), 6.72 (d, J=8.0Hz, H), 6.58 (d, J=8.0Hz, H), 6.40 (s, H), 4.55 (m, 2H), 3.68 (s, 6H), 3.10 (t, J=8.4 Hz, 2H), 2.52 (m, 2H), 2.43 (m, 4H), 2.12 (t, J=8.2, 2H), 1.75 (t, J=7.0Hz, 6H);

13

C NMR (500 MHz, CDCl3) δ: 169.5, 154.5, 148.0, 146.9, 145.1, 136.8, 132.0, 129.2,

128.5, 125.5, 124.9, 124.1, 118.9, 98.9, 95.2, 72.5, 56.9, 56.1, 52.0, 48.2, 25.5, 20.6, 15.1; HRMS (ESI) m/z: 488.98 [M+H]+, Microanalysis calculated for C25H30ClN3O5 (487.98), C: 61.53%, H: 6.20%, N: 8.61%. Found C: 61.55%, H: 6.25%, N: 8.55%. 2-(2-Bromo-3,4,5-trimethoxy-benzylidene)-8-nitro-tetralone-O-(2-diethylamino-ethyl)oxime (11ag) Yield: 42%; Mp: 119-120 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.24 (s, H), 7.05 (d, J=8.0Hz, H), 6.90 (s, H), 6.68 (d, J=8.0Hz, H), 6.42 (s, H), 4.59 (m, 2H), 3.64 (s, 9H), 3.12 (t, J=8.4 Hz, 2H),



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Page 26 of 48

2.58 (m, 2H), 2.41 (m, 4H), 2.10 (t, J=8.2, 2H), 1.72 (t, J=7.0Hz, 6H);

13

C NMR (500 MHz,

CDCl3) δ: 169.9, 153.5, 148.2, 146.8, 145.9, 136.2, 131.3, 129.9, 128.4, 126.5, 125.2, 124.0, 119.1, 98.5, 94.0, 72.4, 56.9, 56.4, 56.0, 52.1, 48.1, 25.7, 20.2, 14.9; HRMS (ESI) m/z: 563.44 [M+H]+, Microanalysis calculated for C26H32BrN3O6 (562.45), C: 55.52%, H: 5.73%, N: 7.47%. Found C: 55.59%, H: 5.92%, N: 7.62%. MTT assay To investigate the effect of synthesized compounds on mammary epithelial cells (MCF-10A), MTT assay was performed30. The cells were propagated in medium consisting of Ham's F-12 medium/ Dulbecco's modified Eagle's medium (DMEM) (1:1) supplemented with 10% foetal calf serum, 2 mM glutamine, insulin (10 µg/mL), hydrocortisone (500 ng/mL) and epidermal growth factor (20 ng/mL). Trypsin ethylenediamine tetra acetic acid (EDTA) was used to passage the cells after every 2-3 days. 96-well flat-bottomed cell culture plates were used to seed the cells at a density of 104 cells mL-1. The medium was aspirated from all the wells of culture plates after 24 h followed by the addition of synthesized compounds (in 200 µL medium to yield a final concentration of 0.1% (v/v) dimethylsulfoxide) into individual wells of the plates. Four wells were designated to a single compound. The plates were allowed to incubate at 37°C for 96 h.

Afterwards,

the

medium

was

aspirated

and

3-[4,5-dimethylthiazol-2-yl]-2,5-

diphenyltetrazolium bromide (MTT) (0.4 mg/mL) in medium was added to each well and subsequently incubated for 3 h. The medium was aspirated and 150 µL dimethyl sulfoxide (DMSO) was added to each well. The plates were vortexed followed by the measurement of absorbance at 540 nm on a microplate reader. The results were presented as inhibition (%) of proliferation in contrast to controls comprising 0.1% DMSO.


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Assay for antiproliferative effect Propidium iodide fluorescence assay30 was carried out on Panc-1(pancreas cancer), PaCa-2 (pancreatic carcinoma), MCF-7 (breast cancer): A-549 (epithelial): HT-29 (colon cancer), H-460 (lung cancer) and PC-3 (prostate cancer) cell lines to investigate the antiproliferative activity of compounds. Propidium iodide is a fluorescence dye which possesses ability to attach with DNA, therefore providing a precise and quick method for the calculation of total nuclear DNA. PI is incapable of crossing the plasma membrane and its fluorescence signal intensity is directly proportional to the amount of cellular DNA. Thus, cells with damaged plasma membranes or altered permeability are totaled as dead ones. To perform the assay, cells were seeded in 96-well flat-bottomed culture plates at a density of 3000-7500 cells/well in 200 µl medium and incubated at 37 °C for 24 h in humidified 5% CO2/95% air atmosphere. Later, the compounds at 10 µM concentrations (in 0.1% DMSO) were added in triplicate wells while 0.1% DMSO served as control, followed by a 48 h incubation of plates. The medium was removed and 25 µl PI (50 µg/mL in water/medium) was added in each well. The plates were then frozen at -80 °C for 24 h, followed by thawing and equilibration to 25oC. The readings were recorded at excitation and emission wavelengths of 530 and 620 nm using a fluorometer (Polar-Star BMG Tech). Following formula was used to calculate the cytotoxicity (%) of compounds:

Where AC= Absorbance of control and ATC= Absorbance of treated cells. To equate the results, erlotinib was used as positive control.

Tubulin polymerization assay The activity of compounds on tubulin polymerization was investigated by Tubulin Polymerization Assay Kit (Cytoskeleton Inc., Denver, CO, USA), which works via fluorescent



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reporter enhancement48. The fluorescence of compounds (dissolved in DMSO at 5 and 25 µM concentration) was recorded in triplicates using FLUO star OPTIMA. Docetaxel and vincristine (Apoteket AB, Sweden) served as positive stabilizing and destabilizing controls. Both were used at 3 µM concentration in PBS38. EGFR inhibitory assay Baculoviral expression vectors such as pFASTBacHTc and pBlueBacHis2B were separately used to clone 1.6 kb cDNA encoding for EGFR cytoplasmic domain (EGFR-CD, amino acids 645–1186) were cloned into, separately. 5ʹ upstream to the EGFR sequence contained a sequence that encodes (His)6. For protein expression, Sf-9 cells were infected for 3 days. Sf-9 cell pellets were solubilized at pH 7.4 at 0° C for 20 min in a buffer comprising 16 µg/mL benzamidine HCl, 10 µg/mL pepstatin, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 100 µM sodium vanadate, 10 µM ammonium molybdate, 1% Triton, 10 mM NaCl and 50 mM HEPES, followed by 20 min centrifugation. Using an equilibrated Ni-NTA superflow packed column, the crude extract supernatant was passed through and washed first with 10 mM and later with 100 mM imidazole for the elimination of nonspecifically bound material. Histidine-tagged proteins were eluted first with 250 and later with 500 mM imidazole followed by dialysis against 10% glycerol, 20 mM HEPES, 50 mM NaCl, and 1 µg/mL each of pepstatin, leupeptin and aprotinin for 2 h. This purification was carried out either on ice or at 4 °C. On the basis of DELFIA/Time-Resolved Fluorometry, EGFR kinase assay was performed to measure the level of autophosphorylation. DMSO (100%) was used to dissolve the compounds, followed by dilution to suitable concentrations using 25 mM HEPES at pH 7.4. In every well, 10 µL (5 ng for EGFR) recombinant enzyme (1:80 dilution in 100 mM HEPES) was incubated with 10 µL compound at 25oC for 10 min, followed by addition of 10 µL 5X buffer (containing 1 mM DTT, 100 µM


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Na3VO4, 2 mM MnCl2 and 20 mM HEPES) and 20 µL 0.1 mM ATP-50 mM MgCl2 for 1 h. By incubating the enzyme with or without ATP-MgCl2, positive and negative controls were included in every plate. After incubation, liquid was removed, and wash buffer was used to wash the plates thrice. Each well of plate was added with 75 µL (400 ng) europium-labeled antiphosphotyrosine antibody for another 1 h, followed by washing. After adding the enhancement solution, the signal was detected (with excitation and emission at 340 at 615 nm, respectively) using Victor (Wallac Inc.). Following equation was used to calculate the autophosphorylation inhibition (%) by the compounds:

The IC50 was calculated using the curves of inhibition (%) with eight concentrations of compound. Most of the signal detected by antiphosphotyrosine antibody is from EGFR, as the impurities in the enzyme preparation are quite low. BRAF kinase assay Each compound in this study was subjected to V600E mutant BRAF kinase assay in triplicate. 1 µL drug and 4 µL assay dilution buffer were pre-incubated with 7.5 ng mouse full-length GSTtagged BRAFV600E (Invitrogen, PV3849) at 25oC for 1 h. The assay was started by adding 5 µL solution comprising 200 ng recombinant human full length, N-terminal His-tagged MEK1 (Invitrogen), 30 mM MgCl2 and 200 µM ATP in assay dilution buffer, followed by continuation at 25oC for 25 min. Using 5X protein denaturing buffer (LDS) solution (5 µL), the assay was quenched. Further denaturing of protein was performed by heating at 70° C for 5 min. Electrophoresis was performed at 200 V by loading 10 µL of each reaction into a 15-well 4-12% precast NuPage gel plate (Invitrogen). Once the electrophoresis was finished, the front



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(containing additional hot ATP) was cut from the gel and subsequently discarded. A phosphor screen was used to develop the dried gel. A reaction containing no inhibitor was used as positive control whereas a reaction without active enzyme served as negative control. ELISA kits (Invitrogen) were used as per manufacturer’s protocol to investigate the effect of compounds on cell based pERK1/2 activity in cancer cells. MDR-reversal assay PHa MDR1/A retrovirus (20) was used to transfect L5178 mouse T-cell lymphoma cells (ATCC, USA). To maintain the expression of MDR phenotype, the mdr1-expressing cell line was cultured with 60 ng/mL colchicine. McCoy’s 5A medium (supplemented with antibiotics, Lglutamine and 10% heat-inactivated horse serum) was used to culture L5178 (parental) mouse Tcell lymphoma cells and the human mdr1-transfected subline at 37˚C in a 5% CO2 atmosphere. At a density of 2×106/mL, the cells were resuspended in serum-free McCoy’s 5A medium and 0.5 mL aliquots were transferred into eppendorf tubes. The compounds were added in the tubes at various concentrations (ranging from 4.0 and 40 µg/mL), followed by incubation at 25oC for 10 min. 10 µL rhodamine 123 (5.2 µM final concentration) was added to the tubes, incubated again at 37˚C for 20 min, washed twice, followed by resuspension in 0.5 mL PBS. Using a flow cytometer, the fluorescence of cell population was measured. In rhodamine 123 exclusion study, verapamil served as positive control. The fluorescence intensity (%) was recorded for the parental and treated MDR cell lines in comparison with untreated cells. Using the following equation, the activity ratio (R) was calculated, based on the recorded fluorescence readings:


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Statistical analysis All the experiments were performed in triplicates and data presented as the mean ± standard error of mean (SEM). The IC50 values were calculated by using Graph Pad Prism 5 software. Data was analyzed using a one-way analysis of variance (ANOVA) for multiple comparisons. ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org and includes HPLC analytical chromatograms of respective most active purified compounds (7f, 7o, 8af and 8ag). Molecular formula strings (CSV). AUTHOR INFORMATION Corresponding Authors HLQ: [email protected]; SNAB: [email protected]; [email protected] Tel: +6-01123695295, Fax: +6-0326983271 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Acknowledgements This work was supported by the Research Incentive Fund of Universiti Kebangsaan Malaysia Arus Perdana grant (AP-2014-023). HQ is grateful to Wuhan University of Technology for financial support.



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Notes “No author has Competing Financial Interest” ABBREVIATIONS MDR , multidrug resistance; EGFR , epidermal growth factor receptor; NSCLC, non-small cell lung; RTK , receptor tyrosine kinases; MAPK, mitogen-activated protein kinase; ABC, ATPbinding cassette, NF-kB, NF-kappaB; DRS, death receptors; tumor necrosis factor (TFN), PI, propidium iodide; TKIs, tyrosine kinase inhibitors; FAR , fluorescence activity ratio; HRMS, high resolution mass spectra; TLC, thin layer chromatography; UV, ultraviolet; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide.

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A. K.; Wubbenhorst, B.; Xu, X.; Gimotty, P. A.; Kee, D.; Santiago-Walker, A. E.; Letrero, R.; D'Andrea, K.; Pushparajan, A.; Hayden, J. E.; Brown, K. D.; Laquerre, S.; McArthur, G. A.; Sosman, J. A.; Nathanson, K. L.; Herlyn, M. Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell 2010, 18, 683-95. 45.

Li, Q.-S.; Li, C.-Y.; Lu, X.; Zhang, H.; Zhu, H.-L. Design, synthesis and biological

evaluation of novel (E)-α-benzylsulfonyl chalcone derivatives as potential BRAF inhibitors. Eur. J. Med. Chem. 2012, 50, 288-295. 46.

Molnar, J.; Gyemant, N.; Tanaka, M.; Hohmann, J.; Bergmann-Leitner, E.; Molnar, P.;

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Page 39 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O

O R'3 O R'3

R'4

R1 i

+ R'4

O

R'6

R2

R2

R1 (3)

(4a-u)

OCH3 R'3

R4

R4

R'4

O

R3

R3

OCH3 (1)

R1 R'6

R'6 OCH3

(2)

H

R'3

R1 R'6 R5

R5

R'4 OCH3

(5a-ag) HCl . H2N OH ii (6)

R6O

HO N

N

R'3

R'3

R'4

R'6

R1 R'6

OCH3

R'4

iii (9)

OCH3

R1 R'6 R5

11(x af, ag)

R'4

R'6

R2

R'4 OCH3

R1 R'6 7(c,f,o) OH N

R3

R'3

R4

R'3

OCH3

10 (f,o) OR6 R2 N R3

R'3

iii (9)

R'4 OCH3

R'3

R4

R1 R'6

R'4

R5 OCH3 8(f,i,o,u,x,af,ag)

Scheme 1: Synthesis scheme of α, β-unsaturated carbonyl based compounds, oxime and oxime ether analogs. Reagents and conditions: (i) NaOH, EtOH, Room temperature (ii) NH2OH.HCl, pyridine, ethanol, anhyd., reflux; (iii) haloalkylamine (9), K2CO3, Acetone/DMF, anhyd., reflux.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table 1: Structures of new (Cyclohexanone derivatives).

synthetic

α,

Page 40 of 48

β-unsaturated carbonyl based

compounds

O R'3

R'3

R'4

OCH3

1. 2. 3. 4. 5. 6.

No.

Comp. 4a 4b 4c 4d 4e 4f

7.

4g

8.

R1 R'6

R'6

R'4 OCH3

(4a-u)

R1 CH2 CH2 CH2

Rʹ3 H H OCH3 H H OCH3

Rʹ4 H OCH3 OCH3 H OCH3 OCH3

Rʹ6 Cl Cl Br Cl Cl Br

CH

H

H

Cl

4h

CH

H

OCH3

Cl

9.

4i

CH

OCH3

OCH3

Br

10. 11. 12. 13. 14. 15.

4j 4k 4l 4m 4n 4o

H H OCH3 H H OCH3

H OCH3 OCH3 H OCH3 OCH3

Cl Cl Br Cl Cl Br

16.

4p

H

H

Cl

H

OCH3

Cl

OCH3

OCH3

Br

H H OCH3

H OCH3 OCH3

Cl Cl Br

CH CH3 CH CH3 CH CH3

NH NH NH

N N N

CH3 CH3 CH3

N 17.

4q

N 18.

4r

19. 20. 21.

4s 4t 4u

N O O O


Page 41 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2: Structures of new synthetic α, β-unsaturated carbonyl based compounds (Tetralone derivatives). R2

O

R3

R'3

R4

R1 R'6 R5

R'4 OCH3

(5a-ag) No.

22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54.

Comp. 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p 5q 5r 5s 5t 5u 5v 5w 5x 5y 5z 5aa 5ab 5ac 5ad 5ae 5af 5ag

R1 CH2 CH2 CH2 CH-CH3 CH-CH3 CH-CH3 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2

R2 H H H H H H H H H H H H H H H H H H H H H OCH3 OCH3 OCH3 H H H H H H H H H

R3 H H H H H H H H H Cl Cl Cl H H H H H H OCH3 OCH3 OCH3 H H H Br Br Br F F F NO2 NO2 NO2

R4 H H H H H H H H H H H H OCH3 OCH3 OCH3 H H H OCH3 OCH3 OCH3 H H H H H H H H H H H H

R5 H H H H H H OH OH OH H H H H H H OCH3 OCH3 OCH3 H H H OCH3 OCH3 OCH3 H H H H H H H H H


Rʹ3 H H OCH3 H H OCH3 H H OCH3 H H OCH3 H H OCH3 H H OCH3 H H OCH3 H H OCH3 H H OCH3 H H OCH3 H H OCH3

Rʹ4 H OCH3 OCH3 H OCH3 OCH3 H OCH3 OCH3 H OCH3 OCH3 H OCH3 OCH3 H OCH3 OCH3 H OCH3 OCH3 H OCH3 OCH3 H OCH3 OCH3 H OCH3 OCH3 H OCH3 OCH3

Rʹ6 Cl Cl Br Cl Cl Br Cl Cl Br Cl Cl Br Cl Cl Br Cl Cl Br Cl Cl Br Cl Cl Br Cl Cl Br Cl Cl Br Cl Cl Br


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 48

Table 3: Structures of new synthetic oxime and oxime ether analogs. HO

R6O

N R'3 R'4

R1 R'6

R'6 OCH3

No.

55. 56,57 58,59

R'3

R'3

R'4

R'4

OCH3

7(c,f,o)

R'3

OCH3

R1 CH2 CH-CH3

Rʹ3 OCH3 OCH3

Rʹ4 OCH3 OCH3

Rʹ6 Br Br

7o, 10o

N-CH3

OCH3

OCH3

Br

N

R'3

R3

R4

R1 R'6

R'4 OCH3

R4

R5

R'4 OCH3

(10-f,o) R6

N

R2

OH

R3

R1 R'6

R'6

Comp. 7c 7f, 10f

R2

N

N

OR6 R'3

R1 R'6 R5

8(f,i,o,u,x,af,ag)

R'4 OCH3

11(x af, ag)

N R6 = No.

60. 61. 62. 63. 64,65 66,67 68,69

Comp. 8f 8i 8o 8u 8x, 11x 8af, 11af 8ag, 11ag

R1 CH-CH3 CH2 CH2 CH2 CH2 CH2 CH2

R2 H H H H OCH3 H H

R3 H H H OCH3 H NO2 NO2

R4 H H OCH3 OCH3 H H H


R5 H OH H H OCH3 H H

Rʹ3 OCH3 OCH3 OCH3 OCH3 OCH3 H OCH3

Rʹ4 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3

Rʹ6 Br Br Br Br Br Cl Br

Page 43 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 4: Inhibitory effects of synthetic cyclohexanone derivatives on the growth of normal (MCF-10A) mammary epithelial cells (cell viability) and different types of human cancer cells. Comp.

Cell viability %

4a

Antiproliferative activity IC50 ± SEM (µM) Panc-1

PaCa-2

90

6.9±0.3

7.2±0.6

4b

90

4.2±2.1

4c

89

4d

MCF-7

A-549

HT-29

H-460

PC-3

6.5±2.1

8.1±0.5

6.1±2.9

5.4±2.7

6.4±0.8

4.2±1.6

4.7±1.4

3.9±1.2

3.7±1.9

4.1±6.5

5.2±0.7

2.4±0.5

2.6±0.5

3.1±0.4

2.6±1.7

2.0±1.5

2.6±4.4

2.9±1.6

93

3.3±2.9

2.9±2.6

2.8±2.2

3.1±0.2

3.4±1.2

4.2±0.4

3.9±0.5

4e

94

3.2±1.7

3.0±1.1

3.2±2.8

4.4±1.2

2.9±1.5

2.9±0.7

3.7±2.6

4f

91

1.6±0.7

1.4±0.8

1.3±1.5

1.8±2.2

1.8±1.4

1.2±1.4

1.1±1.5

4g

93

14.5±1.5

16.7±0.7

15.2±0.3 13.4±0.8 12.1±0.5

13.9±1.8 14.1±2.1

4h

87

12.2±1.3

11.8±1.1

11.9±2.2 12.9±2.1 13.2±1.0

15.3±2.3 12.6±1.2

4i

89

7.2±2.2

7.5±2.9

8.1±1.2

7.6±0.5

6.7±0.2

7.4±2.3

7.2±1.3

4j

94

7.5±1.1

7.7±1.9

8.4±1.4

7.3±0.9

7.4±0.9

7.3±4.1

7.6±1.9

4k

92

6.5±1.6

6.2±0.9

6.4±1.6

7.5±0.5

6.6±1.7

7.2±1.4

7.0±0.5

4l

96

4.2±1.5

4.3±1.1

4.0±1.2

4.4±1.7

4.5±0.4

4.9±0.9

4.8±1.5

4m

91

4.2±0.5

4.6±0.5

4.4±0.6

4.0±2.2

4.4±1.5

4.0±0.7

4.9±1.5

4n

90

3.4±1.5

3.2±1.1

3.3±0.2

3.9±1.8

3.8±2.9

3.5±1.2

3.3±1.2

4o

92

1.8±0.8

1.9±0.8

1.9±0.3

1.3±0.7

1.1±0.2

1.8±1.5

1.6±0.7

4p

89

17.5±3.7

17.0±1.1

17.6±2.9 15.4±2.2 15.9±3.7

14.1±3.9 18.7±4.3

4q

89

15.6±1.7

15.5±4.2

15.4±4.9 16.2±1.9 10.3±2.4

13.9±4.6 15.7±5.2

4r

91

12.2±2.1

11.9±2.4

12.8±3.2 11.1±3.8 12.0±6.2

12.3±1.8 14.6±2.8

4s

88

8.9±1.4

8.7±2.2

7.3±2.1

8.4±2.9

8.8±2.5

7.4±1.6

8.3±2.7

4t

89

5.8±1.3

5.2±2.3

5.6±0.5

5.8±3.2

5.1±1.5

5.3±1.3

5.1±1.3

4u

92

4.9±1.7

4.6±1.5

4.3±1.9

4.1±0.8

4.2±1.8

5.0±2.3

4.6±1.5

Panc-1(pancreas cancer cell line): PaCa-2 (pancreatic carcinoma cell line): MCF-7 (breast cancer cell line): A-549 (epithelial): HT-29 (colon cancer cell line): H-460 (lung cancer cell line): PC-3 (prostate cancer cell line).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 48

Table 5: Inhibitory effects of synthetic tetralone derivatives on the growth of normal (MCF10A) mammary epithelial cells (cell viability) and different types of human cancer cells. Cell Antiproliferative activity IC50 ± SEM (µM) viability Panc-1 PaCa-2 MCF-7 A-549 HT-29 H-460 PC-3 % 5a 92 27.5±1.8 27.9±2.7 26.7±0.9 25.1±3.9 28.2±0.2 25.1±2.5 28.5±3.5 5b 96 24.5±2.4 23.2±0.9 20.5±1.7 23.3±1.2 26.5±3.2 22.9±0.6 23.4±2.9 5c 94 21.3±1.9 23.7±1.9 20.4±4.2 22.6±1.0 21.2±2.6 22.2±5.6 22.0±4.6 5d 98 8.3±2.4 9.8±1.2 8.8±2.9 8.7±1.3 8.4±2.1 9.6±0.5 9.9±1.7 5e 99 7.5±1.2 7.2±2.5 7.2±0.3 7.7±1.2 7.2±2.5 7.9±2.6 7.3±1.3 5f 92 2.4±1.6 2.3±3.5 2.1±2.2 2.6±1.8 2.9±0.5 2.2±1.4 2.4±0.8 5g 96 4.2±0.2 4.9±0.9 4.4±1.5 4.4±0.8 5.1±0.5 4.9±1.8 5.2±0.2 5h 91 3.6±1.4 3.2±4.4 3.1±1.4 3.3±1.4 3.0±1.5 3.8±2.5 3.5±0.9 5i 93 1.9±0.9 1.9±1.2 1.8±1.2 1.6±0.5 1.9±1.6 1.9±0.5 2.2±1.4 5j 94 12.4±1.8 12.8±2.5 14.1±1.5 13.3±0.9 12.6±0.5 13.3±1.2 13.7±0.4 5k 94 10.5±3.1 9.5±0.7 9.1±2.1 10.7±2.9 10.9±4.6 10.6±3.7 10.8±2.2 5l 90 10.2±2.5 10.4±2.4 12.9±0.6 10.7±1.5 9.9±1.1 10.7±1.7 10.2±1.2 5m 95 4.5±0.9 4.0±2.1 5.1±1.6 4.6±0.5 4.4±0.2 4.7±0.7 4.7±1.5 5n 89 2.1±1.5 2.3±0.2 2.6±0.5 2.9±2.6 2.7±0.7 2.6±0.9 2.0±1.7 5o 90 1.4±0.5 1.6±0.7 1.9±0.6 1.9±1.1 1.5±1.4 1.9±2.6 1.5±1.2 5p 93 8.5±1.2 8.7±1.8 9.0±1.1 6.4±0.8 8.4±1.2 9.0±1.5 8.4±3.1 5q 95 6.9±2.4 6.7±2.7 6.4±0.5 6.7±4.2 6.7±3.5 6.4±1.2 7.2±1.4 5r 94 4.2±1.3 4.6±0.5 5.7±2.2 4.5±3.2 3.9±2.8 4.7±0.9 3.9±1.5 5s 91 9.5±2.1 9.9±2.6 10.5±4.9 9.2±2.8 10.4±1.2 9.2±1.5 9.2±1.4 5t 97 5.4±2.4 5.0±1.1 5.4±1.2 5.4±3.2 5.2±1.6 5.8±1.0 5.2±2.7 5u 98 2.6±1.7 2.4±0.8 2.8±0.9 2.2±0.5 2.8±0.4 2.2±0.4 2.0±0.5 5v 98 7.4±0.4 6.7±0.9 7.2±0.3 8.8±1.7 7.10±0.5 7.9±1.5 7.2±2.2 5w 90 4.3±1.8 3.9±1.4 4.1±0.2 4.3±2.1 4.0±1.7 3.8±1.5 4.5±2.9 5x 91 2.5±1.2 2.2±1.7 2.4±1.6 2.6±0.5 1.9±1.6 2.1±3.5 2.2±1.4 5y 94 13.4±1.6 12.8±1.5 14.1±2.6 13.3±0.9 14.0±2.5 12.9±1.3 13.7±1.4 5z 92 10.8±1.2 10.6±0.5 11.1±2.1 10.3±4.2 10.5±2.6 11.1±2.1 10.4±0.8 5aa 92 9.9±1.6 9.0±1.0 10.9±1.6 9.1±1.3 9.2±1.4 10.9±5.6 9.3±2.7 5ab 90 12.4±0.9 15.2±5.6 12.9±1.8 11.1±2.1 12.9±0.2 15.2±4.6 12.6±2.5 5ac 95 9.8±3.2 9.6±2.5 9.8±2.5 10.9±1.6 9.4±0.9 10.6±4.5 9.3±2.9 5ad 93 8.5±1.7 8.9±1.2 10.1±3.5 8.2±1.6 9.6±2.3 8.9±2.6 10.3±2.9 5ae 95 3.5±0.7 3.4±1.1 3.3±1.2 3.6±0.5 3.5±1.7 3.1±2.8 3.1±3.3 5af 90 1.9±0.5 1.7±0.2 2.3±1.3 1.9±0.6 1.9±1.2 1.8±0.4 1.6±0.9 5ag 88 1.2±0.9 1.5±1.2 1.4±2.2 1.3±0.8 1.4±0.8 1.3±0.7 1.4±0.8 Panc-1(pancreas cancer cell line): PaCa-2 (pancreatic carcinoma cell line): MCF-7 (breast cancer cell line): A-549 (epithelial): HT-29 (colon cancer cell line): H-460 (lung cancer cell line): PC-3 (prostate cancer cell line).

Comp.


Page 45 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


Table 6: Inhibitory effects of synthetic compounds on the growth of normal (MCF-10A) mammary epithelial cells (cell viability) and different types of human cancer cells. Cell Antiproliferative activity IC50 ± SEM (µM) viability Panc-1 PaCa-2 MCF-7 A-549 HT-29 H-460 PC-3 % 7c 95 2.2±0.8 2.6±0.5 2.9±0.2 2.6±1.7 2.1±1.4 2.5±0.7 2.5±0.9 98 0.2±0.9 0.7±1.3 0.4±0.9 0.3±0.5 0.4±1.2 0.4±0.7 0.4±0.2 7f 96 0.09±0.01 0.07±0.09 0.06±0.03 0.06±0.04 0.07±0.05 0.08±0.04 0.09±0.02 7o 98 2.0±1.0 2.6±0.5 2.5±1.7 2.2±0.5 2.1±0.5 1.9±0.4 1.9±1.2 8f 90 1.5±0.6 1.2±2.6 1.4±0.2 2.0±0.7 1.1±1.6 1.3±1.2 2.3±2.1 8i 92 1.0±1.2 1.2±2.0 0.8±1.1 1.2±1.4 1.7±0.8 1.1±0.6 1.4±0.2 8o 92 2.1±1.2 2.9±1.4 2.2±1.5 1.9±0.7 2.2±1.6 2.6±0.9 2.2±0.7 8u 89 0.7±0.8 0.6±0.2 0.5±0.7 0.4±0.3 0.6±0.5 0.6±0.8 0.7±1.1 8x 93 0.09±0.03 0.09±0.02 0.07±0.02 0.08±0.02 0.09±0.06 0.08±0.01 0.02±0.04 8af 96 0.02±0.08 0.02±0.07 0.09±0.04 0.02±0.08 0.03±0.07 0.04±0.02 0.02±0.05 8ag 94 4.2±0.9 4.9±0.9 4.1±1.1 4.1±3.2 4.5±2.4 4.1±1.7 4.7±1.8 10f 95 0.09±0.01 0.08±0.01 0.09±0.08 0.07±0.02 0.09±0.05 0.07±0.01 0.08±0.07 10o 95 2.7±0.8 2.1±2.5 2.2±1.6 2.1±1.5 2.9±1.8 2.9±0.6 2.2±2.2 11x 94 6.9±0.3 6.6±1.9 6.6±0.9 7.1±2.1 7.0±2.1 7.2±2.8 6.4±2.1 11af 97 4.2±0.8 4.0±1.4 4.9±2.6 4.3±2.0 4.5±3.2 4.6±2.9 4.4±2.9 11ag Erlotinib 0.02±0.01 0.03±0.02 0.02±0.001 0.02±0.01 0.03±0.01 0.04±0.02 0.02±0.05 Panc-1(pancreas cancer cell line): PaCa-2 (pancreatic carcinoma cell line): MCF-7 (breast cancer cell line): A-549 (epithelial): HT-29 (colon cancer cell line): H-460 (lung cancer cell line): PC-3 (prostate cancer cell line). Comp.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 48

Table 7: Effects of selected synthetic compounds on EGFR, BRAFV600E and MDR.

Comp.

EGFR inhibition IC50 ± SEM (µM)

7f

0.5±0.05

2.7±0.8

15.8

7o

0.07±0.05

1.6±0.3

27.9

8f

3.2±0.4

2.4±0.5

44.6

8i

4.6±2.9

1.7±1.2

41.5

8o

2.1±1.5

1.9±1.1

37.6

8u

8.4±0.2

6.3±1.5

51.4

8x

4.2±0.9

1.8±0.4

52.4

8af

2.0±1.0

0.9±0.4

42.4

8ag

1.9±1.8

1.0±0.6

40.5

10o

0.1±0.05

3.2±1.1

17.2

Erlotinib

0.05±0.02

0.08±0.02

25.9

-

12.8

Verapamil

-

BRAF inhibition IC50 ± SEM (µM)

Fluorescence activity ratio (FAR)


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Figure 1: Effect of selected synthetic compounds on tubulin polymerization activity at concentration of 25 µM. Results are the mean values of three experiments, n=3. While vincristine and docetaxel (3 µM) were used as reference compounds. All compounds showed the inhibition of tubulin as below the horizontal line indicate tubulin inhibition, only docetaxel is above the horizontal line indicating tubulin stabilization and compounds (7o and 10o) close to the horizontal line are considered to be inactive. The y-axis demonstrates tubulin polymerization activity measured at 15 min (arbitrary units) in the growth phase of the tubulin polymerization curve.



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Synthesis of Î±, Î²-Unsaturated Carbonyl-Based Compounds, Oxime

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