{Novel (−)-goniofufurone mimics: synthesis, antiproliferative activity and SAR analysis} J. Serb. Chem. Soc. 84 (12) 1345–1353 (2019) UDC 547.724+547.455.623:547.466.1: JSCS–5268 576+615.9 Original scientific paper 1345 Novel (–)-goniofufurone mimics: Synthesis, antiproliferative activity and SAR analysis BOJANA SREĆO ZELENOVIĆ1#, SLAĐANA KEKEZOVIĆ1#, MIRJANA POPSAVIN1#, VESNA KOJIĆ2, GORAN BENEDEKOVIĆ1# and VELIMIR POPSAVIN1,3*# 1Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia, 2Oncology Institute of Vojvodina, Put dr Goldmana 4, 21204 Sremska Kamenica, Serbia and 3Serbian Academy of Sciences and Arts, Knez Mihajlova 35, 11000 Belgrade, Serbia (Received 12 September, accepted 23 September 2019) Abstract: Divergent syntheses of novel (–)-goniofufurone mimics with an alko- xymethyl group as the side chain have been accomplished from D-glucose in nine synthetic steps and in overall yields 6.7–8.7 %. Their in vitro antiprolife- rative activity was evaluated against eight human tumour cell lines as well as a single normal cell line. All analogues demonstrated powerful to good antipro- liferative effects toward all malignant cell lines under evaluation. Against the HL-60 cell line, all mimics showed increased activities being 27- to 1604-fold more potent than the lead compound, (–)-goniofufurone. Remarkably, the maj- ority of synthesized analogues displayed higher or similar activity to the com- mercial antitumour agent doxorubicin (DOX) against A549 cell line. The most potent compound exhibited 196-fold stronger cytotoxicity than DOX in the culture of this cell line. Keywords: D-glucose; antitumour agents; goniofufurone mimics; cytotoxic lac- tones, structure–activity relationships. INTRODUCTION (–)-Goniofufurone (1) is a synthetic styryl lactone, which represents the opposite enantiomer of naturally occurring (+)-goniofufurone (ent-1, Fig. 1). After isolation of (+)-goniofufurone from the stem bark of Gonithalamus gig- anteus (Annonaceae)1 in 1990, and the confirmation of the absolute stereo- chemistry of both 1 and ent-1,2,3 many syntheses of these compounds with unique and intriguing structures have been published.4–18 Both, 1 and ent-1, demonstrated a remarkable antiproliferative activity toward several human tumour cell lines.19,20 Synthetic molecule 1 and some of * Corresponding author. E-mail: velimir.popsavin@dh.uns.ac.rs # Serbian Chemical Society member. https://doi.org/10.2298/JSC190912104S 1346 SREĆO ZELENOVIĆ et al. its analogues showed more powerful cytotoxicity than the natural product ent-1 and the corresponding analogues from the (+)-series.21 Previous studies in our laboratory showed that dephenylated (–)-gonio- fufurone derivatives bearing an ether group with C9- and C10-hydrocarbon chains (compounds 6 and 7) exhibited in vitro antitumour activity against several human cancer cell lines.22 Fig. 1. Design of (+)-goniofufurone analogues 2–10: i) enantiomerization of ent-1; ii) dephenylation of 1; iii) O-alkylation of HO-7 in 2. As an extension of this work, herein the synthesis and in vitro antitumour activity of six novel (–)-goniofufurone mimics (3–5 and 8–10) and full experi- mental details for the preparation of 6 and 7 are reported. The preparation and cytotoxicity of known23,24 dephenylated (–)-goniofufurone analogue 2 is also disclosed. EXPERIMENTAL General procedures Melting points were determined on a Büchi 510, or on a hot stage microscope Nagema PHMK 05 apparatus and are not corrected. Optical rotations were measured on an Autopol IV (Rudolph Research) automatic polarimeter. The IR spectra were recorded with a FTIR Nexus 670 (Thermo-Nicolet) spectrophotometer. The NMR spectra were recorded on a Bruker AC 250 E or a Bruker Avance III 400 MHz instrument and the chemical shifts are expressed in ppm downfield from tetramethylsilane. Low resolution mass spectra were recorded on a Fin- nigan-MAT 8230 (CI) mass spectrometer. High-resolution mass spectra were taken on a Mic- romass LCT KA111 spectrometer or on LTQ OrbitrapXL (Thermo Fisher Scientific Inc., USA) mass spectrometer. TLC was performed on DC Alufolien Kieselgel 60 F254 (E. Merck). Flash column chromatography was performed using Kieselgel 60 (0.040–0.063 mm, E. Merck). All organic extracts were dried with anhydrous Na2SO4. Organic solutions were concentrated on a rotary evaporator under diminished pressure at a bath temperature below 35 °C. Synthetic procedures General procedure for the synthesis of the intermediates 12–19. To a solution of com- pound 11 (1 eq) in dry Et2O (2 mL) were added Ag2O (2.6 eq), AgOTf (0.3 eq) and the cor- responding alkyl bromide (3 eq). The mixture was stirred under reflux for 8–32 h (Table I). After completion of the reaction, which was detected by TLC, the mixture was purified by flash column chromatography (eluents in Table I). The characterization data for 12–19 are given in the Supplementary material to this paper. NOVEL (−)-GONIOFUFURONE MIMICS 1347 General procedure for the synthesis of the analogues 2–10. A solution of starting com- pound 11–19 (0.1 mmol) in dry EtOH (2 mL) was hydrogenated over 10 % Pd/C (≈10 % of Pd) at room temperature for 18–24 h (Table II). After completion of the reaction (TLC), the mixture was purified by flash column chromatography (eluents in Table II). The character- ization data for 2–10 are presented in the Supplementary material. Cytotoxic activity Cell cultures. Human myelogenous leukaemia (K562), promyelocytic leukaemia (HL- -60), T cell leukaemia (Jurkat), Burkitt’s lymphoma (Raji) and ER+ breast adenocarcinoma (MCF-7) malignant cells were grown in a suspension in RPMI 1640 nutrient medium, while ER− breast adenocarcinoma (MDA-MB 231), cervix carcinoma (HeLa), lung adenocarcinoma epithelial cells (A549), and normal foetal lung fibroblasts (MRC-5) were cultured as a mono- layer in DMEM medium. Both media were supplemented with 10 % of foetal calf serum (FTS, NIVNS) and antibiotics (100 IU mL-1 of penicillin and 100 mg mg-1 of streptomycin). The cell lines were cultured in flasks (Costar, 25 mL) at 37 °C in an atmosphere of 100 % humidity and 5 % of CO2 (Heraeus). Exponentially growing viable cells were used throughout the assays. MTT test.25 Cells were harvested, counted using trypan blue and plated into 96-well mic- rotiter plates (Costar) at the optimal seeding density of 5×103 cells per well to assure a loga- rithmic growth rate throughout the assay period. Viable cells were placed in a volume of 90 μL per well, and preincubated in complete medium at 37 °C for 24 h to allow cell stabilization prior to the addition of the substances. The tested substances, at 10-fold the required final concentration, were added (10 μL well-1) to all wells, except for the control ones, and the microplates were incubated for 72 h. The wells containing cells without tested substances were used as controls. MTT solution (10 μL) was added to all wells 3 h before the end of incubation period. MTT was dissolved in medium at 5 mg mL-1 and filtered to sterilize and remove the small amount of insoluble residue present in some batches of MTT. Acidified 2-propanol (100 μL of 0.04 M HCl in 2-propanol) was added to all wells and mixed thoroughly to dissolve the dark blue crystals of formazan. After a few minutes at room temperature, to ensure that all crystals were dissolved, the plates were read on a spectrophotometer plate reader (Multiscan MCC340, Labsystems) at 540 and 690 nm. The wells without cells contain- ing complete medium and MTT acted as blanks. RESULTS AND DISCUSSION Chemistry The syntheses of intermediates 12–19 is presented in Table I. Starting com- pound 11 was prepared from D-glucose in seven synthetic steps as previously reported.22 O-Alkylation of alcohol 11 with an excess of hexyl bromide in ether, in the presence of silver(I) oxide and silver(I) triflate as catalysts, gave the expected 7-O-hexyl derivative 12 in 69 % yield (Table I, entry 1). Compound 11 under the similar experimental conditions reacted with different alkyl bromides (C7–C13) to afford the corresponding ether derivatives 13–19 in good yields (Table I, entries 2–8). Hydrogenolytic removal of the benzyl ether protective group in 12–19, under standard reaction conditions furnished the target (–)-goniofufurone mimics 3–10 1348 SREĆO ZELENOVIĆ et al. in good to excellent yields (Table II, entries 2–9). Finally, under similar reaction conditions, alcohol 11 gave the known23,24 diol 2 in 97 % yield (entry 1). TABLE I. Preparation of intermediates 12–19 Entry R Reaction time, h Eluent for FCCa Product (yield, %) 1 C6H13 18 3:2 light petroleum/Et2O 12 (69) 2 C7H15 15 1:1 light petroleum/Et2O 13 (71) 3 C8H17 8 1:1 light petroleum/Et2O 14 (77) 4 C9H19 28 3:2 light petroleum/Et2O 15 (33) 5 C10H21 32 3:2 light petroleum/Et2O 16 (54) 6 C11H23 22.5 1:1 light petroleum/Et2O 17 (78) 7 C12H25 12 3:2 light petroleum/Et2O 18 (73) 8 C13H27 17.5 7:3 light petroleum/Et2O 19 (69) aFCC – Flash column chromatography TABLE II. Preparation of final products 2–10 Entry Starting compound R Reaction time, h Eluent for FCCa Product (yield, %) 1 11 H 20 7:3 CH2Cl2/EtOAc 2 (97) 2 12 C6H13 24 7:3 light petroleum/Et2O 3 (96) 3 13 C7H15 20 7:3 light petroleum/Et2O 4 (91) 4 14 C8H17 18 7:3 light petroleum/Et2O 5 (84) 5 15 C9H19 18 Et2O 6 (82) 6 16 C10H21 21 9:1 CH2Cl2/EtOAc 7 (80) 7 17 C11H23 20 7:3 light petroleum/Et2O 8 (86) 8 18 C12H25 20 3:2 light petroleum/Et2O 9 (72) 9 19 C13H27 21 7:3 light petroleum/Et2O 10 (88) aFCC – Flash column chromatography In vitro antiproliferative activity The biological activities of synthesized compounds 2–10 were evaluated by an in vitro cytotoxicity test against a panel of eight human malignant cell lines, including human myelogenous leukaemia (K562), human promyelocytic leu- kaemia (HL-60), T cell leukaemia (Jurkat), Burkitt's lymphoma (Raji), ER+ breast adenocarcinoma (MCF-7), ER− breast adenocarcinoma (MDA-MB 231), cervix carcinoma (HeLa) and lung adenocarcinoma epithelial cells (A549) and NOVEL (−)-GONIOFUFURONE MIMICS 1349 against single normal cell line, foetal lung fibroblasts (MRC-5). Cell growth inhi- bition was evaluated using the standard MTT colorimetric assay after exposure of cells to the test compounds for 72 h.25 (–)-Goniofufurone (1), analogue 2 and the commercial antitumour agent doxorubicin (DOX) were used as positive controls. According to the resulting IC50 values (Table III), of all cell lines tested, four were sensitive to all of the synthesized analogues 2–10 (K562, HL-60, HeLa and A549). Of the remaining four cell lines, Jurkat and Raji cells were sensitive to eight, while MCF-7 and MDA-MB 231 cell lines were sensitive to seven of nine synthesized analogues. The most active compound in A549 cell culture is analogue 10 (IC50 = 0.025 µM) that exhibited 94- and 196-fold higher potency when compared to lead 1 and DOX, respectively. Simultaneously, analogue 10 represents the most active compound described in this paper. TABLE III. In vitro cytotoxicity of (–)-goniofufurone (1), DOX and the analogues 2–10 after 72 h Compound IC50 / µMa K562 HL-60 Jurkat Raji MCF-7 MDA-MB 231 HeLa A549 MRC-5 1 2.96 >100 2.49 23.42 51.27 >100 >100 2.36 >100 2 2.69 9.97 9.51 7.40 9.64 0.24 5.22 31.45 59.88 3 0.70 4.91 8.87 1.11 12.34 15.62 3.54 2.43 >100 4 1.02 1.10 11.53 5.98 2.38 9.76 0.56 4.43 >100 5 0.74 0.68 19.78 4.25 0.34 28.70 3.41 4.19 >100 6 8.61b 1.53b 6.64b 7.25 >100 >100 9.59b 0.92 >100b 7 1.25b 0.14b >100b 76.36 89.36 >100 0.30b 29.05 >100b 8 0.18 1.83 16.26 2.79 2.28 26.57 4.11 7.72 >100 9 3.46 8.25 8.02 3.52 5.31 7.63 2.25 3.96 >100 10 4.87 3.96 4.29 4.88 15.36 36.47 10.32 0.025 >100 DOX 0.25 0.92 0.03 2.98 0.20 0.09 0.07 4.91 0.10 aIC50 is the concentration of compound required to inhibit the cell growth by 50 % compared to an untreated control. The values are means of three independent experiments. The coefficients of variation were less than 10 %; btaken from the literature22 All the synthesized analogues exhibited strong antiproliferative effects on K562 cells with IC50 values in the range of 0.18–8.61 µM. The highest potency against this cell line was recorded after treatment with analogue 8 (IC50 = 0.18 µM), which is approximately as active as DOX (IC50 = 0.25 µM), but 16-fold more active than lead 1. All analogues (2–10) demonstrated powerful to good antiproliferative effects towards HL-60 cells (IC50 values in the range 0.14–9.97 µM), in contrast to lead 1, which was completely inactive against this cell line. Moreover, all analogues with an alkoxymethyl grou(3–10) showed better antiproliferative effects against the HL-60 cell line than diol 2 (IC50 = 9.97 µM). 1350 SREĆO ZELENOVIĆ et al. The most active molecule against HL-60 cell line was analogue 5 (IC50 = = 0.68 µM), which exhibited similar activity as DOX, but was 330-fold more potent than lead 1. Analogue 3 was the most active compound in the culture of Raji cells that exhibited over 2.5-, 6.5- and 21-fold higher potency than control compounds, DOX, 2 and 1, respectively. All novel ether analogues (3–5 and 8–10) demon- strated 4- (4, IC50 = 5.98 µM) to 21-fold (3, IC50 = 1.11 µM) higher activity than lead 1, against Raji cells. The parent compound 1 showed poor activity (IC50 = 51.27 µM) against MCF-7 cells, as did the previously synthesized compounds 6 and 7, which were practically inactive to these cells (IC50 >100 µM and IC50 = 89.36 µM, respect- ively). However, all new analogues (3–5 and 8–10) and compound 2 exhibited good cytotoxic effects toward this cell line with IC50 values in the range 0.34– 15.36 µM, being essentially 3–150-fold more active than 1. The most active compound against the MCF-7 cell line was the ether ana- logue 5. This molecule exhibited submicromolar cytotoxicity (IC50 = 0.34 µM) although its potency was slightly lower than the activity of DOX (IC50 = 0.20 µM). Lead compound 1 was inactive against HeLa and MDA-MB 231 cells, but all novel analogues and dephenylated analogue 2 showed good (0.56–10.32 µM) to moderate inhibitory activity (7.63–36.47 µM), respectively, against these cell lines. All the synthesized ether analogues (3–10) as well as lead compound 1, were completely inactive toward normal MRC-5 cells. Only molecule 2 showed low cytotoxicity (IC50 = 59.88 µM) against this cell line. On the contrary, the com- mercial antitumour agent DOX exhibited a potent cytotoxicity against this cell line. SAR analysis As shown in Table III, replacement of the α-hydroxylbenzyl group in 1 with an alkoxymethyl chain significantly increased the activities of the resulting ana- logues 3–10. (For a graphical presentation see, Fig. S-35A of the Supplementary material). As is further evident from Table III, the introduction of a hydrophobic group, by O-alkylation of OH-7 in 2, resulted in analogues 3–10 of improved cytotoxicity toward the majority of cell lines under evaluation (Fig. S-35B of the Supplementary material). Finally, it was found that the length of the side chain of the analogues is not crucial for antiproliferative activity of the analogues 3–10 (Table III and Fig. S- -35C of the Supplementary material). This is in contrast with previous findings,22 which suggested that one- or two-carbon homologation of the side chain inc- reases the activity of the resulting homologues against most of the cells tested. However, the results of SAR analysis presented in this work are more reliable NOVEL (−)-GONIOFUFURONE MIMICS 1351 because they were conducted on seven pairs of analogues, unlike the previous SAR study that was realized with a lower number of compounds. CONCLUSIONS To summarize, nine novel (–)-goniofufurone analogues were designed and synthesized from D-glucose as the starting compound. The newly synthesized molecules were evaluated for their antiproliferative activity against eight human malignant cell lines. Eight of the nine synthesized analogues showed submicro- molar anticancer activity against at least one of the tested cell lines and five of them were more potent than DOX (10, IC50 = 0.025 µM, and 6, IC50 = 0.92 µM against A549; 7, IC50 = 0.14 µM, and 5, IC50 = 0.68 µM against HL-60 and 8, IC50 = 0.18 µM against K562). SAR analysis showed that the replacement of α-hydroxylbenzyl group in 1 with an alkoxymethyl chain, as well as O-alkylation of OH-7 in 2, may improve the cytotoxicity of the analogues towards the majority of the cell lines under evaluation. All analogues were devoid of any toxicity against a normal human cell line (MRC-5). It is believed that this approach could be used in the search for novel, more potent and selective antitumour agents derived from lead 1. SUPPLEMENTARY MATERIAL Analytical and spectral data, as well as additional experimental data, are available electronically from http://www.shd.org.rs/JSCS/, or from the corresponding author on request. Acknowledgements. This work was supported by research grants from the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant No. OI 172006). This work also received funding from the Serbian Academy of Sciences and Arts under the strategic projects programme (Grant agreement No 01-2019-F65). И З В О Д НОВИ МИМЕТИЦИ (−)-ГОНИОФУФУРОНА: СИНТЕЗА, АНТИПРОЛИФЕРАТИВНА АКТИВНОСТ И SAR АНАЛИЗА БОЈАНА М. СРЕЋО ЗЕЛЕНОВИЋ1, СЛАЂАНА М. КЕКЕЗОВИЋ1, МИРЈАНА ПОПСАВИН1, ВЕСНА В. КОЈИЋ2, ГОРАН И. БЕНЕДЕКОВИЋ1 И ВЕЛИМИР ПОПСАВИН1,3 1Департман за хемију, биохемију и заштиту животне средине, Природно-математички факултет, Трг Доситеја Обрадовића 3, 21000 Нови Сад, 2Онколошки институт Војводине, Пут др Голдмана 4, 21204 Сремска Каменица и 3Српска академија наука и уметности, Кнез Михајлова 35, 11000 Београд Дивергентна синтеза нових миметика (–)-гониофуфурона, са алкоксиметил групом у бочном низу, остварена је полазећи из D-глукозе у девет синтетских фаза са укупним приносима од 6,7 до 8,7 %. Њихова in vitro антипролиферативна активност је испитана према осам хуманих туморских и једној нормалној ћелијској линији. Сви аналози су испољили снажне или добре антипролиферативне ефекте према већини испитиваних ћелијских линија. Према ћелијској линији HL-60, сви миметици су испољили повећану активност и били су 27–1604 пута потентнији од водећег једињења (–)-гониофуфурона. 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