European Journal of Medicinal Chemistry 84 (2014) 555e565
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antitumor activity, and structureeactivity relationship study of trihydroxylated 2,4,6-triphenyl pyridines as potent and selective topoisomerase II inhibitors Radha Karki a, 1, Chanmi Park b, Kyu-Yeon Jun b, Jun-Goo Jee c, Jun-Ho Lee d, Pritam Thapa a, 1, Tara Man Kadayat a, Youngjoo Kwon b, *, Eung-Seok Lee a, * a
College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Global Top 5 Program, Ewha Womans University, Seoul 120-750, Republic of Korea c College of Pharmacy, Kyungpook National University, Daegu 702-701, Republic of Korea d Department of Emergency Medical Technology, Daejeon University, Daejeon 300-716, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 January 2014 Received in revised form 19 June 2014 Accepted 17 July 2014 Available online 18 July 2014
A series of eighteen trihydroxylated 2,4,6-triphenyl pyridines were designed and synthesized which contain hydroxyl groups at ortho, meta or para position of each phenyl rings attached to the central pyridine. They were evaluated for topoisomerase I and II inhibitory activity, and cytotoxicity against several human cancer cell lines for the development of novel anticancer agents. Most of the compounds exhibited strong and selective topoisomerase II inhibitory activity compared to the positive control, etoposide, and also displayed significant cytotoxicity in low micromolar range. Trihydroxylated 2,4,6triphenyl pyridines were more potent than mono- and di-hydroxylated 2,4,6-triphenyl pyridines, which have been previously studied in our research group. Positive correlation between topoisomerase II inhibitory activity and cytotoxicity was observed for the most compounds. Molecular docking study shows qualitatively consistent with the results of biological assays. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Trihydroxylated 2,4,6-triphenyl pyridines Topoisomerase I Topoisomerase II Topo II inhibitory activity Cytotoxicity Anticancer agents Docking study
1. Introduction Topoisomerases play an important role in solving the DNA topological hurdles during DNA replication by their activity of transiently cutting one or both strands of DNA followed by rejoining [1,2]. The human topoisomerase has two types, topoisomerase I (topo I) and topoisomerase II (topo II). Topo I and II inhibitors such as camptothecin, doxorubicin, and etoposide are considered among the most active anticancer agents [3e6]. In the past decades, several natural products and synthetic compounds have been identified as novel topoisomerase inhibitors in order to overcome the limitations of existing topoisomerase inhibitors [7]. Many
* Corresponding authors. E-mail addresses: [email protected] (Y. Kwon), [email protected] (E.-S. Lee). 1 Present address: Division of Hematology & Oncology, Department of Internal Medicine, Kansas University Medical Center, Kansas City, KS 66103, USA. http://dx.doi.org/10.1016/j.ejmech.2014.07.058 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
researchers in medicinal chemistry recently started to develop topo II-selective inhibitors as anticancer agents [8e10]. Topo II is distinct from topo I in that topo II acts as a homodimer, requiring Mg (II) and ATP hydrolysis for enzyme turn over and rapid kinetics; this enables it to cut both DNA strands simultaneously. Furthermore, topo II is the only enzyme available to disentangle the topological problems in chromosomes, which must occur during completion of replication and to decantenate the replicated chromosomes by introducing transient double strand breaks [11]. In addition, one of the subtype of topo II, topo IIa, is cell cycle dependent enzyme, which peaks during G2/M phase and decreases at the end of mitosis. The overexpression of topo IIa is only observed in proliferating cells. Therefore, topo II is a more important factor for cell proliferation than topo I [12e13]. In these reasons, many researchers in medicinal chemistry recently started to develop topo II-selective inhibitors as anticancer agents [8e11]. Polyphenolic phytochemicals are considered to be potentially bioactive compounds. For instance, isoliquiritigenin found in vegetables, tea flavonol epigallocatechin gallate (EGCG) and
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resveratrol found in red wine act as topo II poison. Similarly, myricetin, a naturally occurring flavonol, act as dual inhibitors of topo I and II [11,13e17]. Synthetic compounds such as polyhydroxybenzoyl amide and isoaurostatin derivatives having hydroxyl group in the aromatic ring displayed strong topo I and II inhibition [18,19]. Similarly, several bisphenol analogs were found to be catalytic topo II inhibitor with the exception of a few being topo II poison [20]. In an effort to discover novel anticancer agent as topoisomerase inhibitors, we had previously designed and synthesized several terpyridine bioisosteres bearing 2,4,6-triaryl pyridines. Most of the 2,4,6-triaryl pyridines without substitutions on aryl moiety possessed weak to moderate topoisomerase inhibitory activity and cytotoxicity against several human cancer cell lines [21e23]. From one of the prepared 2,4,6-triaryl pyridines, 2,4,6-triphenyl pyridine (A) did not show any topo I and II inhibitory activity, and cytotoxicity against several human cell lines. For improvement of efficacy of those compounds, we introduced hydroxyl moiety to the phenyl ring since many biologically active natural and/or synthetic compounds possess hydroxyl group in their aromatic molecular skeletons. As anticipated, monohydroxylated 2,4,6-triphenyl pyridines showed relatively stronger topo I and/or II inhibitory activity than those of the non-substituted compounds [24]. With these results, we further attempted to prepare dihydroxylated 2,4,6-triphenyl pyridines, which resulted to enhance topoisomerase II inhibitory activity as well as cytotoxicity against several human cancer cell lines [25]. These results encouraged us to systematically design and prepare trihydroxylated 2,4,6-triphenyl pyridines as shown in Fig. 1. We expected increase of topo II inhibitory activity and selectivity as well as cytotoxicity against several human cancer cell lines since we observed that the increased number of hydroxyl groups in phenyl moiety enhanced the topo II inhibitory activity and selectivity along with cytotoxicity against several human cancer cell lines from previous results. In this study we have described the design and synthesis of trihydroxylated 2,4,6-triphenyl pyridines, and evaluation of topo I and II inhibitory activity, especially selectivity to topo II inhibitory activity, and cytotoxicity against several human cancer cell lines (Fig. 2). Trihydroxylated 2,4,6-triphenyl pyridines are novel compounds which have not been previously reported. They are designed and discovered by our research group possessing strong topo II inhibitory activity, selectivity, and cytotoxicity against
several human cancer cell lines. Structureeactivity relationship study was determined with respect to non-hydroxylated 2,4,6triphenyl pyridine (A), mono- and di-hydroxylated 2,4,6-triphenyl pyridines with trihydroxylated 2,4,6-triphenyl pyridines. The results indicated that topo II inhibitory activity and selectivity were greatly increased, and cytotoxicity was found to be enhanced by the increase in the number of hydroxyl moiety.
2. Chemistry A new series of trihydroxylated 2,4,6-triphenyl pyridines were €hnke synthesis [26,27] as illustrated synthesized using modified Kro in Scheme 1. First, nine hydroxylated chalcone intermediates 3 (R1, R2 ¼ aec) were synthesized using ClaiseneSchmidt condensation reaction as reported earlier [23]. In the second step, three pyridinium iodide salts 4 (R3 ¼ aec) were synthesized in quantitative yield by the treatment of aryl methyl ketone 1 (R1 ¼ aec) with €hnke synthesis, final comiodine in pyridine. Using modified Kro pounds 5 (R1, R2, R3 ¼ aec) were synthesized by the reaction of appropriate hydroxylated chalcone 3 with pyridinium iodide salt 4 in the presence of ammonium acetate and glacial acetic acid in 29.0e88.2% yield. Total eighteen final compounds 6e23 were synthesized from the reaction of nine hydroxylated chalcone intermediates and three pyridinium iodide salts in six different series as shown in Fig. 3. All the compounds contain three hydroxyl moieties substituted at various positions (ortho, meta, or para) of each phenyl rings. Different substitution pattern together with yield, purity and melting point is illustrated in Table 1.
3. Results and discussion Inhibition against relaxation activity of topo I and II was measured by detecting the conversion of supercoiled pBR322 DNA to its relaxed form in the presence of synthesized trihydroxylated 2,4,6-triphenyl pyridines 6e23. Camptothecin and etoposide, clinically used selective topo I and II inhibitors, respectively, were used as references. For cytotoxicity adriamycin (doxorubicin), also clinically using anticancer agent, camptothecin, and etoposide were used as references.
Fig. 1. Structures of 2,4,6-triaryl pyridines, 2,4,6-triphenyl pyridine (A), mono-, di-, and trihydroxylated 2,4,6-triphenyl pyridines.
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Fig. 2. Strategy for the design of trihydroxylated 2,4,6-triphenyl pyridines.
Scheme 1. General synthetic method of trihydroxylated 2,4,6-triphenyl pyridines.
3.1. Topoisomerase I and II inhibitory activity Fig. 4 and Table 2 illustrates the topo I inhibitory activity of prepared trihydroxylated 2,4,6-triphenyl pyridines. None of the compounds possessed topo I inhibitory activity, which indicate that the trihydroxylated 2,4,6-triphenyl pyridines exhibited strong selectivity to topo II inhibition. The effect of trihydroxylated 2,4,6-triphenyl pyridines on human DNA topo II were observed in the relaxation assay using
supercoiled pBR322 plasmid DNA in the presence of ATP. As shown in Fig. 5 and Table 2, most of the compounds (6, 7, 9e15, 17, 18, 21e23) exhibited significant topo II inhibition at 100 mM which were stronger or comparable to positive control, etoposide. Compounds 13 and 17 showed stronger topo II inhibitory activity than that of etoposide at both 100 mM and 20 mM. Compound 13 displayed 90.1% and 48.2% inhibition, and compound 17 possessed 80.6% and 38.5% inhibition, while etoposide displayed 79.6% and 31.1% inhibition at 100 mM and 20 mM, respectively.
Fig. 3. Structures of prepared trihydroxylated 2,4,6-triphenyl pyridines.
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Table 1 Prepared compounds with yield, purity by HPLC, and melting point.
Entry
R1
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
20 20 20 30 30 30 40 40 40 20 20 20 20 20 20 30 30 30
R2 OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH
phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl
2 3 4 2 3 4 2 3 4 2 3 4 2 3 4 2 3 4
OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH
R3 phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl
20 20 20 30 30 30 40 40 40 30 30 30 40 40 40 40 40 40
OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH
phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl
Yield (%)
Purity (%)
Mp ( C)
29.0 80.0 41.8 38.2 61.0 88.2 32.1 85.2 51.8 53.4 78.6 72.7 52.0 43.0 79.3 42.0 60.8 61.3
97 97 97 96 99 98 96 98 97 96 98 98 96 96 100 100 99 100
230e231 259e260 280e281 271e272 253e254 246e247 240e241 279e280 260e261 263e264 278e279 299e300 275e276 330e331 316e317 260e261 236e237 242e243
Also compound 18 exhibited 77.6% and 21.3% inhibition at 100 mM and 20 mM, respectively. Compound 9e12, 22, and 23 displayed 100% topo II inhibition at 100 mM. Similarly, compounds 6, 7, 14, 15, and 21 showed 95.1%, 99.4%, 79.1%, 87.9%, and 92.2% inhibition at 100 mM, respectively, which were higher than that of etoposide. Compounds 8, 19, and 20 exhibited moderate topo II inhibition (46.9%, 31.3%, and 44%, respectively) at 100 mM. Only compound 16 showed weak topo II inhibition of 11.3% at 100 mM.
3.2. Cytotoxicity For the evaluation of cytotoxicity, four human cancer cell lines were utilized: human embryonic kidney 293 cells (HEK293), human prostate tumor cell line (DU145), human colorectal adenocarcinoma cell line (HCT15), and human ductal breast epithelial tumor cell line (T47D). Adriamycin, camptothecin, and etoposide were used as positive controls. Most of the compounds displayed significant cytotoxicity against the tested cell lines at low micromolar concentration compared to the positive controls. The IC50 values (mM) of the prepared trihydroxylated 2,4,6-triphenyl pyridines against those cell lines are shown in Table 2. In general, most
of the compounds displayed strong cytotoxicity (50 4.04 ± 0.2 1.76 ± 0.02 0.98 ± 0.02 1.4 ± 0.03 3.16 ± 0.14 3.98 ± 0.07 >50 3.42 ± 0.02 0.36 ± 0.01 >50 >50 5.37 ± 0.02 1.94 ± 0.04 5.8 ± 0.14
7.74 1.98 2.59 >50 2.18 5.02 >50 5.15 3.95 3.81 1.92 >50 5.12 3.39 >50 5.83 8.24 >50 >50 4.85 3.09 3.52
T47D
100 mM 66.2b/65.7
0.0 95.1 99.4 46.9 100.0 100.0 100.0 100.0 90.1 79.1 87.9 11.3 80.6 77.6 31.3 44.0 92.2 100.0 100.0
HEK293
2.6 0.0 4.9 3.7 0.0 0.0 0.0 0.4 0.0 3.8 0.0 1.9 1.5 0.3 8.4 0.0 0.0 0.0 0.0
1.04 0.28 1.22 8.13 0.14 0.08 >50 0.13 0.74 0.73 0.78 >50 0.79 0.73 >50 7.42 6.99 >50 26.3 5.65 1.56 0.84
± ± ± ± ± ±
0.03 0.01 0.03 0.09 0.01 0.003
± ± ± ±
0.004 0.004 0.01 0.002
± 0.005 ± 0.004 ± 0.3 ± 0.1 ± ± ± ±
1.01 0.08 0.07 0.02
± 0.04 ± 0.08 ± 0.06 ± 0.01 ± 0.08 ± ± ± ±
0.01 0.11 0.07 0.04
± 0.08 ± 0.06 ± 0.06 ± 0.16
± 0.04 ± 0.12 ± 0.06
8.74 ± 0.35 0.75 ± 0.01 3.12 ± 0.02 45.43 ± 1.96 2.51 ± 0.03 3.42 ± 0.04 18.3 ± 0.31 4.57 ± 0.08 2.05 ± 0.02 2.23 ± 0.09 5.35 ± 0.21 4.07 ± 0.12 4.27 ± 0.01 5.76 ± 0.13 >50 4.81 ± 0.03 0.96 ± 0.005 >50 >50 0.65 ± 0.002 3.92 ± 0.00 4.47 ± 0.02
HEK293: human embryonic kidney 293 cells. DU145: human prostate tumor cell line. HCT15: human colorectal adenocarcinoma cell line. T47D: human ductal breast epithelial tumor cell line. a Each data represents mean ± S.D. from three different experiments performed in triplicate. b Positive control value for compounds A and 6e13. c Topo I inhibitory activity and cytotoxicity reported earlier [22].
results showed that most of trihydroxylated 2,4,6-triphenyl pyridines displayed significant and selective topo II inhibition, which was stronger than the positive control, etoposide, and also significant cytotoxicity. All these results suggest that increase in the number of hydroxyl moiety increase topo II inhibition as shown in Fig. 7. Although it was difficult to draw up the correlation between the position of hydroxyl moiety in phenyl ring with topo II inhibition because most of the compounds showed significant activity, we
could drive a trend according to the positions of hydroxyl groups in R1 and R3 based on ortho-, meta-, and para-hydroxy phenyl ring of R2. When R2 is ortho-hydroxy phenyl, the order of topo II inhibition activity with positions of hydroxyl group in R1 and R3 is stated in a way of hydroxy positions in R1 and R3 and % inhibition of topo II at 100 mM in parenthesis followed by the name of compound: parapara (100%, 12) z meta-meta (100%, 9) > ortho-ortho (95.1%, 6) > meta-para (92.2%, 21) > meta-ortho (87.9%, 15) > para-ortho (77.6%, 18); When R2 is meta-hydroxy phenyl, those are meta-para
Fig. 5. Human DNA topo II inhibitory activity of 2,4,6-triphenyl pyridine (A) and 6e23.
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Fig. 6. Structures of monohydroxylated 2,4,6-triphenyl pyridines with % inhibition of topo II at 100 and 20 mM, respectively [24].
Fig. 7. Favorable order of substitution for topo II inhibitory activity.
(100%, 22) z meta-meta (100%, 10) > ortho-ortho (99.4%, 7) > parapara (90.1%, 13) > ortho-para (31.3%, 19) > ortho-meta (11.3%, 16); When R2 is para-hydroxy phenyl, those are meta-para (100%, 23) z meta-meta (100%, 11) > ortho-meta (80.6%, 17) z para-para (79.1%, 14) > ortho-ortho (46.9%, 8) > ortho-para (44.0%, 20). When R2 is ortho-hydroxy phenyl, compounds generally exhibited stronger topo II inhibitory activity than meta- or para-hydroxy phenyl ring in R2. Interestingly, compound 13 which possess para-hydroxyl
moiety at 2- and 6-phenyl ring and meta-hydroxyl moiety at 4phenyl ring displayed the most significant topo II inhibition at both concentrations. Overall, it might be draw out structureeactivity relationship with some exceptions such as: ortho-ortho, meta-meta, and para-para-hydroxyl substitutions in 2- and 6-phenyl ring exhibited better topo II inhibitory activity and cytotoxicities (Fig. 8). In addition, positive correlation was found between topo II inhibition and cytotoxicity for most of the compounds.
Fig. 8. Structureeactivity relationship of trihydroxylated 2,4,6-triphenyl pyridines.
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3.4. Molecular docking studies Compound 13 is the most potent at the low concentration (20 mM) in a cell-free experiment which may imply to be more persuasive topo II inhibitor in cells. Therefore, a series of compounds related to compound 13 were additionally selected from our previous study as depicted in Fig. 9A [24,25] to perform the ensemble docking and clustering study for further comparing the mode of action of compound 13 with that of etoposide. The ensemble docking and clustering with various initial ligand conformers improves the reliability in the docking simulations with AutoDock and AutoDock Vina. In addition to improving accuracy, one can calculate the deviation of energies in each pose, which will be important for the quantitative interpretation. Our calculation revealed that all the docked poses of compounds share the core features as shown in Fig. 9B. It may demonstrate the soundness of our approach. Considering the resemblance of the molecules, it will be reasonable to consider that their binding modes are quite similar. AutoDock Vina docking energies in each pose are as follows: 8.62 ± 0.10, 8.77 ± 0.07, 8.91 ± 0.10, 9.00 ± 0.09, 9.10 ± 0.12, 9.30 ± 0.13 (kcal/mol) for compounds A to E and 13, respectively, and are qualitatively consistent with the results of biological assays except compound E. The lower energies in the molecules that have additional hydroxyl moieties can be explained by the additional hydrophilic contacts with Lys8-OZ, Asp109-OD2, and Ade1486-N3. These interactions reinforce the hydrophobic core contacts with Gly30, Arg55, and Thy1463 (Fig. 9C). It should be underscored that the hydrophilic interactions are rarely overlapped with the contacts by etoposide (Fig. 9D), emphasizing the novelty of the inhibitory mechanism of compound 13. 4. Conclusion We have systematically designed and synthesized eighteen trihydroxylated 2,4,6-triphenyl pyridines by efficient synthetic route, and were evaluated for topo I and II inhibitory activity, and cytotoxicity against several human cancer cell lines. Most of the compounds (6, 7, 9e15, 17, 18, 21e23) showed strong and selective topo
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II inhibitory activity at 100 mM, and significant cytotoxicity. The structureeactivity relationship study revealed that the substitution of hydroxyl group on phenyl ring increase the topo I and II inhibitory activity of compounds, and further increase in the number of hydroxyl moiety enhances selectivity and inhibitory activity of topo II, and cytotoxicity. Also, positive correlation was found between topo II inhibition and cytotoxicity for most of the compounds. Furthermore, molecular docking study shows qualitatively consistent with the results of biological assays.
5. Experimental Compounds used as starting materials and reagents were obtained from Aldrich Chemical Co., Junsei or other chemical companies, and utilized without further purification. HPLC grade acetonitrile (ACN) and methanol were purchased from Burdick and Jackson, USA. Thin-layer chromatography (TLC) and column chromatography (CC) were performed with Kieselgel 60 F254 (Merck) and silica gel (Kieselgel 60, 230e400 mesh, Merck) respectively. Since all the compounds prepared contain aromatic ring, they were visualized and detected on TLC plates with UV light (short wave, long wave or both). NMR spectra were recorded on a Bruker AMX 250 (250 MHz, FT) for 1H NMR and 62.5 MHz for 13C NMR, and chemical shifts were calibrated according to TMS. Chemical shifts (d) were recorded in ppm and coupling constants (J) in hertz (Hz). Melting points were determined in open capillary tubes on electrothermal 1A 9100 digital melting point apparatus and were uncorrected. HPLC analyses were performed using two Shimadzu LC-10AT pumps gradient-controlled HPLC system equipped with Shimadzu system controller (SCL-10A VP) and photo diode array detector (SPD-M10A VP) utilizing Shimadzu Class VP program. Sample volume of 10 mL was injected in Waters X- Terra® 5 mM reverse-phase C18 column (4.6 250 mm) with a gradient elutions of 20%e100% of B in A for 10 min followed by 100%e20% of B in A for 20 min at a flow rate of 1.0 mL/min at 254 nm UV detection, where mobile phase A was double distilled water with 20 mM ammonium
Fig. 9. (A) Structures of compounds A to E and 13 with % topo II inhibition at 100 and 20 mM. Compounds B and C were retrieved from the reference 22 and compounds D and E were from the reference 23. The values in parenthesis are those of positive control, etoposide, extracted from the previous study [24,25]. (B) Overlaid structures of all compounds of (A) to topo IIa (PDB ID: 4FM9) in the calculated binding models. The molecules are colored in cyan. Cartoons in gray and yellow colors mean protein and DNA regions, respectively. (C) Binding mode of compound 13. We labeled only the residues in topo IIa and DNA that contact compound 13 within 3.5 Å, where asterisks indicate DNA residues. (D) Comparison with etoposide. Etoposide is colored in green. All the structural figures were generated by Pymol software (http://pymol.sourceforge.net/). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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formate (AF) and B was 90% ACN in water with 20 mM AF. Purity of compound is described as percent (%). ESI LC/MS analyses were performed with a Finnigan LCQ Advantage® LC/MS/MS spectrometry utilizing Xcalibur® program. For ESI LC/MS, LC was performed with a 2 mL injection volume on a Waters X Terra® 3.5 mm reverse-phase C18 column (2.1ⅹ 100 mm) with a gradient elution from 10% to 80% of B in A for 5 min followed by 80%e10% of B in A for 10 min and 10% B in A for 5 min, at a flow rate of 250 mL/min, where mobile phase A was 100% distilled water with 0.1% formic acid and mobile phase B was 100% ACN. MS ionization conditions were: Sheath gas flow rate: 70 arb, aux gas flow rate: 20 arb, I spray voltage: 4.5 KV, capillary temperature: 215 C, column temperature 40 C, capillary voltage: 21 V, tube lens offset: 10 V. Retention time was given in minutes. 5.1. General method for the preparation of 3 Compounds 3 (R1, R2 ¼ aec) were synthesized either by base catalyzed (KOH/NaOH) or acid catalyzed (BF3-Et2O) ClaiseneSchmidt condensation reaction as reported previously [25]. Total nine hydroxylated chalcone intermediates were synthesized. 5.2. General method for the preparation of 4 A mixture of aryl ketone 1 (R3 ¼ aec), iodine (1.2 equv) and pyridine was refluxed at 140 C for 3 h. Precipitate occurred during reaction which was cooled to room temperature. Then it was filtered and washed with cold pyridine to afford 4 (R3 ¼ aec) in quantitative yield. Three different pyridinium iodide salts were synthesized by this method. 5.3. General method for the preparation of 5 A mixture of hydroxylated chalcone intermediate 3 (R1, R ¼ aec), pyridinium iodide salt 4 (R3 ¼ aec) and anhydrous ammonium acetate in glacial acetic acid were heated at 80e100 C for 12e16 h. The reaction mixture was then extracted with ethyl acetate, washed with water and brine. The organic layer was dried with magnesium sulfate and filtered. The filtrate was evaporated at reduced pressure, which was then purified by silica gel column chromatography with the gradient elution of ethyl acetate/nhexane to afford solid compounds 5 (R1, R2, R3 ¼ aec) in 29.0%e 88.2% yield. Eighteen trihydroxylated 2,4,6-triphenyl pyridine compounds were synthesized by this method. 2
5.3.1. 2,20 ,200 -(Pyridine-2,4-6-triyl)triphenol (6) The same procedure described in section 5.3 was employed with 3 (R1, R2 ¼ a) (0.48 g, 2.00 mmol), dry ammonium acetate (1.54 g, 20.00 mmol), 4 (R3 ¼ a) (0.68 g, 2.00 mmol) and AcOH (2 mL) to yield a yellow solid (0.20 g, 29%, 0.58 mmol). Rf (ethyl acetate/n-hexane 1:2 v/v): 0.21. LC/MS/MS: retention time: 7.56 min, [MH]þ: 356.39. 1 H NMR (250 MHz, DMSO-d6) d 12.32 (s, 2H, 2-phenyl 2-OH, 6phenyl 2-OH), 9.99 (br, 1H, 4-phenyl 2-OH), 8.10 (s, 2H, pyridine H3, H-5), 7.86 (d, J ¼ 7.6 Hz, 2H, 2-phenyl H-6, 6-phenyl H-6), 7.55 (d, J ¼ 7.3 Hz, 1H, 4-phenyl H-6), 7.30 (t, J ¼ 7.8 Hz, 3H, 2-phenyl H-4, 6phenyl H-4, 4-phenyl H-4), 7.04e6.92 (m, 6H, 2-phenyl H-3, H-5, 6phenyl H-3, H-5, 4-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 157.47, 155.07, 154.65, 148.76, 131.07, 130.72, 130.63, 128.69, 125.26, 122.39, 120.43, 119.99, 119.46, 117.52, 116.61. 5.3.2. 2,20 -(4-(3-Hydroxyphenyl)pyridine-2,6-diyl)diphenol (7) The same procedure described in section 5.3 was employed with 3 (R1 ¼ a, R2 ¼ b) (0.36 g, 1.50 mmol), dry ammonium acetate
(1.15 g, 15.00 mmol), 4 (R3 ¼ a) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.42 g, 80%, 1.19 mmol). Rf (ethyl acetate/n-hexane 1:2 v/v): 0.21. LC/MS/MS: retention time: 7.56 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 12.30 (br, 2H, 2-phenyl 2-OH, 6phenyl 2-OH), 9.80 (br, 1H, 4-phenyl 3-OH), 8.13 (s, 2H, pyridine H3, H-5), 7.96 (d, J ¼ 7.7 Hz, 2H, 2-phenyl H-6, 6-phenyl H-6), 7.37e7.28 (m, 5H, 2-phenyl H-4, 6-phenyl H-4, 4-phenyl H-2, H-5, H-6), 6.99e6.93 (m, 5H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5, 4phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.26, 157.50, 155.48, 150.05, 139.20, 131.22, 130.51, 128.88, 122.23, 119.45, 118.31, 117.87, 117.51, 116.70, 114.26. 5.3.3. 2,20 -(4-(4-Hydroxyphenyl)pyridine-2,6-diyl)diphenol (8) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ a, R2 ¼ c) (0.24 g, 1.00 mmol), dry ammonium acetate (0.77 g, 10.00 mmol), 4 (R3 ¼ a) (0.34 g, 1.00 mmol) and AcOH (1 mL) to yield a yellow solid (0.14 g, 41.8%, 0.41 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.42. LC/MS/MS: retention time: 7.33 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 12.35 (br, 2H, 2-phenyl 2-OH, 6phenyl 2-OH), 9.93 (br, 1H, 4-phenyl 4-OH), 8.13 (s, 2H, pyridine H3, H-5), 7.96 (d, J ¼ 7.7 Hz, 2H, 2-phenyl H-6, 6-phenyl H-6), 7.86 (d, J ¼ 8.4 Hz, 2H, 4-phenyl H-2, H-6), 7.30 (t, J ¼ 7.5 Hz, 2H, 2-phenyl H-4, 6-phenyl H-4), 6.98e6.92 (m, 6H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5, 4-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 159.35, 157.61, 155.47, 149.77, 131.22, 129.06, 128.94, 128.16, 122.44, 119.47, 117.57, 116.98, 116.31. 5.3.4. 3,30 -(4-(2-Hydroxyphenyl)pyridine-2,6-diyl)diphenol (9) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ b, R2 ¼ a) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ b) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.20 g, 38.2%, 0.57 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.51. LC/MS/MS: retention time: 6.71 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 9.89 (br, 1H, 4-phenyl 2-OH), 9.60 (br, 2H, 2-phenyl 3-OH, 6-phenyl 3-OH), 7.95 (s, 2H, pyridine H-3, H-5), 7.65e7.53 (m, 5H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6, 4-phenyl H-6), 7.34e7.25 (m, 3H, 2-phenyl H-5, 6-phenyl H-5, 4phenyl H-4), 7.03 (d, J ¼ 8.1 Hz, 1H, 4-phenyl H-3), 6.95 (t, J ¼ 7.7 Hz, 1H, 4-phenyl H-5), 6.86 (d, J ¼ 6.1 Hz, 2-phenyl H-4, 6phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 157.98, 155.80, 155.00, 148.25, 140.68, 130.60, 130.32, 129.98, 125.48, 119.94, 119.25, 117.70, 116.58, 116.29, 113.76. 5.3.5. 3,30 ,300 -(Pyridine-2,4-6-triyl)triphenol (10) The same procedure described in section 5.3 was employed with 3 (R1, R2 ¼ b) (0.48 g, 2.00 mmol), dry ammonium acetate (1.54 g, 20.00 mmol), 4 (R3 ¼ b) (0.68 g, 2.00 mmol) and AcOH (2 mL) to yield a white solid (0.43 g, 61%, 1.22 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.36. LC/MS/MS: retention time: 6.63 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 9.69 (br, 1H, 4-phenyl 3-OH), 9.62 (s, 2H, 2-phenyl 3-OH, 6-phenyl 3-OH), 7.99 (d, J ¼ 1.3 Hz, 2H, pyridine H-3, H-5), 7.71e7.67 (m, 4H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6), 7.39e7.29 (m, 5H, 2-phenyl H-5, 6-phenyl H-5, 4phenyl H-2, H-5, H-6), 6.92e6.85 (m, 3H, 2-phenyl H-4, 6-phenyl H-4, 4-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.25, 158.03, 156.60, 149.82, 140.46, 139.50, 130.46, 130.01, 118.25, 117.91, 116.75, 116.49, 114.21, 113.93.
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5.3.6. 3,30 -(4-(4-hydroxyphenyl)pyridine-2,6-diyl)diphenol (11) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ b, R2 ¼ c) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ b) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.47 g, 88.2%, 1.32 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.36. LC/MS/MS: retention time: 6.30 min, [MH]þ: 356.34. 1 H NMR (250 MHz, DMSO-d6) d 9.60 (s, 3H, 2-phenyl 3-OH, 6phenyl 3-OH, 4-phenyl 4-OH), 7.99 (s, 2H, pyridine H-3, H-5), 7.88 (d, J ¼ 6.8 Hz, 4-phenyl H-2, H-6), 7.70e7.67 (m, 4H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6), 7.32 (t, J ¼ 7.9 Hz, 2H, 2-phenyl H-5, 6phenyl H-5), 6.93e6.85 (m, 4H, 4-phenyl H-3, H-5, 2-phenyl H-4, 6-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.93, 157.92, 156.45, 149.34, 140.61, 129.82, 128.74, 128.39, 117.82, 116.29, 116.06, 115.80, 113.89. 5.3.7. 4,40 -(4-(2-hydroxyphenyl)pyridine-2,6-diyl)diphenol (12) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ c, R2 ¼ a) (0.48 g, 2.00 mmol), dry ammonium acetate (1.54 g, 20.00 mmol), 4 (R3 ¼ c) (0.68 g, 2.00 mmol) and AcOH (2 mL) to yield a yellow solid (0.22 g, 32.1%, 0.64 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.31. LC/MS/MS: retention time: 5.86 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 9.77 (br, 3H, 2-phenyl 4-OH, 4phenyl 2-OH, 6-phenyl 4-OH), 8.06 (d, J ¼ 8.6 Hz, 4H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6), 7.81 (s, 2H, pyridine H-3, H-5), 7.51 (dd, J ¼ 7.5, 1.3 Hz, 1H, 4-phenyl H-6), 7.26 (td, J ¼ 7.2, 1.7 Hz, 1H, 4phenyl H-4), 7.01 (d, J ¼ 7.8 Hz, 1H, 4-phenyl H-3), 6.94 (t, J ¼ 7.5 Hz, 1H, 4-phenyl H-5, 6.90 (d, J ¼ 8.7 Hz, 4H, 2-phenyl H-3, H5, 6-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 158.66, 155.65, 154.98, 148.07, 130.59, 130.37, 130.09, 128.31, 125.99, 119.85, 117.19, 116.51, 115.70. 5.3.8. 4,40 -(4-(3-hydroxyphenyl)pyridine-2,6-diyl)diphenol (13) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ c, R2 ¼ b) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ c) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a light yellow solid (0.45 g, 85.2%, 1.27 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.33. LC/MS/MS: retention time: 5.98 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 9.71 (br, 3H, 2-phenyl 4-OH, 4phenyl 3-OH, 6-phenyl 4-OH), 8.14 (d, J ¼ 8.6 Hz, 4H, 2-phenyl H2, H-6, 6-phenyl H-2, H-6), 7.85 (s, 2H, pyridine H-3, H-5), 7.35e7.27 (m, 3H, 4-phenyl H-2, H-5, H-6), 6.91 (d, J ¼ 8.6 Hz, 4H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5), 6.90e6.85 (m, 1H, 4phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.79, 158.14, 156.41, 149.51, 139.85, 130.28, 130.10, 128.44, 118.10, 115.64, 114.45, 114.11. 5.3.9. 4,40 ,400 -(pyridine-2,4-6-triyl)triphenol (14) The same procedure described in Section 5.3 was employed with 3 (R1, R2 ¼ c) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ c) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.27 g, 51.8%, 0.77 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.26. LC/MS/MS: retention time: 5.49 min, [MH]þ: 356.38. 1 H NMR (250 MHz, DMSO-d6) d 9.64 (br, 3H, 2-phenyl 4-OH, 4phenyl 4-OH, 6-phenyl 4-OH), 8.12 (d, J ¼ 8.6 Hz, 4H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6), 7.84 (s, 2H, pyridine H-3, H-5), 7.83 (d, J ¼ 8.6 Hz, 2H, 4-phenyl H-2, H-6), 6.93e6.87 (m, 6H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5, 4-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 158.81, 158.73, 156.33, 149.09, 130.32, 128.81, 128.69, 128.46, 116.03, 115.63, 113.66.
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5.3.10. 2,20 -(6-(3-hydroxyphenyl)pyridine-2,4-diyl)diphenol (15) The same procedure described in Section 5.3 was employed with 3 (R1, R2 ¼ a) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ b) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.28 g, 53.4%, 0.80 mmol). Rf (ethyl acetate/n-hexane 1:2 v/v): 0.18. LC/MS/MS: retention time: 7.70 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 14.49 (s, 1H, 2-phenyl 2-OH), 9.83 (br, 2H, 4-phenyl 2-OH, 6-phenyl 3-OH), 8.28 (s, 1H, pyridine H-3), 8.11 (d, J ¼ 8.0 Hz, 1H, 2-phenyl H-6), 8.00 (s, 1H, pyridine H5), 7.61 (dd, J ¼ 7.6, 1.4 Hz, 1H, 4-phenyl H-6), 7.44e7.27 (m, 5H, 2phenyl H-4, 4-phenyl H-4, 6-phenyl H-2, H-5, H-6), 7.05 (d, J ¼ 8.1 Hz, 1H, 2-phenyl H-3), 7.01e6.89 (m, 4H, 2-phenyl H-5, 4phenyl H-3, H-5, 6-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 159.36, 158.33, 156.75, 155.10, 153.84, 149.64, 139.39, 131.62, 130.79, 130.55, 127.53, 124.98, 119.97, 119.83, 119.35, 119.20, 118.74, 118.05, 117.65, 116.94, 116.63, 113.46. 5.3.11. 3,30 -(6-(2-hydroxyphenyl)pyridine-2,4-diyl)diphenol (16) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ a, R2 ¼ b) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ b) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.41 g, 78.6%, 1.17 mmol). Rf (ethyl acetate/n-hexane 1:2 v/v): 0.21. LC/MS/MS: retention time: 7.59 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 14.40 (br, 1H, 2-phenyl 2-OH), 9.77 (br, 2H, 4-phenyl 3-OH, 6-phenyl 3-OH), 8.32 (s, 1H, pyridine H-3), 8.27 (d, J ¼ 8.0 Hz, 1H, 2-phenyl H-6), 8.01 (s, 1H, pyridine H5), 7.50 (t, J ¼ 7.5 Hz, 1H, 2-phenyl H-4), 7.45e7.31 (m, 6H, 6-phenyl H-2, H-5, H-6, 4-phenyl H-2, H-5, H-6), 6.97e6.92 (m, 4H, 2-phenyl H-3, H-5, 6-phenyl H-4, 4-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 159.39, 158.32, 158.26, 157.49, 154.79, 150.98, 139.23, 138.93, 131.79, 130.50, 130.45, 127.94, 119.35, 119.20, 118.49, 118.03, 117.87, 117.21, 117.08, 116.85, 116.37, 114.49, 113.65. 5.3.12. 2-(6-(3-hydroxyphenyl)-4-(4-hydroxyphenyl)pyridin-2-yl) phenol (17) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ a, R2 ¼ c) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ b) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.38 g, 72.7%, 1.09 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.46. LC/MS/MS: retention time: 7.43 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 14.65 (s, 1H, 2-phenyl 2-OH), 9.85 (br, 2H, 4-phenyl 4-OH, 6-phenyl 3-OH), 8.33 (s, 1H, pyridine H-3), 8.29 (d, J ¼ 8.1 Hz, 1H, 2-phenyl H-6), 8.03 (s, 1H, pyridine H5), 7.96 (d, J ¼ 8.5 Hz, 2H, 4-phenyl H-2, H-6), 7.52 (t, J ¼ 7.6 Hz, 1H, 2-phenyl H-4), 7.44e7.30 (m, 3H, 6-phenyl H-2, H-5, H-6), 6.98e6.92 (m, 5H, 2-phenyl H-3, H-5, 4-phenyl H-3, H-5, 6-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 159.54, 159.45, 158.29, 157.42, 154.60, 150.57, 139.37, 131.70, 130.45, 129.21, 127.81, 127.77, 119.27, 119.09, 118.03, 117.86, 116.99, 116.29, 116.15, 115.11, 113.63. 5.3.13. 2,20 -(6-(4-hydroxyphenyl)pyridine-2,4-diyl)diphenol (18) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ a, R2 ¼ a) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ c) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a green solid (0.27 g, 52%, 0.77 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.46. LC/MS/MS: retention time: 7.56 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 14.73 (s, 1H, 2-phenyl 2-OH), 9.96 (br, 2H, 4-phenyl 2-OH, 6-phenyl 4-OH), 8.19 (s, 1H, pyridine H-3), 8.08 (d, J ¼ 8.0 Hz, 1H, 2-phenyl H-6), 7.95 (s, 1H, pyridine H-
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5), 7.87 (d, J ¼ 8.4 Hz, 2H, 6-phenyl H-2, H-6), 7.58 (d, J ¼ 7.4 Hz, 1H, 4-phenyl H-6), 7.35e7.27 (m, 2H, 2-phenyl H-4, 4-phenyl H-4), 7.04 (d, J ¼ 8.2 Hz, 1H, 2-phenyl H-3), 7.00e6.89 (m, 5H, 2-phenyl H-5, 4phenyl H-3, H-5, 6-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 159.48, 159.29, 156.52, 155.06, 153.84, 149.59, 131.51, 130.74, 130.65, 128.71, 128.28, 127.36, 125.22, 119.92, 119.31, 119.11, 118.80, 118.04, 117.66, 116.59, 116.23. 5.3.14. 2-(4-(3-hydroxyphenyl)-6-(4-hydroxyphenyl)pyridin-2-yl) phenol (19) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ a, R2 ¼ b) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ c) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.22 g, 43%, 0.64 mmol). Rf (ethyl acetate/n-hexane 1:2 v/v): 0.16. LC/MS/MS: retention time: 7.45 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 14.68 (s, 1H, 2-phenyl 2-OH), 9.89 (br, 1H, 6-phenyl 4-OH, 9.77 (br, 1H, 4-phenyl 3-OH), 8.26e8.24 (m, 2H, 2-phenyl H-6, pyridine H-3), 7.99 (s, 1H, pyridine H-5), 7.96 (dd, J ¼ 8.8, 1.1 Hz, 2H, 6-phenyl H-2, H-6), 7.42 (t, J ¼ 7.8 Hz, 1H, 2-phenyl H-4), 7.39e7.30 (m, 3H, 4-phenyl H-2, H-5, H-6), 6.97e6.91 (m, 5H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5, 4phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 159.52, 159.44, 158.22, 157.32, 154.71, 150.90, 139.12, 131.69, 130.41, 128.52, 128.50, 127.79, 119.28, 119.11, 118.47, 118.02, 116.75, 116.19, 115.20, 114.49. 5.3.15. 4,40 -(6-(2-hydroxyphenyl)pyridine-2,4-diyl)diphenol (20) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ a, R2 ¼ c) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ c) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.42 g, 79.3%, 1.19 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.43. LC/MS/MS: retention time: 7.29 min, [MH]þ: 356.34. 1 H NMR (250 MHz, DMSO-d6) d 14.87 (br, 1H, 2-phenyl 2-OH), 10.01 (br, 2H, 4-phenyl 4-OH, 6-phenyl 4-OH), 8.26e8.23 (m, 2H, 2phenyl H-6, pyridine H-3), 8.00 (s, 1H, pyridine H-5), 7.94 (d, J ¼ 8.5 Hz, 4H, 6-phenyl H-2, H-6, 4-phenyl H-2, H-6), 7.32 (t, J ¼ 7.6 Hz, 1H, 2-phenyl H-4), 6.96e6.91 (m, 6H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5, 4-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 159.65, 159.36, 157.25, 154.53, 150.46, 131.58, 129.14, 128.66, 128.44, 127.94, 127.66, 119.25, 118.99, 118.01, 116.14, 116.12, 115.18, 114.00. 5.3.16. 2-(2-(3-hydroxyphenyl)-6-(4-hydroxyphenyl)pyridin-4-yl) phenol (21) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ b, R2 ¼ a) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ c) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.22 g, 42%, 0.62 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.42. LC/MS/MS: retention time: 6.37 min, [MH]þ: 356.34. 1 H NMR (250 MHz, DMSO-d6) d 9.75 (br, 3H, 2-phenyl 3-OH, 4phenyl 2-OH, 6-phenyl 4-OH), 8.07 (d, J ¼ 8.6 Hz, 2H, 6-phenyl H-2, H-6), 7.89 (s, 1H, pyridine H-3), 7.86 (s, 1H, pyridine H-5), 7.64 (s, 1H, 2-phenyl H-2), 7.58 (d, J ¼ 7.8 Hz, 1H, 4-phenyl H-6), 7.53 (dd, J ¼ 7.6, 1.4 Hz, 1H, 2-phenyl H-6), 7.32e7.23 (m, 2H, 2-phenyl H-5, 4phenyl H-4), 7.02 (d, J ¼ 8.0 Hz, 1H, 4-phenyl H-3), 6.95 (t, J ¼ 7.6 Hz, 1H, 4-phenyl H-5), 6.91 (d, J ¼ 8.7 Hz, 2H, 6-phenyl H-3, H-5), 6.85 (dd, J ¼ 8.0, 2.2 Hz, 1H, 2-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.74, 157.95, 155.88, 155.52, 154.97, 148.15, 140.82, 130.57, 130.21, 129.94, 128.32, 125.73, 119.89, 118.25, 118.15, 117.64, 116.54, 116.17, 115.72, 113.70.
5.3.17. 3,30 -(6-(4-hydroxyphenyl)pyridine-2,4-diyl)diphenol (22) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ b, R2 ¼ b) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ c) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.32 g, 60.8%, 0.91 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.38. LC/MS/MS: retention time: 6.39 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 9.67 (br, 3H, 2-phenyl 3-OH, 4phenyl 3-OH, 6-phenyl 4-OH), 8.16 (d, J ¼ 8.5 Hz, 2H, 6-phenyl H-2, H-6), 7.95 (s, 1H, pyridine H-3), 7.89 (s, 1H, pyridine H-5), 7.71 (s, 1H, 2-phenyl H-2), 7.68 (d, J ¼ 7.7 Hz, 1H, 2-phenyl H-6), 7.36e7.29 (m, 4H, 2-phenyl H-5, 4-phenyl H-2, H-5, H-6), 6.92 (d, J ¼ 8.4 Hz, 2H, 6phenyl H-3, H-5), 6.89e6.84 (m, 2H, 2-phenyl H-4, 4-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.90, 158.18, 157.94, 156.60, 156.34, 149.63, 140.55, 139.64, 130.32, 129.93, 129.88, 128.48, 118.12, 117.77, 116.30, 115.67, 115.55, 114.12, 113.82. 5.3.18. 4,40 -(6-(3-hydroxyphenyl)pyridine-2,4-diyl)diphenol (23) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ b, R2 ¼ c) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ c) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.32 g, 61.3%, 0.92 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.36. LC/MS/MS: retention time: 5.90 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 9.77 (br, 2H, 4-phenyl 4-OH, 6phenyl 4-OH), 9.55 (br, 1H, 2-phenyl 3-OH), 8.15 (d, J ¼ 8.5 Hz, 2H, 6-phenyl H-2, H-6), 7.95 (s, 1H, pyridine H-3), 7.90 (s, 1H, pyridine H-5), 7.86 (d, J ¼ 8.5 Hz, 2H, 4-phenyl H-2, H-6), 7.70 (s, 1H, 2phenyl H-2), 7.68 (d, J ¼ 7.9 Hz, 1H, 2-phenyl H-6), 7.30 (td, J ¼ 7.9, 3.0 Hz, 1H, 2-phenyl H-5), 6.93 (d, J ¼ 8.4 Hz, 2H, 6-phenyl H3, H-5), 6.91 (d, J ¼ 8.6 Hz, 2H, 4-phenyl H-3, H-5), 6.86 (dd, J ¼ 8.1, 2.9 Hz, 1H, 2-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 162.50, 158.88, 158.80, 157.90, 156.47, 156.24, 149.19, 140.75, 130.11, 129.81, 128.68, 128.57, 128.44, 117.76, 116.03, 115.61, 114.73, 114.66, 113.83. 5.4. Pharmacology 5.4.1. Assay for DNA topoisomerase I inhibition in vitro DNA topo I inhibition assay was determined following the method reported by Fukuda et al. [28] with minor modifications. The test compounds were dissolved in DMSO at 20 mM as stock solutions. The activity of DNA topo I was determined by assessing the relaxation of supercoiled DNA pBR322. The mixture of 100 ng of plasmid pBR322 DNA and 1 unit of recombinant human DNA topoisomerase I (TopoGEN INC., USA) was incubated without and with the prepared compounds at 37 C for 30 min in the relaxation buffer (10 mM TriseHCl (pH 7.9), 150 mM NaCl, 0.1% bovine serum albumin, 1 mM spermidine, 5% glycerol). The reaction in the final volume of 10 mL was terminated by adding 2.5 mL of the stop solution containing 5% sarcosyl, 0.0025% bromophenol blue and 25% glycerol. DNA samples were then electrophoresed on a 1% agarose gel at 15 V for 7 h with a running buffer of TAE. Gels were stained for 30 min in an aqueous solution of ethidium bromide (0.5 mg/mL). DNA bands were visualized by transillumination with UV light and were quantitated using AlphaImager™ (Alpha Innotech Corporation). 5.4.2. Assay for DNA topoisomerase II inhibition in vitro DNA topo II inhibitory activity of compounds were measured as follows [29]. The mixture of 200 ng of supercoiled pBR322 plasmid DNA and 1 unit of human DNA topoisomerase IIa (Usb Corp., USA) was incubated without and with the prepared compounds in the assay buffer (10 mM TriseHCl (pH 7.9) containing 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM ATP, and 15 mg/mL bovine serum
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albumin) for 30 min at 30 C. The reaction in a final volume of 20 mL was terminated by the addition of 3 mL of 7 mM EDTA. Reaction products were analyzed on 1% agarose gel at 25 V for 4 h with a running buffer of TAE. Gels were stained for 30 min in an aqueous solution of ethidium bromide (0.5 mg/mL). DNA bands were visualized by transillumination with UV light and supercoiled DNA was quantitated using AlphaImager™ (Alpha Innotech Corporation). 5.4.3. Cytotoxicity assay Cells were cultured according to the supplier's instructions. Cells were seeded in 96-well plates at a density of 2e4 104 cells per well and incubated for overnight in 0.1 mL of media supplied with 10% Fetal Bovine Serum (Hyclone, USA) in 5% CO2 incubator at 37 C. On day 2, after FBS starvation for 4 h, culture medium in each well was exchanged with 0.1 mL aliquots of medium containing graded concentrations of compounds. On day 4, each well was added with 5 mL of the cell counting kit-8 solution (Dojindo, Japan) then incubated for additional 4 h under the same condition. The absorbance of each well was determined by an Automatic Elisa Reader System (Bio-Rad 3550) at 450 nm wavelength. For determination of the IC50 values, the absorbance readings at 450 nm were fitted to the four-parameter logistic equation. The compounds like adriamycin, etoposide, and camptothecin were purchased from Sigma and used as positive controls. 5.4.4. Molecular docking study The template structure for the structure-based docking was prepared by using the coordinate in the complex of TOPOIIa and DNA (PDB ID: 4FM9) [30]. In order to compare the docked poses of the molecules in the current study with etoposide bound with TOPOIIa (PDB ID: 3QX3) [31], we aligned 3QX3 into 4FM9 by TMalign [32]. Modeller package (ver. 9.9) [33] modeled the missed coordinates in topo IIa due to the lack of electron densities. After PDB2PQR [34] determined the ionization states of Asp, Glu, His, and Lys residues in the structure, AMBER molecular dynamics package [35] minimized the energy of the structure. For faithful docking simulation, we employed both AutoDock [36] and AutoDock Vina [37]. The docking protocol consists of four stages such as preparation of ligand parameters, ensemble generation of ligands, docking and clustering. Antechamber program of AMBER package first prepared force-field for each ligand by semi-quantum calculation. Then 30 different conformers of each ligand were generated with sander program. Once gaining 60 structures by dockings with AutoDock and AutoDock Vina with default parameters, we clustered the resulting conformers by structural similarity that was quantified by pairwise root mean square deviation (RMSD) value. The clusters were further sorted according to AutoDock Vina scores. We chose the centroid structure in the best cluster as a final model. In-house written software and scripts automated all the procedures. Acknowledgments
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.07.058. References [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]
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF2012R1A2A2A01046188).
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antitumor activity, and structureeactivity relationship study of trihydroxylated 2,4,6-triphenyl pyridines as potent and selective topoisomerase II inhibitors Radha Karki a, 1, Chanmi Park b, Kyu-Yeon Jun b, Jun-Goo Jee c, Jun-Ho Lee d, Pritam Thapa a, 1, Tara Man Kadayat a, Youngjoo Kwon b, *, Eung-Seok Lee a, * a
College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Global Top 5 Program, Ewha Womans University, Seoul 120-750, Republic of Korea c College of Pharmacy, Kyungpook National University, Daegu 702-701, Republic of Korea d Department of Emergency Medical Technology, Daejeon University, Daejeon 300-716, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 January 2014 Received in revised form 19 June 2014 Accepted 17 July 2014 Available online 18 July 2014
A series of eighteen trihydroxylated 2,4,6-triphenyl pyridines were designed and synthesized which contain hydroxyl groups at ortho, meta or para position of each phenyl rings attached to the central pyridine. They were evaluated for topoisomerase I and II inhibitory activity, and cytotoxicity against several human cancer cell lines for the development of novel anticancer agents. Most of the compounds exhibited strong and selective topoisomerase II inhibitory activity compared to the positive control, etoposide, and also displayed significant cytotoxicity in low micromolar range. Trihydroxylated 2,4,6triphenyl pyridines were more potent than mono- and di-hydroxylated 2,4,6-triphenyl pyridines, which have been previously studied in our research group. Positive correlation between topoisomerase II inhibitory activity and cytotoxicity was observed for the most compounds. Molecular docking study shows qualitatively consistent with the results of biological assays. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Trihydroxylated 2,4,6-triphenyl pyridines Topoisomerase I Topoisomerase II Topo II inhibitory activity Cytotoxicity Anticancer agents Docking study
1. Introduction Topoisomerases play an important role in solving the DNA topological hurdles during DNA replication by their activity of transiently cutting one or both strands of DNA followed by rejoining [1,2]. The human topoisomerase has two types, topoisomerase I (topo I) and topoisomerase II (topo II). Topo I and II inhibitors such as camptothecin, doxorubicin, and etoposide are considered among the most active anticancer agents [3e6]. In the past decades, several natural products and synthetic compounds have been identified as novel topoisomerase inhibitors in order to overcome the limitations of existing topoisomerase inhibitors [7]. Many
* Corresponding authors. E-mail addresses: [email protected] (Y. Kwon), [email protected] (E.-S. Lee). 1 Present address: Division of Hematology & Oncology, Department of Internal Medicine, Kansas University Medical Center, Kansas City, KS 66103, USA. http://dx.doi.org/10.1016/j.ejmech.2014.07.058 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
researchers in medicinal chemistry recently started to develop topo II-selective inhibitors as anticancer agents [8e10]. Topo II is distinct from topo I in that topo II acts as a homodimer, requiring Mg (II) and ATP hydrolysis for enzyme turn over and rapid kinetics; this enables it to cut both DNA strands simultaneously. Furthermore, topo II is the only enzyme available to disentangle the topological problems in chromosomes, which must occur during completion of replication and to decantenate the replicated chromosomes by introducing transient double strand breaks [11]. In addition, one of the subtype of topo II, topo IIa, is cell cycle dependent enzyme, which peaks during G2/M phase and decreases at the end of mitosis. The overexpression of topo IIa is only observed in proliferating cells. Therefore, topo II is a more important factor for cell proliferation than topo I [12e13]. In these reasons, many researchers in medicinal chemistry recently started to develop topo II-selective inhibitors as anticancer agents [8e11]. Polyphenolic phytochemicals are considered to be potentially bioactive compounds. For instance, isoliquiritigenin found in vegetables, tea flavonol epigallocatechin gallate (EGCG) and
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resveratrol found in red wine act as topo II poison. Similarly, myricetin, a naturally occurring flavonol, act as dual inhibitors of topo I and II [11,13e17]. Synthetic compounds such as polyhydroxybenzoyl amide and isoaurostatin derivatives having hydroxyl group in the aromatic ring displayed strong topo I and II inhibition [18,19]. Similarly, several bisphenol analogs were found to be catalytic topo II inhibitor with the exception of a few being topo II poison [20]. In an effort to discover novel anticancer agent as topoisomerase inhibitors, we had previously designed and synthesized several terpyridine bioisosteres bearing 2,4,6-triaryl pyridines. Most of the 2,4,6-triaryl pyridines without substitutions on aryl moiety possessed weak to moderate topoisomerase inhibitory activity and cytotoxicity against several human cancer cell lines [21e23]. From one of the prepared 2,4,6-triaryl pyridines, 2,4,6-triphenyl pyridine (A) did not show any topo I and II inhibitory activity, and cytotoxicity against several human cell lines. For improvement of efficacy of those compounds, we introduced hydroxyl moiety to the phenyl ring since many biologically active natural and/or synthetic compounds possess hydroxyl group in their aromatic molecular skeletons. As anticipated, monohydroxylated 2,4,6-triphenyl pyridines showed relatively stronger topo I and/or II inhibitory activity than those of the non-substituted compounds [24]. With these results, we further attempted to prepare dihydroxylated 2,4,6-triphenyl pyridines, which resulted to enhance topoisomerase II inhibitory activity as well as cytotoxicity against several human cancer cell lines [25]. These results encouraged us to systematically design and prepare trihydroxylated 2,4,6-triphenyl pyridines as shown in Fig. 1. We expected increase of topo II inhibitory activity and selectivity as well as cytotoxicity against several human cancer cell lines since we observed that the increased number of hydroxyl groups in phenyl moiety enhanced the topo II inhibitory activity and selectivity along with cytotoxicity against several human cancer cell lines from previous results. In this study we have described the design and synthesis of trihydroxylated 2,4,6-triphenyl pyridines, and evaluation of topo I and II inhibitory activity, especially selectivity to topo II inhibitory activity, and cytotoxicity against several human cancer cell lines (Fig. 2). Trihydroxylated 2,4,6-triphenyl pyridines are novel compounds which have not been previously reported. They are designed and discovered by our research group possessing strong topo II inhibitory activity, selectivity, and cytotoxicity against
several human cancer cell lines. Structureeactivity relationship study was determined with respect to non-hydroxylated 2,4,6triphenyl pyridine (A), mono- and di-hydroxylated 2,4,6-triphenyl pyridines with trihydroxylated 2,4,6-triphenyl pyridines. The results indicated that topo II inhibitory activity and selectivity were greatly increased, and cytotoxicity was found to be enhanced by the increase in the number of hydroxyl moiety.
2. Chemistry A new series of trihydroxylated 2,4,6-triphenyl pyridines were €hnke synthesis [26,27] as illustrated synthesized using modified Kro in Scheme 1. First, nine hydroxylated chalcone intermediates 3 (R1, R2 ¼ aec) were synthesized using ClaiseneSchmidt condensation reaction as reported earlier [23]. In the second step, three pyridinium iodide salts 4 (R3 ¼ aec) were synthesized in quantitative yield by the treatment of aryl methyl ketone 1 (R1 ¼ aec) with €hnke synthesis, final comiodine in pyridine. Using modified Kro pounds 5 (R1, R2, R3 ¼ aec) were synthesized by the reaction of appropriate hydroxylated chalcone 3 with pyridinium iodide salt 4 in the presence of ammonium acetate and glacial acetic acid in 29.0e88.2% yield. Total eighteen final compounds 6e23 were synthesized from the reaction of nine hydroxylated chalcone intermediates and three pyridinium iodide salts in six different series as shown in Fig. 3. All the compounds contain three hydroxyl moieties substituted at various positions (ortho, meta, or para) of each phenyl rings. Different substitution pattern together with yield, purity and melting point is illustrated in Table 1.
3. Results and discussion Inhibition against relaxation activity of topo I and II was measured by detecting the conversion of supercoiled pBR322 DNA to its relaxed form in the presence of synthesized trihydroxylated 2,4,6-triphenyl pyridines 6e23. Camptothecin and etoposide, clinically used selective topo I and II inhibitors, respectively, were used as references. For cytotoxicity adriamycin (doxorubicin), also clinically using anticancer agent, camptothecin, and etoposide were used as references.
Fig. 1. Structures of 2,4,6-triaryl pyridines, 2,4,6-triphenyl pyridine (A), mono-, di-, and trihydroxylated 2,4,6-triphenyl pyridines.
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Fig. 2. Strategy for the design of trihydroxylated 2,4,6-triphenyl pyridines.
Scheme 1. General synthetic method of trihydroxylated 2,4,6-triphenyl pyridines.
3.1. Topoisomerase I and II inhibitory activity Fig. 4 and Table 2 illustrates the topo I inhibitory activity of prepared trihydroxylated 2,4,6-triphenyl pyridines. None of the compounds possessed topo I inhibitory activity, which indicate that the trihydroxylated 2,4,6-triphenyl pyridines exhibited strong selectivity to topo II inhibition. The effect of trihydroxylated 2,4,6-triphenyl pyridines on human DNA topo II were observed in the relaxation assay using
supercoiled pBR322 plasmid DNA in the presence of ATP. As shown in Fig. 5 and Table 2, most of the compounds (6, 7, 9e15, 17, 18, 21e23) exhibited significant topo II inhibition at 100 mM which were stronger or comparable to positive control, etoposide. Compounds 13 and 17 showed stronger topo II inhibitory activity than that of etoposide at both 100 mM and 20 mM. Compound 13 displayed 90.1% and 48.2% inhibition, and compound 17 possessed 80.6% and 38.5% inhibition, while etoposide displayed 79.6% and 31.1% inhibition at 100 mM and 20 mM, respectively.
Fig. 3. Structures of prepared trihydroxylated 2,4,6-triphenyl pyridines.
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Table 1 Prepared compounds with yield, purity by HPLC, and melting point.
Entry
R1
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
20 20 20 30 30 30 40 40 40 20 20 20 20 20 20 30 30 30
R2 OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH
phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl
2 3 4 2 3 4 2 3 4 2 3 4 2 3 4 2 3 4
OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH
R3 phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl
20 20 20 30 30 30 40 40 40 30 30 30 40 40 40 40 40 40
OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH
phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl
Yield (%)
Purity (%)
Mp ( C)
29.0 80.0 41.8 38.2 61.0 88.2 32.1 85.2 51.8 53.4 78.6 72.7 52.0 43.0 79.3 42.0 60.8 61.3
97 97 97 96 99 98 96 98 97 96 98 98 96 96 100 100 99 100
230e231 259e260 280e281 271e272 253e254 246e247 240e241 279e280 260e261 263e264 278e279 299e300 275e276 330e331 316e317 260e261 236e237 242e243
Also compound 18 exhibited 77.6% and 21.3% inhibition at 100 mM and 20 mM, respectively. Compound 9e12, 22, and 23 displayed 100% topo II inhibition at 100 mM. Similarly, compounds 6, 7, 14, 15, and 21 showed 95.1%, 99.4%, 79.1%, 87.9%, and 92.2% inhibition at 100 mM, respectively, which were higher than that of etoposide. Compounds 8, 19, and 20 exhibited moderate topo II inhibition (46.9%, 31.3%, and 44%, respectively) at 100 mM. Only compound 16 showed weak topo II inhibition of 11.3% at 100 mM.
3.2. Cytotoxicity For the evaluation of cytotoxicity, four human cancer cell lines were utilized: human embryonic kidney 293 cells (HEK293), human prostate tumor cell line (DU145), human colorectal adenocarcinoma cell line (HCT15), and human ductal breast epithelial tumor cell line (T47D). Adriamycin, camptothecin, and etoposide were used as positive controls. Most of the compounds displayed significant cytotoxicity against the tested cell lines at low micromolar concentration compared to the positive controls. The IC50 values (mM) of the prepared trihydroxylated 2,4,6-triphenyl pyridines against those cell lines are shown in Table 2. In general, most
of the compounds displayed strong cytotoxicity (50 4.04 ± 0.2 1.76 ± 0.02 0.98 ± 0.02 1.4 ± 0.03 3.16 ± 0.14 3.98 ± 0.07 >50 3.42 ± 0.02 0.36 ± 0.01 >50 >50 5.37 ± 0.02 1.94 ± 0.04 5.8 ± 0.14
7.74 1.98 2.59 >50 2.18 5.02 >50 5.15 3.95 3.81 1.92 >50 5.12 3.39 >50 5.83 8.24 >50 >50 4.85 3.09 3.52
T47D
100 mM 66.2b/65.7
0.0 95.1 99.4 46.9 100.0 100.0 100.0 100.0 90.1 79.1 87.9 11.3 80.6 77.6 31.3 44.0 92.2 100.0 100.0
HEK293
2.6 0.0 4.9 3.7 0.0 0.0 0.0 0.4 0.0 3.8 0.0 1.9 1.5 0.3 8.4 0.0 0.0 0.0 0.0
1.04 0.28 1.22 8.13 0.14 0.08 >50 0.13 0.74 0.73 0.78 >50 0.79 0.73 >50 7.42 6.99 >50 26.3 5.65 1.56 0.84
± ± ± ± ± ±
0.03 0.01 0.03 0.09 0.01 0.003
± ± ± ±
0.004 0.004 0.01 0.002
± 0.005 ± 0.004 ± 0.3 ± 0.1 ± ± ± ±
1.01 0.08 0.07 0.02
± 0.04 ± 0.08 ± 0.06 ± 0.01 ± 0.08 ± ± ± ±
0.01 0.11 0.07 0.04
± 0.08 ± 0.06 ± 0.06 ± 0.16
± 0.04 ± 0.12 ± 0.06
8.74 ± 0.35 0.75 ± 0.01 3.12 ± 0.02 45.43 ± 1.96 2.51 ± 0.03 3.42 ± 0.04 18.3 ± 0.31 4.57 ± 0.08 2.05 ± 0.02 2.23 ± 0.09 5.35 ± 0.21 4.07 ± 0.12 4.27 ± 0.01 5.76 ± 0.13 >50 4.81 ± 0.03 0.96 ± 0.005 >50 >50 0.65 ± 0.002 3.92 ± 0.00 4.47 ± 0.02
HEK293: human embryonic kidney 293 cells. DU145: human prostate tumor cell line. HCT15: human colorectal adenocarcinoma cell line. T47D: human ductal breast epithelial tumor cell line. a Each data represents mean ± S.D. from three different experiments performed in triplicate. b Positive control value for compounds A and 6e13. c Topo I inhibitory activity and cytotoxicity reported earlier [22].
results showed that most of trihydroxylated 2,4,6-triphenyl pyridines displayed significant and selective topo II inhibition, which was stronger than the positive control, etoposide, and also significant cytotoxicity. All these results suggest that increase in the number of hydroxyl moiety increase topo II inhibition as shown in Fig. 7. Although it was difficult to draw up the correlation between the position of hydroxyl moiety in phenyl ring with topo II inhibition because most of the compounds showed significant activity, we
could drive a trend according to the positions of hydroxyl groups in R1 and R3 based on ortho-, meta-, and para-hydroxy phenyl ring of R2. When R2 is ortho-hydroxy phenyl, the order of topo II inhibition activity with positions of hydroxyl group in R1 and R3 is stated in a way of hydroxy positions in R1 and R3 and % inhibition of topo II at 100 mM in parenthesis followed by the name of compound: parapara (100%, 12) z meta-meta (100%, 9) > ortho-ortho (95.1%, 6) > meta-para (92.2%, 21) > meta-ortho (87.9%, 15) > para-ortho (77.6%, 18); When R2 is meta-hydroxy phenyl, those are meta-para
Fig. 5. Human DNA topo II inhibitory activity of 2,4,6-triphenyl pyridine (A) and 6e23.
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Fig. 6. Structures of monohydroxylated 2,4,6-triphenyl pyridines with % inhibition of topo II at 100 and 20 mM, respectively [24].
Fig. 7. Favorable order of substitution for topo II inhibitory activity.
(100%, 22) z meta-meta (100%, 10) > ortho-ortho (99.4%, 7) > parapara (90.1%, 13) > ortho-para (31.3%, 19) > ortho-meta (11.3%, 16); When R2 is para-hydroxy phenyl, those are meta-para (100%, 23) z meta-meta (100%, 11) > ortho-meta (80.6%, 17) z para-para (79.1%, 14) > ortho-ortho (46.9%, 8) > ortho-para (44.0%, 20). When R2 is ortho-hydroxy phenyl, compounds generally exhibited stronger topo II inhibitory activity than meta- or para-hydroxy phenyl ring in R2. Interestingly, compound 13 which possess para-hydroxyl
moiety at 2- and 6-phenyl ring and meta-hydroxyl moiety at 4phenyl ring displayed the most significant topo II inhibition at both concentrations. Overall, it might be draw out structureeactivity relationship with some exceptions such as: ortho-ortho, meta-meta, and para-para-hydroxyl substitutions in 2- and 6-phenyl ring exhibited better topo II inhibitory activity and cytotoxicities (Fig. 8). In addition, positive correlation was found between topo II inhibition and cytotoxicity for most of the compounds.
Fig. 8. Structureeactivity relationship of trihydroxylated 2,4,6-triphenyl pyridines.
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3.4. Molecular docking studies Compound 13 is the most potent at the low concentration (20 mM) in a cell-free experiment which may imply to be more persuasive topo II inhibitor in cells. Therefore, a series of compounds related to compound 13 were additionally selected from our previous study as depicted in Fig. 9A [24,25] to perform the ensemble docking and clustering study for further comparing the mode of action of compound 13 with that of etoposide. The ensemble docking and clustering with various initial ligand conformers improves the reliability in the docking simulations with AutoDock and AutoDock Vina. In addition to improving accuracy, one can calculate the deviation of energies in each pose, which will be important for the quantitative interpretation. Our calculation revealed that all the docked poses of compounds share the core features as shown in Fig. 9B. It may demonstrate the soundness of our approach. Considering the resemblance of the molecules, it will be reasonable to consider that their binding modes are quite similar. AutoDock Vina docking energies in each pose are as follows: 8.62 ± 0.10, 8.77 ± 0.07, 8.91 ± 0.10, 9.00 ± 0.09, 9.10 ± 0.12, 9.30 ± 0.13 (kcal/mol) for compounds A to E and 13, respectively, and are qualitatively consistent with the results of biological assays except compound E. The lower energies in the molecules that have additional hydroxyl moieties can be explained by the additional hydrophilic contacts with Lys8-OZ, Asp109-OD2, and Ade1486-N3. These interactions reinforce the hydrophobic core contacts with Gly30, Arg55, and Thy1463 (Fig. 9C). It should be underscored that the hydrophilic interactions are rarely overlapped with the contacts by etoposide (Fig. 9D), emphasizing the novelty of the inhibitory mechanism of compound 13. 4. Conclusion We have systematically designed and synthesized eighteen trihydroxylated 2,4,6-triphenyl pyridines by efficient synthetic route, and were evaluated for topo I and II inhibitory activity, and cytotoxicity against several human cancer cell lines. Most of the compounds (6, 7, 9e15, 17, 18, 21e23) showed strong and selective topo
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II inhibitory activity at 100 mM, and significant cytotoxicity. The structureeactivity relationship study revealed that the substitution of hydroxyl group on phenyl ring increase the topo I and II inhibitory activity of compounds, and further increase in the number of hydroxyl moiety enhances selectivity and inhibitory activity of topo II, and cytotoxicity. Also, positive correlation was found between topo II inhibition and cytotoxicity for most of the compounds. Furthermore, molecular docking study shows qualitatively consistent with the results of biological assays.
5. Experimental Compounds used as starting materials and reagents were obtained from Aldrich Chemical Co., Junsei or other chemical companies, and utilized without further purification. HPLC grade acetonitrile (ACN) and methanol were purchased from Burdick and Jackson, USA. Thin-layer chromatography (TLC) and column chromatography (CC) were performed with Kieselgel 60 F254 (Merck) and silica gel (Kieselgel 60, 230e400 mesh, Merck) respectively. Since all the compounds prepared contain aromatic ring, they were visualized and detected on TLC plates with UV light (short wave, long wave or both). NMR spectra were recorded on a Bruker AMX 250 (250 MHz, FT) for 1H NMR and 62.5 MHz for 13C NMR, and chemical shifts were calibrated according to TMS. Chemical shifts (d) were recorded in ppm and coupling constants (J) in hertz (Hz). Melting points were determined in open capillary tubes on electrothermal 1A 9100 digital melting point apparatus and were uncorrected. HPLC analyses were performed using two Shimadzu LC-10AT pumps gradient-controlled HPLC system equipped with Shimadzu system controller (SCL-10A VP) and photo diode array detector (SPD-M10A VP) utilizing Shimadzu Class VP program. Sample volume of 10 mL was injected in Waters X- Terra® 5 mM reverse-phase C18 column (4.6 250 mm) with a gradient elutions of 20%e100% of B in A for 10 min followed by 100%e20% of B in A for 20 min at a flow rate of 1.0 mL/min at 254 nm UV detection, where mobile phase A was double distilled water with 20 mM ammonium
Fig. 9. (A) Structures of compounds A to E and 13 with % topo II inhibition at 100 and 20 mM. Compounds B and C were retrieved from the reference 22 and compounds D and E were from the reference 23. The values in parenthesis are those of positive control, etoposide, extracted from the previous study [24,25]. (B) Overlaid structures of all compounds of (A) to topo IIa (PDB ID: 4FM9) in the calculated binding models. The molecules are colored in cyan. Cartoons in gray and yellow colors mean protein and DNA regions, respectively. (C) Binding mode of compound 13. We labeled only the residues in topo IIa and DNA that contact compound 13 within 3.5 Å, where asterisks indicate DNA residues. (D) Comparison with etoposide. Etoposide is colored in green. All the structural figures were generated by Pymol software (http://pymol.sourceforge.net/). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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formate (AF) and B was 90% ACN in water with 20 mM AF. Purity of compound is described as percent (%). ESI LC/MS analyses were performed with a Finnigan LCQ Advantage® LC/MS/MS spectrometry utilizing Xcalibur® program. For ESI LC/MS, LC was performed with a 2 mL injection volume on a Waters X Terra® 3.5 mm reverse-phase C18 column (2.1ⅹ 100 mm) with a gradient elution from 10% to 80% of B in A for 5 min followed by 80%e10% of B in A for 10 min and 10% B in A for 5 min, at a flow rate of 250 mL/min, where mobile phase A was 100% distilled water with 0.1% formic acid and mobile phase B was 100% ACN. MS ionization conditions were: Sheath gas flow rate: 70 arb, aux gas flow rate: 20 arb, I spray voltage: 4.5 KV, capillary temperature: 215 C, column temperature 40 C, capillary voltage: 21 V, tube lens offset: 10 V. Retention time was given in minutes. 5.1. General method for the preparation of 3 Compounds 3 (R1, R2 ¼ aec) were synthesized either by base catalyzed (KOH/NaOH) or acid catalyzed (BF3-Et2O) ClaiseneSchmidt condensation reaction as reported previously [25]. Total nine hydroxylated chalcone intermediates were synthesized. 5.2. General method for the preparation of 4 A mixture of aryl ketone 1 (R3 ¼ aec), iodine (1.2 equv) and pyridine was refluxed at 140 C for 3 h. Precipitate occurred during reaction which was cooled to room temperature. Then it was filtered and washed with cold pyridine to afford 4 (R3 ¼ aec) in quantitative yield. Three different pyridinium iodide salts were synthesized by this method. 5.3. General method for the preparation of 5 A mixture of hydroxylated chalcone intermediate 3 (R1, R ¼ aec), pyridinium iodide salt 4 (R3 ¼ aec) and anhydrous ammonium acetate in glacial acetic acid were heated at 80e100 C for 12e16 h. The reaction mixture was then extracted with ethyl acetate, washed with water and brine. The organic layer was dried with magnesium sulfate and filtered. The filtrate was evaporated at reduced pressure, which was then purified by silica gel column chromatography with the gradient elution of ethyl acetate/nhexane to afford solid compounds 5 (R1, R2, R3 ¼ aec) in 29.0%e 88.2% yield. Eighteen trihydroxylated 2,4,6-triphenyl pyridine compounds were synthesized by this method. 2
5.3.1. 2,20 ,200 -(Pyridine-2,4-6-triyl)triphenol (6) The same procedure described in section 5.3 was employed with 3 (R1, R2 ¼ a) (0.48 g, 2.00 mmol), dry ammonium acetate (1.54 g, 20.00 mmol), 4 (R3 ¼ a) (0.68 g, 2.00 mmol) and AcOH (2 mL) to yield a yellow solid (0.20 g, 29%, 0.58 mmol). Rf (ethyl acetate/n-hexane 1:2 v/v): 0.21. LC/MS/MS: retention time: 7.56 min, [MH]þ: 356.39. 1 H NMR (250 MHz, DMSO-d6) d 12.32 (s, 2H, 2-phenyl 2-OH, 6phenyl 2-OH), 9.99 (br, 1H, 4-phenyl 2-OH), 8.10 (s, 2H, pyridine H3, H-5), 7.86 (d, J ¼ 7.6 Hz, 2H, 2-phenyl H-6, 6-phenyl H-6), 7.55 (d, J ¼ 7.3 Hz, 1H, 4-phenyl H-6), 7.30 (t, J ¼ 7.8 Hz, 3H, 2-phenyl H-4, 6phenyl H-4, 4-phenyl H-4), 7.04e6.92 (m, 6H, 2-phenyl H-3, H-5, 6phenyl H-3, H-5, 4-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 157.47, 155.07, 154.65, 148.76, 131.07, 130.72, 130.63, 128.69, 125.26, 122.39, 120.43, 119.99, 119.46, 117.52, 116.61. 5.3.2. 2,20 -(4-(3-Hydroxyphenyl)pyridine-2,6-diyl)diphenol (7) The same procedure described in section 5.3 was employed with 3 (R1 ¼ a, R2 ¼ b) (0.36 g, 1.50 mmol), dry ammonium acetate
(1.15 g, 15.00 mmol), 4 (R3 ¼ a) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.42 g, 80%, 1.19 mmol). Rf (ethyl acetate/n-hexane 1:2 v/v): 0.21. LC/MS/MS: retention time: 7.56 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 12.30 (br, 2H, 2-phenyl 2-OH, 6phenyl 2-OH), 9.80 (br, 1H, 4-phenyl 3-OH), 8.13 (s, 2H, pyridine H3, H-5), 7.96 (d, J ¼ 7.7 Hz, 2H, 2-phenyl H-6, 6-phenyl H-6), 7.37e7.28 (m, 5H, 2-phenyl H-4, 6-phenyl H-4, 4-phenyl H-2, H-5, H-6), 6.99e6.93 (m, 5H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5, 4phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.26, 157.50, 155.48, 150.05, 139.20, 131.22, 130.51, 128.88, 122.23, 119.45, 118.31, 117.87, 117.51, 116.70, 114.26. 5.3.3. 2,20 -(4-(4-Hydroxyphenyl)pyridine-2,6-diyl)diphenol (8) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ a, R2 ¼ c) (0.24 g, 1.00 mmol), dry ammonium acetate (0.77 g, 10.00 mmol), 4 (R3 ¼ a) (0.34 g, 1.00 mmol) and AcOH (1 mL) to yield a yellow solid (0.14 g, 41.8%, 0.41 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.42. LC/MS/MS: retention time: 7.33 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 12.35 (br, 2H, 2-phenyl 2-OH, 6phenyl 2-OH), 9.93 (br, 1H, 4-phenyl 4-OH), 8.13 (s, 2H, pyridine H3, H-5), 7.96 (d, J ¼ 7.7 Hz, 2H, 2-phenyl H-6, 6-phenyl H-6), 7.86 (d, J ¼ 8.4 Hz, 2H, 4-phenyl H-2, H-6), 7.30 (t, J ¼ 7.5 Hz, 2H, 2-phenyl H-4, 6-phenyl H-4), 6.98e6.92 (m, 6H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5, 4-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 159.35, 157.61, 155.47, 149.77, 131.22, 129.06, 128.94, 128.16, 122.44, 119.47, 117.57, 116.98, 116.31. 5.3.4. 3,30 -(4-(2-Hydroxyphenyl)pyridine-2,6-diyl)diphenol (9) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ b, R2 ¼ a) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ b) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.20 g, 38.2%, 0.57 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.51. LC/MS/MS: retention time: 6.71 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 9.89 (br, 1H, 4-phenyl 2-OH), 9.60 (br, 2H, 2-phenyl 3-OH, 6-phenyl 3-OH), 7.95 (s, 2H, pyridine H-3, H-5), 7.65e7.53 (m, 5H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6, 4-phenyl H-6), 7.34e7.25 (m, 3H, 2-phenyl H-5, 6-phenyl H-5, 4phenyl H-4), 7.03 (d, J ¼ 8.1 Hz, 1H, 4-phenyl H-3), 6.95 (t, J ¼ 7.7 Hz, 1H, 4-phenyl H-5), 6.86 (d, J ¼ 6.1 Hz, 2-phenyl H-4, 6phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 157.98, 155.80, 155.00, 148.25, 140.68, 130.60, 130.32, 129.98, 125.48, 119.94, 119.25, 117.70, 116.58, 116.29, 113.76. 5.3.5. 3,30 ,300 -(Pyridine-2,4-6-triyl)triphenol (10) The same procedure described in section 5.3 was employed with 3 (R1, R2 ¼ b) (0.48 g, 2.00 mmol), dry ammonium acetate (1.54 g, 20.00 mmol), 4 (R3 ¼ b) (0.68 g, 2.00 mmol) and AcOH (2 mL) to yield a white solid (0.43 g, 61%, 1.22 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.36. LC/MS/MS: retention time: 6.63 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 9.69 (br, 1H, 4-phenyl 3-OH), 9.62 (s, 2H, 2-phenyl 3-OH, 6-phenyl 3-OH), 7.99 (d, J ¼ 1.3 Hz, 2H, pyridine H-3, H-5), 7.71e7.67 (m, 4H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6), 7.39e7.29 (m, 5H, 2-phenyl H-5, 6-phenyl H-5, 4phenyl H-2, H-5, H-6), 6.92e6.85 (m, 3H, 2-phenyl H-4, 6-phenyl H-4, 4-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.25, 158.03, 156.60, 149.82, 140.46, 139.50, 130.46, 130.01, 118.25, 117.91, 116.75, 116.49, 114.21, 113.93.
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5.3.6. 3,30 -(4-(4-hydroxyphenyl)pyridine-2,6-diyl)diphenol (11) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ b, R2 ¼ c) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ b) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.47 g, 88.2%, 1.32 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.36. LC/MS/MS: retention time: 6.30 min, [MH]þ: 356.34. 1 H NMR (250 MHz, DMSO-d6) d 9.60 (s, 3H, 2-phenyl 3-OH, 6phenyl 3-OH, 4-phenyl 4-OH), 7.99 (s, 2H, pyridine H-3, H-5), 7.88 (d, J ¼ 6.8 Hz, 4-phenyl H-2, H-6), 7.70e7.67 (m, 4H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6), 7.32 (t, J ¼ 7.9 Hz, 2H, 2-phenyl H-5, 6phenyl H-5), 6.93e6.85 (m, 4H, 4-phenyl H-3, H-5, 2-phenyl H-4, 6-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.93, 157.92, 156.45, 149.34, 140.61, 129.82, 128.74, 128.39, 117.82, 116.29, 116.06, 115.80, 113.89. 5.3.7. 4,40 -(4-(2-hydroxyphenyl)pyridine-2,6-diyl)diphenol (12) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ c, R2 ¼ a) (0.48 g, 2.00 mmol), dry ammonium acetate (1.54 g, 20.00 mmol), 4 (R3 ¼ c) (0.68 g, 2.00 mmol) and AcOH (2 mL) to yield a yellow solid (0.22 g, 32.1%, 0.64 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.31. LC/MS/MS: retention time: 5.86 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 9.77 (br, 3H, 2-phenyl 4-OH, 4phenyl 2-OH, 6-phenyl 4-OH), 8.06 (d, J ¼ 8.6 Hz, 4H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6), 7.81 (s, 2H, pyridine H-3, H-5), 7.51 (dd, J ¼ 7.5, 1.3 Hz, 1H, 4-phenyl H-6), 7.26 (td, J ¼ 7.2, 1.7 Hz, 1H, 4phenyl H-4), 7.01 (d, J ¼ 7.8 Hz, 1H, 4-phenyl H-3), 6.94 (t, J ¼ 7.5 Hz, 1H, 4-phenyl H-5, 6.90 (d, J ¼ 8.7 Hz, 4H, 2-phenyl H-3, H5, 6-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 158.66, 155.65, 154.98, 148.07, 130.59, 130.37, 130.09, 128.31, 125.99, 119.85, 117.19, 116.51, 115.70. 5.3.8. 4,40 -(4-(3-hydroxyphenyl)pyridine-2,6-diyl)diphenol (13) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ c, R2 ¼ b) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ c) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a light yellow solid (0.45 g, 85.2%, 1.27 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.33. LC/MS/MS: retention time: 5.98 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 9.71 (br, 3H, 2-phenyl 4-OH, 4phenyl 3-OH, 6-phenyl 4-OH), 8.14 (d, J ¼ 8.6 Hz, 4H, 2-phenyl H2, H-6, 6-phenyl H-2, H-6), 7.85 (s, 2H, pyridine H-3, H-5), 7.35e7.27 (m, 3H, 4-phenyl H-2, H-5, H-6), 6.91 (d, J ¼ 8.6 Hz, 4H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5), 6.90e6.85 (m, 1H, 4phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.79, 158.14, 156.41, 149.51, 139.85, 130.28, 130.10, 128.44, 118.10, 115.64, 114.45, 114.11. 5.3.9. 4,40 ,400 -(pyridine-2,4-6-triyl)triphenol (14) The same procedure described in Section 5.3 was employed with 3 (R1, R2 ¼ c) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ c) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.27 g, 51.8%, 0.77 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.26. LC/MS/MS: retention time: 5.49 min, [MH]þ: 356.38. 1 H NMR (250 MHz, DMSO-d6) d 9.64 (br, 3H, 2-phenyl 4-OH, 4phenyl 4-OH, 6-phenyl 4-OH), 8.12 (d, J ¼ 8.6 Hz, 4H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6), 7.84 (s, 2H, pyridine H-3, H-5), 7.83 (d, J ¼ 8.6 Hz, 2H, 4-phenyl H-2, H-6), 6.93e6.87 (m, 6H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5, 4-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 158.81, 158.73, 156.33, 149.09, 130.32, 128.81, 128.69, 128.46, 116.03, 115.63, 113.66.
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5.3.10. 2,20 -(6-(3-hydroxyphenyl)pyridine-2,4-diyl)diphenol (15) The same procedure described in Section 5.3 was employed with 3 (R1, R2 ¼ a) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ b) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.28 g, 53.4%, 0.80 mmol). Rf (ethyl acetate/n-hexane 1:2 v/v): 0.18. LC/MS/MS: retention time: 7.70 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 14.49 (s, 1H, 2-phenyl 2-OH), 9.83 (br, 2H, 4-phenyl 2-OH, 6-phenyl 3-OH), 8.28 (s, 1H, pyridine H-3), 8.11 (d, J ¼ 8.0 Hz, 1H, 2-phenyl H-6), 8.00 (s, 1H, pyridine H5), 7.61 (dd, J ¼ 7.6, 1.4 Hz, 1H, 4-phenyl H-6), 7.44e7.27 (m, 5H, 2phenyl H-4, 4-phenyl H-4, 6-phenyl H-2, H-5, H-6), 7.05 (d, J ¼ 8.1 Hz, 1H, 2-phenyl H-3), 7.01e6.89 (m, 4H, 2-phenyl H-5, 4phenyl H-3, H-5, 6-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 159.36, 158.33, 156.75, 155.10, 153.84, 149.64, 139.39, 131.62, 130.79, 130.55, 127.53, 124.98, 119.97, 119.83, 119.35, 119.20, 118.74, 118.05, 117.65, 116.94, 116.63, 113.46. 5.3.11. 3,30 -(6-(2-hydroxyphenyl)pyridine-2,4-diyl)diphenol (16) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ a, R2 ¼ b) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ b) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.41 g, 78.6%, 1.17 mmol). Rf (ethyl acetate/n-hexane 1:2 v/v): 0.21. LC/MS/MS: retention time: 7.59 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 14.40 (br, 1H, 2-phenyl 2-OH), 9.77 (br, 2H, 4-phenyl 3-OH, 6-phenyl 3-OH), 8.32 (s, 1H, pyridine H-3), 8.27 (d, J ¼ 8.0 Hz, 1H, 2-phenyl H-6), 8.01 (s, 1H, pyridine H5), 7.50 (t, J ¼ 7.5 Hz, 1H, 2-phenyl H-4), 7.45e7.31 (m, 6H, 6-phenyl H-2, H-5, H-6, 4-phenyl H-2, H-5, H-6), 6.97e6.92 (m, 4H, 2-phenyl H-3, H-5, 6-phenyl H-4, 4-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 159.39, 158.32, 158.26, 157.49, 154.79, 150.98, 139.23, 138.93, 131.79, 130.50, 130.45, 127.94, 119.35, 119.20, 118.49, 118.03, 117.87, 117.21, 117.08, 116.85, 116.37, 114.49, 113.65. 5.3.12. 2-(6-(3-hydroxyphenyl)-4-(4-hydroxyphenyl)pyridin-2-yl) phenol (17) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ a, R2 ¼ c) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ b) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.38 g, 72.7%, 1.09 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.46. LC/MS/MS: retention time: 7.43 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 14.65 (s, 1H, 2-phenyl 2-OH), 9.85 (br, 2H, 4-phenyl 4-OH, 6-phenyl 3-OH), 8.33 (s, 1H, pyridine H-3), 8.29 (d, J ¼ 8.1 Hz, 1H, 2-phenyl H-6), 8.03 (s, 1H, pyridine H5), 7.96 (d, J ¼ 8.5 Hz, 2H, 4-phenyl H-2, H-6), 7.52 (t, J ¼ 7.6 Hz, 1H, 2-phenyl H-4), 7.44e7.30 (m, 3H, 6-phenyl H-2, H-5, H-6), 6.98e6.92 (m, 5H, 2-phenyl H-3, H-5, 4-phenyl H-3, H-5, 6-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 159.54, 159.45, 158.29, 157.42, 154.60, 150.57, 139.37, 131.70, 130.45, 129.21, 127.81, 127.77, 119.27, 119.09, 118.03, 117.86, 116.99, 116.29, 116.15, 115.11, 113.63. 5.3.13. 2,20 -(6-(4-hydroxyphenyl)pyridine-2,4-diyl)diphenol (18) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ a, R2 ¼ a) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ c) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a green solid (0.27 g, 52%, 0.77 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.46. LC/MS/MS: retention time: 7.56 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 14.73 (s, 1H, 2-phenyl 2-OH), 9.96 (br, 2H, 4-phenyl 2-OH, 6-phenyl 4-OH), 8.19 (s, 1H, pyridine H-3), 8.08 (d, J ¼ 8.0 Hz, 1H, 2-phenyl H-6), 7.95 (s, 1H, pyridine H-
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5), 7.87 (d, J ¼ 8.4 Hz, 2H, 6-phenyl H-2, H-6), 7.58 (d, J ¼ 7.4 Hz, 1H, 4-phenyl H-6), 7.35e7.27 (m, 2H, 2-phenyl H-4, 4-phenyl H-4), 7.04 (d, J ¼ 8.2 Hz, 1H, 2-phenyl H-3), 7.00e6.89 (m, 5H, 2-phenyl H-5, 4phenyl H-3, H-5, 6-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 159.48, 159.29, 156.52, 155.06, 153.84, 149.59, 131.51, 130.74, 130.65, 128.71, 128.28, 127.36, 125.22, 119.92, 119.31, 119.11, 118.80, 118.04, 117.66, 116.59, 116.23. 5.3.14. 2-(4-(3-hydroxyphenyl)-6-(4-hydroxyphenyl)pyridin-2-yl) phenol (19) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ a, R2 ¼ b) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ c) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.22 g, 43%, 0.64 mmol). Rf (ethyl acetate/n-hexane 1:2 v/v): 0.16. LC/MS/MS: retention time: 7.45 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 14.68 (s, 1H, 2-phenyl 2-OH), 9.89 (br, 1H, 6-phenyl 4-OH, 9.77 (br, 1H, 4-phenyl 3-OH), 8.26e8.24 (m, 2H, 2-phenyl H-6, pyridine H-3), 7.99 (s, 1H, pyridine H-5), 7.96 (dd, J ¼ 8.8, 1.1 Hz, 2H, 6-phenyl H-2, H-6), 7.42 (t, J ¼ 7.8 Hz, 1H, 2-phenyl H-4), 7.39e7.30 (m, 3H, 4-phenyl H-2, H-5, H-6), 6.97e6.91 (m, 5H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5, 4phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 159.52, 159.44, 158.22, 157.32, 154.71, 150.90, 139.12, 131.69, 130.41, 128.52, 128.50, 127.79, 119.28, 119.11, 118.47, 118.02, 116.75, 116.19, 115.20, 114.49. 5.3.15. 4,40 -(6-(2-hydroxyphenyl)pyridine-2,4-diyl)diphenol (20) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ a, R2 ¼ c) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ c) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.42 g, 79.3%, 1.19 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.43. LC/MS/MS: retention time: 7.29 min, [MH]þ: 356.34. 1 H NMR (250 MHz, DMSO-d6) d 14.87 (br, 1H, 2-phenyl 2-OH), 10.01 (br, 2H, 4-phenyl 4-OH, 6-phenyl 4-OH), 8.26e8.23 (m, 2H, 2phenyl H-6, pyridine H-3), 8.00 (s, 1H, pyridine H-5), 7.94 (d, J ¼ 8.5 Hz, 4H, 6-phenyl H-2, H-6, 4-phenyl H-2, H-6), 7.32 (t, J ¼ 7.6 Hz, 1H, 2-phenyl H-4), 6.96e6.91 (m, 6H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5, 4-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 159.65, 159.36, 157.25, 154.53, 150.46, 131.58, 129.14, 128.66, 128.44, 127.94, 127.66, 119.25, 118.99, 118.01, 116.14, 116.12, 115.18, 114.00. 5.3.16. 2-(2-(3-hydroxyphenyl)-6-(4-hydroxyphenyl)pyridin-4-yl) phenol (21) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ b, R2 ¼ a) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ c) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.22 g, 42%, 0.62 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.42. LC/MS/MS: retention time: 6.37 min, [MH]þ: 356.34. 1 H NMR (250 MHz, DMSO-d6) d 9.75 (br, 3H, 2-phenyl 3-OH, 4phenyl 2-OH, 6-phenyl 4-OH), 8.07 (d, J ¼ 8.6 Hz, 2H, 6-phenyl H-2, H-6), 7.89 (s, 1H, pyridine H-3), 7.86 (s, 1H, pyridine H-5), 7.64 (s, 1H, 2-phenyl H-2), 7.58 (d, J ¼ 7.8 Hz, 1H, 4-phenyl H-6), 7.53 (dd, J ¼ 7.6, 1.4 Hz, 1H, 2-phenyl H-6), 7.32e7.23 (m, 2H, 2-phenyl H-5, 4phenyl H-4), 7.02 (d, J ¼ 8.0 Hz, 1H, 4-phenyl H-3), 6.95 (t, J ¼ 7.6 Hz, 1H, 4-phenyl H-5), 6.91 (d, J ¼ 8.7 Hz, 2H, 6-phenyl H-3, H-5), 6.85 (dd, J ¼ 8.0, 2.2 Hz, 1H, 2-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.74, 157.95, 155.88, 155.52, 154.97, 148.15, 140.82, 130.57, 130.21, 129.94, 128.32, 125.73, 119.89, 118.25, 118.15, 117.64, 116.54, 116.17, 115.72, 113.70.
5.3.17. 3,30 -(6-(4-hydroxyphenyl)pyridine-2,4-diyl)diphenol (22) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ b, R2 ¼ b) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ c) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.32 g, 60.8%, 0.91 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.38. LC/MS/MS: retention time: 6.39 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 9.67 (br, 3H, 2-phenyl 3-OH, 4phenyl 3-OH, 6-phenyl 4-OH), 8.16 (d, J ¼ 8.5 Hz, 2H, 6-phenyl H-2, H-6), 7.95 (s, 1H, pyridine H-3), 7.89 (s, 1H, pyridine H-5), 7.71 (s, 1H, 2-phenyl H-2), 7.68 (d, J ¼ 7.7 Hz, 1H, 2-phenyl H-6), 7.36e7.29 (m, 4H, 2-phenyl H-5, 4-phenyl H-2, H-5, H-6), 6.92 (d, J ¼ 8.4 Hz, 2H, 6phenyl H-3, H-5), 6.89e6.84 (m, 2H, 2-phenyl H-4, 4-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.90, 158.18, 157.94, 156.60, 156.34, 149.63, 140.55, 139.64, 130.32, 129.93, 129.88, 128.48, 118.12, 117.77, 116.30, 115.67, 115.55, 114.12, 113.82. 5.3.18. 4,40 -(6-(3-hydroxyphenyl)pyridine-2,4-diyl)diphenol (23) The same procedure described in Section 5.3 was employed with 3 (R1 ¼ b, R2 ¼ c) (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 4 (R3 ¼ c) (0.51 g, 1.50 mmol) and AcOH (2 mL) to yield a yellow solid (0.32 g, 61.3%, 0.92 mmol). Rf (ethyl acetate/n-hexane 1:1 v/v): 0.36. LC/MS/MS: retention time: 5.90 min, [MH]þ: 356.35. 1 H NMR (250 MHz, DMSO-d6) d 9.77 (br, 2H, 4-phenyl 4-OH, 6phenyl 4-OH), 9.55 (br, 1H, 2-phenyl 3-OH), 8.15 (d, J ¼ 8.5 Hz, 2H, 6-phenyl H-2, H-6), 7.95 (s, 1H, pyridine H-3), 7.90 (s, 1H, pyridine H-5), 7.86 (d, J ¼ 8.5 Hz, 2H, 4-phenyl H-2, H-6), 7.70 (s, 1H, 2phenyl H-2), 7.68 (d, J ¼ 7.9 Hz, 1H, 2-phenyl H-6), 7.30 (td, J ¼ 7.9, 3.0 Hz, 1H, 2-phenyl H-5), 6.93 (d, J ¼ 8.4 Hz, 2H, 6-phenyl H3, H-5), 6.91 (d, J ¼ 8.6 Hz, 2H, 4-phenyl H-3, H-5), 6.86 (dd, J ¼ 8.1, 2.9 Hz, 1H, 2-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 162.50, 158.88, 158.80, 157.90, 156.47, 156.24, 149.19, 140.75, 130.11, 129.81, 128.68, 128.57, 128.44, 117.76, 116.03, 115.61, 114.73, 114.66, 113.83. 5.4. Pharmacology 5.4.1. Assay for DNA topoisomerase I inhibition in vitro DNA topo I inhibition assay was determined following the method reported by Fukuda et al. [28] with minor modifications. The test compounds were dissolved in DMSO at 20 mM as stock solutions. The activity of DNA topo I was determined by assessing the relaxation of supercoiled DNA pBR322. The mixture of 100 ng of plasmid pBR322 DNA and 1 unit of recombinant human DNA topoisomerase I (TopoGEN INC., USA) was incubated without and with the prepared compounds at 37 C for 30 min in the relaxation buffer (10 mM TriseHCl (pH 7.9), 150 mM NaCl, 0.1% bovine serum albumin, 1 mM spermidine, 5% glycerol). The reaction in the final volume of 10 mL was terminated by adding 2.5 mL of the stop solution containing 5% sarcosyl, 0.0025% bromophenol blue and 25% glycerol. DNA samples were then electrophoresed on a 1% agarose gel at 15 V for 7 h with a running buffer of TAE. Gels were stained for 30 min in an aqueous solution of ethidium bromide (0.5 mg/mL). DNA bands were visualized by transillumination with UV light and were quantitated using AlphaImager™ (Alpha Innotech Corporation). 5.4.2. Assay for DNA topoisomerase II inhibition in vitro DNA topo II inhibitory activity of compounds were measured as follows [29]. The mixture of 200 ng of supercoiled pBR322 plasmid DNA and 1 unit of human DNA topoisomerase IIa (Usb Corp., USA) was incubated without and with the prepared compounds in the assay buffer (10 mM TriseHCl (pH 7.9) containing 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM ATP, and 15 mg/mL bovine serum
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albumin) for 30 min at 30 C. The reaction in a final volume of 20 mL was terminated by the addition of 3 mL of 7 mM EDTA. Reaction products were analyzed on 1% agarose gel at 25 V for 4 h with a running buffer of TAE. Gels were stained for 30 min in an aqueous solution of ethidium bromide (0.5 mg/mL). DNA bands were visualized by transillumination with UV light and supercoiled DNA was quantitated using AlphaImager™ (Alpha Innotech Corporation). 5.4.3. Cytotoxicity assay Cells were cultured according to the supplier's instructions. Cells were seeded in 96-well plates at a density of 2e4 104 cells per well and incubated for overnight in 0.1 mL of media supplied with 10% Fetal Bovine Serum (Hyclone, USA) in 5% CO2 incubator at 37 C. On day 2, after FBS starvation for 4 h, culture medium in each well was exchanged with 0.1 mL aliquots of medium containing graded concentrations of compounds. On day 4, each well was added with 5 mL of the cell counting kit-8 solution (Dojindo, Japan) then incubated for additional 4 h under the same condition. The absorbance of each well was determined by an Automatic Elisa Reader System (Bio-Rad 3550) at 450 nm wavelength. For determination of the IC50 values, the absorbance readings at 450 nm were fitted to the four-parameter logistic equation. The compounds like adriamycin, etoposide, and camptothecin were purchased from Sigma and used as positive controls. 5.4.4. Molecular docking study The template structure for the structure-based docking was prepared by using the coordinate in the complex of TOPOIIa and DNA (PDB ID: 4FM9) [30]. In order to compare the docked poses of the molecules in the current study with etoposide bound with TOPOIIa (PDB ID: 3QX3) [31], we aligned 3QX3 into 4FM9 by TMalign [32]. Modeller package (ver. 9.9) [33] modeled the missed coordinates in topo IIa due to the lack of electron densities. After PDB2PQR [34] determined the ionization states of Asp, Glu, His, and Lys residues in the structure, AMBER molecular dynamics package [35] minimized the energy of the structure. For faithful docking simulation, we employed both AutoDock [36] and AutoDock Vina [37]. The docking protocol consists of four stages such as preparation of ligand parameters, ensemble generation of ligands, docking and clustering. Antechamber program of AMBER package first prepared force-field for each ligand by semi-quantum calculation. Then 30 different conformers of each ligand were generated with sander program. Once gaining 60 structures by dockings with AutoDock and AutoDock Vina with default parameters, we clustered the resulting conformers by structural similarity that was quantified by pairwise root mean square deviation (RMSD) value. The clusters were further sorted according to AutoDock Vina scores. We chose the centroid structure in the best cluster as a final model. In-house written software and scripts automated all the procedures. Acknowledgments
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.07.058. References [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]
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF2012R1A2A2A01046188).
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