Article pubs.acs.org/jmc
Design and Synthesis of New α‑Naphthoflavones as Cytochrome P450 (CYP) 1B1 Inhibitors To Overcome Docetaxel-Resistance Associated with CYP1B1 Overexpression Jiahua Cui,‡ Qingqing Meng,‡ Xu Zhang, Qing Cui, Wen Zhou,* and Shaoshun Li* School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, China S Supporting Information *
ABSTRACT: CYP1B1 is recognized as a new target in cancer prevention and therapy. Taking α-naphthoflavone as a lead, a series of 6,7,10-trimethoxy-α-naphthoflavones (4a−o) were synthesized and evaluated for their inhibitory potency against CYP1B1 and selectivity over CYP1A1 and 1A2. SAR analysis indicated that introducing methoxy groups at C(6), C(7), and C(10) on the naphthalene part and a fluoro atom at C(3′) on the B-ring, could sharply increase the efficiency toward CYP1B1 inhibition. Among the prepared derivatives, compound 4c is the most potent and selective CYP1B1 inhibitor ever reported. More effort was taken to acquire water-soluble α-naphthoflavone derivatives for further cell-based study of overcoming anticancer drug-resistance. Finally, we obtained water-soluble naphthoflavone (11f) which could obviously eliminate the docetaxelresistance caused by the enhanced expression of CYP1B1 in MCF-7/1B1 cells. It could be envisaged that the discovery of new αnaphthoflavones as CYP1B1 inhibitors is clinically important for overcoming CYP1B1-mediated drug-resistance in cancer therapeutics.
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INTRODUCTION Cytochrome P450 (CYP) 1B1 is a member of the CYP1 family and mainly expressed in extrahepatic organs. It is a hemecontaining enzyme which catalyzes the bioactivation of exogenous procarcinogens such as the polycyclic aromatic hydrocarbons (PAHs),1,2 aromatic amines,1 and nitropolycyclic hydrocarbons.3 The metabolism of these chemicals by CYP1B1 protein has been recognized as the initial step in the carcinogenic action of these chemicals. CYP1B1 has also been identified as a key enzyme that participates in the carcinogenic action of 17-β-estradiol (E2),4 which undergoes CYP1B1-mediated C(4) hydroxylation to generate 4-hydroxyestradiol (4-OHE2), leading to estrogen− related tumorigenesis (Figure 1).5,6 The other two members in the human CYP1 family (CYP1A1 and CYP1A2) prefer the 2hydroxylation of E2 to produce 2-hydroxyestradiol that is not mutagenic.7,8 A great deal of research has confirmed the high and consistent expression of CYP1B1 in several kinds of tumors but not existent in the corresponding normal tissues.9−12 CYP1B1 could metabolically inactivate a range of structurally diverse © 2015 American Chemical Society
anticancer agents such as paclitaxel, docetaxel, tamoxifen, doxorubicin, and mitoxantrone.13−15 The inactivation of those compounds has been deemed as a reason for drug-resistance in certain cancer cell lines. All of these findings make CYP1B1 an important drug target, and inhibition of CYP1B1 is obviously a new cancer prevention/therapeutic strategy.16,17 Naturally occurring flavonoids, which contain the chroman4-one backbone, could effectively inhibit tumor initiation in animal models.18 The inhibition of CYP1 enzymes has been considered as one of the mechanisms by which these natural products exert their chemoprevention activity. The natural phenolic trans-stilbenes with inhibition against the CYP1 family have also been deemed as a series of potential chemopreventive phytochemicals.19,20 α-Naphthoflavone (ANF, Figure 2), a synthetic flavonoid, is recognized as a strong inhibitor of the CYP1B1 enzyme and has been extensively utilized in the characterization of the CYP1B1mediated specific metabolic reaction.21 Shimada et al. tested its Received: January 17, 2015 Published: March 23, 2015 3534
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Figure 1. Physiological functions of CYP1B1 in tumor initiation and drug-resistance.
Herein we report the discovery of water-soluble αnaphthoflavone derivatives as selective CYP1B1 inhibitors over CYP1A1 and 1A2. These α-naphthoflavone derivatives were capable of overcoming the docetaxel-resistance associated with CYP1B1 overexpression in cancer treatment. By optimizing the structure of ANF based on the X-ray cocrystal structure of the CYP1B1−ANF complex, we obtained 4c as the strongest CYP1B1 inhibitor ever reported. However, the solubility of 4c was not satisfactory. To further establish the drug-resistance reversal ability of prepared CYP1B1 inhibitors in cell-based investigation, we introduced various amino acid groups via a carbon linker on C(3) of compound 4c by analyzing the molecular docking model of 4c with CYP1B1 enzyme. The resulting water-soluble CYP1B1 inhibitor 11f could significantly increase the sensitivity of docetaxel-resistant cancer cells to this anticancer agent.
Figure 2. Chemical structure of α-naphthoflavone.
inhibition of 7-ethoxyresorufin O-deethylation (EROD) activity using recombinant human CYP1 enzymes expressed in E. coli membranes.22 The results demonstrated that ANF showed more potent inhibitory effects on human CYP1B1 and 1A2 (IC50 of 5 and 6 nM, respectively) but was comparatively less active toward CYP1A1 inhibition (IC50 of 60 nM). The studies undertaken by McFadyen and colleagues implied that ANF as an inhibitor of CYP1B1 could overcome certain anticancer drug-resistance in a CYP1B1-expressing cancer cell line.13 Meanwhile, this inhibitor was nontoxic at all tested concentrations in vitro. However, its poor water solubility and limited selectivity toward CYP1B1 over other CYP1 isoforms confined its further development. All of these findings defined ANF as a promising lead compound in the discovery of more potent and selective CYP1B1 inhibitors against anticancer drug-resistance.
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RESULTS AND DISCUSSION Chemistry. In the synthesis of α-naphthoflavone derivatives (4a−o, Scheme 1), compound 1 served as the starting point which was easily prepared from 1,5-dihydroxynaphthalene according to our reported procedure.23 The benzoylation of the hydroxyl group of 1 and subsequent Baker−Venkataraman rearrangement of esters (2a−o) with KOH as the catalyst afford diketones (3a−o) in high yield. Then the diketones were 3535
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Scheme 1a
a Reagents and conditions: (a) DCC, DMAP, DCM, rt for 12 h; (b) KOH, Py, reflux, 1 h; (c) H2SO4, EtOH, reflux, 1 h; (d) NaH, MOMCl, DMF, rt for 1 h; (e) KOH, EtOH, rt for 3−5 h; (f) H2O2, NaOH, MeOH, 5 °C for 12 h; (g) HCl, MeOH, 55 °C for 1 h; (h) H2O2, NaOH, MeOH, rt for 48 h.
However, the yield was rather disappointing because of the instability of these dibenzoylmethane intermediates. Since flavanonols were widely accepted as the plausible intermediates in the course of the AFO reaction,24,29 we used 6,7,10-trimethoxy-α-naphthoflavanonol (7a−g, Scheme 1) as the raw material which was successfully oxidized by a large excess of hydrogen peroxide under alkali conditions to give the corresponding naphthoflavonols in high yield. The reaction might proceed through the oxidation of C(2) and further dehydration as illustrated in Figure 4. For the preparation of 6,7,10-trimethoxy-α-naphthoflavanonols (7a−g), compound 1 was also employed as the starting material (Scheme 1). The protection of the hydroxyl group with methoxy methyl ether and further Claisen−Schmidt condensation afforded benzochalcones (6a−g) under mild conditions and with high yield. The epoxidation of the chalcones with alkaline hydrogen peroxide and the formation of the flavanonol skeleton in methanolic hydrogen chloride solution gave the desired compounds. The appropriately designed route for the synthesis of naphthoflavonol amino acid esters (11a−f) is shown in Scheme 2. The nucleophilic substitution between the most active naphthoflavonol (8c) and 2-(2-bromoethyl)tetrahydro-2Hpyran (Scheme 2, n = 1) in the presence of potassium carbonate and further deprotection afforded compound 10a as the key intermediate. Subsequent condensation of 10a with NBoc amino acids and ultimate deprotection step gave compounds 11a−c in high yield. Compounds 11d−f were
converted to 6,7,10-trimethoxy-α-naphthoflavones (4a−o) in the presence of sulfuric acid. The most commonly used method for the synthesis of flavonols was the Algar−Flynn−Oyamada (AFO) reaction that employed the corresponding chalcones as the starting material.24−27 However, in the synthesis of 6,7,10-trimethoxy-αnaphthoflavonols (8a−g), this oxidative cyclization strategy using 2′-hydroxyl benzochalcones (9a−g, Figure 3) failed to
Figure 3. Plausible mechanism for the oxidation of chalcones bearing methoxyl groups.
give any desired naphthoflavonols. The obtained product was characterized as ring-substituted cinnamic acid. This might be due to the electron-donating effects of these methoxy groups on the naphthalene part that led to the oxidation of aromatic rings. The cleavage of the bond between the carbonyl carbon and the naphthalene part gave cinnamic acid derevatives. Fougerousse’s method28 that used Baker−Venkataramen rearrangement of dibenzoylmethanes, which had an oxygen atom connected to C(α) of compound 3a−g, was also tried.
Figure 4. Plausible mechanism for the preparation of α-naphthoflavonols through oxidation. 3536
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Scheme 2a
a Reagents and conditions: (a) Br(CH2CH2)nOTHP, K2CO3, Me2CO, TBAB, rt for 36 h; (b) HCl, MeOH, rt for 6 h; (c) DCC, DMAP, DCM, rt for 12 h; (d) HCl, EtOAc, rt for 12 h.
Table 1. Inhibitory Potency of 6,7,10-Trimethoxy-α-naphthoflavonols (4a−o) against CYP1B1, 1A1, and 1A2
IC50 values (nM)
IC50 ratio
compd
R′
CYP1B1
CYP1A1
CYP1A2
CYP1A1/1B1
CYP1A2/1B1
4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n 4o ANF
H 2′-F 3′-F 4′-F 2′-Cl 3′-Cl 4′-Cl 2′-OCH3 4′-OCH3 3′,4′-OCH3 3′,4′,5′-OCH3 4′-OBn 3′,4′-OBn 4′-OH 2′-OH H
0.2 0.2 0.043 0.3 1.1 0.3 0.2 9.0 1.1 2.5 9.8 771.4 >100000 642.9 62.9 1.3
4.8 5.9 11.5 9.8 19.2 6.4 1.8 18.8 12.9 11.5 22.5 67.8 535.1 113.7 12.1 11.9
16.1 22.1 36.7 15.4 58.0 61.1 27.7 13.5 16.4 19.3 23.5 43.0 96.9 58.4 50.6 3.8
24 29.5 267 33 17.5 21 9 2.1 12 4.6 2.3 0.09 − 0.18 0.19 9.2
81 111 853 51 53 204 139 1.5 15 7.7 2.4 0.06 − 0.09 0.80 2.9
high binding affinity of ANF with human CYP1B1 (Figure 5). The crystal structures of CYP1A1 and 1A2 were also unveiled recently with ANF in their active site cavities,33,34 in which ANF bound in distinctly different orientations from that observed for CYP1B1 enzyme. Taking ANF as a lead compound, we first introduced methoxy groups to C(6), C(7), and C(10) on the scaffold of the lead compound to increase the electron-density of the naphthalene part and thus to intensify the π−π stacking with Phe231. In addition, B-ring modification with univalent bioisosteres was also conducted according to the different orientations of ANF in CYP1 isoforms. In the enzymatic assay, almost all prepared compounds in Table 1 were identified as selective inhibitors of CYP1B1. 6,7,10-Trimethoxy-α-naphthoflavone (4a, IC50 of 0.2 nM) was a 6-fold more potent inhibitor than the lead compound (IC50 of 1.3 nM) which indicated that the introduction of methoxy groups at C(6), C(7), and C(10) on the skeleton of ANF greatly increased its inhibitory efficiency against CYP1B1. As an electron-donating substituent, the methoxy group could increase the electron density of the naphthalene part of ANF via conjugation of the unshared electron pair on the oxygen with the aromatic ring. It could be envisaged that the increased
prepared with the same synthetic strategy, using different brominated alcohols as the reactant in the nucleophilic substitution (Scheme 2). Structure−Activity Relationships of α-Naphthoflavone Analogues against CYP1B1. The inhibitory effects of target compounds against recombinant human CYP1B1, 1A1, and 1A2 enzymes were evaluated using the standard EROD assay30 which has been widely used as a method for the evaluation of CYP1 activity. ANF prepared according to Mahal’s method31 was employed as the positive control in the EROD assay (HPLC purity >99%). The enzymatic inhibitory potency of tested compounds and ANF was displayed by IC50 values indicated from Table 1 to Table 3. The crystal structure of human CYP1B1 with ANF in its active site cavity has been well elucidated recently by X-ray crystallography in 2.7 Å resolution.32 This study suggested that ANF as a small and planar molecule closely fitted the slot-like binding cavity of this enzyme. It lays in the cavity mainly in an optimal orientation with the 2-phenyl group (B-ring) toward the heme prosthetic group. The π−π stacking between the naphthalene part of ANF and a phenylalanine residue (Phe231) along with the hydrogen bond formed by the ketone group of ANF with Asp326 at certain pH values all contributed to the 3537
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compound 4c was characterized as the most potent and selective inhibitor for CYP1B1 ever reported, and this observation indicated that the fluoro atom on C(3′) played a pivotal role in CYP1B1 inhibition. The study that focused on the crystal structure of CYP1B132 demonstrated that C(3′) on the 2-phenyl group of ANF was near the heme prosthetic group (Figure 6). On the contrary, the 4′-carbon on the exocyclic phenyl ring of the lead compound oriented toward the catalytic heme iron in CYP1A1 and 1A2. Thus, the presence of a substituent on C(3′) or C(4′) might influence the formation of Fe(V)−oxo complex during the cytochrome P450 catalytic cycle.35 The SAR analysis confirmed that the introduction of a 3′-fluoro atom on the exocyclic phenyl ring of 4a greatly influenced the catalytic efficiency of CYP1B1. The result of in silico docking study was also consistent with the assumption that this halogen atom probably affected the efficiency of the heme. On the contrary, the 4′-halogen-substituted α-naphthoflavones (4d and 4g) were more potent inhibitors against CYP1A1 and 1A2 than the corresponding 3′-fluoro and 3′-chloro analogues (4c and 4f). Analogue 4l with a 4′-benzyloxy substituent was weakly active toward CYP1B1 inhibition. The substitution of hydrogens on the B-ring at C(3′) and C(4′) with benzyloxy groups (4m) resulted in complete loss of inhibitory activity. A plausible mechanism for the significant decrease in inhibitory efficiency might be the steric hindrance of the benzyloxy group which was a bulkier substituent than the hydrogen or halogen atom. This detrimental effect of bulky substitutions on the B-ring of compounds 4l and 4m was also observed in CYP1A1 and 1A2 inhibition but not as remarkably as that in CYP1B1 inhibitory activity. Introducing a methoxy group on the exocyclic phenyl ring at C(2′) or C(3′) or C(4′) also resulted in a sharp decrease in the efficacy toward CYP1B1 inhibition, since the inhibitory potentials of 4h, 4i, 4j, and 4k were diminished significantly as compared to that of 4a. It also could be observed that a drop in inhibitory potency against CYP1B1 was associated with an increase in the number of methoxy groups on the B-ring. These
Figure 5. X-ray cocrystal structure of the CYP1B1−ANF complex (PDB ID 3PM0). ANF (light blue) and the heme prosthetic group (violet) are shown in sticks. Phe231 (orange) and Asp326 (yellow) are shown in lines.
electron density possibly led to the enhanced π−π interaction of resulting compounds with the side chain of Phe231. In addition, the methoxy group as a small substitution is not sterically large enough to influence the size and shape of the naphthalene part. The substitutions on the B-ring also greatly influenced the inhibitory efficiency of those naphthoflavones. The introduction of a halogen (chlorine or fluorine) atom on the exocyclic phenyl group of 4a was more effective toward CYP1B1 inhibition than the modification of the phenyl ring by the addition of a methoxy or a benzyloxy group. Compound 4c exhibited an IC50 of 0.043 nM for CYP1B1 inhibition and showed a much less inhibitory potency against CYP1A1 and 1A2 enzymes (IC50 of 11.5 and 36.7 nM for CYP1A1 and 1A2, respectively). Among the prepared new naphthoflavones,
Figure 6. B-ring of ANF near the heme prosthetic group of (a) CYP1B1 (PDB ID 3PM0), (b) CYP1A1 (PDB ID 4I8V), and (c) CYP1A2 (PDB ID 2HI4). 3538
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The obtained naphthoflavonols (8a−g) that bear a halogen atom on their B-rings exhibited less inhibitory potency against CYP1B1 than the corresponding naphthoflavones (4a−g), but the selectivity between CYP1B1 and CYP1A subfamily members was retained (Table 2). This change was in agreement with an earlier observation that a hydroxyl group on C(3) was not an essential ingredient for the CYP1B1 inhibitory activity of flavones.17 As we expected, the 3′-fluoro analogue (8c) was the most active compound against CYP1B1 and exhibited the highest selectivity among compounds 8a−8g. Therefore, 8c was selected for further modifications to prepare water-soluble naphthoflavones. Then saturated carbon linkers were introduced to the most active naphthoflavonol (8c), and amino acids were connected to the other end of the linker. The resulting naphthoflavonol amino acid esters (11a−f) exhibited excellent water solubility and were tested for their inhibition of CYP1. Compared with compounds 8a−g mentioned above, naphthoflavonol amino acid esters (11a−f) all exhibited decreased inhibitory potency against the CYP1B1 enzyme. The analogues with larger alkyl groups (11b and 11c) showed decreased inhibitory potency compared with 11a. When ANF was bound in the active site of CYP1B1, the cavity was closed without any solvent or substrate access channels. It was predicted by Mole program that the B′−C loop (Figure 7) was one of the regions that was flexible.32,36 The changes in torsion angle motion of this region may allow the access of a ligand to the cavity. Therefore, in the CYP1B1 enzyme−inhibitor (11a−c) complex, the ethylene bridge that was connected to the C-ring of 11a was probably near the B′− C loop, and the drop in binding affinity of amino acid esters 11b and 11c might be ascribed to the collision of the C(α) alkyl group of the amino acid with the B′−C loop. An increase in the size of the alkyl group on C(α) also resulted in the decrease in efficiency toward CYP1A1 and 1A2 inhibition. Additionally, the length of the alkyl chain also greatly influenced the inhibitory activity of these compounds. The elongation of the chain from the ethylene bridge (11a) to an nhexane backbone (11e) caused a sharp decrease in the potency toward CYP1B1 inhibition (Table 3). The inhibitory activity against CYP1A2 decreased more significantly along with the elongation of the chain length. However, when this chain was extended from an n-hexane backbone (11e) to an n-heptane scaffold (11f), an increase in the inhibitory property against
results illustrated that the number of B-ring substituents was a major factor for the inhibitory efficiency of these new naphthoflavones. Additionally, the introduction of a hydroxyl group on the Bring caused detrimental effects on CYP1B1 inhibition, since both compound 4n and 4o showed much less inhibitory potency against CYP1B1 compared with the corresponding methoxy-substituted ones (4i and 4h). Although we have found the strongest and the most selective CYP1B1 inhibitor (4c), the poor solubility of this compound limited its further application in cell-based study. Since C-ring of ANF was near the flexible helix B′−C loop (B′−C loop, Figure 7) of CYP1B1,32 the introduction of a hydrophilic group
Figure 7. Carbon atom on the C-ring of ANF near the B′−C loop of CYP1B1.
to C(3) on the C-ring might largely increase the water solubility without loss of CYP1B1 inhibitory activity. According to the primary SAR analysis of α-naphthoflavones (4a−o) as CYP1B1 inhibitors, halogen-containing naphthoflavonols (8a− g) which have a functionizable hydroxyl group at C(3) on the C-ring were synthesized and evaluated for their inhibition of CYP1B1.
Table 2. Inhibitory Potency of 6,7,10-Trimethoxy-α-naphthoflavonols (8a−g) against CYP1B1, 1A1, and 1A2
IC50 values (nM)
IC50 ratio
compd
R′
CYP1B1
CYP1A1
CYP1A2
CYP1A1/1B1
CYP1A2/1B1
8a 8b 8c 8d 8e 8f 8g
H 2′-F 3′-F 4′-F 2′-Cl 3′-Cl 4′-Cl
2.3 1.2 0.3 0.8 1.6 3.5 5.0
31.6 27.5 66.9 11.8 32.9 46.0 26.9
34.4 29.4 26.5 57.6 49.6 42.5 83.3
14 23 223 15 21 13 5.4
15 25 88 72 31 12 17
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Table 3. Effect of Chain Length on Potency of 6,7,10-Trimethoxy-α-naphthoflavonol Amino Acid Esters (11a−f) against CYP1
IC50 values (nM)
IC50 ratio
compd
n
R
CYP1B1
CYP1A1
CYP1A2
CYP1A1/1B1
CYP1A2/1B1
11a 11b 11c 11d 11e 11f
1 1 1 2 3 4
H CH3 CH(CH3)2 H H H
19.6 98.4 114.5 37.02 68.81 12.30
13.6 39.4 192.0 70.24 36.16 20.16
56.2 85.9 68.3 121.6 119.7 129.0
0.69 0.40 1.7 1.9 0.53 1.6
2.9 0.87 0.60 3.3 1.7 10
Figure 8. Docking pose of compound 4c (green sticks) in the CYP1B1−ANF complex (PDB ID 3PM0). Phe231 (orange), Asp326 (yellow), and Ser131 (blue-purple) are shown in lines. The heme prosthetic group is shown in violet sticks. Hydrogen bonds are shown by dashed lines.
formation of Fe(V)−oxo complex during CYP1B1 catalytic cycle. Reversal of Drug-Resistance in Cancer Therapy. Docetaxel as an effective chemotherapeutic agent has been used for the treatment of solid tumors in clinics, including breast, ovarian, prostate, lung, and gastro-esophageal cancer. However, because of inherent or acquired resistance to docetaxel, many patients do not respond to this drug. One reason for docetaxel-resistance has been ascribed to the enhanced expression of the CYP1B1 enzyme in cancer,13,37 and the use of ANF as a CYP1B1 inhibitor could eliminate the drug-resistance in a CYP1B1-expressing cancer cell line. The result inspired us to employ 11f as the most active watersoluble compound for cell-based drug-resistance reversal experiment compared with ANF. The expression of the CYP1B1 enzyme could be induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cell line with a marked increase in the level of CYP1B1 mRNA.38−40 We obtained the CYP1B1-expressing MCF-7 cells (referred to as MCF-7/1B1) by treatment of parental MCF-7 human breast cancer cells with a low dose of TCDD according to the reported method.39 Expression of CYP1B1 protein in MCF-7/1B1 and parental MCF-7 cells was measured
CYP1B1 was observed. Among these compounds (11a−f), 11f exhibited the most potent inhibition of CYP1B1 with excellent water solubility. Molecular Docking. To gain insight into the molecular determinants that modulate the inhibitory activity of these new α-naphthoflavone analogues, molecular docking simulations for compound 4c to human CYP1B1 enzyme were performed using the docking program in MOE 2008 software based on the X-ray cocrystal structure of CYP1B1-ANF complex (PDB ID 3PM0). As shown in Figure 8, compound 4c tightly fitted the active site of CYP1B1. The D-ring of 4c was covered by the phenyl ring of Phe231, and the increased π−π interaction should be one of the explanations for the enhanced binding affinity of the inhibitor with CYP1B1. Besides the expected hydrogen bond between the carbonyl group with Asp326, the oxygen of the carbonyl group also seemed to highly specifically form a hydrogen bond with the hydroxyl group in Ser131. This hydrogen bond also made contributions on the strong inhibitory potency of compound 4c. The distance between the fluoro atom on the B-ring and the heme ion was only 1.84 Å, and this observation indicated that the presence of the fluoro atom might greatly influence the 3540
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by western-blot techniques with the recombinant human CYP1B1 enzyme as the positive control (Figure 9). The
Figure 9. Expression of CYP1B1 detected by western-blot in MCF-7/ 1B1 and MCF-7 cells.
enhanced expression of CYP1B1 protein was detectable in MCF-7/1B1 cells compared with parental MCF-7 cells. This result was also confirmed by cell-based EROD assay in which the EROD activity of MCF-7/1B1 cells was obviously higher than that of MCF-7 cells (Figure 10). Figure 11. Survival curve of cells treated with docetaxel. The cell viability was calculated by the percent of surviving cells at each drug concentration relative to the control group in MTT assay. IC50 values of docetaxel for MCF-7/1B1 and MCF-7 cells were 139.8 ± 11.5 and 27.6 ± 4.9 μM, respectively. The results represent the average of three parallel experiments.
Figure 10. EROD activity of MCF-7 and MCF-7/1B1 cells. The fluorescence intensity of the negative control was defined as the origin of the vertical axis.
The influence of CYP1B1 on the antiproliferative activity of docetaxel was investigated in MCF-7/1B1 and MCF-7 cell lines. A significant decrease in sensitivity toward docetaxel was observed in the former, since the IC50 value of docetaxel for MCF-7/1B1 cells was about 5-fold higher than that for the parental MCF-7 cells (Figure 11). The use of ANF and 11f at low concentrations (5 and 10 μM) could obviously eliminate the resistance in MCF-7/1B1 cells. The increase in the concentration of CYP1B1 inhibitors led to an increase in sensitivity of MCF-7/1B1 cells to docetaxel, and neither compound exhibited cytotoxicity at any of the tested concentrations. Since CYP1B1 expression has been considered as a reason for the resistance to docetaxel in CYP1B1transfected cells,13 the inhibition of CYP1B1 by ANF and compound 11f could be the explanation for the elimination of resistance in MCF-7/1B1 cells. Additionally, the water-soluble naphthoflavone 11f was found to be more active than ANF in eliminating CYP1B1mediated drug-resistance (Figure 12). This was mainly ascribed to the poor solubility of ANF in cell culture, which precipitated out at tested concentrations (5 and 10 μM) during the incubation. The precipitation could be clearly identified by observation under an optical microscope (400×). With increased water solubility, compound 11f could obviously eliminate the resistance to docetaxel in CYP1B1-expressing
Figure 12. Elimination of anticancer drug-resistance in MCF-7/1B1 cells. IC50 values of docetaxel shown as means ± SD of three independent measurements were labeled on the top of each column. Significant differences from IC50 values: *, P < 0.001.
cancer cells. The coincubation of these cells with 11f (10 μM) resulted in complete reversal of docetaxel-resistance since the IC50 value of the drug was 25.3 ± 3.6 μM, which was comparable to that in the parental MCF-7 cells (IC50 value of 27.6 ± 4.9 μM). When the concentration of the inhibitor was reduced to 5 μM, decreased sensitivity of docetaxel was detected in MCF-7/ 1B1 cells. The reason might be the inefficient uptake of compound 11f in MCF-7/1B1 cells, and at a concentration of 5 μM in cell culture, the concentration of 11f in cancer cells was insufficient to completely suppress the catalytic activity of CYP1B1. Further studies which include a detailed in vitro and in vivo biological evaluation toward other kinds of drug3541
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Journal of Medicinal Chemistry
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The previous ether was dissolved in dry pyridine (5 mL), powered KOH (112 mg, 2.0 mmol) was quickly added, and then the reaction mixture was refluxed under nitrogen atmosphere until all the starting material was consumed. After cooling, the mixture was diluted with cold water (20 mL) and acidified with glacial acetic acid to pH 5.0. The precipitate was collected by simple filtration and underwent column chromatography to afford the diketones (3a−o) as orange solids, which were immediately used in further cyclization. To a solution of the diketone in ethanol (10 mL) was slowly added concentrated sulfuric acid (1.0 mL). After addition, the mixture was heated to reflux for 1 h. The reaction mixture was concentrated to onehalf volumes, and ethyl acetate (EtOAc, 30 mL) was added. The organic layer was separated, washed with brine, and concentrated. The yellow solid was purified by column chromatography, and further crystallization from ethanol gave the title compounds (4a−o) as yellow crystals. 6,7,10-Trimethoxy-α-naphthoflavone (4a). Yield: 19%. 1H NMR (300 MHz, CDCl3): δ 8.21−8.10 (m, 2H, H-C(2′), H-C(6′)), 7.60− 7.50 (m, 4H, H-C(3′), H-C(4′), H-C(5′), H-C(5)), 7.15 (d, J = 9.0 Hz, 1H), 7.09 (d, J = 9.0 Hz, 1H), 7.02 (s, 1H, H-C(3)), 4.10 (s, 3H, OCH3), 4.09 (s, 3H, OCH3), 3.96 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3): δ 177.73 (quat, CO), 162.91, 154.47, 151.68, 151.27, 149.17, 132.32, 131.32, 128.93, 126.35, 122.31, 121.53, 118.43, 113.00, 109.04, 107.02, 98.90, 58.14 (OCH3), 56.65 (OCH3), 56.61 (OCH3). ESI-HRMS: Calcd for C22H19O5 363.1232; found 363.1224 [M + H]+. Purity: >99% (HPLC). 2′-Fluoro-6,7,10-trimethoxy-α-naphthoflavone (4b). Yield: 17%. 1 H NMR (300 MHz, CDCl3): δ 8.44−8.34 (m, 1H, H-C(6′)), 7.56− 7.45 (m, 2H), 7.35 (t, J = 7.6 Hz, 1H), 7.24−7.17 (m, 2H), 7.13 (d, J = 9.0 Hz, 1H), 7.05 (d, J = 9.0 Hz, 1H), 4.07 (s, 3H, OCH3), 4.04 (s, 3H, OCH3), 3.94 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3): δ 177.69 (quat, CO), 161.98, 159.45, 157.71, 154.45, 151.46, 151.24, 149.14, 132.53, 132.44, 129.39, 124.40, 124.37, 122.26, 121.26, 120.56, 120.48, 118.20, 116.88, 116.65, 113.02, 112.26, 112.11, 108.95, 98.76, 58.04 (OCH3), 56.56 (OCH3), 56.39 (OCH3). ESI-HRMS: Calcd for C22H18FO5 381.1138; found 381.1127 [M + H]+. Purity: 99% (HPLC). 3′-Fluoro-6,7,10-trimethoxy-α-naphthoflavone (4c). Yield: 13%. 1 H NMR (300 MHz, CDCl3): δ 8.04−7.95 (m, 1H, H-C(6′)), 7.81 (d, J = 7.8 Hz, 1H), 7.56−7.46 (m, 2H), 7.22 (dd, J = 8.2, 1.9 Hz, 1H), 7.15 (d, J = 8.8 Hz, 1H), 7.08 (d, J = 8.8 Hz, 1H), 6.99 (s, 1H, HC(3)), 4.12 (s, 3H, OCH3), 4.07 (s, 3H, OCH3), 3.95 (s, 3H, OCH3). 13 C NMR (101 MHz, CDCl3): δ 177.37 (quat, CO), 164.32, 161.88, 161.00, 154.54, 151.38, 151.16, 148.82, 134.43, 134.35, 130.42, 130.34, 122.21, 121.52, 121.50, 121.43, 118.03, 117.82, 113.65, 113.41, 112.90, 108.65, 107.15, 98.64, 57.95 (OCH3), 56.50 (OCH3), 56.20 (OCH3). ESI-HRMS: Calcd for C22H18FO5 381.1138; found 381.1117 [M + H]+. Purity: >99% (HPLC). 4′-Fluoro-6,7,10-trimethoxy-α-naphthoflavone (4d). Yield: 16%. 1 H NMR (400 MHz, CDCl3): δ 7.99 (dd, J = 8.6, 5.3 Hz, 2H, H− C(2′), H-C(6′)), 7.39 (s, 1H, H-C(5)), 7.15 (t, J = 8.6 Hz, 2H, HC(3′), H-C(5′)), 7.03 (d, J = 8.8 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H), 6.81 (s, 1H, H-C(3)), 4.01 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 3.90 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3): δ 177.44 (quat, C O), 165.83, 163.32, 161.75, 154.47, 151.42, 151.26, 148.89, 128.41, 128.33, 122.21, 121.32, 118.18, 116.11, 115.89, 112.89, 108.94, 106.54, 98.74, 58.01 (OCH3), 56.53 (OCH3), 56.48 (OCH3). ESI-HRMS: Calcd for C22H18FO5 381.1138; found 381.1121 [M + H]+. Purity: 99% (HPLC). 2′-Chloro-6,7,10-trimethoxy-α-naphthoflavone (4e). Yield: 19%. 1 H NMR (300 MHz, CDCl3): δ 8.06−7.96 (m, 1H, H-C(6′)), 7.60− 7.50 (m, 2H), 7.49−7.42 (m, 2H), 7.13 (d, J = 8.7 Hz, 1H), 7.06 (s, 1H, H-C(3)), 7.03 (d, J = 8.7 Hz, 1H), 4.08 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.92 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3): δ 177.36 (quat, CO), 160.73, 154.57, 151.77, 151.23, 149.64, 132.73, 131.85, 131.51, 131.06, 130.96, 126.93, 122.32, 121.29, 118.43, 113.23, 113.00, 109.37, 98.76, 58.12 (OCH3), 56.63 (OCH3), 56.53 (OCH3). ESI-HRMS: Calcd for C22H18ClO5 397.0843; found 397.0846 [M + H]+. Purity: 98% (HPLC).
resistant cancer cell lines in addition to chemical optimizations are currently in progress. We believe this study will lead to the discovery of new candidates for the therapy of anticancer drugresistance in malignant diseases.
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CONCLUSION The structure modification of ANF gave new naphthoflavones able to inhibit CYP1B1 with increased potency and selectivity. SAR analysis demonstrated that the methoxy group-containing naphthalene part and substituents on the B-ring were key ingredients for the inhibitory activity of the new naphthoflavones. The fluorine-containing naphthoflavone 4c exhibited an excellent selectivity for CYP1B1 against other CYP1 isoforms and was identified as the most potent inhibitor for CYP1B1 ever reported. Further structural modification afforded naphthoflavonol amino acid esters 11a−f, which showed excellent water solubility and moderate inhibitory potency toward CYP1B1. To rationalize the pharmacological results and gain more information on the binding mode of the new naphthoflavones, a docking study was performed using the Xray cocrystal structure of the CYP1B1−ANF complex. The in silico study implied that the reinforced π−π interaction and the presence of the 3′-fluoro atom both contributed to the high binding affinity of compound 4c with this enzyme. The inherent or acquired resistance to docetaxel is recognized as a new challenge in cancer therapy. The enhanced expression of CYP1B1 has already been accepted as one of the mechanisms for the resistance to docetaxel. The use of 11f as a CYP1B1 inhibitor could obviously eliminate the docetaxelresistance in a CYP1B1-expressing cancer cell line. Meanwhile, compared with ANF, compound 11f was more active in overcoming this acquired resistance in cancer cells. This result indicated that further development of these new naphthoflavones as specific CYP1B1 inhibitors might lead to the discovery of promising candidates in the reversal of CYP1B1mediated anticancer drug-resistance.
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EXPERIMETAL SECTION
Chemistry. Reagents and solvents were obtained from commercial suppliers. Solvents were dried and purified using standard techniques.41 Column chromatography was conducted on silica gel (100−200 mesh) from Qingdao Ocean Chemical Factory. 1H NMR and 13C NMR spectra were recorded on a Varian Mercury 300 spectrometer (300 MHz) or a Bruker Avance 400 spectrometer (400 MHz). Chemical shifts of 1H and 13C spectra were recorded with TMS and solvents as the internal standard, respectively. HRMS spectra were measured on a Waters Q-TOF Premier mass spectrometer. The purity of all target compounds was confirmed to be greater than 95% by HPLC analysis conducted on an Agilent 1260 liquid chromatograph system. The chromatography was carried out at room temperature using a Discovery C18 column (150 × 4.6 mm, 5 μm) and a gradient elution. For compounds 4a−o and ANF, the mobile phase consisted of (A) H2O and (B) MeCN (for compounds 8a−g, (A) H2O and (B) MeOH; for compounds 11a−f, (A) H2O containing 20 mM HCOONH4 and (B) MeOH). The detection wavelength was set at 310 nm. General Procedures for 6,7,10-Trimethoxy-α-naphthoflavones (4a−o). Compound 1 (1.0 mmol), the carboxylic acid (1.5 mmol), DCC (2.0 mmol), and DMAP (30 mg) were dissolved in dry DCM (5 mL), and the mixture was stirred at room temperature overnight under nitrogen atmosphere. After the completion of the reaction, the mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to give ethers (2a−o) as light-yellow crystals. 3542
DOI: 10.1021/acs.jmedchem.5b00265 J. Med. Chem. 2015, 58, 3534−3547
Journal of Medicinal Chemistry
Article
3′-Chloro-6,7,10-trimethoxy-α-naphthoflavone (4f). Yield: 14%. H NMR (300 MHz, CDCl3): δ 8.31 (s, 1H), 7.85 (d, J = 7.2 Hz, 1H), 7.54−7.41 (m, 3H), 7.14 (d, J = 8.7 Hz, 1H), 7.07 (d, J = 8.7 Hz, 1H), 6.97 (s, 1H, H-C(3)), 4.14 (s, 3H, OCH3), 4.07 (s, 3H, OCH3), 3.94 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3): δ 177.4 (quat, CO), 160.90, 154.61, 151.54, 151.21, 148.95, 135.18, 133.93, 130.99, 130.09, 126.73, 123.89, 122.33, 121.54, 118.09, 113.12, 108.65, 107.21, 98.76, 77.48, 77.16, 76.84, 58.10 (OCH3), 56.60 (OCH3), 56.27 (OCH3). ESI-HRMS: Calcd for C22H18ClO5 397.0843; found 397.0840 [M + H]+. Purity: 97% (HPLC). 4′-Chloro-6,7,10-trimethoxy-α-naphthoflavone (4g). Yield: 13%. 1 H NMR (300 MHz, CDCl3): δ 8.06 (d, J = 8.4 Hz, 2H, H-C(2′), HC(6′)), 7.55−7.48 (m, 3H, H-C(3′), H-C(5′), H-C(5)), 7.13 (d, J = 9.0 Hz, 1H), 7.08 (d, J = 9.0 Hz, 1H), 6.95 (s, 1H, H-C(3)), 4.07 (s, 6H, OCH3), 3.95 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3): δ 177.49 (quat, CO), 161.63, 154.61, 151.56, 151.38, 148.99, 137.46, 130.77, 129.20, 127.52, 122.35, 121.53, 118.30, 113.06, 109.08, 107.01, 98.84, 58.14 (OCH3), 56.63 (OCH3). ESI-HRMS: Calcd for C22H18ClO5 397.0843; found 397.0827 [M + H]+. Purity: >99% (HPLC). 2′,6,7,10-Tetramethoxy-α-naphthoflavone (4h). Yield: 21%. 1H NMR (300 MHz, CDCl3): δ 8.45 (dd, J = 7.8, 1.5 Hz, 1H, H-C(6′)), 7.55 (s, 1H, H-C(5)), 7.53−7.45 (m, 2H), 7.17 (d, J = 7.5 Hz, 1H), 7.14−7.04 (m, 3H), 4.09 (s, 3H, OCH3), 4.05 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 3.95 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3): δ 178.24 (quat, CO), 160.02, 158.17, 154.17, 151.65, 151.24, 149.35, 132.16, 129.69, 122.28, 121.17, 120.90, 120.58, 118.51, 112.96, 112.52, 111.65, 108.99, 99.05, 58.16 (OCH3), 56.64 (OCH3), 56.53 (OCH3), 55.64 (OCH3). ESI-HRMS: Calcd for C23H21O6 393.1338; found 393.1325 [M + H]+. Purity: >99% (HPLC). 4′,6,7,10-Tetramethoxy-α-naphthoflavone (4i). Yield: 20%. 1H NMR (400 MHz, CDCl3): δ 7.95 (d, J = 8.5 Hz, 2H, H-C(2′), HC(6′)), 7.45 (s, 1H, H-C(5)), 7.02 (d, J = 8.7 Hz, 1H), 6.98−6.91 (m, 3H), 6.81 (s, 1H, H-C(3)), 4.03 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.87 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3): δ 177.56 (quat, CO), 162.93, 162.14, 154.28, 151.59, 151.24, 148.92, 127.95, 124.59, 122.22, 121.35, 118.37, 114.24, 112.82, 108.87, 105.58, 99.03, 58.09 (OCH3), 56.58 (OCH3), 56.53 (OCH3), 55.50 (OCH3). ESI-HRMS: Calcd for C23H21O6 393.1338; found 393.1333 [M + H]+. Purity: 99% (HPLC). 3′,4′,6,7,10-Pentamethoxy-α-naphthoflavone (4j). Yield: 23%. 1H NMR (400 MHz, CDCl3): δ 7.83 (dd, J = 8.4, 2.1 Hz, 1H, H-C(6′)), 7.55 (d, J = 2.1 Hz, 1H, H-C(2′)), 7.54 (s, 1H, H-C(5)), 7.13 (d, J = 9.0 Hz, 1H), 7.09 (d, J = 9.0 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H, HC(5′)), 6.91 (s, 1H, H-C(3)), 4.08 (s, 6H, OCH3), 4.01 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 3.95 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3): δ 177.57 (quat, CO), 163.01, 154.35, 151.92, 151.66, 151.36, 149.18, 148.96, 124.84, 122.16, 121.37, 120.21, 118.54, 112.69, 111.04, 109.56, 109.12, 106.05, 98.89, 58.03 (OCH3), 57.04 (OCH3), 56.59 (OCH3), 56.28 (OCH3), 56.11 (OCH3). ESI-HRMS: Calcd for C24H23O7 423.1444; found 423.1430 [M + H]+. Purity: 99% (HPLC). 3′,4′,5′,6,7,10-Hexamethoxy-α-naphthoflavone (4k). Yield: 23%. 1 H NMR (300 MHz, CDCl3): δ 7.54 (s, 1H, H-C(5)), 7.37 (s, 2H, HC(2′), H-C(6′)), 7.14 (d, J = 8.7 Hz, 1H), 7.12 (d, J = 8.7 Hz, 1H), 6.92 (s, 1H, H-C(3)), 4.08 (s, 3H, OCH3), 4.06 (s, 3H, OCH3), 4.00 (s, 6H, OCH3), 3.96 (s, 6H, OCH3). 13C NMR (101 MHz, CDCl3): δ 177.60 (quat, CO), 163.18, 154.60, 153.72, 151.92, 151.73, 149.09, 141.49, 127.90, 122.36, 121.54, 118.91, 113.01, 110.73, 107.31, 104.69, 98.94, 61.13 (OCH3), 58.16 (OCH3), 57.76 (OCH3), 56.76 (OCH3), 56.67 (OCH3). ESI-HRMS: Calcd for C25H25O8 453.1549; found 453.1527 [M + H]+. Purity: 99% (HPLC). 4′-Benzyloxy-6,7,10-trimethoxy-α-naphthoflavone (4l). Yield: 18%. 1H NMR (400 MHz, CDCl3): δ 8.03 (d, J = 8.8 Hz, 2H, HC(2′), H-C(6′)), 7.50 (s, 1H, H-C(5)), 7.49−7.38 (m, 4H), 7.38− 7.32 (m, 1H), 7.11−7.06 (m, 3H), 7.01 (d, J = 8.8 Hz, 1H), 6.87 (s, 1H, H-C(3)), 5.15 (s, 2H, OCH2), 4.05 (s, 3H, OCH3), 4.04 (s, 3H, OCH3), 3.92 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3): δ 177.69 (quat, CO), 163.03, 161.38, 154.38, 151.73, 151.36, 149.05, 136.43, 128.81, 128.34, 128.10, 127.60, 124.98, 122.33, 121.47, 118.54, 115.21,
112.95, 109.11, 105.83, 99.13, 70.25 (s, 2H, OCH2), 58.22 (s, 3H, OCH3), 56.74 (s, 3H, OCH3), 56.70 (s, 3H, OCH3). ESI-HRMS: Calcd for C23H21O6 469.1651; found 469.1637 [M + H]+. Purity: >99% (HPLC). 3′,4′-Bis(benzyloxy)-6,7,10-trimethoxy-α-naphthoflavone (4m). Yield: 20%. 1H NMR (400 MHz, DMSO-d6): δ 7.78 (dd, J = 8.5, 2.0 Hz, 1H, H-C(6′)), 7.72 (d, J = 2.0 Hz, 1H, H-C(2′)), 7.53 (s, 1H, H-C(5)), 7.51−7.45 (m, 4H), 7.44−7.29 (m, 5H), 7.13 (d, J = 8.8 Hz, 1H), 7.09 (d, J = 8.5 Hz, 1H, H-C(5′)), 7.08 (d, J = 8.8 Hz, 1H), 6.86 (s, 1H, H-C(3)), 5.27 (s, 2H, OCH2), 5.25 (s, 2H, OCH2), 4.07 (s, 3H, OCH3), 4.05 (s, 3H, OCH3), 3.95 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3): δ 177.57 (quat, CO), 162.79, 154.37, 152.20, 151.68, 151.34, 148.96, 148.89, 136.93, 136.65, 128.69, 128.66, 128.13, 127.60, 127.30, 125.34, 122.23, 121.41, 120.98, 118.50, 114.05, 113.99, 112.81, 109.35, 106.06, 98.98, 72.15 (OCH2), 70.95 (OCH2), 58.09 (OCH3), 56.86 (OCH3), 56.62 (OCH3). ESI-HRMS: Calcd for C36H31O7 575.2070; found 575.2068 [M + H]+. Purity: >99% (HPLC). 4′-Hydroxy-6,7,10-trimethoxy-α-naphthoflavone (4n). Yield: 11%. 1H NMR (400 MHz, DMSO-d6): δ 10.25 (s, 1H, OH), 8.04 (d, J = 8.1 Hz, 2H, H-C(2′), H-C(6′)), 7.22 (s, 2H, H-C(5)), 7.13 (s, 2H, H-C(8), H-C(9)), 6.97 (d, J = 8.1 Hz, 2H, H-C(3′), H-C(5′)), 6.92 (s, 1H, H-C(3)), 3.99 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.80 (s, 3H, OCH3). 13C NMR (101 MHz, DMSO-d6): δ 175.82 (quat, CO), 162.55, 160.62, 153.84, 150.61, 150.58, 147.80, 128.14 (2 CArH, C(2′), C(6′)), 122.19, 120.96, 120.56, 117.34, 115.83 (2 CArH, C(3′), C(5′)), 112.61, 109.39, 104.35, 98.03, 57.25 (OCH3), 56.40 (OCH3), 55.98 (OCH3). ESI-HRMS: Calcd for C22H19O6 379.1176; found 379.1162 [M + H]+. Purity: >99% (HPLC). 2′-Hydroxy-6,7,10-trimethoxy-α-naphthoflavone (4o). Yield: 12%. 1H NMR (400 MHz, CDCl3): δ 10.73 (s, 1H, OH), 8.23 (d, J = 7.7 Hz, 1H, H-C(6′)), 7.71−7.67 (m, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.42−7.36 (m, 3H), 6.90−6.78 (m, 2H, H-C(8), H-C(9)), 4.05 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 3.91 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3): δ 178.96 (quat, CO), 161.04, 156.44, 151.79, 151.00, 149.91, 149.39, 133.43, 125.62, 124.90, 123.94, 121.60, 118.07, 117.66, 112.94, 112.88, 109.29, 108.48, 106.68, 58.26 (OCH3), 57.47 (OCH3), 57.12 (OCH3). ESI-HRMS: Calcd for C22H19O6 379.1176; found 379.1180 [M + H]+. Purity: 96% (HPLC). 1-(4,5,8-Trimethoxy-1-(methoxymethoxy)naphthalen-2-yl)ethanone (5). To a solution of compound 1 (2.76 g, 10 mmol) in dry DMF (16 mL) was added NaH (0.48 g, 12 mmol, 60% dispersion in mineral oil) in portions. Then the mixture was stirred vigorously for another 3 min. Freshly distilled methoxy methyl chloride (MOMCl, 0.96 g, 12 mmol) was dropwise added over 15 min under nitrogen atmosphere to the reaction mixture which was cooled in a water−ice bath. After the addition, the temperature of the solution was allowed to rise to room temperature and the mixture was stirred for 1 h. After completion of the reaction, the mixture was poured into a saturated NaHCO3 solution (150 mL) at 0 °C. The light-yellow powder that precipitated out of the solution was collected by filtration and was carefully washed with cold water. After being dried in a vacuum, 3.07 g of 5 as light-yellow powder was obtained and it could be used for the next step without any purification. An analytical sample was obtained by simple crystallization of the crude product from anhydrous ethanol. Yield: 96%. 1H NMR (300 MHz, CDCl3): δ 6.99 (s, 1H, H-C(2)), 6.93 (d, J = 8.7 Hz, 1H), 6.88 (d, J = 8.7 Hz, 1H), 5.01 (s, 2H, OCH2O), 3.96 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.41 (s, 3H, CH2OCH3), 2.78 (s, 3H, COCH3). 13C NMR (101 MHz, CDCl3): δ 202.12 (quat, CO), 153.48, 151.44, 150.92, 146.31, 132.18, 122.35, 122.22 (7 CAr, quat, C(1), C(2), C(4), C(5), C(8), C(4a), C(8a)), 110.51, 108.31, 105.37 (3 CArH, C(3), C(6), C(7)), 101.84 (OCH2O), 58.29 (OCH3), 57.86 (OCH3), 56.77 (OCH3), 56.73 (OCH3), 31.75 (CCH3). General Procedures for 6,7,10-Trimethoxy-α-naphthoflavonols (8a−g). The previous ketone 5 (0.32 g, 1.0 mmol) was suspended in 10% KOH−ethanol solution (w/v, 3 mL) in a water−ice bath under nitrogen atmosphere, and a solution of aldehyde (2.0 mmol) in anhydrous ethanol (2 mL) was dropwise added. The mixture was stirred at room temperature until all the starting material
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3543
DOI: 10.1021/acs.jmedchem.5b00265 J. Med. Chem. 2015, 58, 3534−3547
Journal of Medicinal Chemistry
Article
1H, OH), 7.76 (dd, J = 7.2, 1.3 Hz, 1H), 7.68 (d, J = 7.6 Hz, 1H), 7.62−7.51 (m, 2H), 7.34 (s, 1H, H-C(5)), 7.27 (d, J 8.8 Hz, 1H), 7.21 (d, J = 8.8 Hz, 1H), 3.97 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.76 (s, 3H, OCH3). 13C NMR (101 MHz, DMSO-d6): δ 171.41 (quat, CO), 153.63, 151.18, 150.78, 147.98, 145.10, 139.62, 132.88, 132.03, 131.57, 130.45, 129.79, 126.97, 120.85, 119.30, 117.68, 113.51, 110.89, 97.56, 57.46 (OCH3), 56.78 (OCH3), 56.20 (OCH3). ESIHRMS: Calcd for C22H18ClO6 413.0792; found 413.0782 [M + H]+. Purity: 99% (HPLC). 3′-Chloro-6,7,10-trimethoxy-α-naphthoflavonol (8f). Yellow powder; yield: 13%. 1H NMR (400 MHz, DMSO-d6): δ 10.03 (s, 1H, OH), 8.47 (s, 1H, H-C(2′)), 8.44 (d, J = 8.0 Hz, 1H, H-C(6′)), 7.64 (t, J = 8.0 Hz, 1H, H-C(5′)), 7.57 (d, J = 7.5 Hz, 1H, H-C(4′)), 7.29 (s, 2H, H-C(8), H-C(9)), 7.28 (s, 1H, H-C(5)), 4.11 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.84 (s, 3H, OCH3). 13C NMR (101 MHz, DMSO-d6): δ 171.50 (quat, CO), 153.64, 150.79, 150.70, 147.43, 142.76, 140.18, 133.92, 133.38, 130.52, 129.11, 126.37, 126.01, 120.98, 118.61, 117.13, 113.62, 109.91, 97.49, 57.46 (OCH3), 56.35 (OCH3), 56.14 (OCH3). ESI-HRMS: Calcd for C22H18ClO6 413.0792; found 413.0783 [M + H]+. Purity: >99% (HPLC). 4′-Chloro-6,7,10-trimethoxy-α-naphthoflavonol (8g). Yellow powder; yield: 12%. 1H NMR (400 MHz, DMSO-d6): δ 9.97 (s, 1H, OH), 8.46 (d, J = 8.6 Hz, 2H, H-C(2′), H-C(6′)), 7.73 (d, J = 8.6 Hz, 2H, H-C(3′), H-C(5′)), 7.31 (s, 3H, H-C(5), H-C(8), H-C(9)), 4.09 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.85 (s, 3H, OCH3). 13C NMR (101 MHz, d5-pyridine): δ 173.37 (quat, CO), 155.20, 152.96, 152.80, 149.43, 145.01, 141.94, 135.39, 132.68, 130.42 (2 CArH, C(3′), C(5′)), 129.47 (2 CArH, C(2′), C(6′)), 123.41, 120.38, 119.62, 115.64, 111.01, 100.54, 59.12 (OCH3), 57.42 (OCH3), 57.28 (OCH3). ESI-HRMS: Calcd for C22H18ClO6 413.0792; found 413.0768 [M + H]+. Purity: 99% (HPLC). General Procedures for 3′-Fluoro-6,7,10-trimethoxy-αnaphthoflavonol Amino Acid Esters (11a−f). 3′-Fluoro-3-(2hydroxyethoxy)-6,7,10-trimethoxy-α-naphthoflavone (10a). To a solution of the naphthoflavonol 8c (260 mg, 0.66 mmol) in acetone (10 mL) were added anhydrous potassium carbonate (910 mg, 6.6 mmol), 2-(2-bromoethoxy)tetrahydro-2H-pyran (690 mg, 3.3 mmol), and TBAB (30 mg). After the addition, the reaction mixture was stirred at room temperature for 36 h until all the starting material (8c) was consumed. EtOAc (60 mL) was added, and the organic layer was washed with brine and concentrated. The residue was purified by column chromatography, and the ether was obtained as a yellow powder. Then the powders were dissolved in methanol (10 mL), and concd hydrochloride (0.5 mL) was added. The reaction mixture was stirred under nitrogen atmosphere for 6 h. Compound 10a as a yellow powder was collected by simple filtration and could be used directly in the next step. An analytical sample was obtained by recrystallization of the crude product from methanol. Yield: 78%. 1H NMR (400 MHz, CDCl3): δ 8.27 (d, J = 8.0 Hz, 1H, H-C(2′)), 8.20 (d, J = 10.5 Hz, 1H, H-C(6′)), 7.57−7.51 (m, 1H), 7.49 (s, 1H, H-C(5)), 7.23 (dd, J = 8.0, 2.4 Hz, 1H), 7.18 (d, J = 8.7 Hz, 1H), 7.10 (d, J = 8.7 Hz, 1H), 5.08 (s, 1H, OH), 4.17−4.13 (m, 2H, OCH2), 4.10 (s, 3H, OCH3), 4.07 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.92−3.87 (m, 2H, OCH2). 13C NMR (101 MHz, DMSO-d6): δ 172.84 (quat, CO), 163.28, 160.87, 153.95, 151.89, 151.86, 150.70, 150.44, 147.20, 140.68, 133.24, 133.15, 130.62, 130.54, 124.51, 124.48, 120.98, 120.88, 117.44, 117.23, 116.99, 115.04, 114.79, 113.21, 109.81, 97.49, 73.57 (OCH2), 60.33 (OCH2), 57.26 (OCH3), 56.25 (OCH3), 56.04 (OCH3). 2-(2-(3′-Fluoro-6,7,10-trimethoxy-α-naphthoflavonol)ethoxy)-2oxoethanaminium Chloride (11a). DCC (2.0 mmol) was added to a solution of 10a (440 mg, 1 mmol), N-Boc glycine (350 mg, 2 mmol), and DMAP (30 mg) in DCM (8 mL) under nitrogen atmosphere. After the addition, the reaction mixture was stirred at room temperature overnight. Then the mixture was filtered, and the filtrate was concentrated at reduced pressure. The residue was subjected to column chromatography with petroleum ether−EtOAc (5:1, V/V) as the eluent to afford the ester as a yellow powder. The powder were then dissolved in EtOAc (5 mL), and concd HCl (0.3 mL) was added. The resulting solution was stirred at room temperature overnight and the product precipitated as reddish brown solid. The solid (11a) was
5 was consumed. Then a saturated NH4Cl solution was added to quench the reaction. The standard extractive workup and further column chromatography afforded the chalcones (6a−g) as red-brown solids. The chalcone was suspended in methanol (5 mL), and the mixture was cooled in a water−ice bath. Then a NaOH solution (16%, w/v, 0.6 mL) and hydrogen peroxide (30%, w/v, 0.5 mL) were dropwise added in turn. After the addition, the reaction mixture was stirred at 5 °C overnight. Then the yellow precipitate was filtered, washed with cold methanol, and transferred to a three-neck round-bottom flask. Then methanol (9 mL) and concd HCl (12 M, 0.3 mL) were subsequently added. After the mixture was stirred at room temperature for 5 h under nitrogen atmosphere, the reaction temperature was raised to 50 °C for 1 h. After cooling, the crude product was collected by simple filtration and was crystallized from EtOAc to afford the 6,7,10-trimethoxy-αnaphthoflavanonols (7a−g) as light yellow crystals. To a suspension of the previous flavanonol (1.0 mmol) in methanol (15 mL) cooled in a water−ice bath were added hydrogen peroxide (30%, w/v, 1.5 mL) and a NaOH solution (16%, w/v, 1.2 mL) dropwise. After the addition, the reaction temperature was allowed to rise to room temperature, and the mixture was stirred until all the starting material was consumed. After acidification of the mixture, the product precipitated out of the solution. Pure product was obtained by simple filtration and washed with water and cold methanol. 6,7,10-Trimethoxy-α-naphthoflavonol (8a). Yellow powder; yield: 29%. 1H NMR (400 MHz, DMSO-d6): δ 9.80 (s, 1H, OH), 8.52−8.43 (m, 2H, H-C(2′), H-C(6′)), 7.69−7.63 (m, 2H, H-C(3′), H-C(5′)), 7.59−7.48 (m, 1H, H-C(4′)), 7.33 (s, 3H, H-C(5), H-C(8), H-C(9)), 4.09 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 3.85 (s, 3H, OCH3).13C NMR (101 MHz, DMSO-d6): δ 171.46 (quat, CO), 153.58, 150.85, 150.79, 147.51, 144.69, 139.59, 131.89, 129.60, 128.55, 127.38, 120.98, 118.64, 117.33, 113.61, 109.99, 97.66, 57.53 (OCH3), 56.54 (OCH3), 56.19 (OCH3). ESI-HRMS: Calcd for C22H19O6 379.1182; found 379.1177 [M + H]+. Purity: >99% (HPLC). 2′-Fluoro-6,7,10-trimethoxy-α-naphthoflavonol (8b). Yellow powder; yield: 15%. 1H NMR (400 MHz, DMSO-d6): δ 9.49 (s, 1H, OH), 7.86 (t, J = 6.9 Hz, 1H), 7.67−7.59 (m, 1H), 7.47−7.39 (m, 2H), 7.33 (s, 1H, H-C(5)), 7.27 (d, J = 8.6 Hz, 1H), 7.22 (d, J = 8.6 Hz, 1H), 3.96 (s, 3H, OCH3), 3.84 (s, 6H, OCH3). 13C NMR (101 MHz, DMSO-d6): δ 171.26 (quat, CO), 160.44, 157.92, 153.59, 151.12, 150.71, 148.07, 142.37, 142.36, 139.84, 132.30, 132.21, 130.98, 130.95, 124.31, 124.27, 120.84, 119.53, 119.39, 119.11, 117.50, 116.29, 116.08, 113.50, 110.46, 97.53, 57.43 (OCH3), 56.56 (OCH3), 56.15 (OCH3). ESI-HRMS: Calcd for C22H18FO6 397.1087; found 397.1082 [M + H]+. Purity: >99% (HPLC). 3′-Fluoro-6,7,10-trimethoxy-α-naphthoflavonol (8c). Yellow powder; yield: 13%. 1H NMR (400 MHz, DMSO-d6): δ 10.03 (s, 1H, OH), 8.33 (d, J = 7.9 Hz, 1H, H-C(2′)), 8.23 (d, J = 11.4 Hz, 1H, H-C(6′)), 7.68 (dd, J = 14.7, 7.9 Hz, 1H, H-C(5′)), 7.40−7.34 (m, 1H, H-C(4′)), 7.31 (s, 2H, H-C(8), H-C(9)), 7.30 (s, 1H, H-C(5)), 4.09 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 3.85 (s, 3H, OCH3). 13C NMR (101 MHz, d5-pyridine): δ 173.34 (quat, CO), 164.88, 162.47, 154.84, 152.24, 152.21, 148.89, 144.38, 144.35, 142.60, 130.95, 130.87, 124.86, 124.83, 122.82, 120.24, 118.98, 116.64, 116.43, 115.29, 115.05, 114.31, 110.05, 99.37, 58.33 (OCH3), 56.72 (OCH3), 56.66 (OCH3). ESI-HRMS: Calcd for C22H18FO6 397.1087; found 397.1069 [M + H]+. Purity: 99% (HPLC). 4′-Fluoro-6,7,10-trimethoxy-α-naphthoflavonol (8d). Yellow powder; yield: 15%. 1H NMR (400 MHz, DMSO-d6): δ 9.76 (s, 1H, OH), 8.52−8.42 (m, 2H, H-C(2′), H-C(6′)), 7.47 (t, J = 8.7 Hz, 2H, H-C(3′), H-C(5′)), 7.27 (s, 1H, H-C(5)), 7.26 (s, 2H, H-C(8), H-C(9)), 4.05 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.84 (s, 3H, OCH3). 13C NMR (101 MHz, DMSO-d6): δ 171.43 (quat, CO), 163.74, 161.28, 153.60, 150.78, 147.39, 143.93, 139.29, 129.80, 129.72, 128.46, 128.43, 120.95, 118.65, 117.26, 115.77, 115.55, 113.57, 109.97, 97.63, 57.51 (OCH3), 56.57 (OCH3), 56.18 (OCH3). ESI-HRMS: Calcd for C22H18FO6 397.1087; found 397.1075 [M + H]+. Purity: 98% (HPLC). 2′-Chloro-6,7,10-trimethoxy-α-naphthoflavonol (8e). Yellow powder; yield: 18%. 1H NMR (400 MHz, DMSO-d6): δ 9.43 (s, 3544
DOI: 10.1021/acs.jmedchem.5b00265 J. Med. Chem. 2015, 58, 3534−3547
Journal of Medicinal Chemistry
Article
successfully collected by filtration, recrystallized from EtOAc−MeOH (9:1, V/V), washed with EtOAc, and dried in vacuo. Yield: 73%. 1H NMR (400 MHz, DMSO-d6): δ 8.43 (s, br, 3H, NH3), 8.24 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 11.2 Hz, 1H), 7.76−7.69 (m, 1H), 7.50−7.44 (m, 1H), 7.29 (s, 2H, H-C(8), H-C(9)), 7.28 (s, 1H, H-C(5)), 4.49 (m, 4H, OCH2), 4.04 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.76 (s, 2H, CH2N). 13C NMR (101 MHz, DMSO-d6): δ 172.41 (quat, C(4)O), 167.61 (quat, CO, Gly), 163.16, 160.75, 153.93, 151.88, 150.61, 150.32, 147.13, 139.93, 132.99, 132.90, 130.67, 130.59, 124.43, 124.41, 120.90, 120.81, 117.54, 117.34, 116.83, 114.87, 114.62, 113.13, 109.67, 97.30, 69.09 (OCH2), 64.74 (OCH2), 57.18 (OCH3), 56.12 (OCH3), 55.95 (OCH3), 39.43 (NCH2). ESI-HRMS: Calcd for C26H25FNO8 498.1564; found 498.1536 [M − Cl]+. Purity: 99% (HPLC). (S)-1-(3′-Fluoro-6,7,10-trimethoxy-α-naphthoflavonol)ethoxy)-1oxopropan-2-aminium Chloride (11b). Reddish brown solid; yield: 28% (calcd from compd 10a). 1H NMR (400 MHz, DMSO-d6): δ 8.79 (s, br, 3H, NH3), 8.12 (d, J = 7.7 Hz, 1H), 8.00 (d, J = 11.1 Hz, 1H), 7.68−7.57 (m, 1H), 7.45−7.36 (m, 1H), 7.07−6.96 (m, 3H, HC(5), H-C(8), H-C(9)), 4.58−4.39 (m, 4H, OCH2), 4.08−3.98 (m, 1H, CHN), 3.86 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 1.45 (d, J = 7.1 Hz, 3H, CH3). 13C NMR (101 MHz, DMSOd6): δ 172.21 (quat, CO), 170.05 (quat, CO, Ala), 163.14, 160.73, 153.73, 151.52, 150.43, 150.07, 146.89, 139.86, 133.02, 132.94, 130.52, 130.44, 124.33, 124.31, 120.73, 120.59, 117.38, 117.17, 116.57, 114.77, 114.53, 112.65, 109.17, 97.04, 68.97 (OCH2), 65.05 (OCH2), 56.93 (OCH3), 55.81 (OCH3), 55.72 (OCH3), 47.86 (CH), 15.51 (CH3). ESI-HRMS: Calcd for C27H27FNO8 512.1721; found 512.1695 [M − Cl]+. Purity: >99% (HPLC). (S)-1-(2-(3′-Fluoro-6,7,10-trimethoxy-α-naphthoflavonol)ethoxy)-3-methyl-1-oxobutan-2-aminium Chloride (11c). Reddish brown solid; yield: 29%. 1H NMR (400 MHz, DMSO-d6): δ 8.70 (d, J = 4.2 Hz, 3H, NH3), 8.17 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 10.9 Hz, 1H), 7.67 (dd, J = 14.4, 8.0 Hz, 1H), 7.48−7.41 (m, 1H), 7.14 (d, J = 8.8 Hz, 1H), 7.11 (d, J = 8.8 Hz, 1H), 7.10 (s, 1H, H-C(5)), 4.63− 4.36 (m, 4H, OCH2), 3.93 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.83− 3.72 (m, 4H, OCH3, CHN), 2.24−2.11 (m, 1H, CH), 1.03−0.90 (m, 6H, CH3). 13C NMR (101 MHz, DMSO-d6): δ 172.37 (quat, C(4) O), 169.01 (quat, CO, Val), 163.22, 160.81, 153.92, 151.73, 151.70, 150.59, 150.27, 147.09, 140.13, 133.09, 133.00, 130.72, 130.63, 124.47, 124.45, 120.91, 120.74, 117.57, 117.36, 116.77, 114.89, 114.65, 112.96, 109.57, 97.22, 69.24 (OCH2), 65.19 (OCH2), 57.30 (NCH), 57.11 (OCH3), 56.07 (OCH3), 55.92 (OCH3), 29.30 (CH), 18.16 (CH3), 17.69 (CH3). ESI-HRMS: Calcd for C29H31FNO8 540.2034; found 540.2040 [M − Cl]+. Purity: 96% (HPLC). Compounds 11d−f were prepared according to the same synthetic procedure, in which 2-(4-bromobutoxy)tetrahydro-2H-pyran, 2-(6bromohexyloxy)tetrahydro-2H-pyran and 2-(8-bromooctyloxy)tetrahydro-2H-pyran were used as the starting materials separately instead of 2-(2-bromoethoxy)tetrahydro-2H-pyran. 2-(4-(3′-Fluoro-6,7,10-trimethoxy-α-naphthoflavonol)butoxy)-2oxoethanaminium Chloride (11d). Dark brown solid; yield: 52% (calcd from compd 8c). 1H NMR (400 MHz, DMSO-d6): δ 8.36 (s, br, 3H, NH3), 8.20 (d, J = 7.8 Hz, 1H), 8.12 (d, J = 11.0 Hz, 1H), 7.72 (dd, J = 14.8, 7.8 Hz, 1H), 7.50−7.43 (m, 1H), 7.30 (s, 3H, H-C(5), H-C(8), H-C(9)), 4.27−4.22 (m, 2H, OCH2), 4.20−4.15 (m, 2H, OCH2), 4.04 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.84−3.79 (m, 2H, CH2N), 1.82 (s, 4H, CH2). 13C NMR (101 MHz, DMSO-d6): δ 172.52 (quat, C(4)O), 168.91 (quat, C O, Gly), 167.69, 163.20, 160.79, 153.85, 151.88, 151.87, 150.60, 150.32, 147.10, 140.48, 133.21, 133.12, 130.71, 130.63, 124.21, 120.95, 120.76, 117.43, 117.22, 116.87, 114.66, 114.41, 112.96, 109.57, 97.35, 70.89 (OCH2), 65.11 (OCH2), 57.13 (OCH3), 56.08 (OCH3), 55.91 (OCH3), 39.62 (NCH), 25.99 (CH2), 24.76 (CH2). ESI-HRMS: Calcd for C28H29FNO8 526.1877; found 526.1865 [M − Cl]+. Purity: 98% (HPLC). 2-(4-(3′-Fluoro-6,7,10-trimethoxy-α-naphthoflavonol)hexyloxy)2-oxoethanaminium Chloride (11e). Reddish brown solid; yield: 57%. 1H NMR (400 MHz, DMSO-d6): δ 8.39 (s, br, 3H, NH3), 8.19 (d, J = 7.8 Hz, 1H), 8.11 (d, J = 11.2 Hz, 1H), 7.75−7.66 (m, 1H),
7.45 (t, J = 8.3 Hz, 1H), 7.28 (s, 3H, H-C(5), H-C(8), H-C(9)), 4.19−4.12 (m, 4H, OCH2), 4.03 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.82−3.77 (m, 2H, CH2N), 1.77−1.67 (m, 2H, CH2), 1.65−1.57 (m, 2H, CH2), 1.48−1.31 (m, 4H, CH2). 13C NMR (101 MHz, DMSO-d6): δ 172.63 (quat, C(4)O), 168.97 (quat, C O, Gly), 167.73, 163.20, 160.79, 153.93, 152.00, 151.98, 150.68, 150.44, 147.20, 140.59, 133.29, 133.20, 130.70, 130.62, 124.29, 124.26, 121.03, 120.84, 117.46, 117.25, 116.99, 114.81, 114.56, 113.13, 109.75, 97.47, 71.50 (OCH2), 65.37 (OCH2), 57.23 (OCH3), 56.21 (OCH3), 56.01 (OCH3), 39.60 (CH2N), 29.47 (CH2), 28.01 (CH2), 25.18 (CH2), 24.97 (CH2). ESI-HRMS: Calcd for C30H33FNO8 554.2190; found 554.2175 [M − Cl]+. Purity: >99% (HPLC). 2-(4-(3′-Fluoro-6,7,10-trimethoxy-α-naphthoflavonol)octyloxy)2-oxoethanaminium Chloride (11f). Reddish brown solid; yield: 49%. 1H NMR (400 MHz, DMSO-d6): δ 8.29 (s, br, 3H, NH3), 8.21 (d, J = 7.9 Hz, 1H), 8.13 (d, J = 11.0 Hz, 1H), 7.71 (dd, J = 14.6, 7.9 Hz, 1H), 7.51−7.41 (m, 1H), 7.31 (s, 3H, H-C(5), H-C(8), H-C(9)), 4.15 (m, 4H, OCH2), 4.05 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.81 (s, 2H, CH2N), 1.75−1.65 (m, 2H, CH2), 1.65− 1.55 (m, 2H, CH2), 1.43−1.33 (m, 2H, CH2), 1.33−1.22 (m, 6H, CH2). 13C NMR (101 MHz, DMSO-d6): δ 172.59 (quat, C(4)O), 168.94 (quat, CO, Gly), 167.71, 163.17, 160.76, 153.88, 151.92, 151.89, 150.64, 150.41, 147.15, 140.58, 133.28, 133.19, 130.62, 130.54, 124.25, 124.23, 121.00, 120.81, 117.39, 117.19, 116.96, 114.80, 114.55, 113.07, 109.68, 97.44, 71.54 (OCH2), 65.41 (OCH2), 57.20 (OCH3), 56.17 (OCH3), 55.96 (OCH3), 39.63 (CH2N), 29.54 (CH2), 28.64 (CH2), 28.03 (CH2), 25.46 (CH2), 25.16 (CH2). ESI-HRMS: Calcd for C32H37FNO8 582.2503; found 582.2488 [M − Cl]+. Purity: >99% (HPLC). Enzymatic Assay. The recombinant human CYP1B1, 1A1, and 1A2 enzymes, each equipped with P450 reductase (Supersomes), were purchased from BD Biosciences. 7-Ethoxyresorufin was obtained from Sigma-Aldrich. NADP+, glucose 6-phosphate (G-6-P), glucose-6phosphate dehydrogenase (G-6-PD), and fatty acid-free BSA were purchased from Shanghai Seebio Biotech. Other solvents and reagents used in the biological evaluation were of the highest quality and commercial availability. A Thermo Scientific Varioskan Flash apparatus was used for recording the fluorescence intensity in the enzyme assay, with excitation and emission filters at 545 and 590 nm, respectively. The in vitro activity of the prepared compounds against the CYP1B1, 1A1 and 1A2 was determined using the standard EROD assay,30 in which the concentration of 7-ethoxyresorufin was fixed at 150 nM in the incubation system. Meanwhile, a NADPH regeneration system containing 1.3 mM NADP+, 3.3 mM G-6-P, and 0.5 U/mL G6-PD was used instead of NADPH (1.67 mM). The time of incubation for the system containing CYP1B1, 1A1, and 1A2 was 35, 15, and 50 min, respectively. The IC50 value for each compound was obtained by Probit analysis using IBM SPSS statistics V21 Software. Molecular Docking. Compound 4c as the most potent inhibitor in enzymatic assay was docked into the active site cavity of CYP1B1 (PDB ID 3PM0) using the docking program in MOE 2008. All water molecules in the crystal structure were removed before the experiment. Hydrogens and partial charges were added with the protonate 3D application. Those residues closed to ANF with a radius of 8 Å were defined as the docking site. The initial 3D conformation of compound 4c was optimized in ChemBio3D Ultra using the MM2 energy minimization method. For docking, we employed the default values of parameters, except for the first scoring function, where ASE Scoring was used instead of the defaulting London dG. The best pose was characterized by the scoring results. To test whether the docking program is feasible for the ligand binding to human CYP1B1, the CYP1B1−ANF complex (PDB ID 3PM0) was initially chosen and the docking structure of ANF was comparable to its crystallographic structure. This result implied that the MOE docking program used is suitable for the identification of binding between α-naphthoflavones and CYP1B1 Cell-Based EROD Assay and Western-Blot. RPMI-1640 culture medium, fetal bovine serum, and PBS (Hyclone) were purchased from Thermo Fisher Scientific. TCDD was obtained from AccuStandard. MCF-7 cells were maintained in RPMI-1640 medium supplemented 3545
DOI: 10.1021/acs.jmedchem.5b00265 J. Med. Chem. 2015, 58, 3534−3547
Journal of Medicinal Chemistry
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with 10% fetal bovine serum at 37 °C in a humidified atmosphere with 5% CO2. Cells were subcultured every 3 days with a subcultivation ratio of 1:5. MCF-7/1B1 cells, which were obtained by coincubation of parental MCF-7 cells with TCDD (10 nM) for 96 h, and referred to as MCF-7/1B1 cells. Medium renewal was conducted every day with freshly prepared medium containing 10 nM TCDD. EROD activity in cancer cells was evaluated according to the reported procedure42 with minor changes. Cells were seeded at 5000 cells per well in an opaque 96-well plate and incubated with 400 nM 7ethoxyresorufin in PBS (100 μL) at 37 °C/5% CO2 for 1 h. The incubation was terminated by adding ice-cold acetonitrile, and the fluorescence was determined at 545 nm excitation and 590 nm emission. The wells in which no cancer cells were planted were set up as the negative control. The fluorescence intensity (recorded value minus negative control) was relevant to the EROD activity in cells. Immunoblots were conducted according to the standard protocol,43 employing the following antibodies: rabbit polyclonal antibody to CYP1B1 (Abcam) and mouse monoclonal antibody to β-actin (Abcam). Antirabbit and antimouse secondary antibodies were coupled to horseradish peroxides (Santa Cruz Biotechnology). Proteins were visualized using an enzyme-linked chemiluminescence detection kit. Cytotoxicity Assay. The in vitro cytotoxicity of docetaxel and the combination of this drug with CYP1B1 inhibitors was evaluated by the standard MTT assay,44 in which the incubation period was extended to 48 h after the addition of freshly prepared concentrations of docetaxel with or without inhibitors. Each 96-well plate included five concentrations of the appropriate drugs with four replicates at each concentration, and there were three replicate plates for each experiment. In the evaluation of the combination of docetaxel with inhibitors (ANF or 11f), wells that only contained the inhibitor (10 μM) were used as the negative control. At this concentration, no inhibition of cell growth was observed. IC50 values of docetaxel were calculated by Probit analysis with SPSS V21. In statistical analysis, Student’s t tests were performed to compare IC50 values of docetaxel using SPSS V21 software. P values
