Young-Jin
Chun
a,
Chaemin
Lim
b,
Seul Ong
Ohk
a,
Ji Min
Lee
b,
Jin Hee
Lee
c,
Sun
Choi
c and
Sanghee
Kim
*b
aCollege of Pharmacy, Chungang University, 221 Huksuk, Seoul, 156-756, Korea
bCollege of Pharmacy, Seoul National University, San 56-1, Shilim, Kwanak, Seoul, 151-742, Korea. E-mail: pennkim@snu.ac.kr; Fax: +82 2 888 0649; Tel: +82 2 880 2487
cCollege of Pharmacy, Division of Life & Pharmaceutical Sciences, and National Core Research Center for Cell Signaling & Drug Discovery Research, Ewha Womans University, 11-1 Daehyun, Seodaemun, Seoul, 120-750, Korea
First published on 8th March 2011
On the basis of our previous insights into the structural requirements of stilbenoids for the inhibition of cytochrome P450 1B1 (CYP1B1), a series of 2,4-dimethoxy group-containing stilbenes was prepared and evaluated for their inhibitory effects on the activity of CYP1s with the ultimate goal of identifying a potent and selective CYP1B1 inhibitor. Among the thirteen derivatives prepared, five compounds exhibited similar or greater potency compared to the previous lead compound, COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compound2,4,3′,5′-tetramethoxystilbene (2,4,3′,5′-TMS), in inhibiting CYP1B1. In particular, 2,2′,3′,4,6′-pentamethoxystilbene was found to be a more selective and more potent CYP1B1 inhibitor than 2,4,3′,5′-TMS. 2,4,2′,6′-TMS showed remarkably potent inhibitory activity against CYP1B1 (IC50 = 1.77 ± 0.14 nM) and also had a very high selectivity toward CYP1 isoenzymes. Molecular modeling was performed to determine the key molecular interactions with the CYP1B1 and CYP1A2 structures. On the basis of these structural and biological studies, the design of more potent and more selective drug-like derivatives can be envisaged.
Several studies have shown that CYP1B1 is expressed at a higher level in malignant tumor cells in the kidney, breast, brain, and lungs than in normal cells.13–16 Overexpression of CYP1B1 has also been detected in colorectal adenomas and premalignant prostate tumors. Moreover, a recent study revealed that CYP1B1 up-regulation plays an important role in endometrial carcinogenesis by targeting multiple pathways.17 Because CYP1B1 is up-regulated in premalignant and malignant tumors, CYP1B1 might be involved in the early development of cancer. In addition, CYP1B1 could be involved in anticancer drug resistance because the metabolism of various anticancer agents by CYP1B1 causes the suppression of the pharmacological effects of drugs.18 Because CYP1B1 is thought to be more detrimental than beneficial,19 the availability of potent and selective inhibitors of CYP1B1 would facilitate the development of CYP1B1-targeted cancer chemotherapy and prevention drugs.
Owing to the important role of CYP1B1 in chemical carcinogenesis, various studies have attempted to identify potent and selective inhibitors from natural sources in recent years.1,20–23 For instance, the inhibitory effects of natural stilbenoids on CYP1 activity have been studied.3,24 It was reported that resveratrol 1 (Fig. 1) inhibited CYP1A1 and CYP1B125–27 and that rhapontigenin 2 exhibited a strong and selective inhibition of CYP1A1 over CYP1B1.3Pinostilbene 3, desoxyrhapontigenin 4, and pterostilbene 5 also appear to be inhibitors of CYP1B1, with Ki values of 0.9, 2.1, and 0.9 μM, respectively, although these compounds showed more potent inhibition of CYP1A1 (Ki = 0.12, 0.16, and 0.57 μM, respectively).28
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Fig. 1 Structures of natural stilbenoids that inhibit CYP1s. |
On the basis of studies of natural stilbenoids showing that the selectivities and inhibitory potencies of the compounds were sensitive to the substitution patterns on the trans-stilbene template, we have previously prepared and evaluated a series of lipophilic stilbenoid compounds of general structure 6 (Fig. 2) in which the phenyl ring at site A contains dimethoxy groups at the 3′ and 5′ positions.29 This study allowed us to identify a highly potent and selective competitive inhibitor of CYP1B1 with an IC50 value of 6 nM: 2,4,3′,5′-tetramethoxystilbene 7 (2,4,3′,5′-TMS).30 2,4,3′,5′-TMS also showed suppression of CYP1B1 induction by TCDD in MCF-7 cells and HL-60 cells.31 In addition, our previous studies revealed interesting structure–activity relationships (SAR) in terms of the importance of substituents at the ortho position of the phenyl ring at site B for discriminating between CYP1As and CYP1B1.
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Fig. 2 General structures 6 and 8 and the structure of COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound2,4,3′,5′-tetramethoxystilbene (7). |
Our prior SAR study led us to analyze stilbenoid compounds with general structure 8 (Fig. 2), in which the phenyl ring at site B contains dimethoxy groups at the 2 and 4 positions, as a new lead template for further chemical optimization and structure–activity studies. As part of our ongoing program to develop potent and selective CYP1B1 inhibitors, we now describe the biological evaluation of a series of 2,4-dimethoxy group containing stilbenes.
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Scheme 1 Synthesis of trans-stilbene derivatives 8a–m: Reagents and conditions: (a) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound18-crown-6, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundKOH, CH2Cl2, rt; (b) 12, AcOH, CH2Cl2, rt; (c) cat. I2, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundheptane, reflux; (d) NaHSO3, aqueous work-up. |
Compdb | R | IC50/nMc | Ratio (1A1/1B1) | Ratio (1A2/1B1) | ||
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1A1 | 1A2 | 1B1 | ||||
a
Enzyme activities were measured as described in the ESI.†
b Compounds 7, 8a, 8d, 8e, 8g, 8i, 8j, and 8k were previously known.
c The IC50 values are the means ± range of two separate experiments determined using a quadratic expression of nonlinear regression methods using Graph-Pad Prism software (San Diego, CA). Control activities in the absence of chemical were 20, 5, and 2 nmol of COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundresorufin formed min−1 (nmol of P450)−1 for CYP1A1, CYP1A2, and CYP1B1, respectively. d Taken from ref. 29. |
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7 d | See Fig. 2 | 300 ± 20 | 3100 ± 880 | 6 ± 2 | 50 | 517 |
8a | H | 493 ± 104 | 326 ± 64 | 25.0 ± 1.0 | 20 | 13 |
8b | 2-Methoxy | 336 ± 61 | 792 ± 136 | 15.5 ± 1.6 | 22 | 51 |
8c | 3-Methoxy | 98.2 ± 24.4 | 903 ± 71 | 15.2 ± 1.5 | 6.5 | 59 |
8d | 4-Methoxy | 666 ± 242 | 1060 ± 239 | 57.5 ± 25.1 | 12 | 18 |
8e | 3,4-Dimethoxy | 190 ± 76 | 337 ± 30 | 41.8 ± 7.9 | 4.6 | 8.1 |
8f | 2,5-Dimethoxy | 350 ± 83 | 302 ± 28 | 3.51 ± 0.40 | 100 | 86 |
8g | 2,4-Dimethoxy | 222 ± 22 | 204 ± 23 | 8.81 ± 1.52 | 25 | 23 |
8h | 2,3-Dimethoxy | 192 ± 21 | 247 ± 36 | 60.3 ± 11.0 | 3.2 | 4.1 |
8i | 2,6-Dimethoxy | 350 ± 49 | 170 ± 32 | 1.77 ± 0.14 | 194 | 95 |
8j | 3,4,5-Trimethoxy | 301 ± 83 | 4690 ± 220 | 71.0 ± 5.0 | 4.2 | 66 |
8k | 2,4,5-Trimethoxy | 78.4 ± 8.8 | 236 ± 18 | 5.46 ± 2.03 | 14 | 43 |
8l | 2,4,6-Trimethoxy | 148 ± 14 | 188 ± 35 | 4.93 ± 1.50 | 30 | 38 |
8m | 2,3,6-Trimethoxy | 420 ± 104 | 1740 ± 220 | 3.30 ± 0.70 | 127 | 526 |
Several compounds exhibited similar or improved potencies compared to the previous lead compound 7 in inhibiting CYP1B1. Compound 8m, 2,2′,3′,4,6′-pentamethoxystilbene, was found to be a more selective and more potent CYP1B1 inhibitor than 7. It showed a potent inhibitory effect on CYP1B1 (IC50 = 3.30 ± 0.70 nM) and, to a much lesser extent, on CYP1A1 (IC50 = 420 ± 104 nM) and CYP1A2 (IC50 = 1740 ± 220 nM). The differences between CYP1A1, CYP1A2, and CYP1B1 inhibition are statistically significant as determined by ANOVA (p < 0.05). Compound 8i, 2,4,2′,6′-TMS, showed a remarkably potent inhibitory activity against CYP1B1 (IC50 = 1.77 ± 0.14 nM) and had a very high selectivity toward the CYP1 isozymes. It exhibited the greatest selectivity (194-fold) of 1B1 over 1A1, but the selectivity between 1B1 and 1A2 was diminished compared to that of 8m (95-fold vs. 526-fold).
The mono-methoxylated phenyl ring-containing compounds, 8b–8d, as well as the non-substituted phenyl ring-containing compound 8a, also preferentially inhibited 1B1 over 1A1 and 1A2. However, the inhibitory potentials and selectivities were diminished substantially compared to those of 8m and 8i. Among the mono-methoxylated compounds, 3-methoxyphenyl ring-containing compound 8c had the lowest selectivity for CYP1B1 over CYP1A1, and 4-methoxylated compound 8d had the lowest selectivity for CYP1B1 over CYP1A2. These results suggest that the substituent at the 3 or 4 position might have a negative effect on the selectivity for CYP1B1 inhibition. This detrimental effect of the methoxy group at the 3 or 4 position was further evidenced by the 3,4-dimethoxylated compound 8e, which exhibited a very low selectivity towards the CYP1 isoenzymes.
Among the dimethoxylated compounds, compound 8h, having dimethoxy groups at the 2 and 3 position, was the least active against CYP1B1 and exhibited the lowest selectivity. However, compound 8i, which differed structurally from 8h by only the position of one methoxy group (C-3vs.C-6) showed remarkably greater inhibitory activity and selectivity. These observations demonstrated that the exact locations of methoxy groups are a very important feature for selectivity.
The tri-methoxylated compounds generally showed potent inhibition of CYP1B1; 8j was the exception. The selectivities varied and were largely dependent on the position of the methoxy groups. Of the tri-methoxylated compounds examined, compound 8m exhibited the greatest selectivity and potency.
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Fig. 3 Predicted binding modes of 8m in (a) CYP1B1 and (b) CYP1A2. The key interacting residues are marked and displayed in capped sticks with carbon atoms in white. The ligand 8m is depicted as a ball-and-stick structure with carbon atoms in purple. Hydrogen bonds are shown by yellow dashed lines. The Van der Waals surfaces of the ligand were generated by MOLCAD and are colored based on hydrogen bonding properties (blue: H-bond-accepting regions). The Fast Connolly surfaces of the proteins were generated by MOLCAD and are colored based on lipophilic potential properties, ranging from brown (highest lipophilic area) to blue (highest hydrophilic area). Protein surfaces are Z-clipped, and non-polar hydrogens are undisplayed for clarity. |
In contrast, the docking result of 8m in CYP1A2 revealed a flipped binding mode (Fig. 3b). In CYP1A2, the binding pocket near the heme is narrower than that of CYP1B1. This narrowness might be why the bulky trimethoxy phenyl ring of 8m was docked in the opposite direction, thus avoiding the steric repulsion that would otherwise be encountered at the binding pocket. Although compound 8m could occupy the active site of CYP1A2, there is no hydrogen bonding between 8m and the residues of CYP1A2. This lack of a vital hydrogen bonding might be the reason for the weak inhibitory activity of 8m against CYP1A2. Our docking model successfully illustrated the selectivity of 8m for CYP1 inhibition and offered important information for developing new stilbenoid compounds targeting CYP1B1.
Footnote |
† Electronic supplementary information (ESI) available: Detailed experimental methods and full characterization for all new compounds. 1H and 13C NMR spectra for selected compounds. See DOI: 10.1039/c0md00242a/ |
This journal is © The Royal Society of Chemistry 2011 |