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. Author manuscript; available in PMC: 2015 Jan 7.
Published in final edited form as: J Undergrad Chem Res. 2013 Sep;12(4):92–95.

DESIGN, SYNTHESIS, AND EVALUATION OF CARBAZOLE ANALOGS AS POTENTIAL CYTOCHROME P450 INHIBITORS

Maryam Foroozesh 1, Quan Jiang 1, Jayalakshmi Sridhar 1, Jiawang Liu 1, Brandan Dotson 1,*, Erika McClain 1,*
PMCID: PMC4286351  NIHMSID: NIHMS631377  PMID: 25580095

Abstract

Carbazoles are a class of nitrogen-containing aromatic heterocyclic compounds. They not only have various biological activities (e.g. antibacterial, anti-inflammatory, antitumor), but also exhibit useful properties as organic materials due to their special structures. Cytochrome P450 enzymes are a superfamily of hemoproteins involved in the metabolism of endogenous and exogenous compounds including many drugs and environmental chemicals. Some aryl and arylalkyl acetylenes, and propargyl ethers have been shown to act as inhibitors of certain P450s. In an attempt to improve the potency and selectivity of inhibition, we have focused our attention on the design and synthesis of a new series of carbazole analogs, a few of which contain a propargyl ether functional group. For this project, eight carbazole analogs have been synthesized and characterized.

Keywords: Carbazoles, Cytochrome P450 enzymes, Acetylenes, Organic synthesis, Enzyme inhibitors, Suicide inhibition, Metabolism

Introduction

Carbazoles are heterocyclic aromatic compounds which have attracted considerable attention in biological and material sciences due to their ubiquitous structural motif with wide-ranging physiological and photophysical properties (1,2). Cytochrome P450 enzymes are the main superfamily of enzymes involved in the metabolism of endogenous and xenobiotic compounds including polycyclic aromatic hydrocarbons and arylamines to more polar molecules during detoxification. However, such metabolism may lead to cytotoxic, mutagenic, or carcinogenic metabolites (3-9). Certain aryl and arylalkyl acetylenes and propargyl ethers have been previously shown to act as inhibitors of these enzymes (10-12). When these compounds fit into the active sites of the P450 enzymes with the correct orientation, the triple bond can be oxidized and bind irreversibly and covalently to the protein (suicide inhibition), thus deactivating the enzymes. The degree and type of inhibition, as well as the selectivity towards different P450 enzymes, greatly depend on the size and shape of the ring system and the placement of the triple bond in the molecule. Based on these observations, a series of 8 carbazole analogs, 4-methoxy-9H-carbazole (I), 4-ethoxy-9H-carbazole (II), 4-propoxy-9H-carbazole (III), 4-(prop-2-yn-1-yloxy)-9H-carbazole (IV), 2-methoxy-9H-carbazole (V), 2-ethoxy-9H-carbazole (VI), 2-propoxy-9H-carbazole (VII), and 2-(prop-2-yn-1-yloxy)-9H-carbazole (VIII) were designed and synthesized (Scheme I). The target compounds were characterized through nuclear magnetic resonance spectroscopy, and gas chromatography/mass spectrometry, and are presently being studied in vitro as potential mechanism-based irreversible (suicide) inhibitors of a number of P450 enzymes. Among these compounds, carbazole propargyl ethers (IV, VIII) are predicted to be good inhibitors of cytochrome P450s in the 1A subfamily. This prediction is based on the fact that the addition of a carbon-carbon triple bond has been previously shown to lead to P450 inhibition in many compounds (as described above) (8,9). These compounds also contain an oxygen atom on the substituent leading to a change in the polarity of the compounds and the orientation of the triple bond.

Scheme 1.

Scheme 1

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Syntheses of the carbazole analogs.

The goal in this project is to find selective suicide inhibitors for certain P450 enzymes involved in the activation of procarcinogens. Such inhibitors could be used to inhibit the metabolism of specific procarcinogens into ultimate carcinogenic forms. They could also be used as probes into the active sites of the target enzymes, as models for anticancer drugs, or in protein labeling studies.

Experimental

Reagents

2-Hydroxycarbazole was purchased from Aldrich Chemical Co. (WI, USA). 4-Hydroxycarbazole was purchased from Acros Organics (NY, USA). Other reagents and solvents (ethyl acetate, hexanes) were purchased from Fisher Scientific (Pittsburg, PA).

Syntheses of I -VIII

The starting material 4-hydroxycarbazole (0.50 g, 2.7 mmol), or 2-hydroxycarbazole (0.50 g, 2.7 mmol)) was dissolved in 10 mL of methyl sulfoxide under nitrogen atmosphere, before the addition of potassium carbonate (0.48 mg, 3.5 mmol, 1.3 eq.). The reaction mixture was stirred at room temperature for 30 minutes before the dropwise addition of the corresponding halide (1.2 eq., methyl iodide, bromoethane, 1-bromopropane, or propargyl bromide respectively). The reaction mixture was stirred at room temperature overnight. The reaction mixture was then poured over 10 mL of deionized ice water, and extracted twice, each time with 30 mL of ethyl acetate. The organic layers were combined and washed with 20 mL of brine, and dried over anhydrous magnesium sulfate. The different analogs were purified by column chromatography using 5-20% ethyl acetate in hexanes as the eluent.

Results and Discussion

4-Methoxy-9H-carbazole [I] (13) (45%, yield). GC-MS showed >99% purity; m/z: 197, 182, 154, 127. 1H-NMR (CDCl3, 300 MHz): δ = 4.02(s, 3H), 6.63(d, J = 8.4 Hz, 1H), 6.64(d, J = 8.4 Hz, 1H), 7.175-7.365(m, 4H), 7.90(br. s, 1H), 8.28(d, J=8.7 Hz, 1H). 13C-NMR (CDCl3, 75 MHz): δ =55.5, 100.4, 103.6, 110.0, 112.6, 119.6, 122.7, 123.0, 125.0, 126.7, 138.7, 140.9, 156.3.

4-Ethoxy-9H-carbazole [II] (14) (43%, yield). GC-MS showed >99% purity; m/z: 211, 183, 154, 127. 1H-NMR (CDCl3, 300 MHz): δ =1.51(t, J = 6.9 Hz, 3H), 4.18(q, 2H), 6.57(d, J = 8.4 Hz, 1H), 6.80(d, J = 8.4 Hz, 1H), 7.14-7.34(m, 4H), 7.63(br. s, 1H), 8.33(d, J=7.5 Hz, 1H). 13C-NMR (CDCl3, 75 MHz): δ =15.0, 63.5, 101.0, 103.4, 110.0, 112.5, 119.5, 122.6, 123.0, 124.8, 126.6, 138.6, 140.8, 155.5.

4-Propoxy-9H-carbazole [III] (37%, yield). GC-MS showed >99% purity; m/z: 225, 183, 154, 127. 1H-NMR (CDCl3 MHz): δ =1.17(t, J = 7.2 Hz, 3H), 1.99(q, 2H), 4.16(t, 2H), 6.64(d, J = 8.4 Hz, 1H), 6.97(d, J = 8.4 Hz, 1H), 7.20-7.36(m, 4H), 7.94(br. s, 1H), 8.32(d, J=7.5 Hz, 1H). 13C-NMR (CDCl3, 75 MHz): δ =11.0, 22.9, 69.5, 101.1, 103.3, 109.9, 112.7, 119.6, 122.8, 123.1, 124.9, 126.7, 138.7, 140.9, 155.8.

4-(Prop-2-yn-1-yloxy)-9H-carbazole [IV] (34%, yield). GC-MS showed >99% purity; m/z: 221, 191, 110. 1H-NMR (CDCl3, 300 MHz): δ =2.55(t, J = 2.1 Hz, 1H), 4.94(d, J=2.4 Hz, 2H), 6.76(d, J = 7.8 Hz, 1H), 7.05(d, J = 7.8 Hz, 1H), 7.21-7.39(m, 4H), 8.01(br. s, 1H), 8.33(d, J=7.5 Hz, 1H). 13C-NMR (CDCl3, 75 MHz): δ = 58.2, 77.7, 81.1, 103.9, 106.5, 112.1, 115.1, 121.9, 124.6, 125.4, 127.3, 128.6, 140.9, 143.2, 156.3.

2-Methoxy-9H-carbazole [V] (15) (67%, yield). GC-MS showed >99% purity; m/z: 197, 183, 154, 127. 1H-NMR (CDCl3, 300 MHz): δ = 3.92(s, 3H), 6.86-6.89(dd, J = 1.9, 6.3 Hz, 1H), 6.93(d, J = 1.9 Hz, 1H), 7.20-7.50(m, 1H), 7.33-7.42(m, 2H), 7.94-8.00(m, 3H). 13C-NMR (CDCl3, 75 MHz): δ = 55.6, 94.7, 108.2, 110.3, 117.3, 119.5, 119.6, 121.1, 123.5, 124.6, 139.5, 140.8, 159.1.

2-Ethoxy-9H-carbazole [VI] (16) (63 %, yield). GC-MS showed >99% purity; m/z: 211, 183, 154, 127. 1H-NMR (CDCl3, 300 MHz): δ = 1.49(t, J = 6.9 Hz, 3H), 4.12(q, 2H), 6.86(d, J = 2.1 Hz, 1H), 6.89(d, J = 2.4 Hz, 1H), 7.20-7.25(m, 1H), 7.33-7.40(m, 2H), 7.93-8.00(m, 3H). 13C-NMR (CDCl3, 75 MHz): δ = 15.0, 63.8, 96.4, 108.7, 110.3, 117.1, 119.4, 119.5, 121.0, 123.6, 124.5, 139.5, 140.8, 158.4.

2-Propoxy-9H-carbazole [VII] (43%, yield). GC-MS showed >99% purity; m/z: 225, 183, 154, 127. 1H-NMR (CDCl3, 300 MHz): δ = 1.10(t, J = 7.2 Hz, 3H), 1.87(q, 2H), 4.03(t, 2H), 6.86-6.89(dd, J = 2.1, 6.4 Hz, 1H), 6.92(d, J = 1.8Hz, 1H), 7.19-7.24(m, 1H), 7.32-7.41(m, 2H), 7.93-8.00(m, 3H). 13C-NMR (CDCl3, 75 MHz): δ = 10.6, 22.7, 70.0, 95.4, 108.7, 110.3, 117.1, 119.4, 119.5, 121.0 123.58, 124.5, 139.5, 140.8, 158.6.

2-(Prop-2-yn-1-yloxy)-9H-carbazole [VIII] (28%, yield). GC-MS showed >99% purity; m/z: 221, 191, 110, 95. 1H-NMR (CDCl3, 300 MHz): δ = 2.53(s, 1H), 4.76(d, J=2.4 Hz, 2H), 6.90(dd, J = 2.3, 8.6 Hz, 1H), 6.98(d, J = 2.2 Hz, 1H), 7.17-7.22(m, 1H), 7.30-7.35(m, 2H), 7.95(t, J = 8.4 Hz, 3H). 13C-NMR (CDCl3, 75 MHz): δ = 56.4, 75.5, 78.8, 96.3, 108.7, 110.4, 118.0, 119.6, 121.1, 123.4, 124.8, 139.6, 140.5, 156.9.

2-Hydroxycarbazole, 4-hydroxycarbazole, and compounds I-VIII were tested at five effective concentrations as potential inhibitors of 7-ethoxyresorufin O-deethylation (EROD), 7-methoxyresorufin O-demethylation (MROD), and 7-pentoxyresorufin O-depentylation (PROD) activities in human P450s 1A1 and 1A2 and rat P450 2B1. These assays have been successfully used for determining the type and extent of inhibition of P450s 1A1, 1A2, and 2B1 (17).

2-Hydroxycarbazole inhibited P450s 1A1, 1A2 and 2B1 with IC50 values of 70.5, 59.7 and 41.6 μM respectively. 4-Hydroxycarbazole inhibited P450s 1A1 and 1A2 with IC50 values of 36.8 and 7.33 μM respectively, but did not inhibit P450 2B1 (IC50 > 100 μM). The substituted carbazoles showed inhibition potency towards P450 1A1, with 4-propargyl substituted compound, IV, having the highest potency with an IC50 value of 6.64 μM. Compounds I, II, III, IV, V and VI showed good inhibition potency for P450 1A2 with IC50 values in the range of 0.110-0.375 μM, with 4-propargyl substituted compound IV exhibiting the highest potency at 0.110 μM. Compounds I, II, III and IV showed good inhibition potency for P450 2B1 with 4-propyl substituted compound, III, having the highest potency with an IC50 value of 0.659 μM.

Based on the above data, we report the synthesis and purification of the eight target compounds. These carbazole analogs were synthesized based on their predicted potential inhibitory effects on a number of cytochrome P450 enzymes especially P450s 1A subfamily. Our previous studies on aryl and arylalkyl acetylenes, and various propargyl ethers have shown that a number of these compounds exhibit inhibition towards P450s 1A1 and 1A2 (6,7). Structure-activity studies of this family of carbazole analogs are ongoing and will provide us with valuable information about the enzyme active sites and modes of action. Our preliminary studies have shown that some of the above carbazole analogs are inhibitors of the target enzymes. However, further studies are in progress in order to determine the potency, selectivity, and type of inhibition of each compound and will be published in the near future.

Table 1.

Inhibition data in supersomes for carbazoles and compounds I-VIII measured using human P450 enzymes (1A1, 1A2) and rat P450 enzyme (2B1).

IC50 (μM)
Compounds P450 1A1 P450 1A2 P450 2B1
2-hydroxycarbazole 70.5 59.7 41.6
4-hydroxycarbazole 36.8 7.33 >100
I 21.5 0.350 1.83
II 10.3 0.206 1.14
III 8.73 0.302 0.659
IV 6.64 0.110 1.78
V 35.0 0.375 13.3
VI 15.6 0.309 8.43
VII 26.4 6.35 10.6
VIII 8.65 2.70 5.26
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Acknowledgement

We thank NIH-MBRS SCORE (grant number S06 GM 08008) for support of the preliminary work done on this project, and the NIH-NIGMS supported RISE and MARC Programs at Xavier University (grant numbers 2R25GM060926-09 and 2T34GM007716) for the support of our undergraduate students working on this project. We also thank NIH RCMI (grant number 8G12MD007595-04) for support of the Major Instrumentation Core at Xavier University of Louisiana.

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