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Published in final edited form as: J Undergrad Chem Res. 2013 ; 12(4): 91–94.
DESIGN, SYNTHESIS, AND EVALUATION OF A FAMILY OF PROPARGYL PYRIDINYL ETHERS AS POTENTIAL CYTOCHROME P450 INHIBITORS Maryam Foroozesh, Quan Jiang, Jayalakshmi Sridhar, Jiawang Liu, Minaruzzaman, Brandan Dotson, and Erika McClain Department of Chemistry, Xavier University of Louisiana, New Orleans, Louisiana 70125 Maryam Foroozesh: [email protected]
Abstract NIH-PA Author Manuscript
Cytochrome P450 enzymes are a superfamily of hemoproteins involved in the metabolism of endogenous and exogenous compounds including many drugs and environmental chemicals. In our previous research, we have determined that certain aryl and arylalkyl acetylenes act as inhibitors of these enzymes. Here we report a family of propargyl ethers containing a pyridine ring system. Five new compounds, 2,4-dimethyl-3-(prop-2-yn-1-yloxy)pyridine(I), 2,4-dimethyl-3((prop-2-yn-1-yloxy) methyl)pyridine(II), 2,3-dimethyl-4-((prop-2-yn-1yloxy)methyl)pyridine(III), 2-methyl-4-((prop-2-yn-1-yloxy)methyl)pyridine (IV), 2-methyl-4(prop-2-yn-1-yloxy)pyridine (V) (Figure 1) have been synthesized and characterized.
Keywords Acetylenes; Organic synthesis; Propargyl pyridinyl ethers; Cytochrome P450 enzymes; Enzyme inhibitors; Suicide inhibition; Metabolism
Introduction NIH-PA Author Manuscript
Cytochrome P450 enzymes are the main superfamily of enzymes involved in the metabolism of endogenous and xenobiotic compounds to more polar molecules that can be more easily conjugated and excreted (1–7). However, such metabolism may lead to cytotoxic, mutagenic, or carcinogenic metabolites (1–7). Some aryl and arylalkyl acetylenes have been shown to act as inhibitors of some of these enzymes (8–10). When these compounds fit into the active sites of the P450 enzymes with the correct orientation, the triple bond can be oxidized to a reactive intermediate that binds 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. As part of a more extensive project, five new acetylenic compounds, 2,4-dimethyl-3(prop-2-yn-1-yloxy)pyridine (I), 2,4-dimethyl-3-((prop-2-yn-1-yloxy)methyl)pyridine (II),
Correspondence to: Maryam Foroozesh, [email protected].
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2,3-dimethyl-4-((prop-2-yn-1-yloxy)methyl)pyridine (III), 2-methyl-4-((prop-2-yn-1yloxy)methyl)pyridine (IV), and 2-methyl-4-(prop-2-yn-1-yloxy)pyridine (V) have been synthesized (Scheme 1) and characterized through nuclear magnetic resonance spectroscopy, and gas chromatography/mass spectrometry. These compounds are presently being studied in vitro as potential mechanism-based irreversible inhibitors of a number of P450 enzymes. Propargyl pyridinyl ethers are predicted to be good inhibitors of P450s. This prediction is based on the fact that pyridines are substrates of these enzymes, and the addition of the carbon-carbon triple bond has previously been shown to make the compounds into inhibitors (6–7). This group of compounds also contain an oxygen atom on the substituent chain leading to a change in the polarity and orientation of the triple bond. 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, and thus act as anticarcinogenic “blocking agents”. They could also be used as models for anticancer drugs, or in protein labeling studies.
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Experimental Reagents 2,4-Dimethyl-3-hydroxypyridine was obtained from Combi-Blocks, Inc. Ethyl 2,4dimethylnicotinate was obtained from Alfa Aesar. 2,3-Lutidine and 2,4-lutidine were obtained from Acros Organics (NY, USA). Other reagents and solvents (ethyl acetate, hexanes, carbon tetrachloride, dichloromethane, and tetrahydrofuran) were purchased from Fisher Scientific (Pittsburg, PA). Synthesis of 2,4-dimethyl-3-(prop-2-yn-1-yloxy)pyridine (I)
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A suspension of 350 mg of 95% sodium hydride (14.0 mmol, 1.8 eq.) in 15 mL of dry tetrahydrofuran (THF) was cooled to 0°C, before the dropwise addition of 1.0 g of 2,4dimethyl-3-hydroxypyridine (8.1 mmol) in 20 mL of THF. After 30 minutes of stirring at 0°C, 1.0 mL of propargyl bromide (80wt% solution in toluene, 9.0 mmol, 1.1 eq.) was added dropwise. The ice bath was removed, and the reaction mixture was heated to reflux overnight. It was then allowed to cool to room temperature, quenched with brine, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate. 2,4-Dimethyl-3-(prop-2-yn-1-yloxy) pyridine was purified (in 57% yield) with column chromatography using 5% ethyl acetate in hexanes as eluent. GC-MS showed >99% purity; m/z: 161, 146, 132, 94. 1HNMR (CDCl3, 300 MHz) δ = 2.34(s, 3H), 2.52(t, J=2.4Hz, 1H), 2.55(s, 3H), 4.56(d, J=2.7Hz, 2H), 6.97(d, J=4.8Hz, 1H), 8.15 (d, J=4.8Hz, 1H). 13CNMR (CDCl3, 75 MHz) δ=16.16, 19.73, 60.07, 75.78, 78.55, 124.10, 140.26, 144.77, 151.63, 152.62. Synthesis of 2,4-dimethyl-3-(prop-2-yn-1-yloxy)methyl) pyridine (II) A solution of 8.7 mL of ethyl 2,4-dimethylnicotinate (48 mmol) in 20 mL of THF was added dropwise to a suspension of 2.0 g of lithium aluminum hydride (53 mmol, 1.1 eq.) in 10 mL of THF. After the addition was complete, the mixture was heated to reflux for 2 hours. It
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was then allowed to cool to room temperature, quenched by cautious sequential addition of 2 mL of deionized water, 2 mL of saturated sodium hydroxide solution, and 6 mL of deionized water. The organic layer was separated and dried over anhydrous magnesium sulfate, concentrated on vacuum, and purified by column chromatography using 2% ethyl acetate in hexanes as eluent to afford the known intermediate, (2,4-dimethylpyridin-3-yl) methanol (11), as a colorless liquid in 77% yield. GC-MS showed >99% purity; m/z: 137.
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A suspension of 387 mg of 95% sodium hydride (15.0 mmol, 2.1 eq.) in 15 mL of tetrahydrofuran was cooled to 0°C before the dropwise addition of 1.0 g of (2,4dimethylpyridin-3-yl)methanol (7.0 mmol) in 20 mL of THF. Thirty minutes after the addition, 1.0 mL of propargyl bromide (80wt% solution in toluene, 9.0 mmol, 1.3 eq.) was also added dropwise at 0°C. The ice bath was removed, and the reaction mixture was heated to reflux overnight. It was allowed to cool to room temperature, quenched with brine, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate. 2,4-Dimethyl-3-((prop-2-yn-1-yloxy) methyl)pyridine was purified with column chromatography using 5% ethyl acetate in hexanes as eluent (43% yield). GCMS showed >99% purity; m/z: 175, 145, 120, 106. 1HNMR (CDCl3, 300 MHz) δ = 2.41(s, 3H), 2.52(t, J=2.4Hz, 1H), 2.64(s, 3H), 4.22(d, J=2.4Hz, 2H), 4.65(s, 2H), 6.96(d, J=5.1Hz, 1H), 8.29(d, J=5.1Hz, 1H).13CNMR (CDCl3, 75 MHz) δ=19.00, 22.32, 57.66, 65.32, 74.91, 79.51, 123.58, 129.07, 147.44, 148.49, 158.21. Synthesis of 2,3-dimethyl-4-((prop-2-yn-1-yloxy)methyl) pyridine (III) (12–13) To 5.39 mL of 2,3-lutidine (46.7 mmol) was carefully added 50 mL of oleum while stirring at 0°C. After the addition, the reaction flask was fitted with a reflux condenser and heated to reflux in an oil bath. 4.3 mL of bromine (83.4 mmol, 0.9 eq.) was added in portions of 0.5 mL over 5 hours, and stirring was continued at 155–175°C for 20 hours. The reaction mixture was allowed to cool to room temperature, poured over 200 g of ice, neutralized with sodium carbonate, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and concentrated on vacuum. The residue was subjected to column chromatography using 5% ethyl acetate in hexanes as eluent to afford 4-bromo-2,3dimethylpyridine. GC-MS showed >99% purity; m/z: 185, 141, 106. 1HNMR (CDCl3, 300 MHz) δ = 2.26 (s, 3H), 2.45(s, 3H), 7.54 (d, J=2.1Hz, 1H), 8.38(d, J=2.1Hz, 1H). 13CNMR (CDCl3, 75 MHz) δ=19.05, 22.10, 117.68, 133.37, 139.28, 147.23, 155.73.
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A mixture of 1.3 g of 4-bromo-2,3-dimethylpyridine (7 mmol) and 1.64 g of copper(I) cyanide (18 mmol, 2.6 eq.) in 25 mL of N,N-dimethylformamide was heated at 140°C for 6 hours. The mixture was poured into 40 mL of deionized ice water and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated by vacuum. The residue was purified with column chromatography using 5% ethyl acetate in hexanes as eluent to obtain the known intermediate 2,3dimethylisonicotinonitrile (14) in 43% yield. GC-MS showed >99% purity; m/z: 132. A solution of 0.3 g of 2,3-dimethylisonicotinonitrile (2.1 mmol) was dissolved in 10 mL of methanol, and cooled to 0°C with an ice-bath before the dropwise addition of 5 mL of 2M sodium hydroxide solution. The reaction mixture was stirred overnight at room temperature. The mixture was then neutralized with dilute hydrochloric acid, and extracted with ethyl J Undergrad Chem Res. Author manuscript; available in PMC 2014 December 24.
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acetate. The organic layer was dried over anhydrous magnesium sulfate, and concentrated on vacuum. The residue was purified with column chromatography using 10% ethyl acetate in hexanes as eluent to obtain the next known intermediate 2,3-dimethylisonicotinic acid (15) in 91% yield. GC-MS showed >99% purity; m/z: 151. A solution of 0.3 g of 2,3-dimethylisonicotinic acid (1.9 mmol) in 10 mL of THF was added dropwise to a suspension of 0.2 g of lithium aluminum hydride (5.3 mmol, 2.8 eq.) in 5 mL of THF. After the addition, the mixture was heated to reflux for 2 hours. It was then allowed to cool to room temperature, quenched by cautious sequential addition of 2 mL of deionized water, 2 mL of saturated sodium hydroxide solution, and 6 mL of deionized water. The organic layer was separated and dried over anhydrous magnesium sulfate, concentrated by vacuum, and purified by column chromatography using 2% ethyl acetate in hexanes as eluent to afford the known intermediate (2,3-dimethyl-pyridin-4-yl)methanol (16) as a colorless liquid in 77% yield. GC-MS showed >99% purity; m/z: 137.
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A suspension of 0.1 g of 95% sodium hydride (4.0 mmol, 2.6 eq.) in 15 mL of THF was cooled to 0°C, before the dropwise addition of 0.2 g of (2,3-dimethylpyridin-4-yl) methanol (1.5 mmol) in 20 mL of THF. Thirty minutes after the addition, 0.5 mL of propargyl bromide (80wt% solution in toluene, 4.5 mmol, 3.0 eq.) was also added dropwise at 0°C. The ice bath was removed, and the reaction mixture was heated to reflux overnight. It was then allowed to cool to room temperature, and quenched with brine, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate. 2,3-Dimethyl-4((prop-2-yn-1-yloxy) methyl)pyridine was purified with column chromatography using 5% ethyl acetate in hexanes as eluent in 43% yield. GC-MS showed >99% purity; m/z: 175, 145, 120, 106. 1HNMR (CDCl3, 300 MHz) δ = 2.29(s, 3H), 2.49(t, J=2.4Hz, 1H), 2.50(s, 3H), 4.19(d, J=2.4Hz, 2H),4.57(s,2H), 7.44(d, J=1.2Hz, 1H), 8.30 (d, J=1.2Hz, 1H) 13CNMR (CDCl3, 75 MHz) δ=19.08, 22.34, 57.25, 68.89, 74.93, 79.33, 130.09, 131.30, 137.14, 146.16, 157.01. Synthesis of 2-methyl-4-((prop-2-yn-1-yloxy)methyl)pyridine (IV)
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(2-methylpyridin-4-yl)methanol was prepared according to the reported procedure (17). A suspension of 375 mg of 95% of sodium hydride (14.8 mmol, 2.1 eq.) in 15 mL of THF was cooled to 0°C before the dropwise addition of 1.0 g of (2-methylpyridin-4-yl) methanol (8.1 mmol) in 20 mL of THF. Thirty minutes after the addition, 1.0 mL of propargyl bromide (80wt% solution in toluene, 9.0 mmol, 1.1 eq.) was added dropwise also at 0°C. The ice bath was removed, and the reaction mixture was heated to reflux overnight. It was then allowed to cool to room temperature, quenched with brine, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate. 2-Methyl-4-((prop-2-yn-1yloxy)methyl) pyridine was purified with column chromatography using 5% ethyl acetate in hexanes as eluent in 43% yield. GC-MS showed >99% purity; m/z: 161, 146, 120. 1HNMR (CDCl3, 300 MHz) δ = 2.50(t, J=2.4Hz, 1H), 2.55(s, 3H), 4.22 (m, 2H), 4.58 (s, 2H), 7.07(d, J=5.1Hz, 1H), 7.14 (s, 1H), 8.45(d, J=5.1Hz, 1H). 13CNMR (CDCl3, 75 MHz) δ=24.37, 57.82, 69.88, 75.18, 79.05, 119.16, 121.52, 146.80, 149.15, 158.58.
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Synthesis of 2-methyl-4-(prop-2-yn-1-yloxy)pyridine (V) (18)
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A mixture of 3.0 g of 2,4-lutidine (28.0 mmol), 5.0 g of N-bromosuccinimide (28.0 mmol, 1.0 eq.), and 0.9 g of azoisobutyronitrile (AIBN) (5.5 mmol, 0.2 eq.) in 300 mL of carbon tetrachloride was heated to reflux overnight. The reaction mixture was allowed to cool to room temperature, filtered, concentrated on vacuum, and purified by column chromatography using 5% ethyl acetate in hexanes as an eluent to get 4-(bromomethyl)-2methylpyridine in 27 % yield. GC-MS showed >99% purity; m/z: 185, 141, 106. 1HNMR (CDCl3, 300 MHz) δ = 2.27(s, 3H), 4.43(s, 2H), 6.95 (d, J=2.4Hz, 1H), 7.20 (s, 1H), 8.39(d, J=2.4Hz, 1H). 13CNMR (CDCl3, 75 MHz) δ=21.44, 34.13, 124.48, 124.82, 149.08, 149.57, 156.79.
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A suspension of 300 mg of 95% sodium hydride (12.0 mmol, 2.2 eq.) in 5 mL of THF was cooled to 0°C before the dropwise addition of 1 mL of propargyl alcohol (80wt% solution in toluene, 9.0 mmol, 1.7 eq.) in 20 mL of THF. Thirty minutes after the addition, 1.0 g of 4(bromomethyl)-2-methylpyridine (5.4 mmol, 1.0 eq.) was also added dropwise at 0°C. The ice bath was removed, and the reaction mixture was heated to reflux overnight. It was then allowed to cool to room temperature, quenched with brine, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and concentrated on vacuum. 2-Methyl-4-(prop-2-yn-1-yloxy)pyridine was purified with column chromatography using 5% ethyl acetate in hexanes as eluent in 43% yield. GC-MS showed >99% purity; m/z: 147, 106. 1HNMR (CDCl3, 300 MHz) δ = 2.38(s, 3H), 2.50(t, J=2.4Hz, 1H), 4.30 (d, J=3.2Hz, 2H), 4.71 (s, 2H), 7.04(d, J=4.5Hz, 1H), 7.26(s, 1H), 8.43(d, J=4.5Hz, 1H). 13CNMR (CDCl3, 75 MHz) δ=21.11, 58.05, 72.54, 74.90, 122.54, 123.56, 147.95, 148.96, 157.25.
Results and Discussion
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Based on the above data, we report the synthesis and purification of the five target compounds. These compounds were synthesized based on their predicted potential inhibitory effect on a number of cytochrome P450 enzymes especially P450s 2A6 and 2B1. This observation confirms that pyridine derivatives containing a carbon-carbon triple bond are useful tools in the study of selective mechanism-based inhibition of various P450 enzymes. Structure-activity studies of this family of compounds are ongoing and provide us with valuable information about the enzyme active sites and modes of action. Our preliminary studies have shown that the above propargyl pyridinyl ethers 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.
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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References NIH-PA Author Manuscript NIH-PA Author Manuscript
1. de Montellano, Ortiz, editor. Cytochrome P450 Structure, Mechanism, and Biochemistry. 2nd Ed.. New York: Plenum Press; 1995. p. 1-652. 2. Estabrook RW. FASEB J. 1996; 10:202–204. [PubMed: 8641552] 3. Masters BSS. FASEB J. 1996; 10:205. [PubMed: 8641553] 4. Bhattacharya KK, Brake PB, Elton SE, Otto SA, Jefcoate CR. J. Biol. Chem. 1995; 270:11595– 11602. [PubMed: 7744798] 5. Schenkman, JB.; Greim, H., editors. Cytochrome P450 handbook of Experimental Pharmacology. Vol. 105. Berlin: Springer-Verlag; 1993. p. 1-739. 6. Hasemann CA, Kurumbail RG, Boddupalli SS, Peterson JA, Deisenhofer J. Current Biology. 1995; 3:41–62. 7. Alexander DL, Jefcoate CR. Proc. Amer. Assoc. Cancer. Res. 1995; 36:152. abstract 905. 8. Foroozesh M, Primrose VL, Guo Z, Bell LC, Alworth WL, Guengerich FP. Chem. Res. in Toxicol. 1997; 10(1):91–102. [PubMed: 9074808] 9. Shimada T, Yamazaki H, Foroozesh M, Hopkins NE, Alworth WL, Guengerich FP. Chem. Res. in Toxicol. 1998; 11(9):1048–1056. [PubMed: 9760279] 10. Strobel SM, Szklarz GD, He Y, Foroozesh M, Alworth WL, Roberts ES, Hollenberg PF, Halpert JR. JPET. 1999; 290:445–451. 11. Li J, Zhang J, Chen J, Luo X, Zhu W, Shen J, Liu H, Shen X, Jiang H. J. of Comb. Chem. 2006; 8(3):326–337. [PubMed: 16677001] 12. Thalhammer A, Mecinović J, Loenarz C, Tumber A, Rose NR, Heightman TD, Schofield CJ. Org. Biomol. Chem. 2011; 9:127–135. [PubMed: 21076780] 13. Rivkin A, Kim YR, Goulet MT, Bays N, Hill AD, Kariv I, Krauss S, Ginanni N, Strack PR, Kohl NE, Chung CC, Varnerin JP, Goudreau PN, Chang A, Tota MR, Munoz B. Bio. & Med. Chem. Lett. 2006; 16(17):4620–4623. 14. Hibino S, Sugino E. J. of Het. Chem. 1990; 27(6):1751–1755. 15. Kondrat’eva GY, Huan CH. Doklady Akademii Nauk SSSR. 1965; 164(4):816–819. 16. Epling GA, Lin KY. J. of Het.. Chem. 1987; 24(3):853–857. 17. Ragan JA, Jones BP, Meltz CN, Teixeira JJ Jr. Synthesis. 2002; (4):483–486. 18. Gu J, Chen J, Schmehl RH. JACS. 2010; 132(21):7338–7346.
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Figure 1.
The structures of the synthesized propargyl pyridinyl ethers.
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Scheme 1.
Syntheses of the propargyl pyridinyl ethers.
J Undergrad Chem Res. Author manuscript; available in PMC 2014 December 24.
NIH-PA Author Manuscript
Published in final edited form as: J Undergrad Chem Res. 2013 ; 12(4): 91–94.
DESIGN, SYNTHESIS, AND EVALUATION OF A FAMILY OF PROPARGYL PYRIDINYL ETHERS AS POTENTIAL CYTOCHROME P450 INHIBITORS Maryam Foroozesh, Quan Jiang, Jayalakshmi Sridhar, Jiawang Liu, Minaruzzaman, Brandan Dotson, and Erika McClain Department of Chemistry, Xavier University of Louisiana, New Orleans, Louisiana 70125 Maryam Foroozesh: [email protected]
Abstract NIH-PA Author Manuscript
Cytochrome P450 enzymes are a superfamily of hemoproteins involved in the metabolism of endogenous and exogenous compounds including many drugs and environmental chemicals. In our previous research, we have determined that certain aryl and arylalkyl acetylenes act as inhibitors of these enzymes. Here we report a family of propargyl ethers containing a pyridine ring system. Five new compounds, 2,4-dimethyl-3-(prop-2-yn-1-yloxy)pyridine(I), 2,4-dimethyl-3((prop-2-yn-1-yloxy) methyl)pyridine(II), 2,3-dimethyl-4-((prop-2-yn-1yloxy)methyl)pyridine(III), 2-methyl-4-((prop-2-yn-1-yloxy)methyl)pyridine (IV), 2-methyl-4(prop-2-yn-1-yloxy)pyridine (V) (Figure 1) have been synthesized and characterized.
Keywords Acetylenes; Organic synthesis; Propargyl pyridinyl ethers; Cytochrome P450 enzymes; Enzyme inhibitors; Suicide inhibition; Metabolism
Introduction NIH-PA Author Manuscript
Cytochrome P450 enzymes are the main superfamily of enzymes involved in the metabolism of endogenous and xenobiotic compounds to more polar molecules that can be more easily conjugated and excreted (1–7). However, such metabolism may lead to cytotoxic, mutagenic, or carcinogenic metabolites (1–7). Some aryl and arylalkyl acetylenes have been shown to act as inhibitors of some of these enzymes (8–10). When these compounds fit into the active sites of the P450 enzymes with the correct orientation, the triple bond can be oxidized to a reactive intermediate that binds 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. As part of a more extensive project, five new acetylenic compounds, 2,4-dimethyl-3(prop-2-yn-1-yloxy)pyridine (I), 2,4-dimethyl-3-((prop-2-yn-1-yloxy)methyl)pyridine (II),
Correspondence to: Maryam Foroozesh, [email protected].
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2,3-dimethyl-4-((prop-2-yn-1-yloxy)methyl)pyridine (III), 2-methyl-4-((prop-2-yn-1yloxy)methyl)pyridine (IV), and 2-methyl-4-(prop-2-yn-1-yloxy)pyridine (V) have been synthesized (Scheme 1) and characterized through nuclear magnetic resonance spectroscopy, and gas chromatography/mass spectrometry. These compounds are presently being studied in vitro as potential mechanism-based irreversible inhibitors of a number of P450 enzymes. Propargyl pyridinyl ethers are predicted to be good inhibitors of P450s. This prediction is based on the fact that pyridines are substrates of these enzymes, and the addition of the carbon-carbon triple bond has previously been shown to make the compounds into inhibitors (6–7). This group of compounds also contain an oxygen atom on the substituent chain leading to a change in the polarity and orientation of the triple bond. 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, and thus act as anticarcinogenic “blocking agents”. They could also be used as models for anticancer drugs, or in protein labeling studies.
NIH-PA Author Manuscript
Experimental Reagents 2,4-Dimethyl-3-hydroxypyridine was obtained from Combi-Blocks, Inc. Ethyl 2,4dimethylnicotinate was obtained from Alfa Aesar. 2,3-Lutidine and 2,4-lutidine were obtained from Acros Organics (NY, USA). Other reagents and solvents (ethyl acetate, hexanes, carbon tetrachloride, dichloromethane, and tetrahydrofuran) were purchased from Fisher Scientific (Pittsburg, PA). Synthesis of 2,4-dimethyl-3-(prop-2-yn-1-yloxy)pyridine (I)
NIH-PA Author Manuscript
A suspension of 350 mg of 95% sodium hydride (14.0 mmol, 1.8 eq.) in 15 mL of dry tetrahydrofuran (THF) was cooled to 0°C, before the dropwise addition of 1.0 g of 2,4dimethyl-3-hydroxypyridine (8.1 mmol) in 20 mL of THF. After 30 minutes of stirring at 0°C, 1.0 mL of propargyl bromide (80wt% solution in toluene, 9.0 mmol, 1.1 eq.) was added dropwise. The ice bath was removed, and the reaction mixture was heated to reflux overnight. It was then allowed to cool to room temperature, quenched with brine, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate. 2,4-Dimethyl-3-(prop-2-yn-1-yloxy) pyridine was purified (in 57% yield) with column chromatography using 5% ethyl acetate in hexanes as eluent. GC-MS showed >99% purity; m/z: 161, 146, 132, 94. 1HNMR (CDCl3, 300 MHz) δ = 2.34(s, 3H), 2.52(t, J=2.4Hz, 1H), 2.55(s, 3H), 4.56(d, J=2.7Hz, 2H), 6.97(d, J=4.8Hz, 1H), 8.15 (d, J=4.8Hz, 1H). 13CNMR (CDCl3, 75 MHz) δ=16.16, 19.73, 60.07, 75.78, 78.55, 124.10, 140.26, 144.77, 151.63, 152.62. Synthesis of 2,4-dimethyl-3-(prop-2-yn-1-yloxy)methyl) pyridine (II) A solution of 8.7 mL of ethyl 2,4-dimethylnicotinate (48 mmol) in 20 mL of THF was added dropwise to a suspension of 2.0 g of lithium aluminum hydride (53 mmol, 1.1 eq.) in 10 mL of THF. After the addition was complete, the mixture was heated to reflux for 2 hours. It
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was then allowed to cool to room temperature, quenched by cautious sequential addition of 2 mL of deionized water, 2 mL of saturated sodium hydroxide solution, and 6 mL of deionized water. The organic layer was separated and dried over anhydrous magnesium sulfate, concentrated on vacuum, and purified by column chromatography using 2% ethyl acetate in hexanes as eluent to afford the known intermediate, (2,4-dimethylpyridin-3-yl) methanol (11), as a colorless liquid in 77% yield. GC-MS showed >99% purity; m/z: 137.
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A suspension of 387 mg of 95% sodium hydride (15.0 mmol, 2.1 eq.) in 15 mL of tetrahydrofuran was cooled to 0°C before the dropwise addition of 1.0 g of (2,4dimethylpyridin-3-yl)methanol (7.0 mmol) in 20 mL of THF. Thirty minutes after the addition, 1.0 mL of propargyl bromide (80wt% solution in toluene, 9.0 mmol, 1.3 eq.) was also added dropwise at 0°C. The ice bath was removed, and the reaction mixture was heated to reflux overnight. It was allowed to cool to room temperature, quenched with brine, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate. 2,4-Dimethyl-3-((prop-2-yn-1-yloxy) methyl)pyridine was purified with column chromatography using 5% ethyl acetate in hexanes as eluent (43% yield). GCMS showed >99% purity; m/z: 175, 145, 120, 106. 1HNMR (CDCl3, 300 MHz) δ = 2.41(s, 3H), 2.52(t, J=2.4Hz, 1H), 2.64(s, 3H), 4.22(d, J=2.4Hz, 2H), 4.65(s, 2H), 6.96(d, J=5.1Hz, 1H), 8.29(d, J=5.1Hz, 1H).13CNMR (CDCl3, 75 MHz) δ=19.00, 22.32, 57.66, 65.32, 74.91, 79.51, 123.58, 129.07, 147.44, 148.49, 158.21. Synthesis of 2,3-dimethyl-4-((prop-2-yn-1-yloxy)methyl) pyridine (III) (12–13) To 5.39 mL of 2,3-lutidine (46.7 mmol) was carefully added 50 mL of oleum while stirring at 0°C. After the addition, the reaction flask was fitted with a reflux condenser and heated to reflux in an oil bath. 4.3 mL of bromine (83.4 mmol, 0.9 eq.) was added in portions of 0.5 mL over 5 hours, and stirring was continued at 155–175°C for 20 hours. The reaction mixture was allowed to cool to room temperature, poured over 200 g of ice, neutralized with sodium carbonate, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and concentrated on vacuum. The residue was subjected to column chromatography using 5% ethyl acetate in hexanes as eluent to afford 4-bromo-2,3dimethylpyridine. GC-MS showed >99% purity; m/z: 185, 141, 106. 1HNMR (CDCl3, 300 MHz) δ = 2.26 (s, 3H), 2.45(s, 3H), 7.54 (d, J=2.1Hz, 1H), 8.38(d, J=2.1Hz, 1H). 13CNMR (CDCl3, 75 MHz) δ=19.05, 22.10, 117.68, 133.37, 139.28, 147.23, 155.73.
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A mixture of 1.3 g of 4-bromo-2,3-dimethylpyridine (7 mmol) and 1.64 g of copper(I) cyanide (18 mmol, 2.6 eq.) in 25 mL of N,N-dimethylformamide was heated at 140°C for 6 hours. The mixture was poured into 40 mL of deionized ice water and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated by vacuum. The residue was purified with column chromatography using 5% ethyl acetate in hexanes as eluent to obtain the known intermediate 2,3dimethylisonicotinonitrile (14) in 43% yield. GC-MS showed >99% purity; m/z: 132. A solution of 0.3 g of 2,3-dimethylisonicotinonitrile (2.1 mmol) was dissolved in 10 mL of methanol, and cooled to 0°C with an ice-bath before the dropwise addition of 5 mL of 2M sodium hydroxide solution. The reaction mixture was stirred overnight at room temperature. The mixture was then neutralized with dilute hydrochloric acid, and extracted with ethyl J Undergrad Chem Res. Author manuscript; available in PMC 2014 December 24.
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acetate. The organic layer was dried over anhydrous magnesium sulfate, and concentrated on vacuum. The residue was purified with column chromatography using 10% ethyl acetate in hexanes as eluent to obtain the next known intermediate 2,3-dimethylisonicotinic acid (15) in 91% yield. GC-MS showed >99% purity; m/z: 151. A solution of 0.3 g of 2,3-dimethylisonicotinic acid (1.9 mmol) in 10 mL of THF was added dropwise to a suspension of 0.2 g of lithium aluminum hydride (5.3 mmol, 2.8 eq.) in 5 mL of THF. After the addition, the mixture was heated to reflux for 2 hours. It was then allowed to cool to room temperature, quenched by cautious sequential addition of 2 mL of deionized water, 2 mL of saturated sodium hydroxide solution, and 6 mL of deionized water. The organic layer was separated and dried over anhydrous magnesium sulfate, concentrated by vacuum, and purified by column chromatography using 2% ethyl acetate in hexanes as eluent to afford the known intermediate (2,3-dimethyl-pyridin-4-yl)methanol (16) as a colorless liquid in 77% yield. GC-MS showed >99% purity; m/z: 137.
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A suspension of 0.1 g of 95% sodium hydride (4.0 mmol, 2.6 eq.) in 15 mL of THF was cooled to 0°C, before the dropwise addition of 0.2 g of (2,3-dimethylpyridin-4-yl) methanol (1.5 mmol) in 20 mL of THF. Thirty minutes after the addition, 0.5 mL of propargyl bromide (80wt% solution in toluene, 4.5 mmol, 3.0 eq.) was also added dropwise at 0°C. The ice bath was removed, and the reaction mixture was heated to reflux overnight. It was then allowed to cool to room temperature, and quenched with brine, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate. 2,3-Dimethyl-4((prop-2-yn-1-yloxy) methyl)pyridine was purified with column chromatography using 5% ethyl acetate in hexanes as eluent in 43% yield. GC-MS showed >99% purity; m/z: 175, 145, 120, 106. 1HNMR (CDCl3, 300 MHz) δ = 2.29(s, 3H), 2.49(t, J=2.4Hz, 1H), 2.50(s, 3H), 4.19(d, J=2.4Hz, 2H),4.57(s,2H), 7.44(d, J=1.2Hz, 1H), 8.30 (d, J=1.2Hz, 1H) 13CNMR (CDCl3, 75 MHz) δ=19.08, 22.34, 57.25, 68.89, 74.93, 79.33, 130.09, 131.30, 137.14, 146.16, 157.01. Synthesis of 2-methyl-4-((prop-2-yn-1-yloxy)methyl)pyridine (IV)
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(2-methylpyridin-4-yl)methanol was prepared according to the reported procedure (17). A suspension of 375 mg of 95% of sodium hydride (14.8 mmol, 2.1 eq.) in 15 mL of THF was cooled to 0°C before the dropwise addition of 1.0 g of (2-methylpyridin-4-yl) methanol (8.1 mmol) in 20 mL of THF. Thirty minutes after the addition, 1.0 mL of propargyl bromide (80wt% solution in toluene, 9.0 mmol, 1.1 eq.) was added dropwise also at 0°C. The ice bath was removed, and the reaction mixture was heated to reflux overnight. It was then allowed to cool to room temperature, quenched with brine, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate. 2-Methyl-4-((prop-2-yn-1yloxy)methyl) pyridine was purified with column chromatography using 5% ethyl acetate in hexanes as eluent in 43% yield. GC-MS showed >99% purity; m/z: 161, 146, 120. 1HNMR (CDCl3, 300 MHz) δ = 2.50(t, J=2.4Hz, 1H), 2.55(s, 3H), 4.22 (m, 2H), 4.58 (s, 2H), 7.07(d, J=5.1Hz, 1H), 7.14 (s, 1H), 8.45(d, J=5.1Hz, 1H). 13CNMR (CDCl3, 75 MHz) δ=24.37, 57.82, 69.88, 75.18, 79.05, 119.16, 121.52, 146.80, 149.15, 158.58.
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Synthesis of 2-methyl-4-(prop-2-yn-1-yloxy)pyridine (V) (18)
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A mixture of 3.0 g of 2,4-lutidine (28.0 mmol), 5.0 g of N-bromosuccinimide (28.0 mmol, 1.0 eq.), and 0.9 g of azoisobutyronitrile (AIBN) (5.5 mmol, 0.2 eq.) in 300 mL of carbon tetrachloride was heated to reflux overnight. The reaction mixture was allowed to cool to room temperature, filtered, concentrated on vacuum, and purified by column chromatography using 5% ethyl acetate in hexanes as an eluent to get 4-(bromomethyl)-2methylpyridine in 27 % yield. GC-MS showed >99% purity; m/z: 185, 141, 106. 1HNMR (CDCl3, 300 MHz) δ = 2.27(s, 3H), 4.43(s, 2H), 6.95 (d, J=2.4Hz, 1H), 7.20 (s, 1H), 8.39(d, J=2.4Hz, 1H). 13CNMR (CDCl3, 75 MHz) δ=21.44, 34.13, 124.48, 124.82, 149.08, 149.57, 156.79.
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A suspension of 300 mg of 95% sodium hydride (12.0 mmol, 2.2 eq.) in 5 mL of THF was cooled to 0°C before the dropwise addition of 1 mL of propargyl alcohol (80wt% solution in toluene, 9.0 mmol, 1.7 eq.) in 20 mL of THF. Thirty minutes after the addition, 1.0 g of 4(bromomethyl)-2-methylpyridine (5.4 mmol, 1.0 eq.) was also added dropwise at 0°C. The ice bath was removed, and the reaction mixture was heated to reflux overnight. It was then allowed to cool to room temperature, quenched with brine, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and concentrated on vacuum. 2-Methyl-4-(prop-2-yn-1-yloxy)pyridine was purified with column chromatography using 5% ethyl acetate in hexanes as eluent in 43% yield. GC-MS showed >99% purity; m/z: 147, 106. 1HNMR (CDCl3, 300 MHz) δ = 2.38(s, 3H), 2.50(t, J=2.4Hz, 1H), 4.30 (d, J=3.2Hz, 2H), 4.71 (s, 2H), 7.04(d, J=4.5Hz, 1H), 7.26(s, 1H), 8.43(d, J=4.5Hz, 1H). 13CNMR (CDCl3, 75 MHz) δ=21.11, 58.05, 72.54, 74.90, 122.54, 123.56, 147.95, 148.96, 157.25.
Results and Discussion
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Based on the above data, we report the synthesis and purification of the five target compounds. These compounds were synthesized based on their predicted potential inhibitory effect on a number of cytochrome P450 enzymes especially P450s 2A6 and 2B1. This observation confirms that pyridine derivatives containing a carbon-carbon triple bond are useful tools in the study of selective mechanism-based inhibition of various P450 enzymes. Structure-activity studies of this family of compounds are ongoing and provide us with valuable information about the enzyme active sites and modes of action. Our preliminary studies have shown that the above propargyl pyridinyl ethers 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.
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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References NIH-PA Author Manuscript NIH-PA Author Manuscript
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Figure 1.
The structures of the synthesized propargyl pyridinyl ethers.
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Scheme 1.
Syntheses of the propargyl pyridinyl ethers.
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