In the Laboratory
Synthesis and Cytotoxic Properties of Chalcones: An Interactive and Investigative Undergraduate Laboratory Project at the Interface of Chemistry and Biology
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John Dickson, Lloyd Flores, Michelle Stewart, Regan LeBlanc, Hari N. Pati, and Moses Lee*,† Department of Chemistry, Furman University, Greenville, SC 29613; *
[email protected] Herman Holt Department of Chemistry, University of North Carolina at Asheville, Asheville, NC 28804
A highly integrated laboratory project that emphasizes techniques of chemistry and biology is described. Laboratory exercises of this nature are becoming increasingly important as interdisciplinary scientific research improves our ability to meet some of the grand challenges in chemistry laid out by the National Research Council in 2003 (1, 2). These experiments combine synthetic organic chemistry, cell biology, and computational chemistry. In the synthesis portion of this sequence, students will learn basic organic laboratory techniques of filtration, recrystallization, thin-layer and column chromatography, melting point determination, and spectroscopy (infrared, nuclear magnetic resonance, and ultraviolet–visible). For the biology component, the instructor should partner with a cell biologist to aid in conducting the experiment. The instructors should decide how involved the students will be in conducting the cytotoxicity testing. Depending on the level of involvement, the students could have an opportunity to use a hemocytometer, prepare a solution of a specific concentration, use a micropipette, and culture cells. The molecular modeling aspect of this laboratory sequence will provide the students with experience in building molecules in the modeling program and optimizing the structure and conformation of the molecule using molecular mechanics and molecular dynamics. At the end of the experiments, students can investigate the relationship between the structure of the molecules and the extent of their biological activity. The students will be synthesizing and testing substituted chalcones (1,3-diaryl-2-propen-1-ones), which are analogs of combretastatin A4 (3, 4). The biological activity of chalcones and combretastatin A4 (Figure 1) can be traced to their ability to bind to or close to the colchicine binding site of tubulin (3, 5), thereby inhibiting tubulin polymerization and microtubule formation (5, 6). Owing to this interaction with tubulin, chalcones have been shown to exhibit cytotoxic properties, including anticancer activity (7).
O
A
B chalcone
CH3O
OH
CH3O
OCH3
OCH3 combretastatin A4 NHAc
O CH3O OCH CH3O
OCH3 colchicine
Figure 1. Structures of chalcone, combretastatin A4, and colchicine. (The rings in chalcone are labeled A and B as per the discussion in the text.)
O
O CH3
R
+
H
R′
NaOH 95% EtOH
Synthesis of Chalcones O
This Journal has published several articles describing the synthesis of chalcones via a Claisen–Schmidt condensation (Scheme I) of substituted acetophenones and benzaldehydes (7–11). The focus of this article, however, is not limited to † Current address: Department of Chemistry, Hope College, Holland, MI 49423
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R
R′
Scheme I. Generic chalcone reaction. R and R’ are various substituents (H, methoxy, nitro, hydroxy, methyl, and chloro).
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In the Laboratory
synthesis, but also the cytotoxicity testing and the investigation of the relationship between molecular structure and biological activity. In order to adequately identify structure–activity relationships, a large number of chalcones must be synthesized. In our case, 30 compounds were prepared. This can be accomplished by allowing each student to choose a different chalcone to synthesize. Doing so also makes this experiment more interesting to students since not everyone is conducting the same reaction. A wide variety of chalcones can be synthesized by combining relatively few acetophenones and benzaldehydes, thus making this experiment cost effective. A list of possible acetophenones and benzaldehydes is provided in the Supplemental Material.W The crude product can be purified by recrystallization from 95% ethanol if it is a solid. In cases where the product does not solidify, the chalcone can be purified by extraction followed by column chromatography. One laboratory period will be needed to perform the synthesis and purification portion of the project. Spectroscopic Analysis Each student should characterize his or her chalcone by IR, 1H NMR, and UV–vis spectroscopy. The student should assign the major peaks in the IR and NMR spectra and calculate the molar absorptivity based on the UV–vis spectrum. From the NMR spectrum, students should be able to determine the purity (> 95%) of the products and the geometry about the carbon–carbon double bond, which is trans based on the coupling constant of the vinylenic hydrogens ( J ≈ 15–16 Hz) (12). The melting point of the chalcones should also be determined.
be more like the structure of combretastatin A4. This explains the link between the s-trans conformation and higher activity reported by Ducki and colleagues (13). This procedure should be repeated using molecular dynamics. The data obtained from these experiments should be used in conjunction with the biological data to determine the relationship between structure and activity. Structure–Activity Relationship The purpose of this exercise is to engage students in determining the most effective way to design potential drugs. Using data from cytotoxicity studies, they should ascertain the structural attributes responsible for activity. For example, studies have shown that the presence of a 3´,4´,5´-trimethoxy substitution on the A ring is beneficial to biological activity (12). The students who took part in this project also noted that as the number of methoxy groups increased, the cytotoxicity increased. But, the presence of a nitro group generally resulted in a poor activity. This exercise provides students with an opportunity to analyze information generated from biological and computational experiments, and to learn how molecular structure can be related to biological activity. Hazards Sodium hydroxide is caustic. Ethanol is flammable. The substituted acetophenones and benzaldehydes should be handled in a manner consistent with the information available on each of the material safety data sheets (MSDS). [Editor’s Note: CLIPs are available in J. Chem. Educ. for NaOH (2001, 78, 447) and ethanol (2004, 81, 1414).]
Cytotoxicity Testing
Conclusion
The cytotoxicity testing of the chalcones utilizes a colorimetric based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. This study is performed on B16 murine melanoma cells. After incubating the cells with each chalcone continuously for 72 hours, the inhibition in cell growth is determined spectrophotometrically using a plate reader. It will be necessary for the instructor to have access to a cell biology facility or to coordinate this experiment with a professor in the biology department. The activity for each chalcone at 1.0 × 10᎑5 M is classified according to three categories, active, slightly active, or inactive, based on the absorbance at 570 nm. A table containing these data is distributed to the students for use in the structure–activity relationship study.
Owing to the interdisciplinary nature of this set of experiments, it is ideal for a curriculum that incorporates chemistry and biology. The techniques presented in this laboratory sequence and the thoughtful interpretation of the data obtained from the experiments is designed to prepare students for scientific research. Collectively, these laboratory exercises are not only an effective way to educate students, but to train the next generation of scientists.
Molecular Modeling Molecular modeling studies can provide valuable information in elucidating the structure–activity relationship. Students should build and optimize the structure of combretastatin A4 and their chalcone using molecular mechanics. The chalcone should also be locked in the s-cis and s-trans conformations about the bond between the C⫽C and C⫽O bonds and then the energy minimized for both conformations. These two conformations can then be superimposed on combretastatin A4. The s-trans conformation will
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Supplemental Material
Detailed student instructions, instructor notes, lists of chemicals and their hazards, synthetic and biological results are available in this issue of JCE Online. Acknowledgments We thank A. Abbott, K. Baxi, A. Bernardo, K. Blackmon, A. Bryant, W. Bu, J. Carriker, M. Felts, L. Glish, C. Groat, G. Hendrickson, S. Horick, M. Isenhower, J. Jones, D. Matthews, T. Neely, A. Pendley, L. Ramsey, R. Smalls, K. Smith, A. Sutterfield, M. Turlington, S. Vandiver, P. Weisbruch, and T. Wilson for their input in the development of these experiments. Support from the NSF (REU program) is acknowledged.
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Literature Cited 1. Borman, S. Chem. Eng. News 2003, October 6, 28–29. Van Hecke, G. R.; Karukstis, K. K.; Haskell, R. C.; McFadden, C. S.; Wettack, F. S. J. Chem. Educ. 2002, 79, 837–844. Walsh, E. J. J. Chem. Educ. 1996, 73, A305–A306. Moore, J. W. J. Chem. Educ. 2003, 80, 591–592. 2. Moore, J. W. J. Chem. Educ. 2002, 79, 1287–1288. National Research Council’s Board on Chemical Sciences and Technology. Beyond the Molecular Frontier: Challenges for Chemistry and Chemical Engineering; National Academies Press: Washington, DC, 2003; available online http://books.nap.edu/catalog/10633.html (accessed Mar 2006). 3. Lawrence, N. J.; Rennison, D.; McGown, A. T.; Ducki, S.; Gul, L. A.; Hadfield, J. A.; Khan, N. J. Comb. Chem. 2001, 3, 421–426. 4. Getahun, Z.; Jurd, L.; Ping, S. C.; Chii, M. L.; Hamel, E. J. Med. Chem. 1992, 35, 1058–1067.
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5. Gaukroger, K.; Hadfield, J. A.; Hepworth, L. A.; Lawrence, N. J.; McGown, A. T. J. Org. Chem. 2001, 66, 8135– 8138. 6. Chii, M. L.; Ho, H. H.; Pettit, G. R.; Hamel, E. Biochemistry 1989, 28, 6984–6991. 7. Palleros, D. R. J. Chem. Educ. 2004, 81, 1345–1347. 8. Vyvyan, J. R.; Pavia, D. L.; Lampman, G. M.; Kriz, G. S. J. Chem. Educ. 2002, 79, 1119–1120. 9. Wachter–Jurcsask, N.; Zamani, H. J. Chem. Educ. 1999, 76, 653–654. 10. Dixon, C. E.; Pyne, S. G. J. Chem. Educ. 1992, 69, 1032– 1033. 11. Moloney, G. P. J. Chem. Educ. 1990, 67, 617–618. 12. Lawrence, N. J.; McGown, A. T.; Ducki, S.; Hadfield, J. A. Anti–Cancer Drug Design 2000, 15, 135–141. 13. Ducki, S.; Forrest, R.; Hadfield, J. A.; Kendall, A.; Lawrence N. J.; McGown, A. T.; Rennison D. Bioorg. Med. Chem. Lett. 1998, 8, 1051–1056.
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