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The inhibition of tumor growth by anti-angiogenic
agents is a relatively new area of study in cancer treatment. Angiogenesis
is the formation of new circulatory vessels provides oxygen and
nutrients that cancer cells utilize to grow and metastasize to different
parts of the body. Anti-angiogenic agents prohibit this process
thereby limiting the amount of growth and movement by tumor cells.
The combination of anti-angiogenic treatment with other cancer treatments
can help to eradicate the cancer from the human body. The Liotta
group studied efficient ways to make angiogenesis inhibitors using
structures found in Mate tea as a reference. The combination of
trans-1 2-diaminocyclohexane and different forms of cinnamic acid
leads to the synthesis of amides. It is thought that the products
of these reactions will be less susceptible to hydrolysis in the
human body compared to the Mate tea molecules. The ability of these
molecules to stop tumor growth while being relatively stable will
be tested in the upcoming months. If these molecules prove to inhibit
the process of angiogenesis and are significantly more stable in
the body the eradication of tumor cells may be possible.
Dr. Jack Arbiser and his lab at Emory University have
previously shown that certain molecules found in Mate tea leaves
have anti-angiogenic effects when placed in cultures containing
tumor cells. The difficulty in using these molecules as angiogenesis
inhibitors lies in the fact that they are very susceptible to hydrolysis
in the body. Cleavage at various parts of the molecules was observed
when placed near cancerous cells. Our project focused on creating
molecules that mimicked the anti-angiogenic effects of the tea molecules
while being more stable in the body. The most important difference
involved replacing the ester functional groups with amides which
presumably would be less susceptible to hydrolysis. Further reactions
included adding methoxy substituents to the aromatic ring of the
compound.
Reaction 1
To a solution of trans-1 2-diaminocyclohexane (1.5
mL 12.48 mmol) and triethylamine (9 mL 60.67 mmol) in toluene (150
mL) was added at 0°C under nitrogen a solution of cinnamoyl
chloride (4.31 g 25.8 mmol) in toluene (5 mL). The reaction mixture
was stirred overnight. A milky white solution was observed upon
observation. A Thin Layer Chromatography (TLC) test was taken and
it was observed that the starting materials had disappeared. The
product was then worked up using dichloromethane and water. A separatory
funnel was used to remove the organic layer. The organic layer was
then dried using magnesium sulfate. After filtering the product
the solvent was evaporated using a Rotovapor machine. A white solid
was left in the flask. An 1H NMR test revealed that the desired
product had formed. A mass spectrometer test was done and showed
promising results. The significant peak at 381. 4 m/z corresponds
to the exact molecular weight of the desired product 374.4 g plus
seven the standard constant used for lithium spectrometer readouts.
2.2939 grams of product were recovered and the product was submitted
to Dr. Arbiser’s lab for further testing.
Reaction 2
Reaction 2 involved adding methoxy groups to the starting
acid in order to produce an amide that may be more stable than the
first product. Step 1 of reaction 2 was accomplished by adding sulfonyl
chloride (.607 mL 8.32 mmol) drop wise to a solution of 3 4 dimethoxycinnamic
acid (1.87 g 8.32 mmol) in toluene (100 mL). A TLC was taken and
noticeable changes were observed between starting material and intermediate.
The newly formed chloride was added to a solution of trans-1 2-diaminocyclohexane
(5mL 4.16 mmol) and triethylamine (3 mL 20.8 mmol) in toluene (100
mL) at 0°C under nitrogen. The solution was allowed to stir
overnight. A pale yellow precipitate was noticed upon observation.
The product was then worked up using dichloromethane and water.
A separatory funnel was used to remove the organic layer. The organic
layer was then dried using magnesium sulfate. After filtering the
product the solvent was evaporated using a Rotovapor. An 1H NMR
test showed that significant impurities were still present. The
product was purified by column chromatography with a 1:4 hexane:ethyl
acetate mixture used as the solvent. An 1H NMR of the purified product
proved that the desired product was present.
The peak at 381.4 m/z indicates the presence of the
desired amide based on the hypothetical molecular weight. Peaks
ranging from 6.8-7.5 ppm indicate the hydrogens on aromatic ring
while the multitude of peaks from 1.5-1.8 indicate the presence
of the diaminocyclohexane on the same molecule.
The compounds desired were attained through the reactions described
above. While it remains to be seen whether these molecules are less
susceptible to hydrolysis in the human body it is now known that
forming amide derivatives of these molecules can be accomplished.
The purification of these molecules was extremely important and
considered throughout the project. The technique of column chromatography
requires immense patience and accuracy in measurement. If further
studies reveal a lack of angiogenic inhibition by the products formed
in the above reactions further purification may be needed. The future
direction of this project lies in the testing of these molecules
as stable angiogenesis inhibitors. These tests will be performed
under the direction of Dr. Arbiser. The molecules will be tested
using transformed SVR endothelial cells that have been implanted
with tumor genes. If the amide products prove to inhibit angiogenesis
in tumor cells and are less susceptible to hydrolysis then our project
would be considered a success
References
Alexakis A. Chauvin, A. Stouvenel, R. Vrancken, E. Mutti S., and
Mangeney P. Tetrahedron: Asymmetry. (2001) 12 1171-1178.
Acknowledgements
This material is based upon work supported by the Howard Hughes
Medical Institute under Grant No. 52003727 and by the Department
of Chemistry at Emory University. Special thanks to Chang Wu for
NMR tutorials.
I basically spent time doing reactions that turned
carbonyl chlorides and carboxylic acids into amides. A nitrogen
coming off of a carbon-oxygen double bond is more stable than an
ester functional group so hopefully it will be more stable in the
body. The point was to make stable molecules that have anti-cancer
effects. The effects we are trying to have are to stop cancer cells
from making new blood vessels that would provide them with the nutrients
to grow and travel to different parts of the body. If we can stop
cancer cells from spreading we can control the harmful effects that
cancer has on the human body.
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