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Honors Theses AY 16/17
Undergraduate Honors Theses
Spring 5-12-2017
Reactions of Nitric Oxide with BetaHydroxylimino Esters
Elayna M. Mahone
University of Wyoming, emahone@uwyo.edu
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Mahone, Elayna M., "Reactions of Nitric Oxide with Beta-Hydroxylimino Esters" (2017). Honors Theses AY 16/17. 36.
http://repository.uwyo.edu/honors_theses_16-17/36
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Reactions of Nitric Oxide with -Hydroxylimino Esters
Elayna Mahone
Under the guidance of Dr. Navamoney Arulsamy
Spring 2017
Honors Program
and
Department of Chemistry
University of Wyoming
ABSTRACT
Nitric oxide (NO) and its one electron reduction product nitroxyl (HNO) are known
to act as vasodilators in mammals, including humans, and are considered a possible cure to
cardiovascular diseases. However, their instability and high reactivity with atmospheric
oxygen preclude the direct use of them as drugs. Recent work has shown that NO donor
molecules can be used as pro-drugs to deliver NO to the appropriate bio-sites. Therefore,
there is considerable research interest in the synthesis and studies of donors capable of
releasing either of the molecules under physiological conditions. As part of our research
effort towards the synthesis of new NO- and HNO-donors, we have synthesized new nitroxy
derivatives from the reaction of two -hydroxylimino esters namely, methyl 3hydroxyliminobutanoate and methyl 3-hydroxyliminopentanoate, with potassium hydroxide
and NO. The isolated products are characterized as potassium 3-methyl- and 3-ethyl-5isoxazolone-4-diazeniumdiolate, as well as potassium 4-acetyltriazolium-1,2,3-triolate. The
new diazeniumdiolates are first examples of diazeniumdiolate-substituted isoxazolone.
Under modified conditions, the reactions yield the potassium salts of 5-acetyl- and 5propionylsydnonate-N-oxides.
Introduction
This experiment was aimed at the synthesis of nitric oxide (NO) donor pro-drugs. Nitric
oxide and its one electron reduction product nitroxyl (HNO) are known to have physiological
roles in the body, where nitroxyl can act as a deterrent to alcoholism, and nitric oxide can act as a
cure to cardiovascular diseases by acting on epithelial tissue. Compounds containing both nitric
oxide and nitroxyl are known to be versatile, where they can release NO and HNO under
controlled physiological conditions. However, due to the versatility of these compounds, nitric
oxide can react with oxygen in air, causing explosive properties. Further study of these
decomposition properties can suggest how compounds release nitric oxide and how they will
react under physiological conditions.
The addition of nitric oxide (NO) is assumed to be dimeric. This dimeric addition is used
for the formation of sydnone-N-oxide and diazeniumdiolate products. The dimeric addition can
add in a cis- or trans- configuration, with the majority of products having added cis-. In our
synthesis, we assume the possibility of a trans- addition for the sydnone product. Depending on
the amount of NO available and added, the biological effect is different. Figure 1 below
demonstrates the possible additions of dimeric nitric oxide in each configuration, ultimately
creating the same product:
2
Figure 1: Dimeric addition of nitric oxide for the formation of sydnone-N-oxide and
diazeniumdiolate products. Upper path adds in a trans- formation with the condensation and loss
of methanol. The cis- formation first rotates on a single bond before condensing.
By creating a reaction between a β-ketoxime ester, such as methyl acetoacetoxime (R = Me), and
nitric oxide, products can be created as NO releasing compounds. By using ketoximes with both
R groups of methyl and ethyl, the nitric oxide is expected to react with the acidic CH2 group on
the ketoxime. Figure 2 shows what the expected products could be under the conditions
mentioned in the figure, as well as in the following experimental section:
Figure 2: Under given conditions, this reaction of a ketoxime is expected to produce one or all of
these four products with diazeniumdiolate groups attached at the acidic CH2 groups.
Experimental
The required oxime substrate was not commercially available, and was generated in situ
without being isolated. The following Figure 3 shows previous research of this substrate being
generated and isolated under the condition of pyridine. During this synthesis, hot methanol was
used in the place of pyridine.

Figure 3: The synthesis of hydroxylamine hydrochloride and potassium chloride under the
conditions of hot methanol to produce the required oxime substrate to use for continued synthesis
and addition of nitric oxide.
3
Two different methods were used for the synthesis of products. The initial steps included
the addition of about 150 mL of ethanol, one equivalent of hydroxylamine hydrochloride, and
one equivalent of the required substrate (either ethyl acetoacetate (R=Me) or ethyl
propionylacetate (R=Et).
The first method allowed the reaction mixture to sit for 24 hours before adding one
equivalent of potassium hydroxide. After the 24 hours at room temperature, the mixture was
degassed and treated with NO for six hours.
For the second method, one equivalent of potassium hydroxide was added immediately to
treat the acidic CH2 group. This was cooled to -10°C, then de-gassed with N2 in order to remove
oxygen, as NO is reactive with air. The following contraption was used for degassing the
mixture:
Figure 4: Degassing contraption and NO reactor.
Figure 4 also shows the setup for the NO reactor, as the mixture was treated with NO after being
de-gassed. This second procedure treated the mixture with NO for six hours as well.
Results and Discussion
The β-ketoxime reactions formed two unexpected products under the two different
methods. The products from the different methods were found to be different through dissimilar
UV-Visible and NMR spectra. A diazeniumdiolate product and a sydnone product were both
observed. Mechanisms and spectra are both shown below.
The first method that required a 24-hour reaction time created a diazeniumdiolate product. Only
the ethyl derivative was used for this synthesis, resulting in the product of potassium 3-ethyl-5isoxazolone-4-diazeniumdiolate, as shown below in Figure 5:
4
Figure 5: The synthesis of potassium 3-ethyl-5-isoxazolone-4-diazeniumdiolate (R = Et)
through a 24-hour reaction mixture time and nitric oxide treatment.
Previous research shows that over an extended period of time, this condensation is possible to
create an isoxazolone ring, as shown in Figure 6:
Figure 6: The condensation and creation of an isoxazolone ring under given conditions.
Condensation mechanism was used for further analysis of this product.
The following UV-Visible and NMR spectra are for confirmation of the structure of this
diazeniumdiolate product, or EtC3HNO2N2O2K. Diazeniumdiolate products will have a single
peak at 245 with one addition of a diazeniumdiolate, 258 with two additions, or 265 nm with
three additions.
Figure 7: UV-Visible spectra of the ethyl derivative of the diazeniumdiolate product with a
single peak at 245 nm.
Figure 7 shows evidence of a diazeniumdiolate product with only one addition, contrary to the
original predicted products for this reaction. This is a result of the diluted product for the UV5
Visible spectrum analysis, showing a possible structure for the diazeniumdiolate. Further
evidence for the structure can be seen in the following NMR spectra.
Figure 8: H1 NMR spectra for the ethyl derivative of the diazeniumdiolate product. Triplet found
at 1.1, and quartet found at 2.3-2.4. Single peak at 4.7 is indicative of reference solution
D2O/DSS.
The above proton spectra for the product EtC3HNO2N2O2K shows a triplet and a quartet, which
is indicative of an ethyl group on the compound. However, no peak is found for the single
hydrogen off of the carbon containing the diazeniumdiolate. This can be explained by the C13
NMR spectra in Figure 9:
6
Figure 9: C13 NMR for EtC3HNO2N2O2K, showing two lower peaks from ethyl group, two
singlets and high ppm values, and five carbons total with one triplet present at 75.6 ppm.
Figure 9 also shows the presence of a triplet around 75.6 ppm. While these values indicate an
isoxasolone ring, the triplet presents the possibility of something else. However, due to the lack
of a hydrogen peak in the H1 NMR, it was found that the reference solution, D2O/DSS had
interfered with the structure. Previous research shows that deuterium can split a carbon into a
triplet. With the deuterium substituting the hydrogen, no peak is found on the proton NMR and a
triplet is found on the C13 NMR, further clarifying the structure of this product.
Due to the previously known condensation of the isoxazolone ring and the structure
suggested from the spectra, the following mechanism for this diazeniumdiolate can be introduced
with the addition of the nitric oxide:
7
Figure 10: Assumed mechanism for isoxozolone ring diazeniumdiolate product, through
condensation over 24-hour reaction time and addition of NO and KOH.
Evidence for the methyl derivative of this product is not shown, as the methyl product has yet to
be purified and further tested.
The second method for this synthesis took place immediately, rather than over a 24-hour
reaction time before adding NO and KOH. Both methyl and ethyl derivatives were available for
analysis, with products assumed to be potassium 4-acetyltriazolium-1,2,3-triolate and potassium
4-propionyltriazolium-1,2,3-triolate:
Figure 11: Synthesis of potassium 4-acetyltriazolium-1,2,3-triolate (R=Me) and potassium 4acetyltriazolium-1,2,3-triolate (R=Et) sydnone-N-oxime products with immediate additions of
KOH and NO.
These products are assumed to be different from the diazeniumdiolate products due to the
additional peaks of the UV-Vis spectra as well as differing NMR spectra. These are assumed to
be sydnone products, as UV-Vis data is somewhat similar to previously recorded sydnone-oxide
UV-Vis, but with one additional peak, as shown below for the methyl derivative
(MeCOC2N3O3K2):
8
Figure 12: UV-Visible data for the methyl sydnone product.
This product was assumed to be a sydnone derivative based on previous literature stating that
sydnone products have two peaks, one at ~230 nm and one at 300 nm. Further data confirmed
the structure of a sydnone derivative, but with an extra nitrogen in the ring, resulting in the third
extra peak in the UV-Visible spectrum. In Figure 13, a peak at 2.402 can be seen for the H1
NMR, indicative of the acetyl group on the product:
Figure 13: H1 NMR for MeCOC2N3O3K2, with a reference solution peak at 4.7 and 0.0 ppm.
Peak at 2.402 indicative of product’s acetyl group.
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Figure 14: C13 NMR spectra for MeCOC2N3O3K2, with four carbons present for overall
Structure (28.16, 118.84, 153.13, and 193.25 ppm.
Figures 13 and 14 show the NMR spectra for the methyl sydnone product, with peaks for an
acetyl group and four carbons. The NMR spectra are somewhat consistent with an expected
sydnone already known in the literature, but the numbers are different from the literature values.
The peak positions of this product are not identical, which leads to the conclusion that this is
indeed a sydnone derivative, but with a different structure, given in each of the spectra above.
This particular synthesis produced crystals that were used for X-ray crystallography and allowed
for an actual structure, as shown below:
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Figure 15: X-ray structure for MeCOC2N3O3K2 with associated ions and bond distances.
Figure 15 allows a look at the overall structure of the compound and the associated ions and
water. The bond distances given suggest neither single nor double bond, but rather delocalization
around the ring. This compound was not found in the literature, meaning the structure was
previously unknown. Based on the delocalized bonds, it can be suggested that aromaticity drives
the condensation of this compound. UV-Visible and NMR data for the ethyl derivative of this
sydnone compound is also given below:
11
Figure 16: UV-Visible spectra for EtCOC2N3O3K2 with associated structure, with extra peak
beyond the basic literature sydnone structure.
Figure 17: H1 NMR for EtCOC2N3O3K2 with expected triplet and quartet and 1.1 and 2.75 ppm
for ethyl indicator. Spectra also has peaks consistent with methanol impurity.
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Figure 18: C13 NMR spectrum for EtCOC2N3O3K2 with five carbon peaks for the carbons
present in the structure.
Figures 16-18 are all associated with the ethyl derivative of the sydnone product, indicating the
ethyl groups as well as the added nitrogen in the sydnone ring. Based on these spectra, the
associated structure can be confirmed. Due to the dimeric addition of nitric oxide (NO), the
mechanism below is associated with the resulting synthesis:
13
Figure 19: The synthesis of the sydnone products potassium 4-acetyltriazolium-1,2,3-triolate and
potassium 4-propionyltriazolium-1,2,3-triolate under the direct and immediate addition of
potassium hydroxide and nitric oxide. Mechanism shows dimeric addition of NO and
condensation.
Figure 19 shows the reaction mechanism for immediate addition of potassium hydroxide and
nitric oxide. It was concluded that the potassium had no time to react with the acidic CH2 group
before the NO reacted with the electron rich nitrogen. After reacting with nitrogen, the reaction
condenses at the acidic CH2, and then reacts with the remaining potassium hydroxide to release
methanol.
Conclusion
All three current products were formed and characterized by UV-Visible and NMR
spectroscopy. Only one of the products allowed the use of X-ray crystallography for
characterization purposes given the crystals produced. The synthesis was successful in creating
NO containing compounds for possible future physiological purposes. Further research for these
compounds should focus on EPR (electron paramagnetic resonance) by creating a nitric oxide
trap and testing the release of NO under physiological conditions in order to confirm if these
products can be used in a medical application. TGA/DSC (Thermogravimetric and Differential
Scanning Calorimetry) should also be included in research in order to test the thermal
decomposition properties and produce an acceptable decomposition mechanism in order to
determine if these products can be explosive or used in a safe and controlled setting. This
synthesis produced 3-4 viable candidates for the use of NO donor pro-drugs, with the products
being potassium 3-ethyl-5-isoxazolone-4-diazeniumdiolate (R = Et), potassium 4acetyltriazolium-1,2,3-triolate (R=Me), and potassium 4-acetyltriazolium-1,2,3-triolate (R=Et).
These diazeniumdiolate and sydnone-N-oxide products can be further characterized and tested,
as well as the methyl derivative of the diazeniumdiolate. More detailed research can be done on
the yield of each of these products under the given conditions, as expected yields were not
calculated due to the nature of this synthesis and the goal of simply creating a viable product.
Acknowledgements
I would like to acknowledge the Honors Program of the University of Wyoming and
allowing this opportunity to conduct research for my undergraduate experience, as well as the
Faculty Grant-In Aid provided. I would like to thank my research advisor, Dr. Navamoney
Arulsamy, for his mentorship and guidance.
14
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