Synthesis of New Nitroxide for use as ORCA in MRI

Elise Ackerman Author

08/04/2020 Added

23 Plays

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This project investigates a synthetic pathway for a new nitroxide radical compound. This compound has potential for use as an MRI contrast agent in order to eliminate the need for metal ions.

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[00:00:00.000]Hello, my name is Elise Ackerman.
[00:00:02.699]I am a senior Chemistry major at the University of Nebraska-Lincoln
[00:00:06.606]and this summer I worked with the Rajca Research group at UNL
[00:00:10.003]to explore synthesis of a new spirocyclohexyl nitroxide
[00:00:13.463]for use as an Organic Radical Contrast Agent in MRI
[00:00:17.157]Magnetic Resonance Imaging typically utilizes Gadolinium-based contrast agents
[00:00:22.705]which introduce a high risk for individuals with kidney disorders
[00:00:26.412]The use of Organic Radical Contrast Agents (ORCAs)
[00:00:29.946]in MRI eliminates the need for Gadolinium-based contrast agents,
[00:00:33.509]thus mitigating any risks of metal toxicity.
[00:00:36.620]The stability of nitroxides
[00:00:38.940]makes them excellent candidates for study as ORCAs.
[00:00:41.634]Nitroxide compounds themselves, however, have a low water relaxivity,
[00:00:45.723]a low water solubility,
[00:00:47.425]and a higher rate of reduction by ascorbate in the bloodstream.
[00:00:50.499]Recent study has introduced an ORCA
[00:00:53.824]consisting of a dendrimer conjugated with
[00:00:55.846]5-membered spirocyclohexyl nitroxides
[00:00:58.348]and polyethylene glycol.
[00:01:00.297]This ORCA exhibits a higher water relaxivity,
[00:01:03.630]a higher water solubility,
[00:01:05.234]and a lower rate of reduction by ascorbate (Vitamin C) in the bloodstream.
[00:01:09.664]Although such advancements have been made,
[00:01:12.011]present research seeks to further increase
[00:01:14.373]water relaxivity and water solubility
[00:01:16.652]within this class of ORCAs.
[00:01:18.256]This project investigates the synthesis of
[00:01:20.767]a novel 5-membered spirocyclohexyl nitroxide
[00:01:23.701]with the potential to further increase
[00:01:25.640]the water solubility and water relaxivity
[00:01:27.653]of the PEGylated dendrimer ORCA.
[00:01:30.187]A synthetic pathway to this new nitroxide
[00:01:32.987]starting from 2,2,6,6-tetramethylpiperidinone-4,
[00:01:37.334]is being evaluated.
[00:01:38.899]Again, the current goal in this field of research
[00:01:42.025]is to develop a nitroxide radical
[00:01:43.983]with a high water solubility,
[00:01:45.331]a high water relaxivity,
[00:01:47.196]and a low rate of reduction by biological
[00:01:49.714]agents like ascorbate.
[00:01:50.957]The nitroxide being synthesized
[00:01:53.064]in this project,
[00:01:53.837]which is shown in Figure 1, contains
[00:01:56.116]several structural characteristics that might
[00:01:58.243]enable accomplishment of these goals.
[00:02:00.020]First, the nitroxide is located in
[00:02:03.187]a five-membered ring.
[00:02:04.349]Five-membered rings have been shown
[00:02:06.147]to resist reduction by ascorbate.
[00:02:08.012]And this is due to the nitrogen atom's
[00:02:10.009]sp2 hybridization.
[00:02:11.423]When the nitroxide is reduced by ascorbate,
[00:02:13.740]the nitrogen's hybridization will change
[00:02:15.831]from sp2 to sp3.
[00:02:17.366]In a 6-membered ring, this change
[00:02:19.603]in hybridization will allow the ring
[00:02:21.337]to adopt a stable chair conformation.
[00:02:23.452]Reduction is thus favored.
[00:02:25.316]In a five-membered ring, changing nitrogen's
[00:02:28.050]hybridization to sp3 will serve to only
[00:02:30.327]increase torsional strain,
[00:02:31.570]thus making reduction unfavorable.
[00:02:34.279]Second, the nitroxide is additionally
[00:02:37.935]protected from reduction by ascorbate
[00:02:40.161]by the presence of spirocyclic groups
[00:02:42.396]at the 2 and 5 position of the 5-membered ring.
[00:02:45.362]These bulky substituents shield the nitroxide,
[00:02:48.464]further preventing reduction.
[00:02:49.789]Finally, the presence of oxygen heteroatoms
[00:02:54.545]on the spirocyclic substituents
[00:02:56.334]increases the nitroxide's
[00:02:57.523]overall polarity.
[00:02:58.537]This will increase water solubility,
[00:03:00.735]and it might result in water molecules
[00:03:02.761]having increased access to the nitroxide radical,
[00:03:05.138]which then will increase water relaxivity.
[00:03:07.626]The methods for this experiment follow
[00:03:11.466]the synthetic scheme shown in Figure 2.
[00:03:13.514]This summer's research focused on
[00:03:15.514]synthesizing Compounds 1, 2, and 3
[00:03:17.929]in the synthetic pathway.
[00:03:19.201]Compound 1 was synthesized according to
[00:03:21.981]literature procedures from 2,2,6,6,tetramethylpiperidinone-4,
[00:03:26.035]which is commercially available.
[00:03:28.243]The desired product was purified using
[00:03:30.153]fractional distillation at 72 C and 2 mm Hg,
[00:03:34.849]and silica gel column chromatography
[00:03:36.986]using a gradient of ethyl acetate and pentane
[00:03:39.932]as the eluent.
[00:03:40.608]The ethyl acetate and pentane ratios
[00:03:43.051]used in this experiment
[00:03:44.735]were 1:3, 1:2, and 1:1.
[00:03:47.259]Compound 2 was synthesized according to
[00:03:50.603]literature procedures from Compound 1.
[00:03:52.539]Literature procedures were modified slightly,
[00:03:55.144]substituting cyclohexanone with the
[00:03:57.464]heterocyclic ketone shown in Figure 2.
[00:03:59.934]The target compound was then isolated
[00:04:02.886]using silica gel column chromatography
[00:04:04.955]using this time dicholoromethane and ethanol
[00:04:07.585]in a 20:1 ratio as the eluent,
[00:04:09.853]and further purified via recrystallization
[00:04:12.167]in ethyl acetate.
[00:04:13.270]Although characterization of Compound 3
[00:04:15.944]is still in progress,
[00:04:17.617]this reaction was carried out again
[00:04:19.297]according to literature procedures.
[00:04:20.655]Compound 2 was dissolved in glacial acetic acid
[00:04:23.773]and then a mixture of bromine and acetic acid
[00:04:26.352]was added dropwise while keeping
[00:04:28.005]the reaction mixture at a constant temperature
[00:04:30.024]using a cool water bath.
[00:04:31.265]During this step, the speed of dropwise
[00:04:34.541]addition of the bromine solution
[00:04:35.930]proved to be a very important step
[00:04:37.630]in the reaction's success.
[00:04:39.110]Analysis of early trials in which
[00:04:41.325]the solution was added quickly showed that
[00:04:43.053]the product was likely not produced.
[00:04:44.807]In the most promising run of this reaction,
[00:04:47.105]however, the bromine solution was added
[00:04:48.892]at a rate of 0.33 mL/min.
[00:04:52.096]The suspected product was then isolated
[00:04:54.643]using a preparative plate
[00:04:55.977]with ethyl acetate and pentane in a 3:1 ratio
[00:04:59.555]as the eluent.
[00:05:00.646]In this project, reaction products
[00:05:04.576]once synthesized, were identified using
[00:05:06.796]nuclear magnetic resonance spectroscopy (NMR)
[00:05:08.903]Figure 3 shows the proton NMR spectrum
[00:05:12.690]of Compound 1 after purification
[00:05:14.647]via column chromatography.
[00:05:16.087]NMR spectra were available
[00:05:18.875]in previously published literature,
[00:05:20.454]and the presence of Compound 1 was confirmed
[00:05:22.789]by comparison to these spectra.
[00:05:24.390]Figure 4 shows the proton NMR spectrum
[00:05:27.977]for Compound 2 after purification
[00:05:29.906]via recrystallization.
[00:05:31.268]NMR spectra for this compound were also
[00:05:33.950]available in previous literature,
[00:05:35.573]and so the presence of Compound 2
[00:05:37.668]was again confirmed via comparison
[00:05:39.481]to published spectra.
[00:05:40.499]Figure 5 shows the proton NMR
[00:05:43.166]for an isolated reaction product
[00:05:44.892]that's suspected to be Compound 3.
[00:05:46.880]Since alpha-monobromination would
[00:05:49.113]cause many protons to become diastereotopic,
[00:05:52.056]the high number of signals shown on this
[00:05:54.213]spectrum is promising.
[00:05:55.747]Future work on this project during the 2020 Academic year
[00:06:00.393]will involve characterization of
[00:06:01.950]Compound 3.
[00:06:02.738]In similar reactions, the monobrominated
[00:06:05.821]product was easily isolated from solution
[00:06:07.469]via filtration, however, when these
[00:06:09.579]procedures were repeated,
[00:06:10.816]proton NMR analysis of the filtered solid
[00:06:13.588]revealed that there was only starting material.
[00:06:15.882]Thus, present efforts focus on extracting
[00:06:18.931]Compound 3 from the filtrate
[00:06:20.807]and identifying it using mass spectrometry,
[00:06:23.228]proton NMR, and infrared spectroscopy.
[00:06:26.321]Once Compound 3 has been successfully
[00:06:30.226]identified, the first three reactions
[00:06:31.766]will be repeated on larger scales
[00:06:33.546]to generate sufficient amounts of Compound 3
[00:06:36.239]in order to proceed to further reactions.
[00:06:38.170]Special thanks in this project to
[00:06:40.914]Shuyang Zhang, the Doctors Rajca,
[00:06:43.630]the Rajca Research Group,
[00:06:45.092]and the UNL Chemistry Department
[00:06:46.762]for continuing instruction and support
[00:06:48.844]on this project.
[00:06:49.745]Additionally, thanks to the National
[00:06:51.883]Institutes of Health for support of this research.

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Synthesis of New Nitroxide for use as ORCA in MRI

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