Synthesis of New Nitroxide for use as ORCA in MRI

Elise Ackerman Author

04/05/2021 Added

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Synthesis of spirocyclohexyl nitroxide for potential use as MRI contrast agent possessing enhanced water relaxivity and water solubility.

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[00:00:01.050]Hello. My name is Elise Ackerman.
[00:00:03.180]I'm a senior chemistry major at the university of Nebraska Lincoln.
[00:00:07.260]And this academic year,
[00:00:08.400]I worked with the Rajca research group at UNL to explore synthesis of a new
[00:00:12.480]Spyrocyclohexyl nitroxide for use as an organic radical contrast
[00:00:17.040]agent in MRI. A vital component of medical analysis,
[00:00:21.360]magnetic resonance imaging,
[00:00:22.770]or MRI, depends on the magnetic qualities of subatomic particles in the human
[00:00:27.030]body. Each hydrogen in the body contains at its core,
[00:00:30.330]a single positively charged proton,
[00:00:32.820]which spins to generate its own magnetic field.
[00:00:35.610]This can be compared to a very small bar magnet.
[00:00:38.640]When an MRI scanner applies a strong magnetic field,
[00:00:41.670]these hydrogen magnets then align with the field. Radio-frequency energy
[00:00:46.560]waves can then be applied in pulses. Now,
[00:00:49.110]when the protons absorb energy from these waves,
[00:00:51.720]they become excited and their alignment with the external magnetic field is
[00:00:55.500]disrupted. When the radio waves are then removed,
[00:00:59.010]the protons gradually release this absorbed energy and they realign with the
[00:01:02.820]magnetic field in a process called "relaxation".
[00:01:06.120]The energy released from protons during relaxation is detected and used as a
[00:01:10.440]signal to generate an image.
[00:01:12.600]Contrast agents work to increase the resolution of MRI images by increasing that
[00:01:17.100]proton signal strength.
[00:01:18.840]These agents possess unpaired electrons resulting in a property called paramagnetism
[00:01:22.680]When paramagnetic compounds are placed in a magnetic field,
[00:01:27.090]they become magnetic and thus they increase the local strength of the magnetic
[00:01:30.900]field.
[00:01:31.710]Now this increased strength causes protons' rate of relaxation to increase,
[00:01:36.240]which then results in the release of a more intense signal to the MRI scanner.
[00:01:41.190]Currently MRI primarily uses Gadolinium based contrast agents,
[00:01:45.900]which introduce a high risk for individuals with kidney disorders.
[00:01:50.430]The use of organic radical contrast agents, or ORCAS, in MRI eliminates the
[00:01:55.260]need for these Gadolinium agents.
[00:01:57.180]Thus mitigating the risks of metal ion toxicity.
[00:02:01.020]The stability of a class of compounds, called nitroxides,
[00:02:04.500]make them excellent candidates for study as ORCAs. Nitroxide compounds
[00:02:08.640]themselves present a low enhancement of water's relaxation rate.
[00:02:12.510]This is also called 1H water relaxivity or r1.
[00:02:16.470]They also have low water solubility and a high rate of reduction by ascorbate
[00:02:20.340]in the bloodstream.
[00:02:21.570]These properties highlight the necessity of structural modification. Recent
[00:02:25.770]study has introduced an ORCA consisting of a PEGylated dendrimer conjugated
[00:02:30.060]with five membered spirocyclohexyl nitroxides. This ORCA exhibits
[00:02:34.230]a higher r1, higher water solubility, and a lower rate of reduction by ascorbate,
[00:02:38.700]or vitamin C, in the bloodstream.
[00:02:41.790]Although these advancements have been made, present research seeks to further
[00:02:45.600]increase r1 and water solubility within this class of nitroxide
[00:02:50.520]orcas. This project investigates the synthesis of a novel five membered
[00:02:55.290]spirocyclohexyl nitroxide with the potential to further increase the water
[00:02:59.980]solubility and r1 of the PEGylated dendrimer ORCA. And in this project,
[00:03:04.810]a synthetic pathway to one new nitroxide is being evaluated.
[00:03:08.620]The nitroxide being synthesized in this project,
[00:03:10.840]which is shown in figure two, contains several structural characteristics
[00:03:15.700]which may protect it from reduction by vitamin C in the bloodstream. First,
[00:03:20.620]the nitroxide is located in a five membered ring.
[00:03:23.590]Five membered rings have been shown to resist reduction by ascorbate.
[00:03:27.190]This is due to the nitrogen atom's sp2 hybridization.
[00:03:31.030]When the nitroxide reacts with vitamin C,
[00:03:33.790]the nitrogens hybridization will change from sp2 to sp3. In a
[00:03:38.550]six membered ring,
[00:03:39.900]this change and hybridization will allow the ring to adopt a more stable
[00:03:44.220]conformation. Reduction is thus favored. In a five membered ring, however,
[00:03:48.930]changing nitrogen's hybridization to sp3 will only serve to increase ring
[00:03:53.440]strain. This makes the reduction reaction unfavorable. Second,
[00:03:58.230]the nitroxide is additionally protected from reduction by ascorbate at the
[00:04:01.860]presence of bulky spirocyclic groups at the two and five position.
[00:04:06.840]These bulky groups shield the nitroxide, further preventing reduction.
[00:04:11.280]Finally,
[00:04:12.000]the presence of oxygen atoms on the spirocyclic rings increases the nitroxide's
[00:04:16.800]overall polarity. This will increase water solubility,
[00:04:20.040]and it might result in water molecules having increased access to the Nitroxide
[00:04:23.760]radical, which will increase water
[00:04:25.710]relaxivity. Methods for this experiment
[00:04:28.470]follow the synthetic schemes shown in figure one.
[00:04:31.950]This year's research focused on synthesizing compounds one,
[00:04:34.920]two and three in the synthetic pathway. Compound
[00:04:37.830]one was synthesized according to literature procedures from
[00:04:41.910]2,2,6,6-tetramethylpiperidinone-4, which is commercially available.
[00:04:46.350]The desired product was then purified using fractional distillation and column
[00:04:50.370]chromatography. Compound two was synthesized
[00:04:53.700]according to literature procedures using compound one. Literature procedures were
[00:04:58.470]modified slightly in this reaction substituting cyclohexanone with the
[00:05:02.880]heterocyclic ketone shown again in figure two,
[00:05:06.120]this compound was isolated using silica gel column chromatography and further
[00:05:10.410]purified via recrystallization in ethyl acetate.
[00:05:14.100]Although characterization of compound three is still in progress,
[00:05:17.130]this reaction was carried out according to the literature procedures.
[00:05:21.210]Compound two was dissolved in glacial acidic acid,
[00:05:23.880]then a mixture of bromine and acetic acid was added dropwise. During this
[00:05:27.630]step, the speed of dropwise addition of the bromine solution has proved to be an
[00:05:31.980]important step in the reaction's success.
[00:05:34.290]In the most promising runs of this reaction,
[00:05:36.270]The bromine solution was added slowly at a rate of approximately 0.33
[00:05:40.170]milliliters per minute.
[00:05:42.030]Reaction products were then identified using nuclear magnetic resonance
[00:05:45.840]spectroscopy. Figure three shows the proton NMR spectrum for compound one.
[00:05:51.030]NMR spectra for this compound were available in previously published
[00:05:54.090]literature.
[00:05:54.930]And so the presence of compound one was confirmed by comparison to these spectra.
[00:05:59.330]Figure
[00:05:59.630]Four shows the proton NMR spectrum from compound two. NMR spectra
[00:06:04.550]for this compound were also available in previous literature.
[00:06:07.400]And so the presence of compound two could be confirmed via comparison to published
[00:06:11.870]spectra.
[00:06:13.010]Future work in this project will involve synthesis and characterization of
[00:06:16.250]compound three. Analysis of the current products suggest the presence of
[00:06:20.470]di-brominated other than mono-brominated compounds.
[00:06:24.160]Thus, present efforts
[00:06:25.690]focus on optimizing reactant ratios and reaction conditions in order to
[00:06:30.460]obtain the desired monobrominated product. After the
[00:06:34.270]monobrominated product is successfully synthesized and isolated, further steps of
[00:06:38.460]the reaction will be continued. Special thanks to Shuyang Zhang,
[00:06:42.490]the Drs. Rajca,
[00:06:43.840]the Rajca research group, and the UNL chemistry department for continuing
[00:06:47.650]instruction and support on this project. Additionally,
[00:06:51.250]thanks to the national institutes of health for supporting this research.

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Synthesis of New Nitroxide for use as ORCA in MRI

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