otz

1. Synthesis of C-OTZ, a C-Cysteine Prodrug for Magnetic
13 13
Resonance Imaging Studies of Drug Uptake and Conversion to
Glutathione in Rat Brain L-cysteine
Figure 3. IR-Spectra of L-
Arif Hussain, and Susan M. Ludeman 1
cysteine,13-OTZ (trial 1), and
1
Albany College of Pharmacy and Health Sciences, Albany, NY 12208 and 13-OTZ (trial 2)
2
North Carolina State University, Raleigh, NC 27695
13-OTZ trial 2
Abstract Figure 2. Synthesis of [13C]-OTZ prodrug
(13C-Cys was used as synthetic precursor to prodrug)
Purpose: The effects of neurodegeneration have been linked to inefficient
detoxification of free radicals due to lowered concentrations of antioxidants, HS * S *
especially glutathione, in the brain. In the biosynthesis of glutathione, cysteine triphosgene
OH OH
concentration is generally the limiting factor. Despite the common contention that H2N O N 13-OTZ trial 1
NaOH H O
antioxidants such as cysteine or glutathione can be taken as oral supplements, O
the ability of these compounds to affect cognitive health is limited due to their [3-13C]-L-Cysteine (13C)- 2
inefficient transport across the blood brain barrier. It was hypothesized that
( * = 13C ) ( 13C-OTZ )
therapeutic increases in intracephalic glutathione concentrations would be
achieved using prodrugs that efficiently cross the blood brain barrier and
metabolize to glutathione. One such compound, 4(R)-[5-13C]- 2-oxothiazolidine-4-
carboxylic acid ([13C]-OTZ), was synthesized using a 13C label for its use with 13C Scheme 2 Synthesis of (4R)-[5-13C]-2-oxothiazolidine-4-carboxylic acid [(13C)-2].
nuclear magnetic resonance (NMR) spectroscopy to monitor in vivo absorption
and metabolism of OTZ to cysteine and glutathione in rat brain.
Methods: L-[3-13C]-Cysteine and triphosgene were reacted under conditions Introduction
which had been optimized with unlabeled materials. The crude product was
The antioxidant glutathione (GSH) is a tripeptide produced from the amino
purified with flash chromatography and material was isolated. Isolated material
acids glutamate, cysteine, and glycine using the enzymes γ-glutamylcysteine
was analyzed with an IR-spectrum along with a melting point comparison to
synthetase and glutathione synthetase (Figure 1). Glutamate and glycine are
determine chemical purity. Impure material was taken up in methanol and filtered
naturally abundant in most tissues, whereas cysteine has a limited supply
to rid of impurities.
which puts a restriction on the amount of glutathione produced.1 GSH and
Figure 4. In Vivo 13C Magnetic Resonance Spectra of
cysteine do not possess the ability to cross the blood brain barrier.2 Injected OTZ in Fat, Brain, and Liver Tissues
Results: The experiment produced product yields of 68-79% [13C]-OTZ. The
Therefore, there is a need for a prodrug .
melting point of the final product was consistent with literature values for optically Figure 4a. (A) A portion of the in vivo 13C magnetic resonance
pure (R)-OTZ. The IR-spectrashowed the product was free of impurities as the spectrum of signals originating from the brain of a rat before bolus
The OTZ prodrug (Figure 1) has a hydrophobic nature, allowing a successful injection of 13C-OTZ (bottom; t = 0:00) and after injection of 1100
expected [13C]-OTZ product had a pure spectrum.
transfer across the blood brain barrier. The OTZ compound has a chemical mg/kg 13C-OTZ. The times given to the right of the top six spectra
structure which was established to be a precursor to cysteine. The compound denote the time elapsed after the 13C-OTZ injection. (B) A portion of
Conclusions: The production of pure [13C]-OTZ and proper analysis of the product the high-resolution 13C magnetic resonance spectrum of the acid
was hypothesized to not only to efficiently cross the blood brain barrier, but the
was successful in that the melting point and IR-Spectra supported the data extract of brain tissue. Signals are detected from natural abundance
conversion to cysteine and eventually glutathione in the brain could be
properly. By successfully labeling OTZ with C-13, further studies were made metabolites and 13C-OTZ (34.2 ppm) and 13C-glutathione (26.5
monitored in vivo. ppm).
possible.
Figure 4b. (A) A portion of the in vivo 13C magnetic resonance
spectrum of signals originating from the brain of a rat after 20 h of
continuous infusion of a dose of 1300 mg/kg [13C]-OTZ. (B) The
Methods and Results natural abundance in vivo 13C magnetic resonance spectrum before
infusion. (C) A portion of the high-resolution 13C magnetic resonance
Synthesis of OTZ (Figure 2) spectrum of the acid extract of brain tissue from the same rat used in
Figure 1. Biosynthesis of Glutathione from OTZ Components of two previously published literature procedures for the synthesis of (A). (D) A portion of the high-resolution 13C magnetic resonance
unlabeled OTZ were modified to convert L‐[3‐13C]‐ cysteine and triphosgene into spectrum of the acid extract of liver tissue from the same rat used in
[13C ]‐OTZ (Figure 2).3,4 The reaction was run under basic conditions. The addition of (A) and spiked with a standard solution of [3-13C]-cysteine. In
triphosgene dissolved in p-dioxane to cooled L‐[3‐13C]‐cysteine in 1M NaOH was addition to OTZ and glutathione, resonances derived from other
OTZ-metabolites were identified: [3-13C]-lactate (21.0 ppm), [1-13C]-
done drop wise while stirring open to air. The reaction mixture was run overnight at taurine (48.5 ppm), [1-13C]-hypotaurine (56.5 ppm). The liver sample
room temperature and concentrated using rotary evaporation. The resultant solid shows a resonance from added [3-13C]-cysteine (26.1 ppm).
was dissolved in water. Silica gel was added and the mixture was lyophilized. A
slurry of the mixture dissolved in CH2Cl2 was made and flash chromatographed on a Figure 4c. A portion of the in vivo 13C magnetic resonance
silica gel column. A mixture of (94.5):(5):(0.5) CH2Cl2 :CH3OH:formic acid was used spectrum showing signals originating from the brain of a rat; (A)
before bolus injection of [13C]-OTZ; (B) 3 h post injection of 1100 mg/
to elute the product. Fractions containing product were identified (TLC), combined kg 13C-OTZ; (C) 7 h post injection of 13C-OTZ. Natural abundance
and concentrated (rotary evaporation) to give the final product as a white solid lipid is visible at 30 ppm and 13C-OTZ at 34.2 ppm.
which, in most cases was pure (IR, m.p). In at least one case, the product was TLC-
pure yet the IR depicted extraneous signals and the m.p was low(158-162 °C ). In
mentioned case, the product was dissolved in methanol and vacuum filtrated to
remove undissolved solute. This mixture was concentrated once again using rotary
Conclusion
evaporation to give the final product. [Trial 1: 0.47 g, 3.2 mmol, 79% yield, mp 167–
The project had two purposes: (1) to synthesize a cysteine prodrug which would cross
172 °C, IR-Spectrum: 1747cm-1 (ketone),. Trial 2: 0.41 g, 2.8mmol, 68% yield, mp
the blood brain barrier more efficiently than its parent and (2) to label the prodrug with
166-171 °C (lit. m.p. for optically pure, unlabeled OTZ: 171–172.5 °C)]. IR-Spectra: 13
C so that its conversion to glutathione and uptake in rat brains could be studied more
1740cm-1 (ketone), see Figure 3.
extensively. Previously, the amount of [13C]-OTZ made was limited and only allowed
Biosynthesis of
for preliminary NMR studies. Since it has been shown that the converison of OTZ to
glutathione in the brain by NMR can be observed, further studies can be initiated to
In vivo NMR Studies (Figure 4)
confirm and expand the project using the newly synthesized OTZ.
Biosynthetic pathway of glutathione and intracellular Preliminary in vivo uptake studies were conducted through collaboration (NC State
integration of OTZ into the reaction as a prodrug for University) using labeled material previously synthesized in our labs. These in vivo References
cysteine. [Entry points for other prodrugs are shown
13
C NMR investigations were conducted using both bolus as well as 20h infusion
administrations of [13C]-OTZ in rat brain1 (Figure 4). Following acquisition of the 3. Meister A, John and Wiley Sons, New York, 1989, pp. 1-48
as well, including an alternative cysteine precursor, 4. Zeevalk G, Manzino L, Sonsalla P, Bernard L, Exp. Neurol. 2007, 203, 512.
N-acetylcysteine (NAC)]. NMR data, animals were sacrificed and tissue extracts were examined (13C NMR). 5. Kaneko T, Shimokobe T, Ota Y, Toyokawa E, Inui T, Shiba T, Bull. Chem. Soc. Jpn 1964, 37,
242.
6. Falb E, Nudelman A, Hassner A, Syn. Comm. 1993, 23, 2839.
.
Acknowledgements
This work was supported in part by Public Health Service Grants R21 AG029994 (MPG/SML)
awarded by the National Institute on Aging and RO1 CA16783 (SML) awarded by the National
Cancer Institute (Department of Health and Human Services). The help of Dr. David Burz
(Department of Chemistry, SUNY Albany) in obtaining NMR spectra is greatly appreciated