Toward hyperpolarized molecular imaging of HIV: Synthesis and longitudinal relaxation properties of 15N-Azidothymidine

Article abstract of DOI:10.1002/jlcr.3220

Previously unreported ¹⁵N labeled Azidothymidine (AZT) was prepared as an equimolar mixture of two isotopomers: 1-¹⁵N-AZT and 3-¹⁵N-AZT. Polarization decay of ¹⁵N NMR signal was studied in high (9.4 T) and low (

Full text of DOI:10.1002/jlcr.3220

Short Note
Received 19 March 2014,
Revised 28 May 2014,
Accepted 30 June 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3220
Toward hyperpolarized molecular imaging of
HIV: synthesis and longitudinal relaxation
15
properties of N-Azidothymidine
a
a,b,c
*
*
Roman V. Shchepin and Eduard Y. Chekmenev
Previously unreported 15N labeled Azidothymidine (AZT) was prepared as an equimolar mixture of two isotopomers: 1-15N-AZT
and 3-15N-AZT. Polarization decay of 15N NMR signal was studied in high (9.4T) and low (~50mT) magnetic fields. 15N T1 values
were 45 5 s (1-15N-AZT) and 37 2 s (3-15N-AZT) at 9.4 T, and 140 16s (3-15N-AZT) at 50mT. 15N-AZT can be potentially 15
N
hyperpolarized by several methods. These sufficiently long 15N-AZT T1 values potentially enable hyperpolarized in vivo imaging
of 15N-AZT, because of the known favorable efficient (i.e., of the time scale shorter than the longest reported here 15N T1) kinetics
of uptake of injected AZT. Therefore, 3-15N-AZT can be potentially used for HIV molecular imaging using hyperpolarized
magnetic resonance imaging.
Keywords: hyperpolarization; contrast agent; azidothymidine; 15N; MRI; AZT; HIV; AIDS
isotopically enriched nuclei biochemically identical to the drug, for
Introduction
example, incorporation of 13C or 15N and (ii) contrast agent
There are approximately 35 million people with HIV worldwide
preparation step, for example, hyperpolarization that makes the
as of 20121 with 1.2 million of them living in the USA. This
enriched molecule activated and useful for molecular imaging.
disease has no cure or vaccine, and HIV/AIDS is managed by
Despite MRI sensitivity gains by orders of magnitude achieved by
hyperpolarization process,7,8 the ultimate limitation of HP approaches
administration of one or more antiretroviral drugs. The course
is still the detection sensitivity,9 which requires a human dose of
of management and the disease progression in monitored by
blood tests to measure the viral and CD4 cell counts in blood.
However, these tests repeated every 3–6 months do not directly
report on the viral load in other tissues such as lymph nodes,
hundreds of milligrams of imaging contrast agent. Therefore, the
use of HP molecular imaging using isotopically enriched treatment
drugs is limited to those injected in relatively large quantities.
Azidothymidine (AZT) or Zidovudine is a Food and Drug
spleen, or brain. Furthermore, when patient’s viral count is
reduced below the detection limit (<50 copies/mL),2 there is
Administration approved nucleoside analog reverse transcriptase
inhibitor, a type of antiretroviral medication.10 It is used for the
no gauge to measure the response to treatment and identify
more effective choice of treatment until the disease manifests
treatment of HIV/AIDS as well as for prevention of the infection
transmission such as mother to child during the period of birth.11 It
again as the elevated level of HIV virus in blood. Information
about disease progression beyond blood tests is critically
important for disease management, progression, response to
treatment, and development of new drugs and vaccines.
Molecular imaging significantly improved patients’ management
has been shown that AZT acts by competing with 3′-deoxythymidine-
5′-triphosphate, for incorporation into DNA by HIV-reverse
transcriptase.12 Incorporation of AZT monophosphate into DNA
by HIV-reverse transcriptase results in chain termination. Because
of its potency as well as the fact that AZT presently available as a
in cancer, where the elevated metabolism of cancer cells can be
imaged with radioactive and hyperpolarized (HP)3 contrast agents.
‘generic drug’, it is used in many combined formulations including
Combivir and Trizivir.13 Furthermore, AZT can be administered in
HIV/AIDS does not have clear metabolic signatures that can be
interrogated by F18-fluorodeoxyglucose positron emission
doses of ~1 g and therefore is a suitable candidate for HP imaging
probe, which can potentially allow for imaging of injected HP
tomography for distinguishing the disease progression.4 Therefore,
other molecular imaging probes are required to report on HIV
progression by targeting HIV/AIDS signatures. Multiple approaches
aDepartment of Radiology, Vanderbilt University Institute of Imaging Science
(VUIIS), Nashville, TN 37232, USA
of molecular imaging beyond metabolite (e.g., glucose, glutamine,
and choline) imaging are feasible including receptor imaging via
targeted positron emission tomography5 and HP molecular
magnetic resonance imaging (MRI)6 probes. An alternative
approach is to image the drug molecule itself. While this can be
bDepartment of Biomedical Engineering, Vanderbilt University, Nashville, TN
37235, USA
challenging if not impossible from the perspective of radioactive cDepartment of Biochemistry, Vanderbilt University, Nashville, TN 37205, USA
isotope incorporation due to sophisticated chemistry and short life
*Correspondence to: Roman V. Shchepin and Eduard Y. Chekmenev, Department
time of biomedically useful nuclei, emerging HP MRI offers clear
of Radiology, Vanderbilt University Institute of Imaging Science (VUIIS), Nashville,
TN 37232, USA.
preparation in two parts: (i) chemical synthesis of the molecule with E-mail: roman.shchepin@vanderbilt.edu; eduard.chekmenev@vanderbilt.edu
benefit of dividing the challenging task of contrast agent
J. Label Compd. Radiopharm 2014
Copyright © 2014 John Wiley & Sons, Ltd.

R. V. Shchepin and E. Y. Chekmenev
contrast agent that has good affinity to HIV’s reverse transcriptase12
and cell membrane permeability enabled by the azide moiety.
Azide group (N3) in the AZT molecular structure can be labeled
with 15N isotope, which can be used for hyperpolarization and
hyperpolarization storage during agent administration and uptake,
Figure 1. Several hyperpolarization approaches can be used for 15N-
AZT hyperpolarization. For instance, dynamic nuclear polarization,14
presently the most widespread hyperpolarization technique, can be
potentially employed similarly to hyperpolarization of other 15N
compounds including choline.15 Additionally, Signal Amplification
by Reversible Exchange16,17 method can provide a good opportunity
for 15N-AZT hyperpolarization, because the unsaturated nitrogens in
azide moiety can be potentially employed for reversible exchange.
Furthermore, parahydrogen induced polarization (PHIP)18,19 can be
also potentially applied, because AZT structure allows for the
incorporation of an unsaturated C¼C bond in the PHIP molecular
hyperpolarization precursor. Similar in complexity, 15N compounds
have been developed20 and 15N HP21 by PHIP. Regardless of
hyperpolarization approach to be applied, 15N isotopic enrichment
of AZT is required, which was successfully demonstrated here.
Moreover, the delivery of HP contrast agent, Figure 1, requires
sufficiently long lifetime of produced contrast agent. The 15N T1
relaxation properties of the produced 15N-AZT that were investigated
here provide critical information for future hyperpolarization and
imaging efforts, Figure 1.
the resulting suspension is chilled for 2 h. The white crystalline precipitate is
isolated by suction and washed with Et2O; yield: 4 g (74% yield).
3′-Azido-3′-deoxy-5′-O-(4-methoxybenzoyl)thymidine
2,3′-Anhydro-5′-O-(4-methoxybenzoyl)thymidine (1.00 g, 2.8 mmol) and
sodium azide-1-15N (273 mg, 4.2 mmol, 1.5 equiv.) are suspended in
DMF (10 mL), and the mixture is heated on an oil bath at 125 °C for 5 h.
The orange homogeneous mixture is poured in H2O (25 mL) containing
5% aq HCl (2.0 mL, 1 equiv.), EtOAc (15 mL) is added. The aqueous layer
is extracted with EtOAc (2 × 10 mL), and the organic extract is washed
with H2O (7 mL), then with brine (7 mL). After drying (Na2SO4), the
solvent is evaporated in vacuo to give 3′-azido-3′-deoxy-5′-O-(4-
methoxybenzoyl)thymidine-1-15N as a foamy solid; yield: 1 g (90%
nominal yield). Crude 3′-azido-3′-deoxythymidine: 3′-azido-3′-deoxy-5′-
O-(4-methoxybenzoyl)thymidine-1-15N (1.0 g, 2.5 mmol) is suspended in
dry MeOH (10 mL), 1 M NaOMe in MeOH (2.9 mL, 1.15 equiv.) is added,
and the mixture is stirred at r.t. overnight (12 h). H2O (15 mL) is added,
and MeOH is evaporated. The aqueous solution is extracted with Et2O
(2 × 10 mL), then Amberlite IRN-77 (H+ form, 1 g) is added, and the
mixture is stirred slowly at r.t. for 15 min. The resin is filtered, washed well
with H2O, and the filtrate is concentrated in vacuo to give a white glassy
solid: 3′-azido-3′-deoxythymidine-1-15N (15N-AZT) yield: 0.270 g (40%).
1H (D2O, 400 MHz) δ: 7.50 (d, 1H, J(HH) = 1.2 Hz), 6.05 (pseudo-t, 1H, J
(HH) = 6.5 Hz), 4.22 (dd, 1H, J(HH) = 6.4 Hz, and J(HH) = 12.0 Hz), 3.87 (m,
1H), 3.73 and 3.63 (dAB, 2 H, J(HH)gem = 12.6 Hz, J(HH)vic = 4.6 Hz), 2.35
(superposition of normal triplet (3-15N-AZT), 1H, J(HH) = 6.5 Hz and
doublet of triplets (1-15N-AZT), 1H, J(HH) = 6.5 Hz and J(H-15N) = 2.6 Hz)
1.74 (d, 3H, J(HH) = 1.1); 13C{1H} (D2O The spectrum was referenced
externally to chloroform-d (77 ppm), 100 MHz) δ: 166.7, 151.7, 137.2,
111.1, 84.7, 83.9, 60.7, 59.6, 35.8, 11.4; 15N{1H} (D2O, The spectrum was
referenced externally to 15N-urea, 76 ppm) 214.9, 75.9 (J(H-15N) = 2.6 Hz).
HR-MS calculated for (M-1): 267.0865; found: 267.0864 (0.4 ppm).
Experimental methods
General
All solvents were purchased from common vendors and were used as
received. New AZT isotopomer was characterized by 1H, 13C, 15N NMR
(Bruker 400 MHz), and high-resolution mass spectrometry (HR-MS). HR
mass spectra were recorded in negative mode using a Synapt hybrid
quadrupole/oa-TOF mass spectrometer (Waters Corp., Milford, MA)
equipped with a dual chemical ionization/electrospray (ESCI) source.
A post-acquisition gain correction was applied using a solution of
3-[(3-holamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS,
FW 614.88) as the lock mass spray.
15N T1 NMR Measurements at 9.4 T. Nuclear magnetic resonance
sample was prepared by dissolving 15N-AZT in 0.50 mL of D2O at the
maximum concentration (~20 mg/mL at 25°C). T1 inversion recovery pulse
sequence (as supplied by Bruker Biospin) was used to measure 15N T1 at
9.4 T. 15N NMR signals of the two 15N sites were integrated (using the same
scale for all the spectra) in resulting set of 1D spectra with varied inversion
recovery delay, Figure 2b and 2c. Mono-experiential decay fitting model
(y = C•exp(ꢀx/T1) + y0) was used for each 15N resonance time series data.
15N T1 NMR Measurements at 50 mT. The aforementioned NMR
15N-AZT preparation
sample was used in the field-cycling 15N T1 experiment, where the NMR
15N-AZT was prepared similar to Czernecki procedure,22 where sodium sample was first polarized inside 400 MHz NMR spectrometer for
azide-1-15N (Sigma-Aldrich, 609374) was used in place of natural isotopic ~5 min, which significantly exceeds T1 at 9.4 T. Then, the sample was
abundance lithium azide. No recrystallization of final product was
performed because of the small synthetic scale.
lifted from 9.4 T magnetic field, and it was exposed to the low fringe field
(B0 = 50 5 mT) of the main NMR magnet. Finally, the sample was quickly
placed back inside the 400 MHz NMR spectrometer, and a single scan 15
N
22
2,3′-Anhydro-5′-O-(4-methoxybenzoyl)thymidine
NMR spectrum was collected without any additional delay. This cycling
operation was repeated with variable low-field sample exposure. Integral
values of 3-15N signal were plotted versus time spent in the low magnetic
field. Mono-experiential decay fitting model (y = C•exp(ꢀx/T1) + y0) was
used for 3-15N resonance time series data. All fitting and error analyses
were performed using Microcal Origin software.
Thymidine (3.63 g, 15 mmol) and Ph3P (5.9 g, 22.5 mmol, 1.5 equiv.)
are dissolved in dimethylformamide (DMF) (30 mL), and the solution
cooled to 15 °C on a water bath. To this stirred mixture, a solution
of diisopropyl azodicarboxylate (4.4mL, 22.5mmol, 1.5 equiv.) in DMF
(7 mL) and 4-methoxybenzoic acid (3.42 g, 22.5 mmol, 1.5 equiv.) is added
drop-by-drop, and stirring is continued at room temperature (r. t.) for
15min. The same quantity of Ph3P and diisopropyl azodicarboxylate is then
added. After 30 min. at r. t., the mixture is poured into Et2O (370mL), and
Results and discussion
In Czernecki procedure22 (Figure 2a), thymidine is protected with
p-methoxybenzyl ether with inversion of 3′-OH center. Lithium azide
with natural isotopic abundance was replaced by sodium 1-15N-azide
substitution step (Figure 2a, II). Because of the double inversion overall
stereochemistry of AZT is the same as that of thymidine. Because
symmetric azide anion was labeled only on one of two ends, the
reaction sequence produced equimolar mixture of two isotopomers
1-15N-AZT and 3-15N-AZT (Figure 2a) in excellent isotopic purity
and satisfactory overall purity (Figures S1–S4).
Figure 1. Overall approach of development of hyperpolarized contrast agents for
molecular imaging.
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Copyright © 2014 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014

R. V. Shchepin and E. Y. Chekmenev
a)
O
N
O
N
O
N
O
N
HN
HN
HN
N
O
O
O
O
HO
PMBO
I
HO
III
PMBO
II
O
O
O
O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
N3
H
H
OH
H
N3
H
15N Peak at 75.9 ppm 1-15N-AZT
15N Peak at 214.9 ppm 3-15N-AZT
15N N
N
50%
N3
=
N N 15N
50%
b)
c)
d)
1-15N-AZT
T1= 45 8s
at 9.4 T
3-15N-AZT
T1= 37 2s
at 9.4 T
3-15N-AZT
T1= 140 16s
at 50 5 mT
6
6
2
3
2
1
0
2
-2
-6
-2
-6
-10
-10
0
40
80
120
0
40
80
120
50
150 250 350 450
Decay Time
(seconds)
Decay Time
(seconds)
Decay Time
(seconds)
Figure 2. (a) 15N-AZT preparation: (I) DIAD (1.5 eq)/Ph3P (1.5 eq), 4-MeOC6H4CO2H (1.5 eq) DMF, DIAD (1.5 eq)/Ph3P (1.5 eq); (II) Sodium 1-15N-azide, DMF; (III) MeONa /
MeOH. (b) Polarization recovery of 15N resonance at 75.9 ppm (1-15N-AZT) at 9.4 T. (c) Polarization recovery of 15N resonance at 214.9 ppm (3-15NAZT) at 9.4 T. (d)
Polarization decay of 15N resonance at 214.9 ppm measured by sample exposure to 50 5 mT magnetic field.
Preparation of 15N-AZT enabled measurement of the background can be attenuated by orders of magnitude in low-
polarization T1 decay of two 15N sites, which is a critical field MRI <0.1 T.26
parameter determining the feasibility of HP imaging and in vivo
The previous work in cellular model31 demonstrated rapid
applications, Figure 1. 15N T1 values were 45 5 s for 1-15N-AZT uptake of AZT by human cell with nearly linear intracellular AZT
(15N peak at 75.9 ppm) and 37 2 s for 3-15N-AZT (15N peak at uptake during first 20 s with peak plateau achieved in less than
214.9 ppm), respectively, at 9.4 T, Figure 2. 15N T1 values were 100 s, which is certainly within 1 × T1 of 15N spin label that is
140 16 s for 3-15N-AZT at 50 mT. 15N T1 values measured at potentially amenable to hyperpolarization. We point out that HP
9.4 T (35–50 s) were significantly shorter than that at 50 mT, compounds are useful for imaging within (1ꢀ 3) × T1 period after
Figure 2. This difference is likely explained by differential their in vivo administration.9 Moreover, this uptake kinetics holds
contribution of 15N chemical shift anisotropy (CSA) at high and true even at high extracellular AZT concentration up to 50 mM.31
low magnetic fields, because 15N CSA over 700 ppm The latter is well below plasma AZT concentration of treated patients
(corresponding to ~30 kHz at this magnetic field) is not with subgram dose. While it remains to be seen (i) if sufficient in vivo
uncommon.23 When the magnetic field is reduced to 50 mT, signal can be detected with HP AZT and (ii) if the in vivo viral uptake
the CSA contribution is negligible, because CSA scales linearly will be sufficient to generate imaging contrast, the kinetics of
with magnetic field, it is reduced by ~200-fold in units of Hertz. hyperpolarization decay (this work) and cellular uptake31 are
15N T1 decay of 1-15N-AZT in the low magnetic field was very certainly promising to support such future studies.
rapid, and T1 was very challenging to quantify, which is related
to the relatively slow speed of NMR sample lifting from the
Conclusions
NMR spectrometer. A possible explanation of significantly
shorter low-field 1-15N-AZT T1 is an accelerated relaxation due Synthetic preparation of 15N analog of antiviral drug AZT has
to the neighboring protons of the five-membered ring.
been demonstrated. While both 15N-AZT isotopomers have
While the high-field 15N T1 values may be sufficient for the sufficiently long high-field 15N T1 (~35–50 s) for potential use in
preparation of HP AZT and its 15N in vivo imaging24, low-field future pre-clinical and clinical studies, 3-15N-AZT is particularly
detection would be clearly advantageous, because of the nearly promising, because it has an ultra-long T1 in excess of 2 min at
threefold increase in T1. It should be noted that low-field HP low magnetic field. This makes 3-15N-AZT a promising candidate
imaging can be more sensitive than high-field HP imaging in for future HP imaging efforts for molecular imaging of HIV/AIDS.
general,25,26 and low-field MRI of HP AZT uptake would be clearly
attractive. Furthermore, the detection sensitivity of HP 15N sites
Acknowledgements
can be further amplified by indirect proton detection of HP
contrast agents.27,28 While the latter is challenging (but certainly
We gratefully acknowledge the financial support from NIH/NCI 5R00
CA134749-03, 3R00CA134749-02S1 and Department of Defense
feasible) in high-field MRI applications, because water protons
generate large background signal,29,30 the water proton CDMRP Era of Hope Award W81XWH-12-1-0159/BC112431.
J. Label Compd. Radiopharm 2014
Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org

R. V. Shchepin and E. Y. Chekmenev
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Conflict of Interest
The authors did not report any conflict of interest.
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Supporting Information
Additional supporting information may be found in the online
version of this article at the publisher’s web-site.
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