15N NMR Hyperpolarization of Radiosensitizing Antibiotic Nimorazole by Reversible Parahydrogen Exchange in Microtesla Magnetic Fields

Article abstract of DOI:10.1002/anie.202011698

Nimorazole belongs to the imidazole-based family of antibiotics to fight against anaerobic bacteria. Moreover, nimorazole is now in Phase 3 clinical trial in Europe for potential use as a hypoxia radiosensitizer for treatment of head and neck cancers. We envision the use of [¹⁵N₃]nimorazole as a theragnostic hypoxia contrast agent that can be potentially deployed in the next-generation MRI-LINAC systems. Herein, we report the first steps to create long-lasting (for tens of minutes) hyperpolarized state on three ¹⁵N sites of [¹⁵N₃]nimorazole with T₁ of up to ca. 6 minutes. The nuclear spin polarization was boosted by ca. 67000-fold at 1.4 T (corresponding to P_15N of 3.2 %) by ¹⁵N?¹⁵N spin-relayed SABRE-SHEATH hyperpolarization technique, relying on simultaneous exchange of [¹⁵N₃]nimorazole and parahydrogen on polarization transfer Ir-IMes catalyst. The presented results pave the way to efficient spin-relayed SABRE-SHEATH hyperpolarization of a wide range of imidazole-based antibiotics and chemotherapeutics.

Full text of DOI:10.1002/anie.202011698

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
International Edition Chemie
www.angewandte.org
Accepted Article
Title: 15N NMR Hyperpolarization of Radiosensitizing Antibiotic
Nimorazole via Reversible Parahydrogen Exchange in Microtesla
Magnetic Fields
Authors: Oleg G Salnikov, Nikita V Chukanov, Alexandra Svyatova,
Ivan A Trofimov, Mohammad S. H. Kabir, Juri G Gelovani,
Kirill V Kovtunov, Igor V Koptyug, and Eduard Y Chekmenev
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202011698
Link to VoR: https://doi.org/10.1002/anie.202011698

Angewandte Chemie International Edition
10.1002/anie.202011698
RESEARCH ARTICLE
1
5
N NMR Hyperpolarization of Radiosensitizing Antibiotic
Nimorazole via Reversible Parahydrogen Exchange in Microtesla
Magnetic Fields
[
a,b,c]
[b,c]
[b,c]
[b,c]
Oleg G. Salnikov,*
Nikita V. Chukanov,
Alexandra Svyatova,
Ivan A. Trofimov,
Mohammad
[
d]
[d,e]
[b,c]
[b,c]
S. H. Kabir, Juri G. Gelovani,
Kirill V. Kovtunov,
Igor V. Koptyug,
and Eduard Y.
[
d,f]
Chekmenev*
[
[
a]
b]
Dr. O. G. Salnikov
Boreskov Institute of Catalysis SB RAS
5
Acad. Lavrentiev Pr., 630090 Novosibirsk, Russia
Dr. O. G. Salnikov, Dr. N. V. Chukanov, A. Svyatova, I. A. Trofimov, Dr. K. V. Kovtunov, Prof. I. V. Koptyug
International Tomography Center SB RAS
3
A Institutskaya St., 630090 Novosibirsk, Russia
E-mail: salnikov@tomo.nsc.ru
[
c]
Dr. O. G. Salnikov, Dr. N. V. Chukanov, A. Svyatova, I. A. Trofimov, Dr. K. V. Kovtunov, Prof. I. V. Koptyug
Department of Natural Sciences
Novosibirsk State University
2
Pirogova St., 630090 Novosibirsk, Russia
[
d]
M. S. H. Kabir, Prof. J. G. Gelovani, Prof. E. Y. Chekmenev
Department of Chemistry, Integrative Biosciences (Ibio), Karmanos Cancer Institute (KCI),
Wayne State University, Detroit, Michigan 48202, United States
E-mail: chekmenevlab@gmail.com
[
[
e]
f]
Prof. J. G. Gelovani
College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, UAE
Prof. E. Y. Chekmenev
Russian Academy of Sciences (RAS)
1
4 Leninskiy Prospekt, 119991 Moscow, Russia
Supporting information for this article is given via a link at the end of the document.
Abstract: Nimorazole belongs to the imidazole-based family of
antibiotics to fight against anaerobic bacteria. Moreover, nimorazole
is now in Phase 3 clinical trial in Europe for potential use as a
hypoxia radiosensitizer for treatment of head and neck cancers. We
imaging of dilute metabolites is a grand challenge despite
potential benefits of providing rich metabolic information, which
is central to diagnosis of diseases with aberrant metabolism
such as cancer, diabetes, etc. NMR hyperpolarization
techniques allow transiently increasing P up to the order of
1
5
3
envision the use of [ N ]nimorazole as a theragnostic hypoxia
[
1, 2]
contrast agent that can be potentially deployed in the next-
generation MRI-LINAC systems. Here, we report the first but very
important steps to create long-lasting (for tens of minutes)
unity.
This massive increase in sensitivity gains by 4-5 orders
of magnitude can be employed for metabolic imaging of
hyperpolarized (HP) compounds, which are typically injected
1
5
15
[3-5] 13
13
hyperpolarized state on three N sites of [ N
3
]nimorazole with T
1
of
and act as metabolic contrast agents.
C-HP [1- C]pyruvate
up to ~6 minutes. The nuclear spin polarization was boosted by
is the most notable metabolite that has been developed so far to
probe upregulated glycolysis in cancer and many other
1
5
15
~
67000-fold at 1.4 T (corresponding to P15N of 3.2%) via N- N
[
6-11]
spin-relayed SABRE-SHEATH hyperpolarization technique relying
diseases.
Numerous clinical trials are now enabled because
1
5
on simultaneous exchange of [ N
3
]nimorazole and parahydrogen on
access to this HP contrast agent became possible via
[
12-14]
polarization transfer Ir-IMes catalyst. The isomeric side product of
dissolution Dynamic Nuclear Polarization (d-DNP).
Despite
over the recent years, d-
DNP has many shortcomings of high equipment- (>$2M) and
1
5
[15-17]
[
N
3
]nimorazole synthesis was hyperpolarized too. The presented
many technology breakthroughs
results pave the way to efficient spin-relayed SABRE-SHEATH
hyperpolarization of a wide range of imidazole-based antibiotics and
chemotherapeutics.
[
12]
operational costs, and long polarization times.
For example,
hyperpolarization of N sites has been a main challenge for
1
5
1
15
conventional d-DNP requiring custom H® N cross-polarization
hardware to achieve efficient hyperpolarization with
number of alternative hyperpolarization
techniques are now under development to address d-DNP
[
18]
P
15N>15%.
A
Introduction
[
1, 19]
limitations.
Signal Amplification by Reversible Exchange (SABRE)
technique relies on the simultaneous chemical exchange of
parahydrogen (p-H ) and to-be-hyperpolarized biomolecule on a
Equilibrium nuclear spin polarization (P) of key biologically-
[20-22]
1
13
15
-6
-
relevant nuclei such as H, C and N is on the order of 10 -10
5
at clinically relevant conditions of body temperature and the
magnetic field of up to 3 T. Since signal-to-noise ratio (SNR) is
directly proportional to P in magnetic resonance (MR), NMR
techniques including most notably MRI have inherently low
detection sensitivity. As a result, clinical MRI is typically limited
to imaging of protons of water and lipids, which have high
physiological concentrations of tens of moles/L. In contrast, MR
2
hexacoordinate Ir complex, usually possessing IMes or a similar
[23]
ligand. At the appropriate magnetic field, nuclear spin order of
parahydrogen-derived protons is transferred to the nuclei of
coordinated substrate. Subsequent dissociation of the complex
liberates HP biomolecule to the solution. SABRE technique
[19, 24]
underwent a rapid development over the past few years,
1
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Angewandte Chemie International Edition
10.1002/anie.202011698
RESEARCH ARTICLE
and the list of amenable biomolecules has been rapidly
Here, we report on synthesis and SABRE-SHEATH
[
25-30]
13
13
15
expanding.
Although [1- C]pyruvate and other
C-
3
hyperpolarization study of [ N ]nimorazole. Although it has been
containing biomolecules are biomedically interesting, their
primary limitation is a relatively short lifetime of HP state with
originally developed as an antibiotic against anaerobic
[
41]
infections,
Europe as a radio-sensitizing agent for the treatment of head
it is currently under a Phase 3 clinical study in
exponential decay time constant T
1
on the order of 1 min in most
HP N sites have garnered a lot of
attention recently because their T can be on the order of 10 min
– as a result, the detection window of HP
[
4, 5, 12]
15
[50, 51]
favorable cases.
and neck cancer.
If successful, nimorazole will be
1
integrated in the first-line of cancer chemotherapy treatment. We
[
28, 31-33]
15
or longer
injectable biomolecules can be significantly expanded.
Although there have been some reports for hyperpolarization
3
envision that HP [ N ]nimorazole can be simultaneously
[
34, 35]
employed for both tumor radiosensitization and also as an
imaging agent to report on tumor hypoxia status.
1
5
of N-labeled biomolecules via d-DNP, this technology is not
well suited due to ultra-long polarization times of several hours,
and low polarization yields on top of the other d-DNP Results and Discussion
[
35, 36]
limitations.
As a result, the progress of biomedical
1
5
translation of HP N has been relatively slow.
Typical SABRE-SHEATH experiments employed ~600 μL of
solution containing ~100 mM of substrate and ~5 mM of SABRE
A variant of SABRE technique called SABRE-SHEATH
[
23, 53]
(
SABRE in SHield Enables Alignment Transfer to Heteronuclei,
pre-catalyst [Ir(IMes)(COD)]Cl
3
in CD OD, placed in a
[
37, 38]
15
Figure 1)
allows for very efficient polarization of N sites in
medium-wall 5 mm NMR tube tightly connected with 1/4 inch
outer-diameter Teflon tube. NMR tube was placed into the flask
filled with water to maintain the constant temperature, and the
flask itself was placed in the MuMETAL magnetic shield with the
ability to control magnetic field inside via an additional solenoid
1
5
N-containing compounds with fast polarization times (1 min or
[
39]
less) and high yields of P15N > 30%.
microtesla magnetic fields are employed to promote polarization
transfer from p-H derived protons to heteronuclei. SABRE-
SHEATH was shown to hyperpolarize metronidazole,
FDA-approved antibiotic that can be safely administered in large
In this technique,
2
[
40]
the
magnet. The SABRE catalyst was activated via bubbling of p-H
2
gas through the solution at 20 standard cubic centimeters per
minute (sccm) flow rate and 7.8 bar pressure for ~2 h. Typical
SABRE-SHEATH experiments were performed using the
[
41, 42]
doses of up to several grams.
This antibiotic is structurally
1
8
similar to many nitroimidazole-based F-labeled radiotracers,
[
43-45]
employed for hypoxia imaging in cancer and other diseases.
2
following procedure. p-H gas was bubbled through the solution
[
46, 47]
More recently, we have demonstrated that spin-relays
can
at 70 sccm flow rate and 7.8 bar pressure for 60 s. Then gas
flow through the solution was terminated by opening the valve
on the by-pass line (Scheme 1). NMR tube was pulled out from
the magnetic shield, wiped with a paper towel and placed into
the probe of 1.4 T Nanalysis NMReady-60PRO bench-top NMR
1
5
15
provide efficient polarization of N- N spin-spin coupled sites in
1
5
metronidazole with – NO
polarization (>16%) and very long T
group undergoes reduction during hypoxia sensing process, but
2
group gaining high levels of
[
48]
1
of ~10 min.
The –NO
2
1
8
1
5
F-labeled radiotracers are not capable of distinguishing
metabolized compounds, and radiotracer’s clearance from
spectrometer for N NMR detection.
1
5
Our initial
N
SABRE-SHEATH experiments using
[
49]
1
5
surrounding tissues takes several hours.
clearance time of ~2 is typically required prior to
nitroimidazole-based positron emission tomography (PET) scan
As a result,
nimorazole with natural abundance of
N
nuclei were
1
h
unsuccessful, and H detection of SABRE-HP samples yielded
1
very small H signal enhancements of ca. 3–4-fold (Figure S1).
1
5
to gain sufficient contrast with hypoxic region.
N-HP
Therefore, we performed the synthesis of isotopically labeled
1
5
15
nitroimidazoles can potentially mitigate this shortcoming, and
enable fast hypoxia scan (<5 min) without ionizing radiation.
[
N
3
]nimorazole to increase N enrichment from ~0.4% (natural
abundance) to ~99% (Scheme 2). In the first step,
1
5
[
2
N ]imidazole was prepared with 50–60% yield via
1
5
15N
condensation of glyoxal, formaldehyde and NH
4
Cl used as a
1
5
[52]
source of N isotope enrichment.
Subsequent nitration with
O
1
5
15
15N
15NO
2
H NO
/H
]imidazole with 30% yield. Next, this compound was
alkylated with 4-(2-chloroethyl)morpholine in the presence of
CO . Due to tautomerism of the imidazole heterocycle, both
nitrogen atoms possess nucleophilic properties, leading to
SO
allowed
to
obtain
4(5)-[ N]nitro-[1,3-
3
2
4
14N
15
O
N
2
H
H
IMes
Ir+
thermally polarized [15N3]NMZ
14N
p-H2
K
2
3
H
NMZ
15N
15
H
15N
unselective alkylation. Thus, the yield of [ N
3
]nimorazole was
15N
NMZ
only 20% while the yield of its isomer (here termed
H
H
o-H2
O
15
15NO2
polarization transfer
15NO
2
15N
3
[ N ]isonimorazole for convenience) was 60% in the last step.
14N
The prevalence of isonimorazole is not unexpected since the
corresponding nitrogen atom is more nucleophilic than the other
one because it is further away from electron accepting nitro
group. Moreover, steric hindrance effects favor the formation of
hyperpolarized [15N ]NMZ
3
[
46]
[37, 38]
15
Figure 1. Schematics of spin-relayed
SABRE-SHEATH
N
1
5
1
5
15
isonimorazole compared to nimorazole. The two N-labeled
isomers were separated chromatographically. See SI for
additional synthetic information and spectral characterization of
the products.
hyperpolarization of [
3 2 3
N ]nimorazole. Both p-H and [ N ]nimorazole are in
chemical exchange with the Ir complex. In the microtesla magnetic field,
1
5
hyperpolarization is transferred from p-H
2
-derived hydride protons to the
N
nuclei of coordinated substrate via spin-relayed mechanism. Dissociation of
1
5
HP
[
N
3
]nimorazole from the Ir complex allows to obtain free HP
1
5
1
5
3
SABRE-SHEATH experiments using [ N ]nimorazole
[
3
N ]nimorazole in solution.
1
5
15
demonstrated efficient N hyperpolarization on all three
N
2
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Angewandte Chemie International Edition
10.1002/anie.202011698
RESEARCH ARTICLE
1
5
hyperpolarized [15
P = 1.9%
nuclei—the representative N NMR spectrum is shown in Figure
a
N ]nimorazole
3
1
5
2
a. The finding that all three N sites have nearly equal %P15N
ranging from 1.9% to 3.2%) is similar to the recent study of
15
~0.14 M
N-3
(
15
bound
N-3
[
46, 47]
2
P = 2.4%
N-1
spin-relayed
(via the network formed by
15N-15N
J )
15
1
5
[48]
polarization transfer in [ N
3
]metronidazole.
Although the
1
5
maximum %P15N level for [ N
3
]nimorazole was lower than that
3
1
5
[48]
P = 3.2%
[15
N ]nimorazole 15N
for [ N
3
]metronidazole by a factor of ~5.3 (3.2% vs. 17% ), the
3
1
5
NO2
concentration of to-be-hyperpolarized substrate (and catalyst)
O
1
5
15N
15NO
was ~4.2-fold greater in [ N
somewhat higher hyperpolarization
]metronidazole may also be tentatively attributed to the
electron donating properties of methyl group (lacking in
3
]nimorazole studies. Moreover, a
2
1
efficiency of
N
1
5
[
N
3
–5
P = 4.7·10 %
neat thermally polarized [15N]pyridine ×128
1
5
b
[
N
3
]nimorazole heterocycle) likely modulating the efficiency of
SABRE-SHEATH polarization transfer process. We conclude
12.4 M
1
5
15
that N- N spin-relayed polarization transfer is a relatively
general phenomenon, which is now documented in the
4
00
350
300
250
N (ppm)
200
150
15
δ
1
5
15
molecular motifs of [ N
3
]nimorazole, [ N
3
]metronidazole and
1
5
15
15
[
N
3
]isonimorazole (see below). In this spin-relayed
Figure 2. (a) Single-scan
N
NMR spectrum of HP
[
N
3
]nimorazole.
[
48] 15
Conditions: ~140 mM substrate, ~7 mM catalyst, 52 °C, 7.8 bar p-H
2
pressure,
mechanism,
polarization transfer from p-H
Second, polarization is transferred from N-3 to N-1 via J15N-
N-3 nuclear spin is hyperpolarized first via
15
7
0 sccm gas flow rate, 0.4 μT magnetic field inside the magnetic shield. (b)
N
2
-derived hydrides (Figure 1).
15
NMR spectrum of thermally polarized neat [ N]pyridine acquired with 16
signal accumulations. Note this spectrum intensity is divided by the number of
signal accumulations and scaled by a factor of 128. The spectra were acquired
1
5
15
2
1
5
15
1
2
5N. Finally, polarization is transferred from N-1 to NO via
1
5
2
[48]
on a 1.4 T bench-top NMR spectrometer. The N NMR spectrum of HP
another J15N-15N (Figure 1).
15
[
3
N ]nimorazole recorded on a 7.05 T NMR spectrometer is presented in
Figure S2.
gauge
to vent
valve
FC
To maximize the polarization levels, >2 h of activation of
backpressure
regulator
[38, 55]
15
SABRE complex
3
in the presence of [ N ]nimorazole is
mass flow
controller
NMR tube
flask
required (Figure 3a). This activation time is comparable to that
1
5
[56]
observed with [ N
The magnetic field profile of polarization transfer in microtesla
3
]metronidazole under similar conditions.
p-H storage tank
2
p-H generator
2
1
5
magnetic shield
field range exhibits a clear maximum at ~0.4 μT for all three
sites (Figure 3b). The maximum position is the same as that for
N
Scheme 1. Scheme of experimental setup.
15
[48]
[
N
3
]metronidazole,
further supporting the similarity in J
1
5
coupling network between the polarizing N spins in these two
molecules. Moreover, the microtesla magnetic field profile is
O
O
15
NH Cl 15N
H15NO /H SO
4
15N
15NO2
15
15
4
3
2
similar for all three N nuclei of [ N
3
]nimorazole, serving as an
CH O
130 oC, 4 h
2
15N
H
15N
additional proof of spin-relayed polarization transfer mechanism
1
5
discussed above. Furthermore, the dynamics of N polarization
H
[15
N ]imidazole
2
(5)-[15N]nitro-[1,3-15N2]imidazole
4
buildup showed that characteristic exponential buildup times are
5
0-60%
15
3
0%
similar (T
= 23–29 s) for the three N nuclei (Figure 3c) at
b
Cl
optimal B
T
T
of ~0.4 μT. Note when sub-optimal B is employed
N
O
15NO
(e.g., <0.04 μT as in Figure S3a), the equilibrium %P15N is
achieved faster (T 3.1–3.5 s) albeit yielding much
lower %P15N by at least several folds.
15N
2
15NO2
15N 15NO2
15N
N
O
b
=
+
15N
O
15N
H
K CO , DMF
15N
N
2
3
15
15
120 oC, 6 h
[15N3]nimorazole
0%
Next, the N T
1 3
relaxation dynamics of HP [ N ]nimorazole
[15
N ]isonimorazole
3
was studied at three relevant static magnetic fields. At ~0.4 μT
2
60%
15
(
the optimal polarization transfer field for [ N
3
]nimorazole), the
1
5
1
5
15
signals of three N nuclei decayed with approximately the same
Scheme 2. Synthesis of
[ N ]nimorazole (4-(2-(5-[ N]nitro-1H-[1,3-
3
1
5
15
15
N
2
]imidazol-1-yl)ethyl)morpholine) and side product [
N
3
]isonimorazole (4-
characteristic relaxation time T
clustering at ~0.4 μT (and similar type of clustering at ~10 μT
in Figure 3e, and < 0.04 μT in Figure S3b) is important for two
1
= 29–33 s (Figure 3d). This
N
1
5
15
(
2-(4-[ N]nitro-1H-[1,3- N ]imidazol-1-yl)ethyl)morpholine). Blue and red
2
T
1
1
5
colors denote the sources of N isotope label.
1
5
reasons. First, it is consistent with the fact that these three
N
nuclei are strongly coupled at microtesla magnetic fields and
1
5
thus continuously share magnetization with each other via N-
1
5
[47, 48]
N spin relays.
Second and more importantly, our recent
1
5
15
study of [ N
demonstrated the detrimental role of N quadrupolar nucleus
3 2
] and [ N ]metronidazole isotopologues clearly
1
4
[
56]
presence in the spin-relayed network.
In this case, two-bond
1
5
14
N- N spin-spin coupling results in
a
significantly lower
1
5
15
effective
N
T
1
for all (remaining)
N
sites in
3
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Angewandte Chemie International Edition
10.1002/anie.202011698
RESEARCH ARTICLE
1
5
15
15
15
rate and 0.4 μT. (d) Relaxation kinetics of HP [
3
N ]nimorazole at 0.4 μT. (e)
[
N
2
]metronidazole versus [ N
3 1
]metronidazole: N T clustering
1
5
Relaxation kinetics of HP [
N
3
]nimorazole at ~10 μT. (f) Relaxation kinetics of
of 8–9 seconds versus 23–31 seconds respectively under similar
1
5
HP [
SHEATH polarization of [
N ]nimorazole at 1.4 T. (g) The gas flow rate dependence of SABRE-
3
[
56]
(1:20) catalyst-to-substrate ratio as the one studied here.
1
15
3
N ]nimorazole nitro group obtained at different
5
[
N
3
]nimorazole T
1
relaxation cluster of 29–33 seconds (Figure
pressures, 20 °C and 0.4 μT. The inset shows pressure dependence of nitro
group N polarization obtained at 99 sccm gas flow rate. (h) Temperature
1
5
[56]
15
3
d) overlaps well with [ N
3
]metronidazole cluster rather than
2
]metronidazole. We rationalize this very important
1
5
1
5
dependence of SABRE-SHEATH polarization of [
.8 bar p-H pressure, 70 sccm gas low rate and 0.4 μT.
3
N ]nimorazole obtained at
with [ N
observation in a way that while two-bond N- N spin-spin
7
2
1
5
14
1
4
coupling results in efficient
N-rendered quadrupolar
depolarization effects, the corresponding three-bond coupling
has a negligible effect. This is advantageous in the context of
Once SABRE-SHEATH polarization transfer was completed,
the samples were transferred from the microtesla field to the 1.4
T bench-top NMR spectrometer through the Earth’s magnetic
1
5
development of N HP contrast agents with SABRE-SHEATH
1
5
approach as remote N-sites don’t require N isotopic labeling to
15
field. In this experimental protocol, relaxation of N nuclei of
1
5
maximize polarization efficiency of the N spins.
15
[
3
N ]nimorazole at the Earth’s field (ca. 10 μT in our lab,
apparently due to presence of various magnetic equipment etc.)
1
5
a
Activation kinetics
b
Magnetic field profile
NO
.6 N-3
N-1
.4
may have an impact on the resultant N polarization levels
detected at 1.4 T due to T relaxation process during the sample
transfer (Figure 3e). At the clinically relevant field of 1.4 T of
T
a
=38±1 min
NO
N-3
.4 N-1
2
0.8
1
0
0
0
0
0
.5
2
0
0
0
T
a
=48±3 min
1
5
15
bench-top NMR spectrometer, N T
1
values for the three
N
.3
.2
.1
T
a
=82±13 min
t
15N=A·[1–exp(–––)]
15
sites were significantly lengthened to 51 ± 4 s for N-3, 109 ± 4
.2
0
1
5
15
2
s for N-1 and 353 ± 13 s for NO sites, respectively. Although
1
5
15
P
slower relaxation for the N nuclei of NO
2
is expected due to
T
a
0
0
-0.2
1
5
relative isolation of this N site from any protons, this long T of
1
40
80
120
0.1
0.2
0.4 0.7
1
1
5
Activation time (min)
Magnetic field (μT)
nearly 6 min is remarkable as it allows storing N polarization
for tens of minutes (Figure 3f).
c
P
15N buildup @ 0.4 μT
d
15N T
1
decay @ 0.4 μT
15
Parahydrogen flow rate dependence of
N SABRE-
T
b
=28±3 s
0
0
.7
.6
1
5
0
0
0
0
0
0
0
.7 NO
2
NO2 N-3 N-1
SHEATH hyperpolarization of [ N
that polarization plateaus after ~50 sccm gas flow rate (Figure
3g). Increase in p-H pressure leads to a nearly linear %P15N
increase (Figure 3g) indicating that %P15N can be further
3
]nimorazole demonstrated
.6 N-3
T
b
=23±3 s
N-1
0.5
.4
.5
T1=33±2 s
T1=29±2 s
T1=29±1 s
0
2
.4
.3
.2
.1
0
T
b
=29±8 s
0.3
0.2
0.1
0
[55]
enhanced at elevated p-H
2
partial pressure. The temperature
t
P15N=A·[1–exp(–––)]
1
5
T
b
dependence of [ N
3
]nimorazole polarization exhibits a clear
0
20
40
60
0
20
40
60
maximum at ~54 °C – with %P15N being an order of magnitude
Buildup time (s)
Decay time (s)
greater than that at ~20 °C. Thus, temperature optimization is
crucial for efficient SABRE-SHEATH hyperpolarization of
15
15
e 0.6 N T decay @ ~10 μT
f 0.8
N T decay @ 1.4 T
1
1
15
[
3
N ]nimorazole. This observation is different from the optimal
0.5
0.4
0.3
0.2
0.1
0
NO
2
N-3 N-1
NO2 N-3 N-1
temperature of nearly all other SABRE-SHEATH substrates
0.6
studied to date with optimal %P15N achieved at room
0.4
T
1
=353±13 s
[25, 27, 38, 40]
T1=31.5±0.8 s
temperature of ~20 °C
with the exception of
T1=51±4 s
T1=38±3 s
T1=34±1 s
15
[57]
0.2
[ N
2
]imidazole
(tOPT > 75 °C)—this is likely explained by the
T1=109±4 s
different kinetics of nimorazole dissociation from Ir complex.
Similar measurements were carried out at lower
0
0
40
80
120
0
400
800
1200
1
5
Decay time (s)
Decay time (s)
concentrations (1 mM catalyst and 20 mM [ N
3
]nimorazole,
Figure S4), and this different concentration regime rendered
g
Flow & pressure profile
[58]
overall similar results. The addition of co-substrate (pyridine
)
0
.6
.3
0
to the initial solution promoted the rate of SABRE complex
activation (Figure S5). However, the maximum in the magnetic
field profile of SABRE-SHEATH polarization shifted from ~0.4 μT
0
0
4
8
h
Temperature profile
NO
N-3
N-1
0
0
0
0
0
0
.6
.5
.4
.3
.2
.1
0
PH2 (bar)
3
.5
2
1.5
1
to ~0.15–0.20 μT, indicating that the active complex likely
2
2
15
3
contained both pyridine and [ N ]nimorazole molecules.
Characteristic polarization buildup and relaxation times at
microtesla magnetic field were similar to those observed without
a co-substrate (compare Figure 3 and Figure S5). On the other
7
5
3
.8 bar
.8 bar
.8 bar
0.5
0
1
5
hand, N relaxation at the Earth’s magnetic field was more
efficient, while relaxation at 1.4 T was slower in the presence of
pyridine (Figure 3 and Figure S5). Taken altogether, addition of
pyridine as a co-substrate led to significant %P15N decrease by
0
20 40 60 80 100
20
40
60
80
p-H flow rate (sccm)
Temperature (°C)
2
1
5
Figure 3. SABRE-SHEATH hyperpolarization of [
catalyst and ~100 mM substrate initial concentrations: (a) SABRE-SHEATH
3
N ]nimorazole at ~5 mM
2
–6-fold.
We have also explored the possibility to hyperpolarize the
1
5
catalyst activation kinetics in the presence of [
bar p-H pressure, 20 sccm gas low rate and 0.4 μT. (b) SABRE-SHEATH
magnetic field profile of [
pressure and 70 sccm p-H
3
N ]nimorazole at 20 °C, 7.8
1
5
15
2
3 3
isomer of [ N ]nimorazole ([ N ]isonimorazole) using SABRE-
1
5
N
3
]nimorazole obtained at 20 °C, 7.8 bar p-H
flow rate. (c) SABRE-SHEATH polarization buildup
]nimorazole obtained at 20 °C, 7.8 bar p-H pressure, 70 sccm gas low
2
SHEATH. In this compound, the nitro group is located closer to
the donor N atom that can coordinate to Ir complex than in
2
1
5
of [
N
3
2
4
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Angewandte Chemie International Edition
10.1002/anie.202011698
RESEARCH ARTICLE
nimorazole and metronidazole. This proximity of nitro group is a1.6
b1.6
expected to complicate coordination of the substrate to the metal
SNRmax = 98
SNRmax = 82
[
54, 59]
3
2
1
0
3
2
1
0
center due to both steric and electronic effects.
Nevertheless, we have demonstrated that SABRE-SHEATH
1
0
0
.2
1.2
0.8
0.4
0
1
5
15
hyperpolarization of all N nuclei of [ N
possible, resulting in up to 0.47% polarization (Figure S6). This
observation paves the way to SABRE-SHEATH
3
]isonimorazole is
.8
.4 catheter
.5×0.5 mm2 pixel size
catheter
axial view
axial view
hyperpolarization of other biologically active nitroimidazole
derivatives containing nitro group in the α position to the nitrogen
atom with a lone electron pair. This range of compounds
includes prospective antitumor prodrug evofosfamide (a.k.a. TH-
0.5×0.5 mm2 pixel size
0
0
0
0.4
0.8
1.2
1.6
0
0.4
0.8
1.2
1.6
x, cm
x, cm
c
d
[
60]
7
6
5
4
3
2
1
0
7
6
5
4
3
2
1
3
02 ),
antibiotics
azomycin
and
benznidazole,
SNRmax = 75
3
2
1
0
SNRmax = 75
3
2
1
0
immunosuppressive drug azathioprine and many others.
Dependence of SABRE-SHEATH polarization efficiency of
1
5
[
3
N ]isonimorazole on magnetic field, polarization buildup time
1
5
and temperature was investigated, as well as relaxation of
N
coronal view
.2×1.2 mm2 pixel size
coronal view
1.2×1.2 mm2 pixel size
hyperpolarization at three different magnetic fields. Magnetic
field profile demonstrated the maximum at 0.25 μT (Figure S8a),
1
0
0
0
1
2
3
4
5
6
7
1
2
3
4
5
6
7
1
5
which is lower than corresponding maximum for [ N
3
]nimorazole.
]isonimorazole
]nimorazole (~12–13 s
vs. 23–29 s). Temperature dependence of SABRE-SHEATH
x, cm
x, cm
1
5
Characteristic polarization buildup time for [ N
3
1
5
15
1
5
Figure 4. N TrueFISP MRI of HP [ N ]nimorazole (~110 mM) in a 5 mm
NMR tube using multi-nuclear 9.4 T MRI scanner. Frequency offset was
3
(
Figure S8b) was also lower than for [ N
3
15
2
adjusted to the signal of NO group. The imaging parameters: spectral width
1
5
polarization for [ N
different: the broad maximum was observed at ~30–43 °C
Figure S8f), in contrast to a sharper peak at ~54 °C for
3
]isonimorazole was also dramatically
(SW) = 5 kHz, repetition time (TR) = 14 ms, echo time (TE) = 7 ms, flip angle =
30˚. (a–b) Axial projection (the first and the second scan): scan acquisition
time = 356 ms, matrix size = 32×32 (zero-filled to 512×512), FOV = 1.6 cm ×
(
2
1
5
1.6 cm, spatial resolution = 0.5×0.5 mm /pixel. (c–d) Coronal projection (the
first and the second scan): scan acquisition time = 684 ms, matrix size =
64×64 (zero-filled to 512×512), FOV = 7.8 cm × 7.8 cm, spatial resolution =
[
3
N ]nimorazole. Comparing the efficiency of hyperpolarization
1
5
15
for the three N sites, NO
2
group exhibited the highest
]nimorazole. However, in case
1
5
2
polarization levels, similar to [ N
3
1.2×1.2 mm /pixel. Full sets of 16 consecutively recorded MR images are
1
5
15
presented in Figure S9 (axial projection) and Figure S10 (coronal projection).
3
of [ N ]isonimorazole two N nuclei in imidazole ring exhibited
1
5
1
5
15
N NMR spectra acquired at the similar conditions demonstrated P15N of
15 15
.78% for the NO group and P15N of 0.33% for the N-3 site (Figure S11).
2
similar polarization levels, while for [ N
was polarized more efficiently.
3
]nimorazole N-1 site
0
1
5
15
Relaxation of N nuclei in [ N
and the Earth’s magnetic field was faster than in
3
]isonimorazole at microtesla
Biomedical Outlook
1
5
[
N
3
]nimorazole. Similar values (19–21 s and ~18–24 s) were
observed at both magnetic fields under study (Figures S8c and
3
The reported herein study was performed in CD OD because it
S8d). On the other hand, at 1.4
T
relaxation of
is readily available and it has been employed by the vast
1
5
15
[
[
N
N
3
]isonimorazole
]nimorazole (Figure S8e): 90 ± 10 s for N-3 site, 128 ± 14
N
nuclei was slower compared to
majority of published in vitro studies. For future in vivo
1
5
15
3
applications, we envision hyperpolarization in
a
more
1
5
15
2
s for N-1 site and 417 ± 27 s for NO group, respectively. The
biocompatible ethanol solvent (SABRE hyperpolarization of
1
5
15
long relaxation time for NO
2
group of [ N
3
]isonimorazole
[55]
nicotinamide in ethanol has been well documented ) followed
signifies that other nitroimidazole compounds yet to be
hyperpolarized by SABRE will likely exhibit a similarly long
[39, 61]
by rapid catalyst removal using functionalized resins
and
reconstitution to aqueous medium via rapid vacuum distillation of
ethanol down to less that 10% content. These two steps should
take less than 0.5 minute based on our experience resulting in
1
5
relaxation times of HP N sites.
The feasibility of 2D sub-second N MRI visualization of HP
1
5
1
5
15
[
N
3
]nimorazole was demonstrated. Figure 4 presents 2D
N
15
relatively small polarization losses for all three N sites. The
TrueFISP MR images of a 5 mm NMR tube containing ~110 mM
presence of 10% ethanol is a generally accepted clinical practice
1
5
HP [ N
3
]nimorazole acquired on a 9.4 T MRI scanner. Note the
in the context of PET tracers’ administration with up to 20 mL
2
very high spatial (0.5×0.5 mm /pixel) and temporal (356 ms)
[62]
dose.
The development of bio-injectable catalyst-free
1
5
resolution that can be potentially achievable with HP N MRI of
formulation is the next key step for SABRE-SHEATH
hyperpolarization technique.
1
5
[
N
3
]nimorazole – first two images in each series are shown for
two different projection views. This proof-of-principle
Although catalyst activation takes ~2 h, it can be performed
well in advance of the hyperpolarization procedure with normal
1
5
demonstration opens the prospects for utilization of SABRE
N
1
5
hyperpolarization of [ N
3
]nimorazole in bioimaging applications
hydrogen (as the materials are stable for many hours), and the
such as reporting on tumor hypoxia status in a manner similar to
15
3
bolus of [ N ]nimorazole can be hyperpolarized in ~1 min.
[
43-45]
that of nitroimidazole-based PET tracers.
Although the optimum polarization temperature is ~50–60 ºC
(
Figure 3h), this is not a translational challenge—our previous
1
3
experience with other parahydrogen-hyperpolarized
C
compounds produced at 68 ºC shows that the HP liquid
equilibrates to below 37 ºC at the time of the actual in vivo
[
63, 64]
injection.
We note that the produced dose of HP
1
5
[
N ]nimorazole in NMR tube of ~50 µmoles is certainly
3
5
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Angewandte Chemie International Edition
10.1002/anie.202011698
RESEARCH ARTICLE
1
5
sufficient for future pilot in vivo studies in small rodents—
with NO
2
group in α position to donor nitrogen atom of
1
3
previous successful studies with HP C injectable agents
imidazole ring opens opportunities for efficient hyperpolarization
of a wide range of other biomedically interesting molecules with
[
9, 64]
employed ~5 µmole dose in mice.
We envision that the
injected nimorazole will undergo cellular uptake within one
minute based on the pharmacodynamics data for structurally
very long-lived HP state retained by NO
2
group. For example,
HP azomycin can potentially act as a suitable pH sensor (pKa ~
[
45]
[57]
[66]
similar
nitroimidazole
compounds.
The
subsequent
7.0), and TH-302 can be employed as a theragnostic agent
for cytotoxic chemotherapeutics selectively activating on hypoxic
tumors, which are typically difficult to treat using conventional
chemotherapies.
metabolism of nitroimidazole compounds in hypoxic cells leads
to formation of nitroso and hydroxylamine intermediates and
[
65]
amino-derivative metabolic products, Figure 5.
The ab initio
1
5
calculations of N chemical shifts using Gaussian’09 (see SI for
details) for nimorazole and its metabolic reduction products
1
5
15
reveal that the associated N chemical shifts of all labelled
N
Acknowledgements
sites are very sensitive to reduction process. While the nitro-
group nitrogen is the most sensitive one (with nearly 800 ppm
dynamic range), it may not necessarily be the most useful
sensing site for hydroxylamino- and amino-derivatives because
O.G.S. thanks the Russian Foundation for Basic Research
(
Grant 19-33-60045) for the support of mechanistic studies with
the use of parahydrogen. E.Y.C. thanks the following awards for
funding support: NSF CHE-1904780 and NIH 1R21CA220137.
N.V.C., A.S., K.V.K. and I.V.K. thank the Russian Foundation for
Basic Research (grants 17-54-33037, 18-33-20019 and 19-53-
1
5
1
of the anticipated decrease in N T due to directly attached
protons. On the other hand, while the dynamic range of the other
two sites is less dramatic (12 ppm and 20 ppm respectively),
1
5
1
these two N sites will likely retain their long T in vivo. To
1
2013) for financial support and the Russian Ministry of Science
1
5
summarize, the relaxation properties and the N chemical shift
and Higher Education (Project AAAA-A16-116121510087-5).
We dedicate this work to the memory of Dr. Kirill V. Kovtunov,
our mentor, colleague and friend.
dispersion of metabolic products bode well for future in vivo
1
5
studies of using HP [ N
sensor.
3
]nimorazole as an injectable hypoxia
Keywords: parahydrogen • hyperpolarization • SABRE-
Nimorazole metabolism in hypoxic environment
SHEATH • nimorazole • radiosensitization
H2O
O
O
O
H
2
O
O
e- + 2H+
-
+
-
+
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2
2e + 2H
2e + 2H
2
015, 21, 3156-3166.
N
N
N
N
[
2] K. V. Kovtunov, E. V. Pokochueva, O. G. Salnikov, S. Cousin, D.
2 2
2O + H O
Kurzbach, B. Vuichoud, S. Jannin, E. Y. Chekmenev, B. M. Goodson,
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A. 2006, 103, 11270-11275.
-
+
1
67
1
2O
NO
45
2
+ 2H
178
1
67
158
15N
15N
15N
15N
15N
5
15NO
15NHOH
15NH2
2
3
790
108
15N
15N
15N
17
[
4] J. Kurhanewicz, D. B. Vigneron, K. Brindle, E. Y. Chekmenev, A.
2
47
242
238
]hydroxylamino- [15
intermediate
235
Comment, C. H. Cunningham, R. J. DeBerardinis, G. G. Green, M.
O. Leach, S. S. Rajan, R. R. Rizi, B. D. Ross, W. S. Warren, C. R.
Malloy, Neoplasia 2011, 13, 81–97.
[15
N
]nimorazole
[15
N
]nitroso- [15
intermediate
N
N
]amino-
3
3
3
3
derivative
[
5] K. M. Brindle, J. Am. Chem. Soc. 2015, 137, 6418-6427.
Figure 5. Metabolism of nitroimidazole compounds on the example of
[
65]
15
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Wolber, K. Golman, J. H. Ardenkjaer-Larsen, K. M. Brindle, Nat. Med.
2
[
nimorazole in hypoxic environment.
Color-coded values of N chemical
shifts (in ppm) were computed for aqueous media using Gaussian’09 ab initio
calculations (see SI for details).
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To summarize, we report on efficient synthesis and SABRE-
1
5
15
SHEATH hyperpolarization of N-labeled [ N
3
]nimorazole. The
HP state with %P15N of up to 3.2% N polarization is created on
1
5
1
5
15
all three N sites, and it persists for tens of minutes with N T
1
[
9] E. Cavallari, C. Carrera, M. Sorge, G. Bonne, A. Muchir, S. Aime,
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of [ N ]nimorazole and other HP nitroimidazoles as cancer
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Keshari, A. Kjaer, C. Laustsen, D. A. Mankoff, M. E. Merritt, S. J.
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21, 1-16.
The relaxation dynamics results presented here provide
valuable insights for the rational development of schemes and
1
5
molecular substrates for efficient spin-relayed N SABRE-
SHEATH hyperpolarization. Although synthesis yields a side
product, we were able to successfully separate and purify both
[
13] J. H. Ardenkjaer-Larsen, B. Fridlund, A. Gram, G. Hansson, L.
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5
compounds. The feasibility of SABRE-SHEATH
N
1
5
3
hyperpolarization of the synthesized isomer of [ N ]nimorazole
6
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.202011698
RESEARCH ARTICLE
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Angewandte Chemie International Edition
10.1002/anie.202011698
RESEARCH ARTICLE
Entry for the Table of Contents
Insert graphic for Table of Contents here.
0
.8
16
8
15N
T = 5.9 min
15N MRI
1
HH
@
1.4 T
O
15N
0.4
15NO
2
2
N
0.5×0.5 mm
pixel size
SABRE
P15N = 3.2%
0
[15
N ]nimorazole
0
3
0
10
20
0
8
16
Decay time (min)
x, mm
1
5
Synthesis of
3
N -enriched antibiotic and hypoxic radiosensitizer nimorazole is reported. Spin-relayed Signal Amplification By
1
5
Reversible Exchange in microtesla magnetic field yielded up to 3.2% N nuclear spin polarization with relaxation time constant of 5.9
0.2 min at 1.4 T. The isomer of nimorazole formed in the synthesis was also hyperpolarized indicating that this approach can be
±
extended to a wide range of nitroimidazole-based drugs.
Institute and/or researcher Twitter usernames: https://twitter.com/mmt_lab, https://twitter.com/waynestatechem,
https://twitter.com/OlegSalnikovNMR
8
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