Achieving High 1H Nuclear Hyperpolarization Levels with Long Lifetimes in a Range of Tuberculosis Drug Scaffolds

Article abstract of DOI:10.1002/chem.201703278

Despite the successful use of isoniazid, rifampicin, pyrazinamide and ethambutol in the treatment of tuberculosis (TB), it is a disease of growing global concern. We illustrate here a series of methods that will dramatically improve the magnetic resonance imaging (MRI) detectability of nineteen TB-relevant agents. We note that the future probing of their uptake and distribution in vivo would be expected to significantly enhance their efficacy in disease treatment. This improvement in detectability is achieved by use of the parahydrogen based SABRE protocol in conjunction with the ²H-labelling of key sites within their molecular structures and the ²H-labelling of the magnetization transfer catalyst. The T₁ relaxation times and polarization levels of these agents are quantified under test conditions to produce a protocol to identify structurally optimized motifs for future detection. For example, deuteration of the 6-position of a pyrazinamide analogue leads to a structural form that exhibits T₁ values of 144.5 s for 5-H with up to 20 % polarization. This represents a >7-fold extension in relaxation time and almost 10-fold improvement in polarization level when compared to its unoptimized structure.

Full text of DOI:10.1002/chem.201703278

A Journal of
Accepted Article
Title: Achieving High 1H Nuclear Hyperpolarization Levels with Long
Lifetimes in a Range of Tuberculosis Drug Scaffolds
Authors: Simon Duckett, Philip Norcott, Peter J Rayner, and Gary G. R.
Green
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To be cited as: Chem. Eur. J. 10.1002/chem.201703278
Link to VoR: http://dx.doi.org/10.1002/chem.201703278
Supported by

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Achieving High 1H Nuclear Hyperpolarization Levels with Long
Lifetimes in a Range of Tuberculosis Drug Scaffolds
Philip Norcott,[a] Peter J. Rayner, [a] Gary G. R. Green, [a] and Simon B. Duckett*[a]
However, while nuclear magnetic resonance (NMR)
spectroscopy and MRI are invaluable analytical techniques for
research and medicine they suffer from an inherent insensitivity
that means they cannot routinely provide access to this type of
Abstract: Despite the successful use of isoniazid, rifampicin,
information.[8] This challenge stems from the fact that even in the
pyrazinamide and ethambutol in the treatment of tuberculosis (TB) it
1.5 T magnetic field of a commonly used hospital scanner the
is a disease of growing global concern. We illustrate here a series of
associated nuclear spin interaction produces a signal that is
methods that will dramatically improve the magnetic resonance
derived positively from just 1 in 105 of the proton nuclei present.
imaging (MRI) detectability of nineteen TB relevant agents and note
Hyperpolarization techniques change the associated Boltzmann
that the future probing of their uptake and distribution in vivo would
spin state populations to improve detectability by several orders
be expected to significantly enhance their efficacy in disease
of magnitude.[9]
treatment. This improvement in detectability is achieved by use of
2
the parahydrogen based SABRE protocol in conjunction with the H-
labelling of key sites within their molecular structures, and the 2H-
labelling of the magnetization transfer catalyst. The T1 relaxation
times and polarization levels of these agents are quantified under
test conditions to produce a protocol to identify structurally optimized
motifs for future detection. For example, deuteration of the 6-position
of a pyrazinamide analogue leads to a structural form that exhibits T1
values of 144.5 s for 5-H with up to 20% polarization. This
Figure 1. Structures of the tuberculosis drugs pyrazinamide (1) and isoniazid
(2).
represents a >7-fold extension in relaxation time and almost 10x
improvement in polarization level when compared to its unoptimized
structure.
Introduction
Tuberculosis (TB) is a bacterial infection that is recognized by
the World Health Organization as one of the most significant
infectious diseases, killing an estimated 1.8 million people in
2015 alone.[1] Pyrazinamide[2] (1) and isoniazid[3] (2), whose
structures are detailed in Figure 1, are two first-line drugs that
are used in conjunction with ethambutol or rifampicin in its
treatment.[4] In the case of pyrazinamide, the proposed active
form, pyrazinoic acid, is produced by the pyrazinamidase
enzyme and accumulates within Mycobacterium tuberculosis.[5]
While its precise mode of therapeutic action remains unclear,
Scheme 1. Representation of the Signal Amplification by Reversible
Exchange (SABRE) process for substrate L where IMes is 1,3-bis(2,4,6-
trimethylphenyl)imidazol-2-ylidene.
the prodrug isoniazid is activated by KatG catalase to form a
complex which binds to the carrier protein InhA to reduce its
activity.[6]
The 11C radiolabeling of pyrazinamide, isoniazid and
rifampicin in conjunction with subsequent imaging by positron
A number of hyperpolarization methods have been developed
which are beginning to find use in the study of living systems.[10]
Those that utilize parahydrogen (p-H2) as the polarization
feedstock are known collective by the term PHIP (parahydrogen
induced polarization) processes.[11] Although p-H2 is itself
undetectable by NMR spectroscopy as it possesses no angular
spin momentum, when it is incorporated into a new molecule
through a chemical reaction that breaks its nuclear symmetry the
resulting environments can produce strongly hyperpolarized MR
signals.[12] Signal Amplification By Reversible Exchange
(SABRE) is a rapidly emerging and inexpensive method for
hyperpolarizing materials that uses p-H2.[13] It achieves this result
through the catalytic transfer of polarization from p-H2 to a target
emission tomography allows access to pharmacokinetic and
drug distribution data that is used in TB diagnosis.[7] Imaging the
uptake and distribution of these therapeutics by magnetic
resonance imaging (MRI) might therefore aid in TB treatment.
[a]
Dr P Norcott, Dr PJ Rayner, Prof. GGR Green, Prof SB Duckett
Centre for Hyperpolarisation in Magnetic Resonance
Department of Chemistry, University of York
YO10 5NY, UK
simon.duckett@york.ac.uk
Supporting information for this article is given via a link at the end of
the document.
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substrate (L) via an iridium complex without changing the
material’s chemical identity.
A common polarization catalyst that is used in the SABRE
approach is formed by the reaction of [IrCl(COD)(IMes)] (where
two agents were prepared at a concentration of 20 mM in a 4:1
ratio with the precatalyst [IrCl(COD)(IMes)] (5 mM). These
samples were placed under 3 bar of p-H2 and a series of SABRE
polarization transfer experiments undertaken which involved
shaking the sample for 10 seconds in the 65 gauss fringe field
IMes
= 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene, and
COD = cis,cis-1,5-cyclooctadiene), with the hyperpolarization
target (L), and p-H2 (Scheme 1).[14] Reversible polarization
transfer then readily occurs through the J-coupling network[15] of
the iridium catalyst from the hydride ligands into the target
substrate at low field, although it can also be driven by radio
frequency excitation at high field.[16] Developments in SABRE
over the last few years have seen significant progress in the
hyperpolarization of 1H, 15N, 31P and 13C nuclei amongst
others.[17]
associated with a 9.4 T NMR spectrometer, followed by
immediate transfer into the magnet for measurement. The
average 1H NMR signal intensities seen for 1 in the resulting
measurements, under SABRE, corresponded to polarization
levels of 1.7%, 2.6% and 2.7% respectively, while those for 3
were 2.4%, 4.3% and 3.9%. Hence, both of these materials are
highly suitable for SABRE. As expected, the measured T1 values
Table 1. T1 values and hyperpolarization levels of 1, 3 and a series of
deuterated methyl pyrazinecarboxylate derivatives.
A
key limitation that is associated with all
hyperpolarization techniques is the rate at which the
hyperpolarized nuclei relax back to their thermally distributed
form.[18] This timescale is often elevated for heteronuclei but the
ubiquity and high natural abundance of 1H nuclei make its
detection highly desirable in MRI. Hence the establishment of
methods to hyperpolarize nuclei, in a long-lived form, reflect a
scientifically challenging but very important research goal that
has involved a range of approaches.[19] T1 relaxation times of 1H
nuclei approaching 2 minutes have been reported in an
isotopically labelled methyl nicotinate in conjunction with
SABRE polarization of up to 50%.[20] Alternatively, 1H singlet
Compound
Site
T1(no cat.) (s)
T1(with cat.) (s)
Polarization
Level (%)
Methanol-d4
1
3-H:
5-H:
6-H:
80.6
20.9
21.2
32.7
12.1
12.3
2.7 ± 0.3
2.8 ± 0.3
1.6 ± 0.1
3-H:
5-H:
6-H:
64.1
23.4
23.5
20.6
9.3
12.0
4.3 ± 0.3
3.9 ± 0.2
2.4 ± 0.2
3
4
5
6
7
8
9
states have been used to store magnetization in
a
5-H:
6-H:
24.9
24.0
10.3
14.1
3.5 ± 0.3
2.8 ± 0.2
functionalized pyridazine with a TLLS value of over 4 minutes.[21]
Both pyrazinamide and isoniazid have already been
reported to participate in SABRE hyperpolarization,[22] but
further optimization is needed prior to their potential in vivo
detection. Herein we develop a strategy to increase the
relaxation times of these key tuberculosis related
pharmaceuticals, and some related structural analogues,
through selective deuteration. By extending the relaxation times
3-H:
6-H:
91.1
84.0
26.0
39.2
6.7 ± 0.3
6.2 ± 0.3
3-H:
5-H:
99.7
144.5
32.9
26.2
11.9 ± 0.6
9.2 ± 0.5
of these substrates, their suitability to SABRE could also
5-H:
6-H:
39.5
98.1
22.6
43.1
7.8 ± 0.2
2.5 ± 0.2
23]
improve.[20,
This hypothesis stems from the fact that each
molecule of p-H2 possesses a specific amount of underlying
polarization and consequently transfer to fewer 1H sites on a
target could be beneficial. Consequently, a further benefit to this
approach lies in the simplified and stronger MR response that
might result.
3-H:
5-H:
53.0
53.1
25.6
21.8
8.8 ± 0.3
7.5 ± 0.2
Ethanol-d6
3-H:
5-H:
6-H:
55.4
17.2
17.2
8.3
4.6
7.4
3.16 ± 0.08
1.96 ± 0.05
0.95 ± 0.04
Results and Discussion
3
4
5
6
SABRE investigation of pyrazinamide (1) and methyl
pyrazine-2-carboxylate (3) We begin our study with the two
pyrazinamide-class substrates pyrazinamide (1) and methyl
pyrazine-2-carboxylate (3, Table 1). The latter, alongside
numerous other ester derivatives, have been recognized as
being active against several strains of M. tuberculosis.[24]
In methanol-d4 solution, 1 was found to exhibit T1 values
of 80.6, 20.9 and 21.2 seconds for protons 3-H, 5-H and 6-H
respectively. In contrast, the methyl ester 3 was found to exhibit
T1 values of 64, 23.4 and 23.5 seconds. NMR samples of these
5-H:
6-H:
11.0
11.7
5.4
7.6
2.35 ± 0.05
0.99 ± 0.02
3-H:
6-H:
18.3
20.9
5.9
13.6
4.4 ± 0.2
3.9 ± 0.3
3-H:
5-H:
21.4
23.8
10.8
11.5
4.7 ± 0.3
2.9 ± 0.2
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and long lived response. This increase in T1 relaxation time is
consistent with the removal of the strong JH-H coupling that is
present within 3.
of 3 under SABRE conditions at 298 K were found to decrease
in accordance with the fact that they reflect a weighted response
from the free and bound forms.[23, 25] The corresponding average
fall in T1 for the sites in 1 under these conditions was 47%, while
that for 3 was 60% which is consistent with the observation of
better polarization transfer in 3 as these differences suggest a
stronger interaction with the catalyst.
3
From an MRI perspective, substrates such as 7 and 8
which contain a single proton, and two 2H labels, would be
desirable probes due to the reduction in chemical shift artefacts
that would be seen in an echo planar image. While these
compounds exhibited higher proton T1 values than 1 they were
not a long as those of 6. However, 7 produced an impressive
SABRE response of 7.8% polarization which is higher than the
2.5% polarization achieved for 8 even though its T1 values are
shorter. These results confirm therefore that substrate binding (L,
Scheme 1) occurs predominately at the nitrogen centre that is
Synthesis of deuterated analogues of pyrazinamide (1) and
methyl pyrazine-2-carboxylate (3) In order to improve on the
hyperpolarization level, a series of deuterated derivatives of 1
and 3 were prepared by the palladium-catalysed reduction of the
26]
corresponding heteroaryl chlorides under D2,[20,
followed by
4
remote to ester, with the associated JH-H polarization transfer
transesterification or amidation as shown in Scheme 2. Full
synthetic procedures and characterization data are presented in
the supplementary information.
coupling in the SABRE catalyst being more efficient than the
5JH-H coupling that would be used for 8.
We sought to further improve these results by replacing
the CH3 group in 6 with a CD3 group to produce 9. Despite our
predictions, this d3-methyl ester performed worse than 6. Upon
replacing the methanol-d4 solvent with the more biologically
amenable solvent ethanol-d6, the T1 values and signal
enhancements achieved across this series of compounds fell,
although the best results were still achieved with 6 (4.7%). This
change is consistent with the associated increase in solvent
viscosity and hence more facile relaxation that results.[27]
SABRE investigations harnessing deuterated catalysts with
deuterated analogues of methyl pyrazine-2-carboxylate (3)
In order to see if the effect of relaxation caused by the SABRE
catalyst can be reduced, this process was re-examined with a
series of deuterated analogues of [IrCl(COD)(IMes)] where the
NHC isotopologues had the form shown in Figure 2. Results with
5 and 6, when the catalyst bears a d22-IMes ligand, proved
optimal with both the T1 values and hyperpolarization levels
increasing as detailed in Table 2 (also see supplementary
information). The polarization level of 13.4% seen for the 3-H
response of 6 corresponds to a ~4200-fold signal enhancement
at 9.4 T. The catalyst bearing the d24-IMes ligand, however, was
not as efficacious, slightly increasing T1 values, but with a loss in
observed polarization. In an effort to further improve the
polarization transfer efficiency, we tested the effect of
temperature. This revealed that when 6 was polarized with the
d22-IMes catalyst at 0°C a ~6300-fold signal gain for H-3 was
realized (corresponding to 20.4% polarization, see supporting
information). The temperature at which the SABRE catalysis is
Scheme 2. Synthetic route to the pyrazine carboxylate and amide
isotopologues 3-12. aCommercially available. bD2O/THF instead of EtOD.
cMethyl 3,6-dibromopyrazine-2-carboxylate used as the starting material and
Na2CO3 used in place of K2CO3.
SABRE studies on deuterated analogues of methyl
pyrazine-2-carboxylate (3) The relaxation and SABRE
polarization data for the methyl ester derivatives 4-6 that contain
a single 2H-label are detailed in Table 1. Substrate 4, with
deuteration at the 3-position, gave a slight increase in the
relaxation times of the remaining 1H nuclei compared to 3,
whilst delivering comparable levels of SABRE polarization to it.
In contrast, compound 5, where the label is in the 5-position,
exhibits significantly improved relaxation times and polarization
levels (6.2‒6.7%). In this case the effective T1 values of the
substrate in the presence of the hyperpolarization catalyst are
25 and 39 seconds, and they increase to 91 and 84 seconds in
its absence. These numbers indicate that relaxation within the
catalyst is therefore reduced by substrate deuteration and
consequently higher hyperpolarization levels result.[20] The most
striking result though was achieved with deuteration at the 6-
position as compound 6 gave 11.9% polarization for 3-H (a
~3700-fold signal gain) and 9.2% polarization for 5-H. The T1
Table 2. Effect of a deuterated catalyst and substrate system in methanol-d4
solution.
Compound
Ligand
Site
T1(with
(s)
Polarization
Level (%)
cat.)
3-H:
6-H:
26.0
39.2
6.7 ± 0.3
6.2 ± 0.3
IMes
5
3-H:
6-H:
40.8
54.0
10.9 ± 0.5
9.0 ± 0.5
d22-IMes
3-H:
5-H:
32.9
26.2
11.9 ± 0.6
9.2 ± 0.5
IMes
6
3-H:
5-H:
37.9
31.3
13.4 ± 0.7
9.5 ± 0.3
d22-IMes
1
values of the H nuclei in 6 are now 100 and 145 seconds and
hence we have achieved our initial goal of producing a strong
conducted has been shown to significantly affect the rate of
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dissociation of the substrate molecule.[14b] As such, the lifetime
of the SABRE active complex will be longer at lower
temperatures, which proves to be more efficient for polarization
Figure 2. Deuterated analogues of the IMes ligand.
Table 3. T1 values and hyperpolarization levels of deuterated pyrazinamide
derivatives
SABRE studies on deuterated analogues of pyrazinamide
(1) Investigations were also conducted on a series of related 2H-
labelled derivatives of pyrazinamide (1), the results of which are
detailed in Table 3. Polarization levels and relaxation times were
again affected, with deuteration at the 6-position (compound 12)
yielding improved levels of polarization, especially in ethanol-d6,
with its two protons exhibiting 6.4 and 7.7% polarization with the
d22-IMes catalyst. Interestingly, the T1 values for 12 in the
presence of this catalyst proved to be remarkably close to that of
the substrate alone.
Compound
Site
T1(no
cat.) (s)
T1(with cat.) (s)
Polarization
Level (%)
Methanol-d4
3-H:
5-H:
6-H:
80.6
20.9
21.2
32.7
12.1
12.3
2.7 ± 0.3
2.8 ± 0.3
1.6 ± 0.1
1
5-H:
6-H:
19.4
20.4
11.3
11.6
3.2 ± 0.2
2.1 ± 0.1
10
11
3-H:
6-H:
34.8
33.8
21.6
12.7
3.4 ± 0.1
2.8 ± 0.02
Systematic study of SABRE utilising the isoniazid-type
substrates 17-19 Our strategy for increasing the SABRE 1H-
polarization levels and T1 relaxation times by isotopic labelling
can also be applied to isoniazid-type substrates. Isoniazid (2)
has a different structural motif to that of 1, with it containing a
single nitrogen centre and substitution in the 4-position.
12
13
14
39.4 (49.6)*
39.3 (49.1)*
3-H:
5-H:
51.2
51.5
2.7 (3.92 ± 0.04)*
2.4 (3.92 ± 0.04)*
6-H:
5-H:
102.8
55.9
39.8
0.9 ± 0.1
Additionally,
2 exhibits more rapid relaxation than 1 and
consequently significantly lower polarization levels are achieved.
2
Optimal, isotopic substitution now results from H labelling at the
2- and 5-positions, as shown in 17, which acts to create two
24.5 (37.0)*
4.1 ± 0.3 (9.3 ± 0.6)*
1
isolated H spin systems that exhibit slow relaxation (ca. 34.3 s)
4
whilst maintaining a JH-H transfer coupling that enables 2.0%
Ethanol-d6
polarization under similar SABRE conditions to those used
previously. 17 was synthesised from dichloroisonicotinic acid 15
by esterification and subsequent palladium catalyzed reduction
under D2 to yield deuterated isonicotinate 16. Reaction with
hydrazine hydrate then gave 2,5-d2-isoniazid 17 that could also
be converted into oxadiazole 18 upon treatment with triethyl
orthoacetate (Scheme 3).[28]
3-H:
5-H:
6-H:
56.5
13.6
13.8
18.3
8.0
8.7
2.6 ± 0.2
2.0 ± 0.2
1.2 ± 0.1
1
5-H:
6-H:
13.4
13.1
6.9
6.8
1.7 ± 0.08
1.1 ± 0.1
10
11
The 2H-labelled derivative 18, was compared to its
unlabelled isotopologue 19 which has shown activity against a
number of strains of M. tuberculosis.[29] As shown in Table 4,
when compared to pyrazinamide (1), the unlabeled compounds
2 and 19 exhibited much shorter T1 values; between 4 and 7
seconds in the presence of the SABRE catalyst. Furthermore,
the signal enhancements produced by SABRE are low. However,
upon deuteration of isoniazid to give 17, these relaxation times
are increased several-fold and polarization levels of 1.9‒2.1%
result in methanol-d4. 18 also exhibited significantly longer
relaxation times in both methanol-d4 and ethanol-d6 when
compared to 19, but the corresponding increase in polarization
level is less than that seen with 17. These values can be
improved by the use of the d22-IMes ligand, with the polarization
level for 17 rising to 3.7%. Higher temperatures also result in
improved responses, such that 14 yields a 9.7% return at 313 K
with the d22-catalyst (see the supporting information). Thus we
suggest that, in contrast to 6, the optimal exchange rate for 17
occurs at higher temperatures. Therefore we note that while
varying the temperature clearly changes the levels of
3-H:
6-H:
25.6
30.3
8.3
4.1
3.0 ± 0.2
2.2 ± 0.1
12
13
14
3-H:
5-H:
32.8
34.9
18.4 (26.1)*
18.8 (26.6)*
4.5 ± 0.3 (7.7 ± 0.4)*
3.3 ± 0.2 (6.4 ± 0.4)*
6-H:
5-H:
57.4
29.0
12.0
2.2 ± 0.1
15.3 (16.9)*
6.1 ± 0.3 (7.6 ± 0.2)*
*[IrCl(COD)(d22-Imes)] used as the catalyst
transfer to 6.
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Table 4. T1 values and hyperpolarization levels of deuterated isoniazid
derivatives
For the mixture of 4 and 6, the H-5 polarization of 4
remains constant but there is a significant fall in the other three
response levels and the associated relaxation times. In contrast,
the polarization level of 13 and 14 both improve, from 0.9% and
4.1% alone to 2.0% and 5.5% in the mixture. The T1 of 13 falls
as a consequence while that of 14 sees little change. This
suggests that it may be possible to improve the levels of
enhancement produced with the substrates that exhibit weaker
polarization by this combination strategy. We therefore
examined a sample of 8 and 13 as a further control and
discovered that while the polarization of 8 fell to from 2.5% to
1.2% that of 13 increased to 1.7% from 0.9%. There were
associated increases in the T1 values of agent 8 and falls in the
value for 13 in accordance with these changes.
Compound
Site
T1(no
T1(with cat.) (s)
Polarization Level (%)
cat.) (s)
Methanol-d4
2
o-H:
m-H:
7.4
7.5
4.9
5.8
1.2 ± 0.02
0.5 ± 0.09
17
18
19
o-H:
m-H:
34.4
34.3
16.5 (17.5)*
19.7 (21.0)*
2.1 ± 0.1 (3.7 ± 0.3)*
1.9 ± 0.1 (3.7 ± 0.4)*
o-H:
m-H:
9.5
40.7
19.3
24.3
0.35 ± 0.04
0.39 ± 0.04
o-H:
m-H:
7.9
9.2
6.4
6.7
0.37 ± 0.02
0.11 ± 0.01
Ethanol-d6
2
o-H:
m-H:
5.2
4.9
4.4
4.4
0.69 ± 0.07
0.06 ± 0.01
17
18
19
1.07 ± 0.09 (1.82 ±
0.08)*
1.38 ± 0.1 (1.41 ±
0.05)*
o-H:
m-H:
24.5
26.2
12.9 (15.5)*
14.0 (17.4)*
Scheme 3. Synthesis of deuterated isoniazid analogues
o-H:
m-H:
12.1
34.7
14.3
18.0
0.88 ± 0.07
0.93 ± 0.08
Conclusions
o-H:
m-H:
5.0
6.2
4.6
5.0
0.51 ± 0.01
0.06 ± 0.01
We have undertaken a systematic SABRE study of the
substrates pyrazinamide (1), methyl pyrazine-2-carboxylate (3)
and isoniazid (2) and a series of their deuterated analogues. As
a consequence, we have shown that the selective partial
deuteration of these tuberculosis drug scaffolds results in both a
dramatically improved SABRE response and significantly
improved relaxation times. Further refinement through the use of
*[IrCl(COD)(d22-Imes)] used as the catalyst
hyperpolarization achieved using SABRE in these systems a
trend was not observed and each agent needs to be
investigated individually to secure an optimal response.
1
a deuterated SABRE polarization transfer catalyst, results in H
signal gains of up to 6300 times those seen under Boltzmann
conditions at 9.4 T result to yield 20.3% net polarization, an
effect which is achieved in a matter of seconds. T1 relaxation
times as high as 145 s were seen in methanol-d4 solution for
agent 6, thereby lengthening the observation window that is
available to the detection of an MR response after the
Seeking improvement, SABRE studies on mixtures of these
agents We hypothesized that if a mixture of two of these agents
were to be employed, it might be possible to improve the
detectability of one at the expense of the other. We therefore
prepared three samples, containing equimolar amounts of 6 and
8, 4 and 6 and 13 and 14. While 6 and 8 produced individual
returns of 9.2, 11.9 and 2.5% respectively this changed to 6.4,
6.6 and 1.8% in the mixture and hence both agents perform
worse when mixed and agrees with the fall in relaxation times
that is also seen.
hyperpolarization transfer step.
6
represents the optimum
3
isotopologue due to removal of the strong JH-H coupling that
facilitates relaxation within 3, whilst also maintaining an efficient
4JH-H coupling to the metal hydrides when bound to the catalyst
enabling efficacious polarization transfer.
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10.1002/chem.201703278
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For the tuberculosis drug pyrazinamide (1), increases in T1
relaxation of up to 340% and increases in average signal
enhancements from SABRE of up to 360% were achieved in the
biologically amenable solvent ethanol-d6, which correspond to
the detection of a 1H signal that has greater than 2500 times the
intensity received under Boltzmann detection conditions. This
Keywords: Hyperpolarization • SABRE • Tuberculosis •
Catalysis • Isotopes
Supporting information via: DOI: 10.15124/5e44a9c2-c968-
42c9-b8f0-885a552bafa0
systematic study of
a series of pyrazine-2-carboxylates,
[1]
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WHO, WHO Press: Geneva, 2016.
pyrazine-2-carboxamides, and isoniazid substrates reveals clear
insight into the desired deuterium substitution patterns required
to optimize the detection of these pharmaceutically important
molecules for SABRE hyperpolarization. In the case of 1 and 3,
retaining a 1H label at the 5-H or 1-H sites produce the optimum
situation in accordance with the fact that SABRE transfer will
proceed into this site via a large JH-H coupling in the associated
catalyst.[15] In view of the fact that tandem treatments with these
agents are also common, we also tested the effect of SABRE on
a series of mixtures of these agents. It is clear from the
presented data that it may be possible to further improve the
response of weakly polarising systems under such conditions.
By extending the relaxation times and increasing
polarization levels of the key TB drugs shown here we open up
the potential for their in vivo detection. As SABRE can be a
continuous process,[30] the polarization level can be maintained
to the point of injection, and thus, despite the T1 times being
reduced in water (see supporting information), these signal
gains and relaxation rates should give enough scope for
clinically relevant information to be collected. We predict that it
will be possible to improve on these responses still further by
either working at higher pressures of p-H2 or utilising alternative
SABRE catalysts in conjunction with the 2H-labelling strategy
exemplified here.[14b, 14c, 23] We hope that ultimately this would
contribute positively to the treatment of tuberculosis.
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Experimental Section
Polarization transfer procedure: The polarization transfer experiments
were conducted in a 5 mm NMR tube that was equipped with a J.
Young’s Tap. Samples were prepared as follows; a 5 mM solution of
[IrCl(COD)(NHC)] and 20 mM of substrate (1-19) in methanol-d4 or
ethanol-d6 (0.6 mL). The samples were degassed prior to the introduction
of parahydrogen at a pressure of 3 bar. Samples were then shaken for
10 s in the specified fringe field of an NMR spectrometer before being
rapidly transported into the magnet for subsequent interrogation by NMR
spectroscopy.
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Synthetic Procedures and characterization: Full experimental
procedures and characterization data can be found in the supplementary
information.
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Acknowledgements
This work was supported by The Wellcome Trust (Grants
092506 and 098335), the MRC, Bruker Biospin UK and the
University of York. We also thank Professor V. H. Perry for
useful discussions.
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10.1002/chem.201703278
Chemistry - A European Journal
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FULL PAPER
The tuberculosis drugs,
Philip Norcott, Peter J. Rayner, Gary
G. R. Green, Simon B. Duckett*
pyrazinamide, isoniazid and their
derivatives have been optimally
hyperpolarized using SABRE.
Through 2H-labelling the drugs and
catalysts T1 lifetimes of over two
minutes and ~20% polarization have
been achieved which opens the door
to in vivo assessment.
Page No. – Page No.
Achieving High 1H Nuclear
Hyperpolarization Levels with Long
Lifetimes in a Range of Tuberculosis
Drug Scaffolds
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