Hydrazo-CEST: Hydrazone-Dependent Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Contrast Agents

Chemistry - A European Journal

10.1002/chem.201801671

DOI: 10.1002/ ((please add manuscript number))

Article type: Full Paper

Hydrazo-CEST: Hydrazone-Dependent Chemical Exchange Saturation Transfer Magnetic

Resonance Imaging Contrast Agents

Trina Dang, Mojmir Suchy, Yen J. Truong, Wendy Oakden, Wilfred W. Lam, Caitlin Lazurko,

*

Glenn Facey, Greg J. Stanisz, and Adam J. Shuhendler

T. Dang, Dr. M. Suchy, Y.T. Truong, C. Lazurko, Dr. G. Facey, Prof. A.J. Shuhendler

Department of Chemistry & Biomolecular Sciences, University of Ottawa, Ottawa, Ontario,

Canada

E-mail: adam.shuhendler@uottawa.ca

Dr. M. Suchy, Prof. A.J. Shuhendler

University of Ottawa Heart Institute, Ottawa, Ontario, Canada

Dr. W. Oakden, Dr. W.W. Lam, Prof. G.J. Stanisz

Department of Physical Sciences, Sunnybrook Research Institute, Toronto, Ontario, Canada

Prof. G.J. Stanisz

Department of Biomedical Physics, University of Toronto, Toronto, Ontario, Canada

Department of Neurosurgery and Pediatric Neurosurgery, Medical University, Lublin, Poland

Keywords: magnetic resonance imaging, chemical exchange saturation transfer, contrast agents,

hydrazones, aldehydes

The rapid formation of hydrazones under physiological conditions was exploited for the

detection of aldehydes through chemical exchange saturation transfer magnetic resonance

imaging (CEST-MRI). A metal-free, diamagnetic contrast agent derived from N-amino

anthranilic acid was introduced, which selectively ‘turned-on’ upon hydrazone formation

through an effect termed Hydrazo-CEST. While the hydrazine form of the probe produced no

CEST-MRI signal enhancement, the formation of the aryl hydrazone resulted in >20% intensity

1

decrease in the bulk water signal through the CEST effect, as measured by 300 MHz H NMR,

3

T and 7T MRI. Both the electronic contributions of the N-amino anthranilate and the aldehyde

binding partner were shown to directly impact the exchange rate of the proton on the ring-

proximal nitrogen, and thus the imaging signal. Additionally, the presence of the carboxylic acid

moiety ortho to the hydrazine was necessary not only for contrast production, but also for rapid

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hydrazone formation and prolonged hydrazone product stability under physiological conditions.

This work provided the first example of an MRI-based contrast agent capable of a ‘turn on’

response upon reaction with bioactive aldehydes, and outlined both the structural and electronic

requirements to expand on Hydrazo-CEST, a novel, hydrazone-dependent subtype of

diamagnetic CEST-MRI.

1

. Introduction

Magnetic resonance imaging (MRI) is a clinical diagnostic technique employing a variety of

contrast mechanisms, some of which are reliant upon the mapping of differential water proton

relaxation following radiofrequency excitation in a magnetic field. MRI has traditionally been

applied to anatomical and functional imaging in the clinical setting, and has been moving toward

molecular-level diagnosis in part through the use of biochemical-targeted contrast

[

1]

agents. Gadolinium-based chelates that reduce the relaxation time of neighboring water protons

have previously been the focus of contrast agent development, however concerns about chelate

[

2]

stability in vivo has motivated a search for metal-free alternatives. Chemical Exchange

[

3,4]

Saturation Transfer (CEST) using organic (i.e. diamagnetic) probes is a recently proposed

metal-free MRI technique offering a promising alternative mode of molecular-level contrast that

[

5]

can overcome the toxicity concerns associated with paramagnetic agents. The CEST

imaging mechanism depends on a contrast agent proton that readily exchanges with water, and

that possesses a sufficiently large resonance frequency offset from water (~1-3 ppm) to allow its

specific spin saturation by radiofrequency irradiation (Figure 1a). A spin saturation effectively

masks the exchangeable proton from detection, reducing the post-saturation amplitude of the

o

water peak (S) relative to its pre-saturation magnitude (S ) (Figure 1b). Ultimately, the

suppression of water signal results in a loss of image signal (i.e. a darkening) in the immediate

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vicinity of the contrast agent. Since solute-water proton exchange rates (ksw) for the CEST effect

2

3

are typically 10 -10 Hz, a few seconds of irradiation can significantly amplify the signal

[

6]

produced from a single contrast agent molecule.

Humans are exposed to toxic aldehydes on a daily basis, both externally through the

environment (e. g. acrolein, formaldehyde) and internally through highly regulated metabolic

processes (e. g. acetaldehyde, 3-aminopropanal). Dysregulation of these metabolic processes,

[

7,8]

[7]

[9]

including lipid peroxidation,

carbohydrate autoxidation, polyamine oxidation,

and

[

7]

myeloperoxidase activity, leads to an increased production of aldehydes during chemical or

mechanical stress, and throughout the course of a range of diseases. Aldehydes have been

suggested to be valuable biomarkers of brain damage, ischemia-reperfusion injury, and

[

10–12]

neurodegenerative disorders,

but their detection in vivo is challenging, and has remained

elusive until a very recent report by Caravan and coworkers demonstrating lung fibrosis imaging

[

13]

by hydroxylamine-modified gadolinium chelates. This initial demonstration of the value of

aldehydes as imaging biomarkers targeted allysine residues on fibrosis-associated oxidized

collagen, employing a mechanism of selective probe retention in aldehyde-rich tissue

environments. However, endogenous aldehydes are not usually protein-bound, but rather are

small (MW < 100), mobile and reactive molecules; consequently, their in vivo detection is not

[

13]

amenable to the ‘probe retention’-based imaging mechanism previously described.

Within the framework of our interest in developing advanced probes for molecular

imaging, we have built on the value of aldehydes as diagnostic biomarkers and designed

molecular probes capable of the in vivo imaging of these small, unbound aldehydes. Two key

design criteria for our probes were established: (1) the probe must react rapidly with endogenous

aldehydes to form stable products; (2) a significant signal enhancement should be observed upon

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reaction of the molecular imaging probe with the aldehydes. To comply with both requirements,

we investigated molecular probes based on the N-amino anthranilic acid (2-hydrazinobenzoic

acid) scaffold. Structurally similar hydrazines have been recently shown to react with aldehydes

-

1 -1

in “click-like” fashion, with a reaction rate > 2 M s that is comparable to that associated with

[

14,15]

strain-promoted alkyne-azide cycloaddition.

Moreover somewhat structurally related N-

substituted (N-phenyl, N-mesyl, N-trifluoroacetyl) anthranilic acids are known to act as

[

16]

diamagnetic CEST MRI contrast agents. Through a detailed investigation of a small library of

N-amino anthranilic acids and corresponding hydrazones, we have expanded the functionalities

available for metal-free contrast agent design, demonstrated that the proton on the ring-proximal

nitrogen of an aryl hydrazone is amenable to detection by CEST-MRI, and present the first

CEST agent that conditionally produces signal only after binding rapidly to aldehydes under

physiological conditions.

2

. Results and Discussion

.1 Synthetic Procedures

2

Intermediates and final products described in this study (Figure 2) have been prepared in

moderate to good yields, often times using modified literature protocols. Synthetic schemes, full

experimental details and NMR spectra associated with the compounds discussed within this

paper can be found in the Supporting Information (SI).

Briefly, anthranilic acids 1a-1f were diazotized (NaNO

2

, HCl), followed by SnCl

2

· 2H

2

O-

as

[

17]

mediated

reduction. Corresponding

hydrazines 2a-2f were obtained

dihydrochlorides. Treatment of 2a-2f with 2-formylbenzenesulfonic acid afforded hydrazones

3

a-3f; when 2a-2f were treated with acetaldehyde, hydrazones 4a-4f were obtained. One notable

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exception is hydrazone 4d, which we were unable to isolate. To expand the library of

compounds, hydrazone 2f was treated with aldehydes or corresponding synthetic equivalents (3-

[

18]

aminopropanal diethyl acetal, crotonaldehyde, racemic glyceraldehyde

and glycolaldehyde

dimer) to furnish hydrazones 5a-5d. Reaction of 2f with acetone or pyruvic acid afforded

hydrazones 6a and 6b.

To prepare the control compounds used in this study, 5-methoxyanthranilic acid (1f) was

[

19]

esterified (Fischer esterification),

followed by a one pot diazotization-reduction

[

17]

cascade. Ester hydrazine 7 was obtained as a dihydrochloride salt. Treatment of 7 with 2-

formylbenzenesulfonic acid furnished hydrazone 8a, while 8b was prepared by reaction of 7 with

acetaldehyde. Reaction of commercially available 4-methoxyphenyl hydrazine dihydrochloride

(MeOPH, 9) with 2-formylbenzenesulfonic acid led to the formation of hydrazone 10a; despite

many attempts we were unable to isolate hydrazone 10b derived from 9 and

acetaldehyde. Finally, when hydrazine 2f was treated with malondialdehyde tetramethyl

[

20]

acetal, pyrazole 11 was obtained. 3-Hydroxybenzoic acid (12) was commercially available.

2

.2 Structural requirements for CEST signal

One limitation of diamagnetic CEST contrast agents is their often-small frequency offsets (~1-3

ppm) from water, making their signal deconvolution from the highly abundant water peak

[

16]

difficult. Anthranilic acids have previously been identified as CEST agents with a significant

4

.8-9.3 ppm CEST signal frequency offset, a result of intramolecular hydrogen bonding between

[

16]

the benzylic amine and a hydrogen bonding partner in the ortho-position. We explored the

effect of converting the weakly nucleophilic aromatic amine of anthranilic acid into an a-

nucleophilic hydrazine with the intention of producing an aldehyde-specific imaging

agent. Interestingly, the formation of the hydrazine nucleophile completely abolished proton

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exchange detectable by CEST methods (e.g. 2f), but this signal was recovered upon conversion

of the probe to a hydrazone (e.g. 4f); only the hydrazone, but not the hydrazine, provided CEST

contrast (Figure 3). To denote the set of probes behaving in a similar manner, we propose the

term Hydrazo-CEST. Probe chemistries that are activated (i.e. produced detectable changes in

image signals) only once bound to, or acted upon by, their intended molecular target of interest

benefit from an inherent decrease of background signal, maximizing the achievable signal-to-

noise ratio and imaging signal reliability. In addition, the ‘turn-on’ mechanism of aldehyde

reporting utilized by Hydrazo-CEST is independent of the anchorage of target aldehydes, and

would be capable of mapping freely diffusing small molecule aldehydes as well as those derived

from oxidized residues of biomacromolecules. The Hydrazo-CEST probes presented here thus

take advantage of activation by endogenous aldehydes, and are an initiating class of non-optical,

activatable imaging agents capable of selectively sensing endogenous reactive carbonyls.

Hydrazo-CEST probes maintain a large 6.4 ppm frequency offset, and exhibit the same

requirements for a hydrogen bonding partner in the ortho-position as demonstrated for

1

6

anthranilic acids. The loss of the carboxylic acid proton through esterification results in a

complete loss of the CEST effect (8b, Figure 4a), and the complete removal of the carboxylic

acid to form a substituted phenylhydrazine (10a, Figure 4b) results in significant broadening and

weakening of the CEST signal, and an approximate 2 ppm upfield shift. While compound 10a

exhibits some CEST signal likely due to interaction of the sulphate with the ring proximal

nitrogen, the loss of the intramolecular hydrogen bonding due to removal of the ortho

carboxylate substantially alters the CEST signal produced. The importance of this intramolecular

hydrogen bond is supported through molecular modelling of aliphatic and aryl aldehyde-derived

[

21]

hydrazones (Figure S1). E/Z isomerization around the hydrazone bond is known, which alters

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the relative position of the proton on the ring-proximal nitrogen, the putative exchangeable

proton producing the Hydrazo-CEST signal. Density functional theory calculations using a

B3LYP force field in water as solvent revealed that the E-isomers of both the aliphatic

acetaldehyde- and aryl 2-formylbenzenesulfonic acid-derived hydrazones adopted conformations

optimally positioning the ring-proximal nitrogen for intramolecular hydrogen bonding with the

neighboring carboxylic acid group. The calculated geometry of the aliphatic aldehyde-derived

hydrazone is that of a planar pseudo-six membered ring, with the putative exchangeable proton

bonding directly with the carboxyl oxygen. The aryl aldehyde-derived hydrazone formed a more

strained pseudo-six membered ring, with the ring-proximal nitrogen hydrogen bonding to the

1

carboxyl proton (Figure S1). H-NMR spectra of the acetaldehyde-derived hydrazone (4f) show

both E- and Z-isomers, with E predominating 2:1 over the Z-isomer, and the cis conformation of

the hydrazone bond predominating 2:1 over the trans conformation. Only the E-isomer is

1

observed in the H-NMR of the 2-formylbenzenesulfonic acid-derived hydrazone (3f), which is

in agreement with our molecular modelling that was unable to minimize the energy associated

with the Z-isomer for this structure (Figure S1).

The characterization of the structural requirements for Hydrazo-CEST continued by the removal

of the a-nucleophile but retention of the carboxylic acid, which nearly completely abolished

Hydrazo-CEST signal production (Figure 4c). Finally, we sought to confirm that the proton on

the ring-proximal nitrogen was actually the proton exchanging with water to induce the Hydrazo-

CEST effect. Complete abolishment of signal production was observed with pyrazole 11

possessing no exchangeable proton on the ring proximal nitrogen (Figure 4d), supporting the

hypothesis that indeed the proton on the ring-proximal nitrogen is the exchangeable proton

giving rise to Hydrazo-CEST. Therefore, both computational and experimental data highlight

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the importance of the conformation of the hydrazone bond to position the exchangeable

hydrogen, which is indeed that on the ring-proximal nitrogen, for hydrogen bonding with the

necessary carboxylic acid group in the ortho position.

2

.3 Kinetics of hydrazone formation & stability studies

In order to take advantage of the enhanced signal-to-noise ratio provided by the aldehyde-

dependent activation of Hydrazo-CEST, the formation of the hydrazone from N-amino

anthranilic acid must occur in situ under physiological conditions. The rates of reaction of

[

14,15]

carbonyls with hydrazine-derived nucleophiles have previously been well-characterized.

It

was found that intramolecular acid/base catalysis significantly enhanced the rate of hydrazone

formation, with the reaction of butanal and 2-hydrazinobenzoic acid (2a) having a pseudo-first-

-

1

order rate constant (kobs) of 1.5 ± 0.2 min in a 1:10 DMF: PBS solution. Repeating the

[

14]

methodology outlined previously, however at 37°C in 100% 1´ PBS, we determined the kobs

for the reaction of 5-methoxy N-amino anthranilic acid (2f) and related analogs with 2-

formylbenzenesulfonic acid (Figure 5, S2) and acetone (Figure S3). The calculated kobs are

summarized in Table S1. Under the conditions employed, the kobs for hydrazone formation from

2

f with 2-formylbenzenesulfonic acid was an order of magnitude faster than with acetone. This

difference in reaction rate was previously observed among carbonyl compounds, with hydrazone

[

14,15]

formation from aldehydes proceeding faster than from ketones.

Likewise, the kobs for

hydrazone formation from 2-formylbenzenesulfonic acid and 7, a methyl ester analog of 2f, was

slowed by three orders of magnitude, which was previously observed and attributed to the

[

14]

increased bulk of the methyl ester relative to the carboxylic acid. Importantly, substantial

differences were observed in the extent of reaction completion and product stability, in addition

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to rate of reaction, between hydrazine structures for a given aldehyde, as well as between

carbonyl types for a given hydrazine (Table S1).

Hydrazone stability was assayed both spectrophotometrically (Figure 5a, S2a vs. b), as well as

by HPLC (Figure 5b). While the formation of hydrazone from 2f and 2-formylbenzenesulfonic

acid reached ~90% completion within 30 min of initiation (Figure 5a, black curve), the absence

of the intramolecular acid/base catalyst, as in the reaction of 4-methoxyphenylhydrazine (9) or 7

with the same aryl aldehyde, substantially reduced reaction completion (<25% maximum;

Figure 5a, red and blue curves, respectively), and reduced stability of the hydrazone product

(

Figure 5b). This data suggests a role for the ortho-carboxylic acid in product stabilization, in

[

14]

addition to the catalytic effect previously observed. A similar difference in reaction

completion was observed for the reaction of 2f with acetone, resulting in <25% product

formation (Figure S3). Under the buffer and temperature conditions studied (i.e. 37°C and 1´

PBS), the rate of the reaction, the extent of reaction completion, and the stability of the

hydrazone product were all substantially improved in the presence of the ortho-carboxylic

acid. Since the signal produced by Hydrazo-CEST depends on the hydrazone form of the probe,

the faster-forming and longer-lived the hydrazone product under physiological conditions, the

better the imaging signal evolved and the more potentially sensitive the imaging technique.

2

.4 Electronic requirements for CEST signal

After demonstrating the structural requirement of the ortho-carboxylic acid moiety for Hydrazo-

CEST signal production and the rapid, stable, and aldehyde-preferential formation of hydrazone

under physiological conditions, the electronic contributions to Hydrazo-CEST were

3 3 2

evaluated. Electron donating (OH, OCH ) and electron-withdrawing (I, SO , NO ) substituents

were appended at the 5-position of N-amino anthranilic acid (compounds 2b-f), both

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acetaldehyde (4b-f) or 2-formylbenzenesulfonic acid-derived hydrazones (3b-f) were prepared,

and Z-spectra were acquired in 1:10 D O:PBS at pH 7.4 on a 300 MHz NMR (Figures S4-5).

2

Hydrazo-CEST signal production (%MTRasym) was normalized to hydrazone concentration, as

different hydrazone species showed different maximum solubility in neutral buffer (Figure S6-

7

). In this way, per molar signal output could be compared across hydrazone series to examine

electronic contributions to Hydrazo-CEST (Figure 6). Importantly, Hydrazo-CEST as a contrast

mechanism produced as much signal as the aspirin metabolite salicylate (Figure 6, grey bar), a

[

22,23]

diamagnetic CEST agent recently shown to have very high signal production.

Ring

deactivating substituents at the 5-position on the N-amino anthranilic acid substantially reduced

Hydrazo-CEST signal relative to the unsubstituted parent compound. For the acetaldehyde-

derived hydrazones 4a-4f, a decrease in Hydrazo-CEST signal relative to the unsubstituted

parent hydrazone was observed with an increasing strength of ring activation. However, the 2-

formylbenzenesulfonic acid-derived hydrazones 3a-3f showed the opposite effect: an increase in

Hydrazo-CEST signal production with an increase in ring activation potential.

In efforts to explain these trends in signal production, the rate of exchange between bulk water

and the proton on the ring-proximal nitrogen, ksw, were measured by varying the saturation pulse

[

24]

power, as previously described, and the values are shown in Figure 6. Indeed, the change in

%MTRasym induced by altering the electronics through the 5-position of the hydrazine ring

paralleled the change in ksw. The importance of the contribution of the aryl hydrazine electronics

to signal generation is supported by the linear free energy relationship between the chemical shift

2

of the exchangeable hydrazone proton and the normalized proton exchange rates (R =0.990)

(

Figure S8). Additionally, the aldehyde binding partner contributes to ksw of the proton on the

ring-proximal nitrogen, likely via the π-conjugation maintained through the hydrazone bond

1

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(

Figure S9). The o-sulfo substituent of the aryl aldehyde is more electron-withdrawing than the

aliphatic carbon of the acetaldehyde, reducing electron density around the hydrazone bond (e.g.

f versus 4f). While the exchange process of this system is complicated by the intramolecular

3

hydrogen bond provided by the ortho carboxylate, overall the data suggests that electronic

contributions of both the aldehyde and hydrazine binding partners affect per molar normalized

imaging signal by modulating the exchange rate of the proton on the ring-proximal nitrogen with

water.

2

.5 Concentration- and pH-dependence of Hydrazo-CEST signal

The MRI contrast produced by CEST probes is dependent upon voxel-wise concentration,

however previous investigations have reported non-linear concentration relationships for CEST

[

25]

contrast agents. In order to evaluate this dependence for Hydrazo-CEST compounds, Z-

spectra were acquired for acetaldehyde (4b-4f) and 2-formylbenzenesulfonic acid-derived

hydrazones (3b-3f) on a 300 MHz NMR from 5 mM to 40 mM, or the maximum solubility of the

compound in neutral buffer (Figure S6-7). The dynamic range for Hydrazo-CEST signal

production from each compound was then plotted using the maximum signal for each compound

(6.4 ± 0.4 ppm). Within the series of acetaldehyde-derived hydrazones, the 5-methoxy- (4f) and

5

-hydroxy-substituted (4e) analogs had the broadest dynamic range for imaging signal (Figure

S10a, green and blue curves, respectively), with the 5-methoxy-derivative showing the higher

absolute %MTRasym and the most linear concentration-dependent signal production

2

(

R =0.98). While the signal production at 20 mM was highest for the unsubstituted hydrazone

(

4a) relative to 4e and 4f, its low sensitivity to changes in concentration below 10 mM and

limited solubility beyond 20 mM substantially narrowed its dynamic range. For the series of 2-

formylbenzenesulfonic acid-derived hydrazones, the 5-hydroxy-substituted (3e) analog displayed

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the best dynamic range with the most linear concentration-dependent signal production

2

(

R =0.98) of all of the analogs examined (Figure S10b, blue curve). In fact, both the

unsubstituted (3a) and 5-methoxy-substituted (3f) analogs displayed non-linear concentration

dependence over the range of concentrations examined (Figure S10b, red and green curves,

respectively). Importantly, the signal production from 4f at 5 mM (MTRasym=9%) has been

[

16,26]

shown to be suitable for detection in vivo by CEST-MRI,

with salicylate, a diamagnetic CEST-MRI contrast agent readily detectable in vivo.

In addition to local probe concentrations, microenvironmental pH can modify CEST signal

and is on par on a per molar basis

[

23]

[

4,27]

production, as pH directly impacts ksw

unsubstituted, 5-hydroxy-, and 5-methoxy-substituted hydrazones derived from acetaldehyde

4a, 4e, 4f) and 2-formylbenzenesulfonic acid (3a, 3e, 3f) (Figure S11). The aliphatic aldehyde-

.

The effect of pH on %MTRasym was evaluated for the

(

derived hydrazones displayed a broad pH range (approx. pH 6 to 8) over which high CEST

signal production was observed, with maximum signal occurring between pH 6.5 and 7.5. The

5

-methoxy-substituted probe (4f) produced significantly higher CEST signal at all pH values

tested. Note that 4f was not soluble at 40 mM at pH 6.5, however both 4a and 4e were

completely soluble from pH 6.0-8.0. In contrast, the %MTRasym from aromatic aldehyde-derived

hydrazones was substantially reduced above pH 6.5-7.0, with maximum signal generation

occurring below pH 6.5. The hydroxy- and methoxy-substituted aryl-hydrazones (3e, 3f) gave

better CEST signal production from pH 6.5-8.0, but both were insoluble at 40 mM at pH

6

.0. The differential pH responses associated with the HydrazoCEST probes studied are justified

by the higher predicted electron density around the ring-proximal nitrogen of the aliphatic versus

aromatic hydrazones (Figure S9), which would favor the protonated form of the hydrazo moiety

[

7]

at higher pH values. Since the majority of endogenously formed aldehydes are non-aromatic,

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the 5-methoxy-substituted analog (2f) was selected as the lead compound for endogenous

aldehyde detection with Hydrazo-CEST due to its superior dynamic range with aliphatic

aldehydes and enhanced signal generation at neutral pH.

2

.6 Detection of endogenous carbonyls

Having optimized Hydrazo-CEST probe chemistry for high signal production in physiological

conditions, signal generation from endogenous carbonyls was explored. Z-spectra were

generated for a variety of hydrazones formed from 2f and endogenous aldehydes and ketones

(

Figure 7): 5a is the hydrazone of 3-aminopropanal, a product of polyamine catabolism

[

11,12]

suggested to be elevated following traumatic brain injury (Figure 7a);

5b is the hydrazone

product of crotonaldehyde, a terminal product of lipid peroxide catabolism following cell stress

[

7]

(

Figure 7b); 5c is the hydrazone formed from glyceraldehyde, the glycolytic intermediate

required for phospholipid biogenesis (Figure 7c); 6b is the hydrazone product of 2f and

pyruvate, a key biochemical intermediate for a variety of biosynthetic and metabolic pathways

including anaerobic glycolysis (Figure 7d). Importantly, there was no hydrazone formation

between 2f and D-glucose under physiological conditions (37°C, 1´ PBS, pH=7.4) within 4 h of

incubation, suggesting glucose will not interfere with aldehyde sensing by Hydrazo-CEST.

2

.7 Hydrazo-CEST MRI

As further support of this novel class of contrast agent for aldehyde-activated imaging, we

validated the performance of Hydrazo-CEST by 3T and 7T MRI (Figure 8). Just as observed in

the NMR, the hydrazine form of the probe (2f) produced no CEST contrast (Figure 8a, left),

however signal production was substantially increased following hydrazone formation from

either aliphatic (4f) or aromatic aldehydes (3f) with a frequency offset from water of 6.4

ppm. The kinetics of Hydrazo-CEST signal production through hydrazone formation were also

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evaluated in the MRI at 37°C in 1´ PBS at pH=7.4 (Figure 8b). Either PBS (red curve) or a

PBS solution of 2-formylbenzenesulfonic acid at a final concentration of 20 mM (blue curve)

were injected into the imaging phantom, and Z-spectra were acquired every 4.5 min to monitor

hydrazone formation. A single saturation peak at 6.4 ppm was observed 4.5 min after aldehyde

injection. The Hydrazo-CEST signal increased rapidly, surpassing 50% maximal signal within

the first 4.5 min, and reaching a maximum plateau (%MTRasym 4.6±0.7) by 18 to 22.5 minutes

after aldehyde addition. This MRI data confirms that Hydrazo-CEST probes are rapidly

activated by aldehydes under physiological conditions, and are readily detectable by 3T and 7T

MRI. Both hydrazine and hydrazone functionalities are utilized in medications broadly spanning

[

29]

indications from anti-infectives and anti-parasitics, to anti-cancer agents. Taken together with

preliminary in vitro toxicity assays demonstrating the initial safety of 2f (Figure S12), the

substantial signal production (approx. 20% MTRasym) by the 3-aminopropanal-derived hydrazone

(

5a), the limited signal production by metabolic aldehyde (glyceraldehyde, 5c), and the limited

hydrazone formation rates between 2f and ketones, such as pyruvate (6b) (Figure S3) supports

that Hydrazo-CEST is potentially valuable as a first-in-class, aldehyde-activated MRI contrast

agent for mapping these endogenous markers of tissue stress.

3

. Conclusion

In conclusion, we have developed a new class of CEST-MRI contrast agents derived from

N-amino anthranilic acids, termed Hydrazo-CEST, which conditionally respond to the presence

of some endogenous aldehydes upon in situ transformation from hydrazine to hydrazone. The

‘turn-on’ mechanism of aldehyde reporting utilized by Hydrazo-CEST is independent of the

anchorage of target aldehydes, and would be capable of mapping freely diffusing small molecule

1

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aldehydes as well as those derived from oxidized residues of biomacromolecules. The

importance of the carboxylic acid ortho to the a-nucleophile was investigated, demonstrating its

absolute requirement for signal production, as well as for rapid hydrazone formation and

extended product stability under physiological conditions. Additionally, the electronic

requirements providing optimal CEST contrast have been identified, with electron-donating

substituents at the 5-position (i.e. hydroxy and methoxy) providing superior performance in

terms of signal generation over the ranges of concentrations and pH expected in living

subjects. The rapid reaction with aldehydes and high CEST MRI signal production at 3T and 7T

support the application of these probes for the detection and mapping of endogenous aldehydes

in living subjects. With Hydrazo-CEST, small molecule, endogenous aldehyde biomarkers of

disease may now be accessible to mapping, allowing chemical biological investigations of these

important biomolecules.

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements

This work was supported by an NSERC Discovery Grant RGPIN 2015-05796 (A.J.S.), the

Canada Research Chairs Program 950-230754 (A.J.S.), the Canadian Foundation for Innovation

(A.J.S.), and the Canadian Institutes of Health Research PJT376892 (A.J.S.).

Received: ((will be filled in by the editorial staff))

Revised: ((will be filled in by the editorial staff))

Published online: ((will be filled in by the editorial staff))

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Chemistry - A European Journal

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Figure 1. Chemical exchange saturation transfer (CEST) imaging works through proton

exchange between contrast agent and water. (A) An exchangeable proton (white circle) on the

CEST contrast agent (green hexagon) has a chemical shift that is different from that of water by

1

H-NMR. This chemical shift difference (+Δω) allows the exchangeable proton on the contrast

agent to be spin saturated, effectively making the exchanged water protons invisible to detection

by magnetic resonance imaging (MRI). (B) The exchangeable proton on the contrast agent is

spin saturated (purple circle) and undergoes exchange with water protons, effectively making

o

water invisible to the MRI (signal peak at S versus that as S+Δω). Since proton exchange occurs

thousands of times per second, and since saturation is induced for approximately 3-5 seconds,

thousands of water protons are “silenced” by a single contrast agent, allow the depression of

water to be mapped by the MRI. The signal intensity (%CEST) is calculated as the signal ratio

with and without contrast agent proton spin saturation.

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Chemistry - A European Journal

10.1002/chem.201801671

Figure 2. Chemical structure of compounds discussed in this study. * Compounds were not

isolatable.

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Chemistry - A European Journal

10.1002/chem.201801671

2

f

e.g. 4f

3

5

0

3

2

5

0

5

1

.00

1

0

.00

.75

.50

.25

.00

25

20

15

0.75

0.50

0.25

S/S

Proton

/ Sa ter

o

S/S_o

Proton

Water

MTR_a_s_y_m(%)

20

15

W

S

o

MTR _s_y_m(%)

Proto

a

n

Water

MTR_a_s_y_m(%)

1

5

0

1

5

0

.00

1

2

8

4

0

-4

-8

-12

1

2

8

4

0

-4

-8

-12

Δω (ppm from H O)

2

Δω (ppm from H O)

2

Figure 3. Hydrazo-CEST is a new mechanism of MRI contrast enhancement, with signal

production selectively turned on in the presence of bioactive carbonyls (e.g. aldehydes and

ketones). The exchangeable hydrazo proton (purple) does not result in the suppression of water

signal when the contrast agent, based on substituted N-amino anthranilic acids, is in the

hydrazine form (i.e. k’sw is not amenable to CEST-MRI). The rapid condensation of the

hydrazine with the aldehyde or ketone to form a hydrazone results in an optimized exchange of

the spin saturated proton with water, significantly reducing the water signal and producing

CEST-MRI contrast enhancement (i.e. ksw is ideally suited to CEST-MRI). Z-spectra were

3

acquired at 37°C, pH 7.4, and 40 mM concentrations. In this example, R=CH .

2

Chemistry - A European Journal

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3

2

5

0

5

35

30

25

20

15

^a⁾1

.00

b) ₁_.₀₀

0

.75

.50

.25

.00

0.75

0.50

0.25

0.00

O

S/S_o

Proton

Water

MTR_a_s_y_m(%)

20

S/S_o

Proton

Water

MTR_a_s_y_m(%)

S/S_o

Proton

Water

NH

N

O

1

5

0

5

0

O

10a_O

MTR_a_s_y_m(%)

O

S

10

5

NH

HO

8

b

N

0

-12

1

2

8

4

0

-4

-8

-12

12

8

4

0

-4

-8

Δω (ppm from H O)

2

3

5

0

5

0

35

30

25

20

c) 1.00

d) 1.00

0.75

3

2

0

.75

.50

.25

.00

S/S_o

Water

O

N

S/S_o

Proton

Water

^M^T^Rasym(%)

S/S_o

O

Proton

0.50

OH

N

15

10

5

MTR_a_s_y_m(%) 15

Water

O

Proton

OH

MTRasym(%)

HO

10

5

11

0.25

10 mM

max. solubility)

(

1

2

0

.00

0

-12

1

2

8

4

0

-4

-8

-12

1

2

8

4

0

-4

-8

Δω (ppm from H O)

2

Figure 4. Z-spectra demonstrating the requirements of both the hydrazone and o-carboxylic acid

for the production of contrast for CEST-MRI. The masking of the o-carboxylic acid as the

methyl ester (a) or its complete absence (b) significantly impairs CEST-MRI signal generation.

Likewise the absence of the hydrazine moiety nearly completely abolishes signal production (c).

The absence of the hydrazone proton through the formation of a pyrazole also completely

abolishes CEST-MRI signal (d). Raw data points (S/S , black circles), exchangeable proton (red)

o

and water (blue) Lorentzian curves, and CEST-MRI signal production (MTRasym(%), black

curve) are shown. All Z-spectra are obtained at 37°C and pH 7.4 with 40 mM compound

2

solutions in 10:1 PBS:D O unless otherwise indicated.

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Chemistry - A European Journal

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SO3^-

O

H

a)

O

R

O

R

NH

N

NH2

R

3

SO3^-

Me 2O fNA COOH

MeOPH

Me 7E ster COOMe

H

1

00

Me

MeOPH

MeEster

2

O

f

NA³

7

5

0

50

100

150

t (min)

Incubation

Time (min)

b)

0

37.5

75

125

1

0

.0

.5

.0

_M_e_O₂_N_f_A3

1.0

0.5

0.0

1.0

0.5

0.0

0

.5

0.0

1

6.5 17.0 17.5 18.0 18.5

16.5 17.0 17.5 18.0 18.5

Retention Time (min)

1

.0

1

0

.0

.5

.0

1.0

0.5

0.0

1.0

0.5

MeOPH

0.5

0.0

1

6.5 17.0 17.5 18.0 18.5 16.5 17.0 17.5 18.0 18.5 16.5 17.0 17.5 18.0 18.5

16.5 17.0 17.5 18.0 18.5

Retention Time (min)

Figure 5. The o-carboxylic acid contributes to substantial reaction completion as well as

extended stability of the product hydrazone under physiological conditions. (a) Kinetic plots for

the reaction of N-amino anthranilic acid (2f), MeOPH (red), or the methyl ester of 2f (compound

7

) (blue) with 2-formylbenzenesulfonic acid are shown, demonstrating that the carboxylic acid

moiety is necessary for driving the reaction to completion, and to maintaining the stability of the

hydrazone product. (b) High-pressure liquid chromatography traces of the reaction of 2f (black)

and MeOPH (red) with 2-formylbenzenesulfonic acid. The hydrazone peak is shown (r.t. = 17.5

min for 2f and 17.2 min for MeOPH) at the time points indicated. All reactions were performed

in 1´ PBS at 37°C, and points represent mean±s.d. for n=3 trials.

2

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Chemistry - A European Journal

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Salicylate

O

OHOHOH

3

5

7

40 Hz

36 Hz

40 Hz

R

6

2 Hz

OH

R

HO

MeO

OCH

50 Hz

3

NH

N

HN

H

3

O

S

N

4

O

Iod

I

o

I

do

Not Soluble

Not Achievable Su

S

l

O

ulfo

3

2

Nitro

Nitr

NO

R

1

.2

1.0

0.8

0.6

0.4

0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

%

MTR_a_s_y_m/mM

% MTR_a_s_y_m/mM

Figure 6. The substitution of both the hydrazine and carbonyl substantially impact the CEST-

MRI signal generation from Hydrazo-CEST contrast agents. The concentration-normalized

Hydrazo-CEST signal (%MTRasym/mM, black bars) is plotted for hydrazones formed by different

substituents at the 5-position of the aryl hydrazine moiety, and with aliphatic (4) or aromatic (3)

aldehydes. Signal production can be directly compared to salicylate, a known diamagnetic CEST

agent (grey). Note: Sulfo-substituted hydrazine did not form a hydrazone with acetaldehyde.

Values shown on plot are ksw in Hz.

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Chemistry - A European Journal

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3

5

0

3

2

5

0

5

a)

b) 1.00

1

0

.00

.75

.50

.25

.00

S/S_o

25

20

0.75

Proton

^S^/^So

S/S

Wa

o

ter

S/S_o

Proton

Water

MTR_a_s_y_m(%)

Water

O

Water

Proton

MTR

20

O

Proton

O

0

.50

OH

MTR_a_s_y_m(%) 15

O

(%) 15

asy mO H

NH

N

10

5

1

5

0

5b

mM

0.25

5

5a

0 mM

4

(

max. solubility)

NH2

0

.00

0

-12

1

2

8

4

0

-4 -8

1

2

8

4

0

-4

-8

-12

Δω (ppm from H O)

2

3

2

5

3

5

0

1

0

.00

c)

d) 1.00

0

5

3

O

25

.75

.50

.25

.00

0

.75

.50

.25

.00

O

^S^/^So

S/S_o

Water

O

OH

S/S_o

Proton

Water

MTRasym(%)

Water

20

2

0

NH

Proton

O

Proton

MTR

OH

N

^M^T^Rasym(%) 15

(%) 15

5

c

asym

NH

N

HO

10

5

1

5

0

6b

HO

OH

O

0

-12

1

2

8

4

0

-4

-8

1

2

8

4

0

-4

-8

-12

Δω (ppm from H O)

2

Δω (ppm from H O)

2

Figure 7. Hydrazo-CEST signal production from endogenous carbonyls. Z-spectra were

acquired on a 300 MHz NMR in 10:1 PBS:D O at 37°C, pH 7.4, at 40 mM, or the indicated

2

concentration providing maximum solubility. Hydrazones were formed between 2f and (a) 3-

aminopropanal, (b) crotonaldehyde, (c) glyceraldehyde, and (d) pyruvate.

2

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Chemistry - A European Journal

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O

OH

O

a)

OH

NH

N

OH

NH

O

S

NH

N

O

NH2

2

f

3f

4f

1

5

at 7T

0

3

5

35

30

25

20

15

10

5

1

0

.00

.75

.50

.25

.00

1.00

0.75

0.50

0.25

0.00

3

2

1

0

5

0

5

S/S

Water

Proton

MTRasym(%)

o

S/S_o

Proton

Water

MTRasym(%)

3f

at 7T)

2

f

10

5

(

at 7T)

0

-8

0

-8

8

4

0

-4

8

4

0

-4

Δω (ppm from H2O)

3

5

0

35

30

25

20

15

10

5

1

.00

.75

.50

.25

.00

1.00

0.75

0.50

0.25

0.00

S/S

Water

Proton

MTRasym(%)

o

25

20

S/So

0

Water

Proton

MTRasym(%)

1

5

0

5

0

4f

at 7T)

4f

(at 3T)

(

0

-12

8

4

0

-4

-8

12

8

4

0

-4

-8

b)

2

Δω (ppm from H O)

2

t = Pre-injection

4.5 min

9 min

13.5 min

2

f + Vehicle

f + Aldehyde

%

MTRasym

2

.6±0.7

3.6±0.7

4.2±0.7

18 min

22.5 min

27 min

31.5 min

4

.1±0.7

4.6±0.7

4.5±0.7

4.7±0.7

Δω (p.p.m. from water)

Figure 8. Hydrazo-CEST probes provide high contrast and rapidly form hydrazones in situ. (a)

Phantom image and Z-spectra of hydrazine 2f and hydrazones 3f and 4f were acquired at the

indicated field strength, 37°C, pH 7.4, and 40 mM concentration. (b) Z-spectra were acquired

following the addition of vehicle (red) or 2-FB (blue) to a 40 mM solution of hydrazine 2f at

3

7°C and pH 7.4. Values indicate the %MTRasym ± the standard deviation of the off-resonance

measurements once the system had reached steady state.

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Chemistry - A European Journal

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Aldehyde and seek! A novel class of contrast agents that conditionally provide chemical

exchange saturation transfer magnetic resonance imaging (CEST-MRI) signal upon in situ

reaction with aldehydes is introduced.

Keyword

T. Dang, M. Suchy, Y. J. Truong, W. Oakden, W. W. Lam, C. Lazurko, G. Facey, G. J. Stanisz,

and A. J. Shuhendler

Hydrazo-CEST: Hydrazone-Dependent Chemical Exchange Saturation Transfer Magnetic

Resonance Imaging Contrast Agents

2

8

Article Doi

Hydrazo-CEST: Hydrazone-Dependent Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Contrast Agents

DOI: 10.1002/chem.201801671

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Article abstract of DOI:10.1002/chem.201801671

Full text of DOI:10.1002/chem.201801671

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