Ratiometric pH-Responsive 19F Magnetic Resonance Imaging Contrast Agents Based on Hydrazone Switches

Hydrazone-based molecular switches serve as efficient ratiometric pH-sensitive agents that can be tracked with 19F NMR/MRI and 1H NMR. Structural changes induced between pH 3 and 4 lead to signal appearance and disappearance at 1H and 19F NMR spectra allowing ratiometric pH measurements. The most pronounced are resonances of the CF3 group shifted by 1.8 ppm with 19F NMR and a hydrazone proton shifted by 2 ppm with 1H NMR.


General
Unless otherwise noted, all reagents and starting materials were purchased from commercial vendors and used without further purification. All experiments were conducted in the air unless otherwise noted. Column chromatography was performed using silica gel (60 Å, 230-400 mesh). Mass analysis was performed by UPLC-MS-MS (Waters Xevo G2 QTof, ESI ionization, high-resolution TOF detection).
NMR spectra were recorded on a 400 MHz Agilent spectrometer and referenced internally using the residual protonated solvent resonances relative to tetramethylsilane ( = 0 ppm), or trifluoroacetic acid ( 19 F NMR,  = -76.5 ppm). UV-Vis spectra were recorded using a JASCO V-650 UV-Vis spectrophotometer.
Saturated NH 4 Cl (aq.) (20 mL) and water (50 mL) were added, and the two layers were separated, and the aqueous layer was extracted with Et 2 O (3 × 30 mL). The combined organic layers were dried with MgSO 4 and evaporated under reduced pressure to give the product (4.5 g, 85%) as a bright yellow oil.

S4
Compounds: Trifluoromethylaniline (1.0 g, 6.2 mmol, 1 eq.) was dissolved in a mixture of 10.0 mL conc. HCl and 10.0 mL of EtOH and stirred in an ice bath for 30 min. A cold solution (8.0 mL) of sodium nitrite (0.4 g, 6.2 mmol, 1 eq.) was then added dropwise to the acidified solution over a period of 30 min. The obtained diazonium salt solution was then added dropwise to a suspension of ethyl-2-pyridylacetate (1.0 mL, 6.2 mmol, 1 eq.) and sodium acetate (3.3 g, 36.7 mmol, 6,4 eq.) in a cooled 40 mL ethanol/water (8: 1) mixture. The resultant reaction mixture was stirred overnight and then washed with methylene chloride. The organic fraction was washed twice with 30 mL saturated sodium bicarbonate solution and dried over magnesium sulfate. The crude product was then subjected to silica gel column chromatography (methanol/methylene chloride 1: 8) to give the pure compound as a bright orange solid.           X-ray quality crystals were obtained by dissolving 30 mg of the pure compounds in a minimum amount of dichloromethane and allowed to slowly crystallize.

S12
The diffraction data for both compounds were collected using an XtaLAB Synergy-I diffractometer with a HyPix3000 hybrid pixel array detector and microfocused PhotonJet-I X-ray source (Cu Kα). The structures were solved using the SHELXT 1 program and refined by the full-matrix least-squares procedure with Olex2.refine in OLEX2 (version 1.5). 2

Computational Methods
DFT calculations were carried out using B3LYP hybrid functional combined with a 6-31G (d, p) basis set, CPCM(Acetonitrile). 4 All calculations were performed using Orca 4.1.1 software. 5 Input files and molecular plots were prepared with Avogadro software. 6

Table S2
Calculated structures and HOMO/LUMO orbitals of isomers.

1-E
The samples for imaging were prepared by dissolving 5 mg of compound 1 or 3 in 1000 L of acetonitrile. Trifluoroethanol (TFE, 20 L) was added as a 19 F reference. The pH was adjusted to 7, 3.5, and 2 by adding calculated amounts of 0.65 M TFA. First, the scanner was tuned to the TFE resonance frequency. Sample shimming and other adjustments were completed using this frequency. Next, using a single-pulse sequence, the 19 F spectrum was acquired, from which relevant frequencies of the -CF 3 group were obtained. For MRI imaging, FLASH (compound 1) and RARE (compound 3) sequences were S15 used. Frequency-selective RF pulses were applied for the selective excitation of individual isomers, thus enabling pH-dependent imaging. Figure S14 shows the 19 F MR images of compound 1 obtained at different pH and different RF transmitter SFO1 frequencies. In the rightmost column, the transmitter frequency shift is provided.
Based on the NMR spectrum, the frequency Δ 1 was set to the resonance frequency of the -CF 3 group (blue rectangle, plot S14-C). Image S14-B was taken with this frequency, while for image S14-A, the frequency was changed by 1.8 ppm to cover the spectral region of the second peak (red rectangle). At a neutral pH, there was only one peak, and image S14-A shows only noise.
In the second row, images at the intermediate pH are shown. In this case, the NMR spectrum contained two peaks for the -CF 3 group. For image S14-E, the frequency Δ 1 was set to the resonant frequency of the right peak. Next, the SFO1 frequency was shifted by 1.8 ppm, and image S14-D was acquired.
Finally, for the third row of the table, SFO1 was set to Δ 1 +1.8 ppm (image S14-G) and next to Δ 1 for the S14-H image. This is how the images that make up the columns (A, D, G and B, E, H) represent pHdependent MRI imaging. The FLASH sequence with the following parameters was used to obtain the images shown in Figure S14: TR 33 ms; TE 16 ms; FA 15°, MTX 32 x 32; FOV 3 x 3 cm; NA 900; transmitter excitation pulse bandwidth 200 Hz; receiver effective spectral bandwidth 50k Hz.
The same principles outlined above were used in the MRI imaging of compound 3, presented in Figure   S15. In this case, since the two isomer peaks are closer than in the case of compound 1, the RF pulses with a narrower bandwidth of 50 Hz and narrower receiver effective spectral bandwidth of 3.2 kHz were used. The RARE sequence with the following parameters was used to obtain the images shown in Figure S15: TR 5000 ms; TE 24 ms; MTX 32 x 32; FOV 3 x 3cm; NA 4; transmitter excitation pulse bandwidth 50 Hz; receiver effective spectral bandwidth 3.2 kHz. S16 Figure S14. 19 F MRI of compound 1.  The solubilities of compounds 1, 2, and 3 were practically identical and are summarized in Table S5.  Figure S20 shows the spatial arrangement of samples with different pH. Figure S20. The arrangement of the samples used for 19 F MRI. 0 eq. (A, neutral pH), 0.6 eq. (B, 3.5 pH) and 1.6 eq. (C, 2 pH) of TFA.
To obtain the 19 F images shown in Figure S21 the RARE sequence with the following parameters was used: TR 5000ms; TE 24ms; MTX 32x32; FOV 3x3cm; rare factor 8; NA 16; transmitter excitation pulse bandwidth 300Hz; receiver effective spectral bandwidth 3.2kHz. To use the pH-dependent resonance frequency shift effect, images were acquired with different RF transmitter SFO1 frequencies. For image S21-A scanner frequency was set to -CF3 group resonance around -62.0 ppm. In this image, a stronger signal from a neutral pH sample and a weaker signal from a 3.5pH sample is observed. To obtain the S21-B image, scanner frequency was moved downfield by 1.7ppm to cover the resonance frequency of the 2pH sample. Indeed in figure S21-B, a stronger signal from a 2 pH sample and a weaker signal from a 3.5pH sample is observed. The images in Figure S21 show how by varying the scanner frequency accordingly, pH-sensitive MRI imaging can be accomplished. Figure S21. 19 F MRI images of three samples obtained with different transmitter frequencies.