A Single‐Pot Template Reaction Towards a Manganese‐Based T 1 Contrast Agent

Abstract Manganese‐based contrast agents (MnCAs) have emerged as suitable alternatives to gadolinium‐based contrast agents (GdCAs). However, due to their kinetic lability and laborious synthetic procedures, only a few MnCAs have found clinical MRI application. In this work, we have employed a highly innovative single‐pot template synthetic strategy to develop a MnCA, MnLMe , and studied the most important physicochemical properties in vitro. MnLMe displays optimized r 1 relaxivities at both medium (20 and 64 MHz) and high magnetic fields (300 and 400 MHz) and an enhanced r 1 b=21.1 mM−1 s−1 (20 MHz, 298 K, pH 7.4) upon binding to BSA (K a=4.2×103 M−1). In vivo studies show that MnLMe is cleared intact into the bladder through renal excretion and has a prolonged blood half‐life compared to the commercial GdCA Magnevist. MnLMe shows great promise as a novel MRI contrast agent.


Table of Contents
hydrogen bonds to unbound water and chloride.The bound chloride acts as a hydrogen bond acceptor to unbound water, and O1 also acts as a hydrogen bond acceptor to unbound water.Both complexes are arranged in layers parallel to (101).Within and between the layers, these hydrogen bonds assemble the complexes and unbound species into a 3−D hydrogen-bonded network.S11 and S12.Parameters from the analysis of 1 H NMRD data.

Experimental Procedures
Materials and Measurements.All of the materials and reagents for the synthesis of mononuclear Mn 2+ and Zn 2+ complexes (MnL Me and ZnL Me ) were purchased from Sig-ma-Aldrich (Dorset, UK) and used without further purification.Solvents were purchased from VWR (Leicestershire, UK).IR spectra were recorded on a Perkin Elmer Rx FTIR x2 with diamond ATR, DRIFT attachment. 1 H-NMR and T1 measurements were carried out on a JEOL JNM-LA400 Spectrometer (400 MHz).Electronic absorption spectral titration experiments were done using a ThermoFisher Scientific Evolution 300 UV-Vis spectrophotometer, and the fluorescence emission studies were carried out on a Horiba Fluoromax-4P spectrofluorometer.High resolution mass spectrometry (HRMS) spectra of the compounds were recorded on Advion MS SOP electrospray ionization (ESI) spectrometer.The pH measurements were carried out by using a Jenway 3520 digital pH meter with a Mettler-Toledo 51343160 glass electrode.
Synthesis of Mn 2+ complex MnL Me (1).2,6-diacetylpyridine (0.163 g, 1.0 mmol) was added to 60 mL of methanol (MeOH), and solid MnCl2 (0.121 g, 0.96 mmol) was added and stirred for an hour at 60 °C.To this mixture, a methanolic solution (20 mL) of acetohydrazide (0.148 g, 2.0 mmol) was added dropwise.The mixture turned slowly from colorless to pale yellow and was refluxed overnight, and hot filtered.The solvent was evaporated in air, and the resultant bright yellow solid was washed with little amounts of isopropanol/diethyl ether mixture and dried.X-Ray Data Collection and Reduction.Single crystal X ray diffraction data were collected in series of ω-scans using a Stoe IPSD2 image plate diffractometer utilizing monochromated Mo radiation (λ = 0.71073 Å).Standard procedures were employed for the integration and processing of the data using X-RED. [1]Samples were coated in a thin film of perfluoropolyether oil and mounted at the tip of a glass fiber located on a goniometer.Data were collected from crystals held at 150 K in an Oxford Cryosystems nitrogen gas cryostream.
Crystal structures were solved using dual space methods implemented within SHELXT. [2]Completion of structures was achieved by performing least squares refinement against all unique F2 values using SHELXL-2018. [3]All non H atoms were refined with anisotropic displacement parameters.Hydrogen atoms were placed using a riding model.Where the location of hydrogen atoms was obvious from difference Fourier maps, C-H and O-H bond lengths were refined subject to chemically sensible restraints.Me and ZnL Me .Both structures crystallise in P21/c but are close to being body centred.Each structure solves and refines very satisfactorily in this space group and the refinement is stable.In neither case is there convincing evidence for a body-centring translation although the symmetry of the primitive cell generates a pseudo-body-centred array of the complexes.However, the strict bodycentring translation is not obeyed; complexes related by the pseudo translation are rotated by approximately 180 degrees about the Zn1-N1 bond.

Pseudosymmetry in the crystal structures of MnL
The diffraction data below demonstrate that the true symmetry is P not I.

Observed diffraction data for MnL Me :
Reflections with h+k+l=2n: The data consistent with bodycentring are thus weak but not systematically absent.The data consistent with bodycentring are thus weak but not systematically absent.

Observed diffraction data for ZnL Me
Relaxometry studies.The longitudinal relaxation times (T1) and water proton relaxation rates (r1 = 1/T1) of the MnL Me were measured on a JEOL JNM-LA 400 Spectrometer (400 MHz).The T1 values were obtained by the inversion-recovery method (180º −  − 90º).The relaxivity of the complex was determined by different concentrations of MnL Me (1.0 to 5.0 mM) at pH = 7.3 (50 mM HEPES buffer, 0.15 M NaCl, 298 ± 0.2 K).The water proton relaxivity r1 of the MnL Me was determined from the slope of the plot of 1/T1 vs. [MnL Me ].Stock solutions of MnL Me (10 mM) and BSA were prepared in MilliQ water.The concentration of BSA was determined spectroscopically based on the tryptophan absorbance at 280 nm (ε280 = 43824 M −1 cm −1 ) in water.The hydration state of the metal was empirically calculated by applying the following equations (1) and ( 2): [4] where, FW = 366.28g/mol; y = 6.05; r1 = 13.9 mM -1 s -1 and q = 2.3.
1 H Nuclear Magnetic Relaxation Dispersion (NMRD) profiles were measured on a Fast-Field Cycling (FFC) Stelar Smart Tracer Relaxometer over a continuum of magnetic field strengths from 0.00024 to 0.25 T (corresponding to 0.01-10 MHz proton Larmor Frequencies).Additional data points in the range 20-120 MHz were obtained with a High Field Relaxometer (Stelar) equipped with the HTS-110 3T Metrology Cryogen-free Superconducting Magnet.The measurements were performed using the standard inversion recovery sequence (20 experiments, 2 scans) with a typical 90° pulse width of 3.5 μs and the reproducibility of the data was within ± 0.5%.The temperature was con-trolled with a Stelar VTC-91 heater airflow equipped with a copper-constantan thermocouple (uncertainty of ± 0.1 K).The Mn(II) concentration was estimated by 1 H-NMR (Bruker Advance III Spectrometer equipped with a wide bore 11.7 T magnet) measurements using Evans's method. [5]The hydration number q was adjusted to 2; the distance between the metal ion and the protons of the bound, rMnH, and outer sphere, aMnH, water molecule were set to 2.74 and 3.6 Å, respectively.The value of the distance rMnH is consistent with the average distances obtained (rMnH = 2.7373 Å) from X-ray crystal structure data of MnL Me . 17O NMR MnL Me : 17 O NMR spectra were acquired on a Bruker Avance III spectrometer (11.7 T) using a 5 mm probe under temperature control.An aqueous solution of the Mn(II) chelate (2.3 mM) in HEPES buffer (pH = 7.4) containing 2.0% 17 O-enriched water (Cambridge Isotope) was prepared and analysed.The transverse relaxation rates were measured from the signal width at half-height, as a function of temperature in the 278-350 K range.A diamagnetic blank solution containing acidified water (pH = 3.0) enriched with H2 17 O (2.0%) was used as reference.Notably, the observed transverse relaxation rates R2 depend on the rate of water exchange at 298 K, 298 kex, and its activation enthalpy HM, the electronic relaxation time 298 T2e, and the 17 O hyperfine coupling constant AO/ħ.MnL Me /BSA: the same procedure was applied to the BSA-bound complex.A 0.12 mM solution of the complex and 1.43 mM of BSA in HEPES containing 2.0% 17 O-enriched water (Cambridge Isotope) was prepared.The diamagnetic blank solution was prepared by dissolving BSA (1.43 mM) in HEPES containing 2.0% of the 17 O isotope (Cambridge Isotope).
The hydration state of Mn(II) (q) was calculated by using the empirical equation [6] reported below: where, r2°max is the maximum 17 O transverse relaxivity and A0/ħ is the hyperfine coupling constant.MR Imaging.Phantoms of 0.1-0.5 mM MnL Me in 50 mM HEPES buffer (pH 7.4) and 0.1-0.5 mM MnL Me with 0.6 mM BSA in 50 mM HEPES buffer (pH 7.4) were prepared in a customized 5 × 5 well-plate (0.25 mL capacity).Phantom and in vivo MR imaging were acquired on a 7 T preclinical MR scanner (Bruker BioSpec 70/30, Bruker BioSpin, Ettlingen, Germany) using an 86-mm diameter 1 H transceiver volume coil (Bruker).The sample temperature was regulated with a heated pad system (Medres medical research GmbH, Cologne, Germany) and monitored with corresponding temperature probe and by 1  HPLC strategies.Liquid chromatography-mass spectrometry (LC-MS) was performed using an Agilent LC-MSD system (LC: 1200 series, MS: 6120 quadrupole) and Daly conversion dynode detector with UV detection at 220, 254 and 280 nm and used the following method to analyse the samples.Luna C18 column (250 × 2 mm), eluent A: CH3CN, eluent B: NH4OAc buffer (pH 7.0, 298 K), gradient (0-95% CH3CN over 18 min, flow rate 0.3 mL/min.Urine samples were preprocessed with a 10 kDa molecular weight cut off filter before injection onto the column. NMR titrations.NMR spectra for transmetalation of MnL Me and [Gd(DTPA)(H2O)] 2-(Magnevist®) with 1 and 25 molar equivalents Zn 2+ were acquired on a 600 MHz Bruker Avance III spectrometer at 298 K. T1 relaxation was measured via an inversion recovery experimentusing 8 inversion times (0.05, 0.075, 0.1, 0.25, 0.35, 0.5, 0.75 and 2 s) before and after addition of Zn 2+ .T2 relaxation was measured using a Carl-Purcell-Meiboom-Gill spin-echo experiment.Relaxivity values (r1, r2) were calculated for each transmetalation experiment from the previously determined concentration.The concentration of paramagnetic complexes in solution was determined from bulk magnetic susceptibility shift in NMR spectra. [5]sults and Discussion

Proton Larmor Frequency / MHz
FigureS16. 1 H NMRD profiles of MnL Me at pH 6 and three different temperatures (283 K ( ), 298 K ( ) and 310 K ( ). [Mn 2+ ] = 4.0 mM.The curves through the data points were calculated with the parameters in Table S11.
Figures.S1-S2. 1 H and 13 C-NMR spectral profiles of ZnL Me Figure S3.Absorption spectra of MnL Me and ZnL Me Figure S4-S5.ESI-Mass spectral profiles of MnL Me and ZnL Me Figure S6.X-Ray crystal structure of ZnL Me .Bound water (O3) forms two hydrogen bonds to unbound water and chloride.The N−H functions of the ligand form

Figure S12 .
Figure S12.Chelating stability of MnL Me was monitored by change in the absorption spectral intensity as a function of time.Absorption spectra of MnL Me (50µM) obtained over 10 days (A), Change in the absorption intensities at 213 and 275 nm (B), and the ratio of Abs275/Abs213 as the function of time (C).

Figure S14 .
Figure S14.Transchelation of MnL Me .(a) no transchelation observed in competition with 25 molar equivalents of DTPA (b), nor with 25 molar equivalents EDTA (c).Solutions were incubated for 1 hour with the competing ligands.

Figure S15 .
Figure S15. Figure S15.LC-MS of mouse urine samples.LC-MS analysis of pure MnL Me (a) and a urine sample from a healthy mouse after intravenous administration of a clinical dose of MnL Me (b).A control urine sample without prior MnL Me administration (c) and a urine sample spiked with MnL Me (d) on an Agilent LC-MS system (LC: 1200 series, MS: 6120 quadrupole).MS chromatogram (e) of the pure MnL Me extracted at m/z+ 329.1 and the ESI-MS spectra at 520 sec (8.6 min).At 40 min post intravenous MnL Me (0.1 mmol/kg bodyweight, 2.5x10 -6 M) injection, 24 % of the contrast agent was excreted intact into the bladder.The asterisk (*) marks a signal from the eluent system.

Figure S19 .
Figure S19.T1 and T2-weighted phantom imaging of MnL Me and Magnevist.(A) T1-weighted imaging of 0.1-0.5 mM MnLMe without and with 0.6 mM BSA in 50 mM HEPES (first and second row, respectively) illustrates highly enhanced contrast enhancement in MnL Me samples with BSA in comparison to MnL Me samples without BSA.Compared to 0.1-0.5 mM Magnevist without and with 0.6 mM BSA in 50 mM HEPES (third and fourth row, respectively), overall contrast enhancement of MnL Me samples is lower.Images were recorded at 7 T and 298 K with a flip angle of 10°.(B) T2-weighted imaging of 0.1-0.5 mM MnL Me without and with 0.6 mM BSA in 50 mM HEPES (first and second row, respectively) shows superior T2 contrast modulation of MnL Me in comparison to 0.1-0.5 mM Magnevist without and with 0.6 mM BSA in 50 mM HEPES (third and fourth row, respectively).

Figure S20 .
Figure S20.Biodistribution of Magnevist and MnL Me : (A) Representative images of Magnevist®-induced contrast enhancement in vivo.(B) Total signal intensity over time of MnL Me represented as the area under the curve (AUC 0-40 min (ΔSI*min)) was comparable to Magnevist in all analyzed organs (mean ± SEM, n = 3 for MnL Me , n = 3 for Magnevist ® ).Percentage of signal intensity enhancement normalized to pre-injection over time for all organs of interest is given in (C) for MnL Me and (D) for Magnevist ® .Scheme S1.MnL Me binds with bovine serum albumin (BSA), resulting in an enhanced relaxivity due to the decreased molecular rotation.
H spectroscopy (PRESS) on ethylene glycol.T1weighted MR images of MnL Me were acquired with a standard 3D T1-FLASH sequences and the following parameters: TE = 2.64 ms, TR = 8.78 ms, flip angle 30°, FOV 60×40×21.21mm³, image size 272×136×96, slice thickness 21.12 mm, resolution 0.220×0.294×0.220mm³, 1 average with a total acquisition time of 1 min 54 s.For comparison of MnL Me to Magnevist in T1-weighted imaging, the flip angle was reduced to 10°.T1 maps were acquired with a 2D RAREVTR sequence (TE 6.705 ms, FOV 50×50 mm², image size 128×128, resolution 0.390×0.390mm², total scan time: 12 min 52 s) with 15 TR values of 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500 and 3000 ms.T2-weighted images were acquired with a standard 2D spin-echo sequence and the following parameters: TE 8.993 ms, TR 4253.613ms, FOV 60×40 mm², image size 200×134, resolution .…(1)….(2) 0.300×0.298mm², 1 average with a total acquisition time of 1 min 8 s.T2-maps were acquired with a standard MSME sequence, variable TEs of 8-200ms in 8 ms steps and the following parameters: TR 2000 ms, FOV 50×50 mm², image size 128×128, resolution 0.300×0.298mm², total scan time 4 min 16 s.For in vivo imaging, healthy female C56BL/6 mice were anesthetized with 1.5 % isoflurane in pure oxygen.For the application of the MR contrast agents, a catheter was placed in a lateral tail vein.T1-weighted pre-and post-contrast in vivo MR images were acquired with a 3D FLASH-sequence and the following parameters: TE = 2.64 ms, TR = 8.78 ms, flip angle 10°, FOV 75×35×21.21mm³, image size 272×136×96, slice thickness 21.12 mm, resolution 0.276×0.257×0.220mm³, total acquisition time 1 min 54 s.After the pre-contrast acquisition, MnL Me (n = 3) or Magnevist (n = 3) were applied at a final dose of 0.1 mmol/Kg body weight and allowed to distribute for 5 min before 16 post-contrast images were acquired back-to-back.For data analysis in PMOD (PMOD Technologies LCC, Bruker), fixed volumes of interest were placed in the kidneys, liver, heart, bladder, and thigh muscle.Post-contrast values were normalized to pre-contrast, and the resulting percent signal chance represented over time.The area under the curve was measured over a period of 1 -40 minutes post-contrast administration and plotted using GraphPad Prism 7. Statistical significance was evaluated using unpaired two-tailed t-tests, p-values < 0.05 were considered significant.All animal procedures were con-ducted following German federal regulations on the use and care of experimental animals and approved by the local authorities (Regierungspräsidium Tübingen).

Figure S6 .Figure S7 .
Figure S6.ORTEP diagram of ZnL Me drawn at 50% ellipsoid level.Bound water (O3) forms two hydrogen bonds to unbound water and chloride.The N−H functions of the ligand form hydrogen bonds to unbound water and chloride.The bound chloride acts as a hydrogen bond acceptor to unbound water, and O1 also acts as a hydrogen bond acceptor to unbound water.Both complexes are arranged in layers parallel to (101).Within and between the layers, these hydrogen bonds assemble the complexes and unbound species into a 3−D hydrogen-bonded network.

Figure S12 .Figure S13 .
Figure S12.Chelating stability of MnL Me was monitored by change in the absorption spectral intensity as a function of time.Absorption spectra of MnL Me (50µM) obtained over 10 days (A), Change in the absorption intensities at 213 and 275 nm (B), and the ratio of Abs275/Abs213 as the function of time (C).To reduce the errors during measurement, the ratio of absorption maxima λmax values was plotted with a function of time

Figure S14 .
Figure S14.Transchelation of MnL Me .No transchelation was observed in competition with 25 molar equivalents of DTPA (b), nor with 25 molar equivalents EDTA (c) in comparison to pure MnL Me (a) Solutions were incubated for 30 mins with the competing ligands.The asterisk (*) marks a signal from the eluent system.

Figure S15 .
Figure S15.LC-MS of mouse urine samples.LC-MS analysis of pure MnL Me (a) and a urine sample from a healthy mouse after intravenous administration of a clinical dose of MnL Me (b).A control urine sample without prior MnL Me administration (c) and a urine sample spiked with MnL Me (d) on an Agilent LC-MS system (LC: 1200 series, MS: 6120 quadrupole).MS chromatogram (e) of the pure MnL Me extracted at m/z+ 329.1 and the ESI-MS spectra at 520 sec (8.6 min).At 40 min post intravenous MnL Me (0.1 mmol/kg bodyweight, 2.5x10 -6 M) injection, 24 % of the contrast agent was excreted intact into the bladder.The asterisk (*) marks a signal from the eluent system.

Figure S19 .
Figure S19.T1 and T2-weighted phantom imaging of MnL Me and Magnevist.(A) T1-weighted imaging of 0.1-0.5 mM MnL Me without and with 0.6 mM BSA in 50 mM HEPES (first and second row, respectively) illustrates highly enhanced contrast enhancement in MnL Me samples with BSA in comparison to MnL Me samples without BSA.Compared to 0.1-0.5 mM Magnevist without and with 0.6 mM BSA in 50 mM HEPES (third and fourth row, respectively), overall contrast enhancement of MnL Me samples is lower.Images were recorded at 7 T and 298 K with a flip angle of 10°.(B) T2-weighted imaging of 0.1-0.5 mM MnL Me without and with 0.6 mM BSA in 50 mM HEPES (first and second row, respectively) shows superior T2 contrast modulation of MnL Me in comparison to 0.1-0.5 mM Magnevist without and with 0.6 mM BSA in 50 mM HEPES (third and fourth row, respectively).

Figure S20 :
Figure S20: Biodistribution of Magnevist and MnL Me : (A) Representative images of Magnevist ® -induced contrast enhancement in vivo.(B) Total signal intensity over time of MnL Me represented as the area under the curve (AUC 0-40 min (ΔSI*min)) was comparable to Magnevist in all analyzed organs (mean ± SEM, n = 3 for MnL Me , n = 3 for Magnevist ® ).Percentage of signal intensity enhancement normalized to pre-injection over time for all organs of interest is given in (C) for MnL Me and (D) for Magnevist ® .Bars represent standard error of the mean; *p-value < 0.05, **p-value < 0.01.

Table S1 .
Crystallographic data for MnL Me andZnL Me

Table S2 .
Details of all of the unique classical hydrogen bonds in MnL Me .

Table S3 .
Atomic coordinates (x 10 4 ) and equivalent isotropic displacement parameters (Å 2 x 10 3 ) for MnL Me .U(eq) is defined as one third of the trace of the

Table S7 .
Atomic coordinates ( x 10 4 ) and equivalent isotropic displacement parameters (Å 2 x 10 3 ) for ZnL Me .U(eq) is defined as one third of the trace of the orthogonalized U ij tensor.