Nickel ( II ) complexes of NCH 2 CF 3 cyclam derivatives as contrast agents for 19 F magnetic resonance imaging †

Magnetic resonance imaging (MRI) is one of the most common techniques in molecular imaging. It is based on the detection of the NMR signal originating from water protons in a tissue. To increase its sensitivity, paramagnetic contrast agents (CAs) are often applied. They affect mainly the longitudinal (T1) relaxation time of the H signal, which leads to an increase in the intensity of the water proton MRI signal. However, essentially all tissues contain water and, thus, the background signal compromises the detection accuracy. This problem can be solved by using non-proton MRI and the F nucleus seems to be the most promising candidate. Natural monoisotopic F has an NMR resonance frequency close to that of H (40.08 MHz T for F compared to 42.58 MHz T for H) and exhibits sensitivity comparable to H (83%). Fluorine concentration in organisms is virtually zero and, therefore, the lack of background in fluorine-based images enables “hotspot” imaging. The wider spectral range of the F nucleus (∼350 ppm) compared to H (∼10 ppm) is also beneficial for some applications. Moreover, only small hardware and software adjustments of standard H scanners are needed for F detection. This makes the nucleus very potent for e.g. cellular tracking of labelled cell cultures. However, the F nucleus present in organic molecules has usually a very long T1 relaxation time requiring a long delay between excitation pulses; this prolongs the total duration of imaging experiments to unrealistic lengths. Shortening of the T1 relaxation time can result in significant shortening of the experimental time. However, it is necessary to take into account the concomitant shortening of the transversal (T2 or T* 2) relaxation time, which leads to signal broadening and can result in very fast loss of signal intensity. It has been shown that the introduction of highly paramagnetic lanthanide(III) ions to the close vicinity of the fluorine atom(s) leads to significant shortening of the relaxation times, and the T* 2=T1 ratio is in the range of 0.3–0.9, which is suitable for MRI measurements. It is known that, despite the low overall electronic spin (S = 1) and magnetic momentum (μeff ∼ 3 B.M.) of Ni(II), this ion can induce a large paramagnetic chemical shift and relaxation enhancement comparable to that of lanthanide(III) ions with higher S and μ. Therefore, some Ni(II) complexes have been studied as MRI agents employable in the Chemical Exchange Saturation Transfer method. Here, we decided to study the F NMR relaxation properties of Ni(II) complexes. The Ni(II) ion fits perfectly in the cavity of 1,4,8,11-tetraazacyclotetradecane (cyclam) and cyclam derivatives are well-known to form Ni(II) complexes with high thermodynamic stability, especially with ligands having coordinating pendant arms enabling octahedral binding to the metal. The 2,2,2-trifluoroethyl side arm was chosen as a group containing a high number of equivalent fluorine atoms. Therefore, ligands 1 and H4te2p-tfe2 (Fig. 1)

Magnetic resonance imaging (MRI) is one of the most common techniques in molecular imaging.It is based on the detection of the NMR signal originating from water protons in a tissue.To increase its sensitivity, paramagnetic contrast agents (CAs) are often applied. 1,2They affect mainly the longitudinal (T 1 ) relaxation time of the 1 H signal, which leads to an increase in the intensity of the water proton MRI signal.However, essentially all tissues contain water and, thus, the background signal compromises the detection accuracy.4][5][6] Natural monoisotopic 19 F has an NMR resonance frequency close to that of 1 H (40.08 MHz T −1 for 19 F compared to 42.58 MHz T −1 for 1 H) and exhibits sensitivity comparable to 1 H (83%). Fluorine concentration in organisms is virtually zero and, therefore, the lack of background in fluorine-based images enables "hotspot" imaging.The wider spectral range of the 19 F nucleus (∼350 ppm) compared to 1 H (∼10 ppm) is also beneficial for some applications.Moreover, only small hardware and software adjustments of standard 1 H scanners are needed for 19 F detection. 6This makes the nucleus very potent for e.g.9][10][11] However, the 19 F nucleus present in organic molecules has usually a very long T 1 relaxation time requiring a long delay between excitation pulses; this prolongs the total duration of imaging experiments to unrealistic lengths.Shortening of the T 1 relaxation time can result in significant shortening of the experimental time.However, it is necessary to take into account the concomitant shortening of the transversal (T 2 or T * 2 ) relaxation time, which leads to signal broadening and can result in very fast loss of signal intensity.It has been shown that the introduction of highly paramagnetic lanthanide(III) ions to the close vicinity of the fluorine atom(s) leads to significant shortening of the relaxation times, 12 and the T * 2 =T 1 ratio is in the range of 0.3-0.9, which is suitable for MRI measurements.12c, 13 It is known that, despite the low overall electronic spin (S = 1) and magnetic momentum (μ eff ∼ 3 B.M.) of Ni(II), this ion can induce a large paramagnetic chemical shift and relaxation enhancement comparable to that of lanthanide(III) ions with higher S and μ. 14 Therefore, some Ni(II) complexes have been studied as MRI agents employable in the Chemical Exchange Saturation Transfer method. 15Here, we decided to study the 19 F NMR relaxation properties of Ni(II) complexes.The Ni(II) ion fits perfectly in the cavity of 1,4,8,11-tetraazacyclotetradecane (cyclam) and cyclam derivatives are well-known to form Ni(II) complexes with high thermodynamic stability, especially with ligands having coordinating pendant arms enabling octahedral binding to the metal.The 2,2,2-trifluoroethyl side arm was chosen as a group containing a high number of equivalent fluorine atoms.Therefore, ligands 1 and H 4 te2p-tfe 2 (Fig. 1) Fig. 1 Ligands studied in this work.† Electronic supplementary information (ESI) available: Synthesis of studied compounds, single-crystal RTG diffraction data, details on potentiometric study, study of acid-assisted dissociation of prepared complexes, relaxation study, were suggested for testing the 19 F NMR parameters of their complexes.
Acylation of 1,8-dibenzylcyclam with ethyl trifluoroacetate or trifluoroacetic anhydride yielded the corresponding bis(amide).It was followed by BH 3 reduction 16 in diglyme at elevated temperature and the benzyl protecting groups were removed by Pd/C hydrogenolysis to obtain ligand 1.The reaction 17 of amine 1 in neat P(OEt) 3 with CH 2 O led to the tetraethyl bis(methylenephosphonate) cyclam derivative.The ethylester groups were removed by transesterification with trimethylsilylbromide 18 followed by the silylester hydrolysis to yield H 4 te2p-tfe 2 , which was isolated in a zwitterionic form after ion exchange chromatography.Synthetic details and results of a single-crystal X-ray diffraction study of However, the cis-[Ni(1)(Cl) 2 ] complex shows extremely low solubility in all solvents.Thus, the presence of chloride ions had to be avoided during the preparation of a water-soluble complex.Therefore, ligand 1 in the form of a free base and Ni(ClO 4 ) 2 was used for further complex preparation.The course of the reaction in the H 2 O : DMSO 1 : 6.5 mixture at 50 °C was followed by 19 F NMR spectroscopy (Fig. S2 †).Such a solvent mixture was used to keep the reaction mixture fully homogeneous right from the beginning as compound 1 is poorly soluble in water.The reaction proceeds (at 50 °C) through an intermediate (δ F = −22.9ppm) and is completed during 90 min to give the final complex with δ F = −29.3ppm (at 50 °C).On cooling to 25 °C, the signal shifts to −26.5 ppm.No further 19 F NMR spectral changes were observed upon heating the solution at 100 °C for several days.
To obtain an aqueous stock solution, prolonged heating (80 °C) of the suspension of ligand 1 with Ni(ClO 4 ) 2 in H 2 O : MeOH 1 : 1 (with subsequent evaporation of MeOH) was used.It led to the formation of a light blue aqueous solution of a single product with δ F = −26.2ppm.When the aqueous solution of the complex prepared in H 2 O : MeOH was mixed with the sample prepared in H 2 O : DMSO, only one symmetric signal in 19 F NMR was observed revealing that the species formed in both experiments are identical complexes.
Despite a number of attempts, we were not able to crystallize this light blue product.However, red single-crystals of trans-[Ni(1)](ClO 4 ) 2 (Fig. 2) were obtained when the blue aqueous solution of the complex was saturated with NaClO 4 and was left standing for a few weeks.In this complex, the cyclam ring is coordinated in the centrosymmetric trans-III configuration 19 (d Ni-N = 1.95 and 1.99 Å for secondary and ter-tiary amino groups, respectively).Consistent with the red colour, only very weak axial interaction with perchlorate anions located in distant positions (d Ni-O = 2.83 Å) was observed.
Dissolution of the red trans-[Ni( 1)](ClO 4 ) 2 complex in water produced a light blue solution with δ F = −19.3ppm and the species slowly isomerizes with first-order kinetics (τ 1/2 ∼3.5 h, 25 °C) to a species with δ F −26.3 ppm (Fig. S3 †).The final species is identical to the original Ni(II)-1 complex, as it was confirmed by 19 F NMR after the addition of a standard.Taking into account the isomerism of metal ion-cyclam complexes, 19 red trans-[Ni(1)](ClO 4 ) 2 with the trans-III configuration probably forms a blue hexacoordinated trans-[Ni(1)(H 2 O) 2 ] 2+ species upon dissolution, and this complex is rearranged in solution to the cis-[Ni(1)(H 2 O) 2 ] 2+ species with the cis-V cyclam conformation.
Typically, the trans-III isomer of cyclam complexes is considered to be the thermodynamically most stable one, 20 and the preference of the cis-V cyclam conformation for the Ni(II)-1 complex is rather surprising.However, the higher stability of the cis-V isomer over the trans-III one was supported also by the isolation of cis-[Ni(1)(H 2 O) 2 ](TsO) 2 (Fig. 2).The structure shows Ni-N distances of 2.07 and 2.09 Å for secondary amino groups, and 2.22 and 2.25 Å for tertiary ones, respectively, with two water molecules coordinated with d Ni-O = 2.07 and 2.10 Å.
Based on the data presented above, one can conclude that the reaction of ligand 1 with Ni(ClO 4 ) 2 leads to the formation of the cis-[Ni(1)(H 2 O) 2 ](ClO 4 ) 2 complex.The geometries of Ni(II) coordination polyhedra found in the solid state structures are compiled in Table S2 † and are comparable to other [Ni(L)(H 2 O) 2 ] complexes of cyclam derivatives. 21,22The Ni-F distances found in all solid-state structures were in the range of 4.8-5.4Å (Table S3 †).
The complex of the second studied ligand, H 4 te2p-tfe 2 , was prepared by heating the ligand together with a slight excess of NiCl 2 in aq.ammonia ( pH 10) at 75 °C for 24 h; the excess of Ni(II) ions was removed by column chromatography.As mentioned above, the course of the reaction was followed by 19 F NMR (Fig. S4 †), which showed a fast drop in the concentration of the free ligand (δ F = −68.3ppm), and the formation of an intermediate (δ F = −41.1 ppm) and its slower rearrangement to the final product (δ F = −26.4ppm).The time-dependence of intensities of all three signals could be satisfactorily fitted using a monoexponential function (Fig. S4 †) and showed comparable rate constants for all three processes (see the ESI †).Such behaviour points to the presence of an equilibrium between the free ligand and the intermediate with an irreversible (rate-determining) reaction step leading to the formation of the final complex.
The final product was isolated in the form of light blue crystals, which were identified as (NH 4 ){trans-[Ni(Hte2ptfe 2 )]}•3.25H 2 O by single-crystal X-ray diffraction.Therefore, the complex species present in solution are expected to be trans-[Ni(H n te2p-tfe 2 )] n−2 (n = 0, 1) depending on pH.The molecular structure of the complex anion is shown in Fig. 3, and geometric parameters of the coordination sphere of Ni(II) and Ni-F distances are listed in Tables S2 and S3.† The cyclam ring exhibits the trans-III configuration 19 (d Ni-N are ∼2.10 and 2.11 Å for amino groups bearing the methylenephosphonate pendant arms, and 2.22 and 2.23 Å for those substituted by trifluoroethyl groups) with the oxygen atoms of phosphonate groups occupying apical positions (Ni-O distances are 2.06 and 2.10 Å, respectively).The molecules of the complex are connected via short hydrogen bonds between the oxygen atoms of protonated and unprotonated phosphonate pendants (d O⋯O = 2.47 Å), forming infinite chains, similar to what was found for analogous complexes of cyclam-methylenephosphonate derivatives. 23,24Bonding distances and the overall molecular structure are very similar to those of Ni(II) complexes of analogous derivatives. 22,24he thermodynamics of complexing properties of the phosphonate H 4 te2p-tfe 2 ligand was studied by potentiometry (see the ESI and Table S4 †).The comparison of ligand stepwise protonation constants (log K 1-4 10.86, 10.09, 5.60 and 4.73) with those of the N 1 ,N 8 -dimethyl-N 4 ,N 11 -bis(methylenephosphonate) analogue 25 (log K 1-4 11.47, 12.17, 7.20 and 6.33, Table S5 †) points to significantly decreased ligand basicity caused by the presence of electron-withdrawing -CH 2 CF 3 groups.Surprisingly, it affects not only the first two protonation constants corresponding to the ring amino groups but also those of the phosphonate moieties, probably as a result of a strong electron-withdrawing effect transferred through intramolecular hydrogen bonds, which are expected to have a geometry analogous to that found for related cyclam derivatives. 25quilibration of the Ni(II)-H 4 te2p-tfe 2 system is relatively slow and, therefore, the out-of-cell titration method had to be used.As the complexation mechanism is not fully straightforward (see above), the samples used for the out-of-cell titration were heated at 50 °C for 2 weeks to ensure quantitative rearrangement of the intermediate to the final trans isomer.The time required for equilibration was checked by 19 F NMR of separate samples.The stability constant, log K NiL = 13.28, is about 2-7 orders of magnitude lower than the constants of complexes with related ligands (Table S5 †), 26,27 mainly as a consequence of the lower ligand basicity.The distribution diagram of the system (Fig. S8 †) shows that full Ni(II) complexation by H 4 te2ptfe 2 is completed at pH 7 and the complex is present at this pH almost entirely in a fully deprotonated form.
For possible in vitro/in vivo utilization, kinetic inertness is a more important parameter than thermodynamic stability.Kinetic inertness is often tested in acidic solutions as acidassisted complex dissociation.Thus, the decomposition of both studied complexes was examined in 1 M aq.HCl at 37 and 80 °C.Both complexes are decomposed relatively slowly by HCl at 37 °C (τ 1/2 ∼8 and ∼10 h for cis-[Ni(1)(H 2 O) 2 ] 2+ and trans-[Ni(H n te2p-tfe 2 )] n−2 , respectively) but the decomplexation of the cis-[Ni(1)(H 2 O) 2 ] 2+ complex is substantially accelerated at the higher temperature (80 °C, τ 1/2 ∼ 3 min compared to ∼5 h for trans-[Ni(H n te2p-tfe 2 )] n−2 , Table S6 †), as the presence of apically coordinated pendant arms in the H 4 te2p-tfe 2 complex enhances kinetic inertness.High inertness has been observed for several Cu(II) complexes with analogous cyclam-based ligands, 28 and highly protonated species of several Ni(II) 22,24 complexes of phosphonated cyclam derivatives have been isolated even in the solid state.These results suggest sufficient complex stability under physiological conditions and warrant the possible use of the Ni(II)-H 4 te2p-tfe 2 complex in in vitro/ in vivo applications.
As the trans-[Ni(te2p-tfe 2 )] 2− complex is kinetically inert and promises reasonable stability in vivo, its 19 F MRI-related parameters were investigated.Although the cis-[Ni(1)(H 2 O) 2 ] 2+ complex is not suitable for any in vivo application due to its low solubility in chloride-containing media, it was studied as well for comparative purposes as there are no related data reported in the literature at all.The 19 F NMR relaxation measurement of both Ni(II) complexes at B 0 = 7.05 T showed extreme shortening of 19 F NMR T 1 relaxation times by 2-3 orders of magnitude compared with the values observed for the free ligands, 1 and H 4 te2p-tfe 2 (Table 1).
The suitability of the trans-[Ni(te2p-tfe 2 )] 2− complex for 19 F MRI was tested by phantom visualization at B = 4.7 T. At this magnetic field, the T 1 of the complex is very short in the millisecond range with a still convenient T * 2 =T 1 relaxation times ratio (Table 1).The observed relaxation times are even slightly shorter than those reported for studied Ln(III) complexesin the cases of highly paramagnetic Tb(III), Dy(III) and Ho(III) complexes with the estimated Ln(III)-F distance lying in the range of 5-7 Å, the reported T 1 is typically in the range of 7-11 ms at 4.7 T and room temperature.12c, 13 The very short relaxation time of the Ni(II) complex required optimization of the fast pulse sequencefor the visualization of the complex, a fast gradient echo sequence with TE = 1.3 ms and TR = 3 ms was used.The slowly relaxing samples (containing the free ligand and trifluoroethanol used as a standard) were best measured using a long turbospin echo sequence employing TE = 40 ms and TR = 2000 ms.For localization of the samples, the 1 H MRI scan (Fig. 4A) was also acquired.Fig. 4 shows the results of the MRI visualization.The brightness of aq.solution of the Ni(II) complex compared to aq. solutions of the free ligand and trifluoroethanol is caused by its paramagnetism, which shortens the T 1 relaxation time of water protons (r 1 (complex) = 0.12 mm −1 s −1 , 4.7 T, 25 °C).As each sample has a different 19 F NMR chemical shift (δ F −26 ppm, −68 ppm and −77 ppm for the complex, free ligand and trifluoroethanol, respectively), each signal can be excited separately.In the case of the fast sequence, only a negligible signal of the free ligand was detected, as virtually no diamagnetic sample relaxation occurs during the sequence time-scale.On the contrary, in the experiment employing the long sequence, no signal of the paramagnetic sample was found as its magnetization relaxes before the start of acquisition.
In conclusion, transition metal ion complexes of fluorinecontaining ligands can be considered a new class of 19 F MRI contrast agents, as shown in the case of Ni(II).The presence of strongly complexing and electron donating phosphonates enhances the kinetic inertness of the studied complexes and compensates the disadvantageous coordination properties of fluorine-containing ligands.Relaxation parameters of the trans-[Ni(te2p-tfe 2 )] 2− complex with fluorine atoms located about 5 Å from the Ni(II) centre are highly suitable for 19 F MRI hot-spot imaging employing fast pulse sequences.As the Ni(II) complexes with coordinated water molecules exhibit useful water proton T 1 -relaxivity, properly designed compounds could be potentially used as dual 1 H/ 19 F MRI contrast agents. 29he work was supported by the Czech Science Foundation (P207-11-1437) and by the project of the Ministry of Health, Czech Republic, for development of research organization IN00023001 (Institutional support, Institute for Clinical and Experimental Medicine).We thank Z. Böhmová and J. Hraníček for potentiometric and AAS measurements, respectively.-0.66(4) s -1 mM -1 -0.12(2) s -1 mM -1 a T 1 was determined using inversion recovery pulse sequence; T * 2 was determined from line-width using Lorentzian-shape fitting of the signal.

Table 1
19F NMR relaxation times a and 1 H relaxivity of the studied compounds ( pH 7, 25 °C)