Hafnium-Based Contrast Agents for X-ray Computed Tomography

Article pubs.acs.org/IC

Hafnium-Based Contrast Agents for X‑ray Computed Tomography Markus Berger,*,† Marcus Bauser,† Thomas Frenzel,† Christoph Stephan Hilger,† Gregor Jost,† Silvia Lauria,‡ Bernd Morgenstern,‡ Christian Neis,‡ Hubertus Pietsch,† Detlev Sülzle,† and Kaspar Hegetschweiler*,‡ †

Drug Discovery, Pharmaceuticals, Bayer AG, 13342 Berlin, Germany Department of Inorganic Chemistry, Saarland University, Campus C4.1, 66123 Saarbrücken, Germany

‡

S Supporting Information *

ABSTRACT: Heavy-metal-based contrast agents (CAs) offer enhanced X-ray absorption for X-ray computed tomography (CT) compared to the currently used iodinated CAs. We report the discovery of new lanthanide and hafnium azainositol complexes and their optimization with respect to high water solubility and stability. Our efforts culminated in the synthesis of BAY-576, an uncharged hafnium complex with 3:2 stoichiometry and broken complex symmetry. The superior properties of this asymmetrically substituted hafnium CA were demonstrated by a CT angiography study in rabbits that revealed excellent signal contrast enhancement.

1. INTRODUCTION Iodinated contrast agents (CAs) have successfully been used for decades in X-ray and computed tomography (CT) diagnostic imaging, despite the fact that iodine is not the most suitable element with respect to X-ray attenuation. In modern CT scanners, tube voltages of 80−140 kV are routinely used, while the K-edge of iodine is only at 33.2 keV.1 High Z elements with a K-edge in the range of 60−80 keV would yield substantially higher X-ray attenuation in the diagnostically used X-ray spectrum (Figure 1). Therefore, the search for hydrolytically

>60 keV, have therefore been selected as the elements for a new class of noniodinated CAs. Mononuclear lanthanide complexes like Gd-DTPA10,11 have proven to be well-tolerated CAs in magnetic resonance imaging (MRI); however, their metal content is too low to be used as effective CAs in CT. This drew our attention to azainositolderived trinuclear complexes 12 of the composition [M3(H−3taci)2]3+, that form unique, sandwich-type cage structures with heavy-metal ions such as bismuth(III)13 or lanthanides like gadolinium(III),14 which had been described in the context of potential MRI applications.

2. RESULTS AND DISCUSSION Synthesis and Characterization of Lanthanide Azainositol Complexes. To circumvent the known instability of [Gd3(H−3taci)2]3+ and avoid the release of toxic, free, solvated gadolinium(III) ions, we attempted to stabilize the complex unit by introducing short carboxylic acids to the azainositol ligand core in a manner reported for an iron(III) 1,3,5-trideoxy1,3,5-tris(methylamino)-cis-inositoltri-N,N′,N″-acetic acid derivative15 in the hope of retaining the 3:2 stoichiometry, which allows for the necessary high metal content of around 40%. We utilized a complex-formation protocol that was generally applicable to late lanthanides and 1,3,5-triamino-1,3,5trideoxy-cis-inositoltri-N,N′,N″-acetic acid/propionic acid (taci-ta/taci-tp) ligands or their N-methylated relatives 1,3,5trideoxy-1,3,5-tris(methylamino)-cis-inositoltri-N,N′,N″-acetic acid/propionic acid (maci-ta/maci-tp) and were delighted to observe formation of the desired lanthanide inositol com-

Figure 1. Mass attenuation coefficients for iodine, lutetium, and hafnium superimposed with a typical 120 kV CT X-ray.

stable and highly water-soluble compounds with a high content of an element, which offers more appropriate X-ray attenuation, is ongoing2−8 but did not result in clinical development candidates so far. Elements such as the lanthanide lutetium and the transition-metal hafnium,9 both with K-edge energies of © 2017 American Chemical Society

Received: February 10, 2017 Published: April 21, 2017 5757

DOI: 10.1021/acs.inorgchem.7b00359 Inorg. Chem. 2017, 56, 5757−5761

Article

Inorganic Chemistry Table 1. Numbering of Lanthanide Complexes with Short Termination and CA-Relevant Propertiesa

a

DTPA comp.: competition test with equimolar amounts of DTPA at 120 °C, 45 min. The recovery of the inositol complex in percent is reported.

Scheme 1. Synthesis of 5a

plexes.16 Although these complexes were hydrolytically stable under hydrothermal conditions at 120 °C for 45 min (typical conditions for heat sterilization of injections), further investigations revealed not sufficient complex stability when challenged with DTPA, a strong ligand for lanthanide ions.17 In this test, the inositol complex was heated in the presence of equimolar amounts of free DTPA. The formation of Ln-DTPA indicated only moderate complex stability of the new Ln3 complex (Table 1). In order to reach our primary optimization goal of a 100% chemically and physiologically stable complex, we varied the ligand and lanthanide. Two clear trends were identified: progression toward lanthanides with higher atomic number and the introduction of shorter carboxylate substituents, e.g., acetate instead of propionate, led to the highly stable complex Na3[Lu3(H−3taci-ta)2] (5) containing lutetium and the taci-ta ligand (Table 1). A synthetic sequence based on all-cis-2,4,6tris(benzyloxy)-1,3,5-cyclohexanetriamine (9)18 as the key intermediate derived from the nickel complex 819 delivered 5 after acetate introduction via tert-butyl bromoacetate, deprotection, and complex formation with the respective lanthanide chloride in the presence of sodium hydroxide in methanol (Scheme 1). Detailed structural determination by X-ray crystallography was possible in the case of the lutetium taci-ta complex as its potassium salt K3[Lu3(H−3taci-ta)2] (5a). X-ray analysis confirmed the trinuclear, sandwich-type structure of the Lu3L2 complex. Each Lu3+ cation is bonded to four alkoxy and two carboxylate oxygen-donor atoms. The six oxygen atoms form a slightly distorted trigonal prism, with two of the rectangular faces being capped by the remaining two aminedonor atoms. A similar, bicapped trigonal prism with coordination number 8 has already been observed for the [M3(H−3taci)2(H2O)6]3+ (M = La, Gd)14 complexes, which inspired this study. However, in contrast to our hydrolytically labile ligand-design starting point, all of the amine donors now each carry an additional acetate substituent, which takes over

a

Reagents and conditions: (a) BnBr, NaOH, DMF, then HCl, 60%; (b) BrCH2CO2tBu, Hünig’s base, CHCl3, 99%; (c) 6 N HCl, reflux, 90%; (d) NaOH, LuCl3·6H2O, MeOH, 75%.

the coordinative duties of the water ligand and becomes stereogenic upon coordination. The NMR data of the initially isolated Na3[Lu3L2] salt indicated a 2:3 ratio of the D3symmetric (R,R,R)2 or (S,S,S)2 form and the C2-symmetric (R,R,S)2 or (S,S,R)2 form, whereas crystals grown from aqueous K3[Lu3L2] solutions (Figure 2) solely contained the C2 isomer. Synthesis and Characterization of Hafnium Azainositol Complexes. The complex anion Lu3(H−3taci-ta)23− has a charge of 3− and therefore carries three sodium counterions. This is undesirable for a CA because it increases the osmotic pressure of the highly concentrated formulations for injection and reduces its tolerability. The next optimization goal therefore was to reduce the osmolality by using a chargeneutral compound (at pH 7.4). The switch from lutetium(III) to hafnium(IV) delivers uncharged inositol complexes with optimal osmotic properties; unfortunately, we observed a decrease in the solubility upon metal exchange (Table 2). Although replacement of acetate (12) with propionate (14) in the inositol ligand brought an improvement in the solubility, 5758


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Inorganic Chemistry

to Hf4+, which was visible by negative-mode electrospray ionization mass spectrometry detection. Because of the limited synthetic accessibility of 16, additional hafnium inositol complexes with broken symmetry were designed by replacing the free hydroxyl group with alcoholates attached to the inositol core by a reductive amination procedure used during ligand synthesis.20 Compounds 17−20 represent all combinations of acetate/propionate at R1 and ethanol/propanediol at R3 and show equally high solubility combined with excellent hydrothermal stability. Only minimal formation of Hf-DTPA was observed in the DTPA competition experiments (Table 2). It is important to note that Hf-DTPA is several orders of magnitude more stable than, e.g., Gd-DTPA16 and that the newly formed Hf3 complexes are obviously even more stable than Hf-DTPA. The combination of acetate and propionate around the azainositol core present in 21 and Hf3(H−3taci-dp-a)2 (BAY576), introduced to break the C3 symmetry, enabled identification of the monoacetate (R3) and dipropionate (R1) ligands 1,3,5-triamino-1,3,5-trideoxy-cis-inositol-N-acetic acidN′,N″-dipropionic acid (taci-dp-a) as the most advantageous for solubility and stability. While compounds 17−20 were synthesized by a statistical alkylation protocol followed by the separation of monoalkyl and dialkylazainositoles, BAY-576 could be synthesized by a more targeted approach (Scheme 2). For protection of two of the three amino groups, 9 was reacted with Staab’s reagent, yielding the bridged urea 22. Alkylation, followed by deprotection, gave the glycine derivative 24. The propionates were introduced by acrylonitrile in aqueous sodium hydroxide, followed by acidic hydrolysis to the hydrochloride

Figure 2. View of the structure of the complex anion, as found in crystals of 5a (C2 isomer).

103 mg of Hf/mL was not sufficient for an applicable CA. Likewise, methylation of the amino functionalities delivered no enhancement in terms of the stability or solubility. A path forward with respect to the solubility presented itself when compound 16 was isolated as a byproduct during the preparation of [Hf3(H−3maci-tp)2] (15), which had lost one propionate ligand by a retro-Michael reaction in the final complex formation step. This breaks the symmetry of the complex and leads in this case to a more than 10-fold higher solubility with sufficient stability. The negative charge of the carboxylate is retained by a free hydroxide anion tightly bound

Table 2. Numbering of Hafnium Complexes with Short Termination and CA-Relevant Propertiesa

a

DTPA comp.: Competition test with equimolar amounts of DTPA at 120 °C, 45 min. The recovery of the inositol complex in percent is reported. 5759


Article

Inorganic Chemistry Scheme 2. Synthesis of BAY-576a

Figure 3. View of the structure of the neutral complex found in a crystal of BAY-576, a (Hf-a-p)2-(Hf-p-p) species.

kg or 500 mg of I/kg, respectively, at 1.5 mL/s in the ear vein followed by 10 mL of saline at the same flow rate by a CTpower injector (Stellant; Bayer). Bolus tracking at the top of the descending aorta (threshold, 50 HU; delay time, 2 s) was used to start the imaging. The animal was then moved into the CT during the scan with a table feed of 3.8 cm/s (pitch = 1) in the cranial-caudal direction, following the bolus along the descending aorta. Images were reconstructed with a slice thickness of 1 mm and a field of view of 35 × 35 cm using a medium soft kernel (B30). The used CT imaging parameters were comparable to standard clinical protocols (CT tube voltage, 120 kV; tube current, 154 mA). Representative images from one animal are shown in Figure 4. The contrast-to-noise

a

Reagents and conditions: (a) 1,1′-carbonyldiimidazole, CHCl3, 80%; (b) BrCH2CO2tBu, Hünig’s base, CHCl3, 90%; (c) 9 N HCl, μW, 150 °C, 90%; (d) prop-2-enenitrile, NaOH, 50 °C, 99%; (e) 6 N HCl, reflux, 90%; (f) HfCl4, NH4OH, water, pH 1, 140 °C, 4 bar, 82%.

salt of 26. A hydrothermal process was used for hafnium complex formation. Highly pure BAY-576 was recovered subsequent to fractionated ultrafiltration, followed by filtration through a C18-HPLC sunfire bed and treatment with charcoal and mixed ion-exchange resin with good overall yield. An osmolality between 860 and 985 mOsmol/kg was measured for batches purified by the above protocol at a concentration of 300 mg of Hf/mL, which only slightly exceeds the values of commercial iodinated nonionic monomeric CAs. BAY-576 could be crystallized despite the broken C3 symmetry in two different crystal forms representing two possible “regioisomers”. In the taci-dp-a ligand of complex BAY-576, one amino group carries an acetate (a) substituent and the other two a propionate (p) substituent, and the three hafnium centers may either receive a (Hf-a-a)-(Hf-p-p)2 or a (Hf-a-p)2-(Hf-p-p) structure. Regarding again the option for an R or S configuration together with the restriction for uniform R,R or S,S hafnium binding, now a total of nine diastereomeric pairs of enantiomers could form (Figure S1, Supporting Informaton). In six of the nine forms, the two ligands of the Hf3L2 unit are related by a 2-fold rotational axis, and the point group is C2. In the other three forms, any element of symmetry is lacking (point group C1). Two C2-symmetric species of complex BAY-576, a (Hf-a-a)-(Hf-p-p)2 species with a rel(R,R,R)2 configuration and a (Hf-a-p)2-(Hf-p-p)-species with an (S,S,S)2 configuration, have been characterized by singlecrystal X-ray diffraction. The molecular structure of the latter is depicted in Figure 3. To demonstrate the high efficacy of the hafnium complex as a CA in CT imaging, a comparative animal study against iopromide (Ultravist, Bayer, Germany) was performed in rabbits.21 Four rabbits (2.5−2.8 kg, male) received an intravenous injection of BAY-576 or iopromide. Imaging was performed in a crossover study design, where each animal was imaged after the injection of each CA with a 1 day delay between injections. The anesthetized animals were placed in the CT, and the CA was injected with a dose of 500 mg of Hf/

Figure 4. CT angiography in a rabbit. Baseline image without CA in the middle, with BAY-576 on the left and iopromide on the right. The brightness and contrast of all images were equally scaled.

ratio in the descending aorta and in the major vessels in the abdomen was clearly higher after the injection of BAY-576 compared to iopromide. Visualization of smaller vessels in the liver and abdomen was also improved by high contrast and sharp borders.

3. CONCLUSIONS A new class of highly stable hafnium and lanthanide azainositol complexes was synthesized and characterized. Especially unsymmetrically substituted hafnium complexes, represented by BAY-576, with their excellent water solubility, are highly suitable as CT CAs. A feasible and selective synthesis was developed that allowed the preparation of multiple gram quantities of BAY-576 for in-depth pharmacological and radiological characterization.21 An animal study with this complex demonstrated increased CT signal intensity and improved visualization of small structures compared to the traditional iodinated agent iopromide at the same dose. These 5760


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interest; National Inst. of Standards and Technology-PL, Gaithersburg, MD (United States). Ionizing Radiation Div. 1995. (10) Janon, E. Gadolinium-DPTA: a radiographic contrast agent. AJR, Am. J. Roentgenol. 1989, 152, 1348−1348. (11) Unger, E.; Gutierrez, F. Ytterbium-DTPA: A Potential Intravascular Contrast Agent. Invest. Radiol. 1986, 21, 802−807. (12) Hegetschweiler, K. A rigid, cyclohexane-based polyaminopolyalcohol as a versatile building block for tailored chelating agents. Chem. Soc. Rev. 1999, 28, 239−249. (13) Hegetschweiler, K.; Ghisletta, M.; Gramlich, V. 1, 3, 5Triamino-1, 3, 5-trideoxy-cis-inositol, a new ligand with a remarkable versatility for metal ions. 4. Preparation, characterization, and x-ray structure of the trinuclear lead (II) and bismuth (III) complexes. Inorg. Chem. 1993, 32, 2699−2704. (14) Hedinger, R.; Ghisletta, M.; Hegetschweiler, K.; Toth, E.; Merbach, A. E.; Sessoli, R.; Gatteschi, D.; Gramlich, V. Trinuclear Lanthanoid Complexes of 1, 3, 5-Triamino-1, 3, 5-trideoxy-cis-inositol with a Unique, Sandwich-Type Cage Structure. Inorg. Chem. 1998, 37, 6698−6705. (15) Welti, G. 1,3,5-Triamino-1,3,5-tridesoxy-cis-inosit-Derivate für die Radioimmunotherapie und zur Behandlung der Thalassaemie. Diss. Naturwissen, ETH Zürich, No. 12613, 1998. (16) Berger, M.; Schmitt-Willich, H.; Sülzle, D.; Pietsch, H.; Frenzel, T.; Jost, G.; Hegetschweiler, K.; Neis, C.; Lauria, S. Bis azainositol heavy metal complexes for X-ray imaging. WO2013/171048, 2013. (17) Martell, A. E.; Smith, R. M. Amino acids. Critical Stability Constants; Springer: Berlin, 1974; Vol. 1. (18) Bartholomä, M.; Gisbrecht, S.; Stucky, S.; Neis, C.; Morgenstern, B.; Hegetschweiler, K. Chelation versus Binucleation: Metal Complex Formation with the Hexadentate all-cis-N1, N2-Bis (2, 4, 6-trihydroxy-3, 5-diaminocyclohexyl) ethane-1, 2-diamine. Chem. Eur. J. 2010, 16, 3326−3340. (19) Ghisletta, M.; Jalett, H.-P.; Gerfin, T.; Gramlich, V.; Hegetschweiler, K. 1, 3, 5-Triamino-1, 3, 5-trideoxy-cis-inositol, a New Ligand with a Remarkable Versatility for Metal Ions. Helv. Chim. Acta 1992, 75, 2233−2242. (20) Berger, M.; Sülzle, D.; Frenzel, T.; Graham, K.; Jost, G.; Neis, C.; Hegetschweiler, K.; Pietsch, H. Bis Azainositol Hafnium Complexes for X-ray Imaging. WO2014/173857, 2016. (21) Frenzel, T.; Bauser, M.; Berger, M.; Hilger, C. S.; HegeleHartung, C.; Jost, G.; Neis, C.; Hegetschweiler, K.; Riefke, B.; Suelzle, D.; Pietsch, H. Characterization of a Novel Hafnium-Based X-ray Contrast Agent. Invest. Radiol. 2016, 51, 776−785. (22) Yeh, B. M.; FitzGerald, P. F.; Edic, P. M.; Lambert, J. W.; Colborn, R. E.; Marino, M. E.; Evans, P. M.; Roberts, J.C.; Wang, Z. J.; Wong, M. J.; Bonitatibus, P. J. Opportunities for new CT contrast agents to maximize the diagnostic potential of emerging spectral CT technologies. Adv. Drug Delivery Rev. 2016. (23) Atak, H.; Shikhaliev, P. M. Dual energy CT with photon counting and dual source systems: comparative evaluation. Phys. Med. Biol. 2015, 60, 8949−8975.

novel hafnium complexes represent a promising new class of Xray CAs and, in addition, because of their high K-edge, may also be highly useful in combination with the newly developed photon-counting CT detectors in K-edge imaging.1,22,23 In combination with its favorable pharmacokinetic and toxicological properties, BAY-576 constitutes a milestone in the development of a technically feasible noniodinated CT CA.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b00359. X-ray crystallographic data in CIF format (CIF) X-ray crystallographic data in CIF format (CIF) X-ray crystallographic data in CIF format (CIF) X-ray crystallographic data in CIF format (CIF) Detailed synthetic procedures, spectroscopic data, descriptions of the experimental methods, and a comparative animal study (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Markus Berger: 0000-0001-6676-6090 Notes

The authors declare the following competing financial interest(s): M.B., M.B., T.F., C.S.H., G.J., H.P., and D.S. are employees of Bayer AG.

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REFERENCES

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Hafnium-Based Contrast Agents for X-ray Computed Tomography

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