Lipophilic X-ray contrast agents diatrizoyl double esters with cholesterol and cholic acid

Potential biodegradable and organ-specific X-ray contrast agents have been prepared by linking diatrizoic acid to cholesterol and cholic acid, respectively, via a spacer group containing a carbonate double ester. In vitro experiments revealed that the cholic acid esters were enzymatically degraded in the presence of pig liver esterase, while the double esters with cholesterol appeared to be stable, possibly due to low solubility in water.


Introduction
X-ray contrast agents for parenteral use today are triiodinated benzene derivatives with hydrophilic substituents to secure high water-solubility.These agents have similar pharmacokinetic properties such as extracellular distribution and renal elimination. 1,2During the past there has been considerable interest in organospecific contrast agents; e.g.agents for liver imaging. 1,38][9] Long residual time in the liver and other organ systems is a major problem with these agents.Bile acid drug conjugates, e.g. the anticancer drug chlorambucil, 10,11 are potential liverspecific targeting drugs.So-called double esters are frequently used in prodrug design because of their high affinity for unspecific esterases. 12The antibiotic pivampicillin is a typical example. 13ouble esters have also been evaluated in particulate contrast agents. 65][16] The preparation of potential biodegradable conjugates between diatrizoic acid and the steroids cholesterol and cholic acid, respectively, and evaluation of their degradability in vitro, constitutes the subject of the present report.

Synthesis
Epimeric cholest-5-en-3β-yl 1(R,S)-chloroethyl carbonates (2) were prepared in quantitative yield by esterification of cholesterol ( 1) with (±)-1-chloroethoxycarbonyl chloride according to Dang et al., 17 cf.Scheme 1.The epimeric double esters 3 were subsequently prepared in 66% yield by esterification of diatrizoic acid as its tetrabutylammonium salt with the chlorocarbonates 2 in DMF, in the presence of catalytic amounts of potassium iodide.t-Butyl cholate (5) was prepared as described by Bonar-Law 18 who first reacted cholic acid (4) with trifluoroacetic anhydride (TFAA) to the corresponding C-24 mixed anhydride and with the C-3 and C-7 hydroxyl groups trifluoromethyl acetylated, cf.Scheme 2. This mixed anhydride was then treated with t-butanol followed by hydrolysis of the C-3 and C-7 trifluoromethyl acetyl groups with aqueous ammonia furnishing t-butyl cholate (5).

Enzymatic degradation
Degradation studies with pig liver esterase revealed that the cholic acid conjugates 8 was a good substrate for the enzyme.The half life of the two epimeric esters 8 was found to be less than three hours.
The cholesterol derived double esters 3 appeared to be stable in similar degradation studies.
The stability might be due to its low solubility in aqueous solutions.

Experimental Section
General Procedures. 1 H NMR spectra were recorded at 200, 300, and 500 MHz using Varian Gemini 200, Varian XL 300 (manual), and Bruker Avance DRX 500 spectrometers. 13C NMR spectra were recorded at 50, 75 and 125 MHz using the above mentioned spectrometers.
Assignments of NMR shifts have been limited to the diagnostically important parts, e.g. the contrast agent segment and the spacer (= the 1-chloro-ethoxycarbonate portion).Similarly, we report only assigned 13 C NMR shifts for selected parts of the molecules comprising the contrast agent segment, the spacer and the resonances of C 3 , C 7 , C 12 , C 21 in the steroid moieties.
Additional resolved resonances are listed without assignments.3][24] Melting points were measured with a Reichert melting point microscope and are uncorrected.FAB and EI spectra were obtained using a Trio-2 Mass Spectrometer, VG Biotech Ltd.The EI spectra were recorded using 70 electronvolt ionizing voltage.Electrospray MS was obtained using a Bruker Apex 4.7 instrument.Elemental analyses were performed by Ilse Beetz, Kronach, Germany.
TLC analyses were performed on silica gel plates (Merck 4500).
The cholesterol derived double esters 3 did not hydrolyze when treated under similar conditions.

of the diatrizoyl double esters 3 and 8 in esterase solutions.
The conjugates 8 (0.10 g, 0.09 mmol) were added to a solution of pig liver esterase (0.8 ml, Sigma E 3128) in ammonium carbonate buffer (500 ml).The solution was shaken at 37 o C for 12 h,