Medicinal Inorganic Chemistry - American Chemical Society

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Chapter 9

Bile Acids at Work: Development of a New Intravascular MRI Contrast Agent 1

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Pier Lucio Anelli , Marino Brocchetta , Vito Lorusso , Giuseppe Manfredi , Alberto Morisetti , Pierfrancesco Morosini , Marcella M u r r u , Daniela Palano , and Massimo Visigalli 1,2

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Milano Research Centre, Bracco Imaging SpA, via E. Folli 50, 20134 Milan, Italy Current address: GSK Italy, via Asolana 68, 43056 S. Polo di Torrile, Parma, Italy

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Complexes of Gd(III) featuring strong binding to human serum albumin (HSA) can be potentially used as intravascular MRI contrast agents. Accordingly, a series of Gd(III) complexes containing a bile acid moiety as the HSA binding subunit were synthesised. Diethylenetriaminepentaacetic acid was used as the chelating moiety of Gd(III) ion for all the conjugates. Derivatives of cholic, chenodeoxycholic, deoxycholic, and lithocholic acids, as well as bile acids in which the 12-a OH group was oxidized to a ketone, were prepared. Preliminary screening, which was based on the evaluation of binding to HSA and in vivo tolerability (in mice), led to the identification of the conjugate containing a deoxycholic subunit (i.e.1c)as the clinical candidate. During development, the route to 1c used for the research phase was largely modified, taking advantage of a different synthetic approach.

© 2005 American Chemical Society

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Gadolinium complexes of polyaminopolycarboxylic ligands are by and large the most preferred species used as contrast agents for Magnetic Resonance Imaging (MRI) (/). Ail the complexes so far approved for administration in humans are so-called extracellular fluid (ECF) contrast agents (2). Such contrast agents are able to cross blood vessel walls. This means that, immediately after intravenous administration, they diffuse from plasma into the interstitial space until equilibrium between the two compartments is reached. These complexes usually do not enter cells. Although the currently approved complexes can be successfully used for some angiographic procedures, they appear not suitable when visualization of coronary arteries is required (J). Indeed, imaging of coronary arteries, which are tiny and highly tortuous vessels, is furtherly complicated by cardiac and respiratory motion effects. In order to fill this gap, contrast agents, which after administration are confined in the vascular space, have been developed by several research groups. Two main classes of compounds have been proposed to avoid, or at least to largely reduce, extravasation: •

•

Species which by virtue of their molecular size, are not able to cross blood vessel walls. Accordingly, supramolecular systems (like micelles and liposomes) (¥), proteins (e.g. albumin) (J) and polymers (6) labelled with a large number of gadolinium complexes have been proposed. Among these species, dendrimers (e.g. Gadomer-17) (7) have proved noticeably promising. Interestingly, also a gadolinium complex (i.e. P792) featuring four large hydrophilic side chains, due to its peculiar structure and size (6473 Da), behaves like diese systems (8). Small Gd(III) complexes (in the 800-1200 Da range) which feature suitable binding to Human Serum Albumin (HSA), the main component of human plasma proteins. It is important to stress that this binding needs to be tuned in such a way to prevent leakage in the interstitial space but at the same time to allow elimination from the body in a reasonable timeframe. MS-325 (i.e. a Gd(III) complex of a diethylenetriaminepentaacetic acid, DTPA, ligand featuring a diphenylcyclohexyl residue and a phosphonate group) represented a breakthrough in this context (9). The compound is presently under late clinical development

We also focused our efforts on the latter class of compounds, the so-called "protein binders". Initially, variously mono-, di- and tri-substituted derivatives of Gd-DTPA " were investigated (10, 11). More recently, we took advantage of the experience gained in the use of bile acids as carriers for the development of hepatocyte-directed Gd(III) complexes (12). At that time, with the aim of addressing specific transporters of the hepatocyte trafficking, a number of 2

Sessler et al.; Medicinal Inorganic Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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conjugates of Gd(III) complexes with bile acids was prepared. In depth investigations involved several structural features of the conjugates like: i) nature of the bile acid; ii) site of conjugation on the bile acid skeleton; Hi) nature of the polyaminopolycarboxylic ligand; iv) global charge of the conjugate. Indeed, during such studies we had found that complex l a iV-methyiglucammonium salt (see Table I), was endowed with promising features in terms of binding to HSA (72% binding extent) and tolerability (LD 7.6 mmol/kg, in mice). Accordingly, this compound became our lead candidate and led us to explore structural modifications aimed at improving the HSA binding extent. It is worth noting that l a incorporates a subunit of cholic acid in which the 3 α-ΟΗ residue has been converted into a 3β·ΝΗ residue for conjugation purposes. Binding of simple bile acids to HSA had been studied more than twenty years ago by Roda et al (13). 50

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Table I. Chemical Structures of Compounds la-g

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Compound la lb lc Id le If Η α

R, α-ΟΗ Η α-ΟΗ Η = 0 = 0 Η

R α-ΟΗ Η Η α-ΟΗ Η α-ΟΗ Η 2

R Η Η Η Η Η Η CH COO'NMGH 3

2

Μ is either sodium or N-methylglucammonium (NMGH).

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They had shown that the binding association constant of bile acids roughly increases 60 times on going from cholic acid to lithochoiic acid, with intermediate values for deoxycholic and chenodeoxycholic acids. This clearly reflects the different lipophilicity of the bile acids, due to the different number and position of the hydroxy groups on die steroidic moiety. Accordingly, we decided to investigate how modifications to the basic structure of l a could affect binding to HSA and tolerability. To minimize the number of variables, the chelating moiety (i.e. the DTPA subunit derived from L-glutamic acid, vide infra) was kept unmodified in all the compounds which were synthesised. Conversely, trying to take advantage of the indications of Roda et al (13), all structural variations were aimed at understanding the effect of modifications in the hydrophilicity/lipophilicity balance of the bile acid moiety. Table I lists the conjugates which were prepared. In all compounds the cholanoic moiety stems from a natural bile acid in which the 3 α-ΟΗ has been converted into a 3/*-NH group. Apart from taking advantage of the different number and position of OH groups on the steroidic moiety, hydrophilicity/lipophilicity of the structures has been tuned either oxidizing one of the OH residues to a ketone (like in conjugates le,f) or introducing an auxiliary charged group (like in lg). 2

Chemistry of Conjugates lb-g The general synthetic route for the preparation of compounds lb-g, with the exception of If, is the same previously used to achieve l a and reported in Figure 1 (14). The key step of such route is the assembly of the skeleton of ligand 2a which occurs by diethoxyphosphoryl cyanide (DEPC) mediated condensation of the versatile intermediate 6 and the 3^-aminocholate derivative 8. On the one hand, die monoacid pentaester 6 is obtained, using a Rapoport-like methodology (15% by double alkylation of L-glutamic /-butyl benzyl diester 3 with bis(/butyl) iV-(2-bromoethyl)iminodiacetate 4, in its tum obtained by alkylation of ethanolamine with /-butyl bromoacetate followed by bromination of the hydroxyl group with iV-bromosuccinimide/triphenylphosphine (NBS/PPh ). Hydrogenolysis of the benzyl ester affords derivative 6. On the other hand, 3ßaminocholate derivative 8 is obtained from methyl etiolate and diphenylphosphoryl azide (DPPA) under Mitsunobu conditions followed by reduction of the azide group with triphenylphosphine/H 0 (Staudinger conditions) (16). After condensation of 6 with 8, all ester functions of intermediate 9 are deprotected in two steps and ligand 2a is complexed with Gd 0 . Complexes lb-d were synthesised according to the same path starting from 3

2

2

3

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OH

Bzjooe

COOMe

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2

HO** (i) PPh , DEAD, DPPA, THF ®PPh ,H 0,THF 3

3

2

COOMe

H ,Pd/C EtOH

ι— 5 R = COOBzl

2

• 6 R = COOH

H N* 2

COOR

ROOC (i)CF COOH, CH Cl2 (S)NaOH,H 0 Gd 0 , ghicamine, H 0 3

2

COOR

j

2

2aR=R* = H .

2

2

la.

3

2

Figure 1. Synthetic Route to la

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the methyl esters of lithocholic, deoxycholic and chenodeoxycholic acid, respectively. Noticeably, it was found that: i) the key condensation can be advantageously carried out replacing diethoxyphosphoryl cyanide with dicyclohexylcaibodiimide/hydroxybenzotriazole; ii) the final hydrolysis of the five /-butyl and of the methyl ester functions can be performed in a single step with a large excess of LiOH in dioxane/H 0. For the synthesis of le, the intermediate 11 was prepared from the 3/2-N derivative 10 of methyl deoxycholate by oxidation of the hydroxy group with Cr0 in l^SCVacetone followed by Pd/C catalyzed hydrogenation of the azide moiety (Figure 2). 2

3

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3

COOMe

Figure 2. Synthesis ofIntermediate 11 To avoid tedious protection/deprotection sequences of the hydroxy groups, for the preparation of If (le. the 7QP-OH analogue of le) a completely different approach was pursued. Indeed, it was found that complex la, in the presence of 12Λ! icr°

3 Na'

Ν

ί\

Θ

la

R, - 3α-ΟΗ "

If

R

1 =

0=

-«

1290 le 3.0(0/10)* 91 If 5.0 (0/5)* 57 1.5 (8/10) >W Determined by means of ultrafiltration. * In mice, after intravenous administration. From ref. 20. Binding was not more precisely assessed in the light of the tolerability data. Single dose toxicity data reported in terms of dose level (dead animals/treated animals). 5

C

e

d

e

α c

d

e

As anticipated, our aim was to increase the lipophilicity of complex l a to achieve a stronger binding to HSA, Indeed, with the lithocholic derivative l b binding to HSA remarkably increased to 98%. However, likely due to such strong binding, the tolerability (LD 0.6 mmol/kg, in mice) and the elimination of the complex proved definitively unacceptable. Complexes characterized by qn intermediate lipophilicity, like le and Id, which derivefromdeoxycholic and chenodeoxycholic acid, respectively, showed HSA binding around 95% and very encouraging tolerability data. Attempts to reduce the HSA binding extent of the lithocholic derivative l b introducing an additional charged residue in the structure led to complex l g which, however, proved only slightly more tolerable than lb. Complex le in which the 12-OH residue of the steroidic moiety has been converted into a ketone is characterized by a good tolerability but a lower HSA binding in comparison with the hydroxylated precursor lc. S0

Sessler et al.; Medicinal Inorganic Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

145 A remarkable weakening of the binding to HSA as a consequence of the conversion of the 12-OH residue of l a into a ketone can also be noticed for complex If in comparison with its hydroxylated precursor la. On the basis of the promising HSA binding and tolerability results, the properties of complex l c werefttrtherlyinvestigated. The strong binding is also responsible for the relatively high relaxivity in human serum (~ 27 mM~V ). Under the experimental conditions used for compounds la-g, MS-325 showed a relaxivity of - 35 mM'V but binding to HSA was reduced to 80% (19). As expected, studies carried out in rats and monkeys showed that, due to the high HSA binding, le features a long plasma half-life if compared to that of routine ECF agents. Nonetheless, the elimination in rats of lc, which mainly occurs through the biliary route, accounted for more than 82% within 24 h and for 94% after 7 days. In the same study, biodistribution confirmed that there was no accumulation of gadolinium in any organs (20). Since size and anatomy of the coronary arteries of pigs are very similar to those of humans, imaging studies were carried out in micropigs. Such studies showed that administration of l c at 0.1 mmol kg" dose level could guarantee a sufficient signal enhancement of blood in the coronary arteries for a suitable time (19,20, 21). On these grounds, le was selected as the candidate for clinical development as a contrast agent for MRI coronary angiography. Preliminary results on the first administration to humans have been reported (22). l

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Process Research and Development of lc Once complex l c was selected for clinical development, a synthetic route amenable to scale up was required. The research route which, as already discussed, roughly follows that of complex l a (see Figure 1) was critically analyzed. A not so appealing scenario appeared: i) the overall yield was very low (i.e. 6%fromL-glutamic acid); ii) a large number of chemical steps (i.e. 12) and several isolated intermediates (i.e. 10), most of which were purified by silica gel chromatography, were involved. Although the latter issues are ordinary life in process research and development, the low overall yield deserves some further comments. The monoacid pentaester 6 is a versatile intermediate, nearly perfect for the synthesis of a series of compounds like those above described. However, its synthesis (14) largely accounts for the low yield observed in the preparation of lc. Furthermore, the conversion of methyl deoxycholate into the corresponding 3/?-NH derivative 14 (see Figure 5; Research route) involves the intermediate production of the 3ß-N derivative 15. Although the latter proved a perfectly safe compound, we must underline that: i) the reagent (i.e. diphenylphosphoryl azide) which is used in the Mitsunobu reaction is extremely expensive; ii) alternative approaches to generate the azide by phase transfer catalysed 2

3

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nucleophilic substitution on the 3-mesyloxy derivative had to rely upon the hazardous NaN . Therefore, a new route to derivative 14 was devised. Again taking advantage of a Mitsunobu reaction, methyl deoxycholate was converted into the corresponding 3/^phthalimido derivative 16. Subsequently, the phthalic residue was removed in two steps using NaBH in dimethylacetamide/MeOH at first and then HCl in MeOH to give 14 (Figure 5; Development route). Additionally, during development the synthesis of the ligand precursor of lc starting from 14 was largely changed. 3

4

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Research route

Development route -COOMe

OH

PPh , DIAD, DPPA, THF 3

PPh , DIAD, phthalimide, THF 3

COOMe

PPh , H 0, THF 3

2

©NaBH*, DMAC ÖD HCl MeOH,

Figure 5. Synthesis ofIntermediate 14 Research vs Development Route

Sessler et al.; Medicinal Inorganic Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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Looking at Figure 6, it is easy to recognize that in the research route the disconnection approach involvesfirstformation of bonds a and then bond b. To circumvent the drawbacks linked to the use of 6, the disconnection was turned the other way around. Accordingly, the development route entails first the formation of bond b and then bonds a. The success of this approach was heavily dependent on the choice of a suitably protected L-glutamic acid derivative of some sort. Methyl iV-Boc-L-pyrogiutamate, 17 (see Figure 7), proved the ideal intermediate in this respect. Indeed, this choice came out of a massive work during which Cbz protected derivatives as well as different esters of Lpyroglutamic acid were screened. The final route to lc, which led to a successful pilot plant campaign, is depicted in Figure 7. The key step is the condensation between derivatives 14 and 17 to afford 18 which occurs in good yield in the absence of any catalyst. The Boc protecting group of 18 is then removed under acidic conditions to give 19. At this stage, according to a Rapoport-like approach, 19 could have been alkylated with the bromo derivative 4 (see Figure 1). However, we found much more convenient to in-situ generate the mesyl derivative of hydroxyethyl derivative 20 and directly proceed to the double alkylation of 19 in w-butyl acetate. The thus obtained hexaester 21 is deprotected to ligand 2c in the presence of a large excess of NaOH in *-PrOH/H 0. Eventually, the ligand is complexed with Gd 0 to give the gadolinium complex lc. It is noteworthy that hexaester 21 differs from the analogous intermediate according to the research route, for the presence of a methyl instead of a i-butyl ester on the central acetic residue of the DTPA moiety. This proved a noticeable advantage in order to minimize racemization during basic hydrolysis of 21 to 2c. According to the synthetic route optimized during scale up, the number of isolated intermediates dropped to 6 and all silica gel chromatographies were eliminated. The overall yield for the new process is roughly 30% (from L-glutamic acid). 2

2

3

COOH

Figure 6. Disconnection Approach to 2c

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COOCH3

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O^OtBu

HN 2

17

COOCH3 toluene, Δ

CHsOOC^^/v^CONH MeS0 H, MeOH

NHR

3

COOtBu \ R

HO'

MsCl, DIEA XOOtBu

w-BuOAc

• COOR'

20

ROOCL

ROOC

N"

ROOC^

CONH

#

>T ^COOR COOR

Η

R = t B u

21 R' = Me

I NaOH I /-PiOH/H 0 = R' = Η ^ , I Gd 0 , NaOH, H 0 2

2

3

2

Figure 7. Syntesis of lc: Final Route

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Conclusions A series of conjugates of Gd(III) complexes with bile acids were synthetised and investigated as MRI intravascular contrast agents. Complex lc, containing a subunit of deoxycholic acid, was selected as a clinical candidate for development. The synthetic route to lc, which had been used during the research phase, was completely changed during scale up to pilot plant m order to achieve an industrially appealing route. The compound is presently undergoing Phase Ha clinical trials.

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References 1. The Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging, Merbach, A.E.; Toth, E. Eds.; John Wiley & sons: Chichester, England, 2001. 2. Dawson, P. In Textbook of Contrast Media, Dawson, P.; Crosgrove, D. O.; Grainger, R. G. Eds; Martin Dunitz: London, 1999; pp 319-321. 3. Li, D.; Zheng, J.; Bae, Κ. T.; Woodward, P. K.; Haacke Ε. M . Invest Radiol 1998, 33, 578-586. 4. Anelli, P. L.; Lattuada, L.; Lorusso, V.; Schneider, M . ; Tournier, H.; Uggeri, F. MAGMA 2001, 12, 114-120. 5. Brasch, R. Magn. Reson. Med 1991, 22, 282-287. 6. Vexier, V.; Clement, O.; Schmitt-Willich, H.; Brasch, R. J. Magn. Reson. Imaging 1993, 4, 381-388. 7. Misselwitz, Β.; Schmitt-Willich, Η.; Ebert, W.; Frenzel, Τ.; Weinmann H.-J. MAGMA 2001,12, 128-131. 8. Port, M.; Corot, C.; Rousseaux, O.; Devoldere, L.; Idee, J. M.; Dencausse, Α.; Le Graneur, S.; Simonot, C.; Meyer, D. MAGMA 2001, 12, 121-127. 9. Caravan, P.; Cloutier, N . J.; Greenfield, Μ. T.; McDermid, S. Α.; Dunham, S. U.; Bulte, J. W. M.; Amedio, Jr., J. C ; Looby, R. J.; Supkowski, R. M.; Horrocks, Jr., W. DeW.; McMurry, T. J.; Lauffer, R. B. J. Am Chem. Soc. 2002, 124, 3152-3162. 10. Anelli, P. L.; Lolli, M.; Fedeli, F.; Virtuani, M . U.S. Patent 6,458,337, 2002. 11. Calabi, L.; Maiocchi, Α.; Lolli, M . ; Rebasti, F. U.S. Patent 6,403,055, 2002. 12. Anelli, P. L.; Calabi, L.; de Haën, C.; Lattuada, L. ; Lorusso, V.; Maiocchi, Α.; Morosini, P.; Uggeri, F. Acta Radiol. 1997, S142, 125-133. 13. Roda, Α.; Cappelleri, G.; Aldini, R.; Roda, E.; Barbara, L. J. Lipid Res. 1982, 23, 490-495. 14. Anelli, P. L.; Fedeli, F.; Gazzotti, O.; Lattuada, L.; Lux, G.; Rebasti F. Bioconjugate Chem. 1999, 10, 137-140. 15. Williams, Μ. Α.; Rapoport, Η. J. Org. Chem. 1993, 58, 1151-1158. 16. Anelli, P. L.; Lattuada, L.; Uggeri, F. Synth. Commun. 1998, 28, 109-117.

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17. Anelli, P. L.; Brocchetta, Μ.; Morosini, P.; Palano, D.; Carrea, G.; Falcone, L.; Pasta, P.; Sartore, D. Biocatal. Biotransform, 2002, 20, 29-34. 18. Weinmann, H.-J.; Press, W.-R.; Gries, H, Invest Radiol 1990, 25, S49-S50. 19. Cavagna, F. Μ.; Anelli, P. L.; Lorusso, V.; Maggioni, F.; Zheng, J.; Li, D.; Abendschein, D. R.; Finn, P. J. Proceedings, ISMRM-ESMRMB Joint Annual Meeting, Glasgow, Scotland, 2001; p. 519. 20. de Haën, C ; Anelli, P. L.; Lorusso, V.; Morisetti, Α.; Maggioni, F.; Uggeri, F.; Cavagna, F. Μ. Invest Radiol submitted. 21. Cavagna, F. M.; Lorusso, V.; Anelli, P. L.; Maggioni, F.; de Hain, C. Acad. Radiol 2002, 9 (Suppl. 2), S491-S494. 22. La Noce, Α.; Stoelben, S.; Scheffler, K.; Hennig, J.; Lenz Η. Μ.; La Ferla R.; Lorusso, V.; Maggioni, F.; Cavagna, F. M . Acad. Radiol 2002, 9 (Suppl. 2), S404-S406.

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