Conjugates of gadolinium complexes to bile acids as hepatocyte-directed contrast agents for magnetic resonance imaging

Article abstract of DOI:10.1021/jm0310683

A series of structurally different Gd(III) conjugates incorporating a bile acid moiety have been prepared. Polyaminopolycarboxylic ligands such as diethylenetriaminepentaacetic acid (DTPA) and 1,4,7,10-tetraazacyclododecane-1, 4,7,10-tetracetic acid (DOTA

Full text of DOI:10.1021/jm0310683

J . Med. Chem. 2004, 47, 3629-3641
3629
Con ju ga tes of Ga d olin iu m Com p lexes to Bile Acid s a s Hep a tocyte-Dir ected
Con tr a st Agen ts for Ma gn etic Reson a n ce Im a gin g
Pier Lucio Anelli,* Luciano Lattuada, Vito Lorusso, Giovanna Lux, Alberto Morisetti, Pierfrancesco Morosini,
Michele Serleti, and Fulvio Uggeri
Bracco Imaging spa, Milano Research Centre, via E. Folli 50, 20134 Milano, Italy
Received October 20, 2003
A series of structurally different Gd(III) conjugates incorporating a bile acid moiety have been
prepared. Polyaminopolycarboxylic ligands such as diethylenetriaminepentaacetic acid (DTPA)
and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA) have been selected as
chelating subunit for the Gd(III) ion. Cholic acid, cholylglycine, and cholyltaurine have been
incorporated as the bile acid moieties. In first generation conjugates the Gd(III) complex is
linked to the carboxyl group of cholic acid. Second generation conjugates feature the attachment
of the Gd(III) complex to the 3 position of the steroidic backbone of the bile acid. Finally, in
third generation conjugates the Gd(III) complex is attached to the ꢀ nitrogen atom of
cholyllysine. The conjugates are eliminated through the biliary route to a various extent (7.5
to 77% in rats) according to their structural features. Among the most promising terms, a
second generation conjugate in which the Gd(III) complex is linked to cholic acid through the
3R hydroxy group seems to enter hepatocytes using the Na+/taurocholate transporter.
Noticeably, some of the second generation conjugates are characterized by very high toler-
abilities (LD50 up to 9.5 mmol/kg) after intravenous administration in mice.
In tr od u ction
cut diagnoses of specific hepatic diseases (e.g. hepato-
cellular carcinoma). Since it is known that several other
bile acids are taken up by hepatocytes through the Na+/
taurocholate transporter, we investigated whether bile
acids can be successfully used as address moieties to
confer specificity to Gd(III) complexes.
In this paper we discuss (i) the structural features
that were taken into account in the design of the Gd(III)
complex/bile acid conjugates, (ii) the synthetic ap-
proaches for the preparation of the conjugates, and (iii)
pharmacokinetic and toxicology evaluation of the con-
jugates.12
In the past decade the use of gadolinium complexes
of simple polyaminopolycarboxylic ligands (e.g. di-
ethylenetriaminepentaacetic acid ) DTPA or 1,4,7,10-
tetraazacyclododecane-1,4,7,10-tetraacetic acid ) DOTA)
as extracellular MRI contrast agents has become more
and more widespread.1 Such agents immediately after
intravenous administration equilibrate between blood
and the interstitial fluid; however, they do not enter
cells.2 In the meantime, the search for complexes, which
are taken up into hepatocytes, has been quite active.3
Noticeably, Gd(III) complexes in which a lipophilic
residue was introduced in the basic unit of DTPA have
been developed as hepatospecific agents (e.g. Gd-
BOPTA,4 Gd-EOB-DTPA5). Compared to the extra-
cellular contrast agents, these “lipophilic” complexes are
taken up to a variable extent by hepatocytes in different
animal species6 as well as in humans.7,8 However, it has
been shown that the transport of such agents through
the basolateral membrane of hepatocytes occurs by
passive diffusion.9
More recently, we became interested in contrast
agents which enter hepatocytes by means of active
transport mechanisms. Conjugation of the DTPA moiety
to a triiodinated carrier (i.e. iopanoic acid)10 led to a
contrast agent which was taken up by hepatocytes
through a carrier-mediated mechanism operated by the
organic anion transporting polypeptide (OATP).9 Indeed,
it has been shown that the Na+/taurocholate transporter
is not expressed in the basolateral membrane of some
human hepatoma cell lines.11 Therefore, the discovery
of a contrast agent which is transported into hepatocytes
by such a transporter could, in principle, allow clear-
Resu lts a n d Discu ssion
Design of th e Con ju ga tes. We studied conjugates
in which a Gd(III) complex is linked to a bile acid
through a spacer of different length. Various chelating
subunits for the coordination of Gd(III) ion were se-
lected. As a general requirement they had to afford
conjugates characterized by thermodynamic and kinetic
stabilities for the Gd(III) complex high enough to allow
in vivo administration. We incorporated in the structure
of the conjugates, DTPA, DTPA monoamide, DOTA
monoamide, and 10-hydroxypropyl-1,4,7,10-tetraaza-
cyclododecane-1,4,7-triacetic acid (HP-DO3A) subunits.
On the basis of thermodynamic studies carried out on
the parent or closely related structures,13 we can assume
that all these chelating subunits are characterized by
log KML > 19.
Among the bile acids for which hepatocyte uptake is
demonstrated to be mediated by the Na+/taurocholate
transporter,14 we chose cholic acid, cholylglycine (glyco-
cholic acid), and cholyltaurine (taurocholic acid).
The site of conjugation of the chelating subunit on to
the bile acid moiety was quite thoroughly explored.
* Corresponding author. E-mail: pier.lucio.anelli@bracco.com.
Phone: +39 02 21772353. Fax: +39 02 21772794.
10.1021/jm0310683 CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/05/2004

3630 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Anelli et al.
Ch a r t 1
Ch a r t 2
Accordingly, three different generations of conjugates
can be recognized.
F ir st Gen er a tion Con ju ga tes. From a purely syn-
thetic perspective the easiest way to functionalize cholic
acid takes advantage of its carboxylic moiety. Indeed, a
cholic acid conjugate of EDTA has been already re-
ported.15 We therefore planned the synthesis of the two
ligands (Chart 1) in which cholic acid is linked to the
chelating subunit through an amide bond. It is worth
noting that upon complexation with Gd(III) such DTPA
and DTPA-monoamide ligands give rise to complexes
characterized by a residual charge of -2 and -1,
respectively. On the basis of previous studies, a negative
charge on the chain linked to C17 on the steroid moiety
seems crucial for bile acids to be taken up by the Na+/
taurocholate transport system.16
Ch a r t 3
Secon d Gen er a tion Con ju ga tes. In light of the
importance of a negative charge on the side chain, the
second approach involved the insertion of the chelating
moiety on to the steroid skeleton of the bile acid. In
particular, we focused on the hydroxy group in position
3, which is more reactive and accessible if compared to
the other two hydroxy groups in positions 7 and 12. This
strategy, already reported by Kramer and co-workers17
for the synthesis of bile acid-drug conjugates for the
specific targeting of liver, was successfully applied to
achieve the DTPA, DOTA, and HP-DO3A derivatives
(Chart 2). A homogeneous series of DTPA monoamides
conjugated to cholic acid, cholylglycine and cholyl-
taurine, i.e. 1c-e, was also synthesized in order to
assess the role played by the bile acid.
Th ir d Gen er a tion Con ju ga tes. Generally speaking,
the last approach involved the coupling of the bile acid
carboxylic moiety to the R-amino group of a function-
alized R-amino acid. Accordingly, cholic acid was coupled
to lysine and after further functionalization led to the
conjugates depicted in Chart 3. This strategy is very
attractive and straightforward because the steroid frag-
ment of the bile acid is not modified and a free carboxylic
moiety in the side chain is still retained. This synthetic
approach has already showed promising results in the
enhancement of the hepatoselectivity of rhodamine,18
thyroxine,19 and peptide20 conjugates.
Ch em istr y
The synthesis of 1a is reported in Scheme 1. A 5-fold
molar excess of diethylenetriamine was reacted with the
bromo derivative 321 in order to maximize the yield in
monoalkylated product 4, which was then fully alkyl-
ated with tert-butyl bromoacetate. The nitro group of 5
was reduced to amino and acylated with Boc-glycine,
using diethyl cyanophosphonate (DEPC) as coupling
reagent.22 Deprotection of Boc and tert-butyl esters with
trifluoroacetic acid gave compound 8, which was coupled
to cholic acid N-hydroxysuccinimidyl ester 923 (Chart
4) to obtain ligand 1a .
Ligand 1b was synthesized by reaction of monoacid
1024 with cholic acid N-(2-aminoethyl) amide 1125 (Chart
4) in the presence of DEPC, followed by hydrolysis of
the methyl esters with NaOH at pH 12 (Scheme 2).
The synthesis of the bile acid intermediates 15c-f,
which are necessary for the achievement of the second

Hepatocyte-Directed MRI Contrast Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3631
Sch em e 1a
Sch em e 2a
a
Reagents and conditions: (a) 11, DEPC, Et3N, DMF; (b)
a
Reagents and conditions: (a) (NH2CH2CH2)2NH, MeCN; (b)
NaOH, H2O-MeOH.
BrCH2COOtBu, ClCH2CH2Cl, DIEA; (c) H2, Pd/C, EtOH; (d) Boc-
Gly, DEPC, Et3N; (e) CF3COOH, PhOMe, CH2Cl2; (f) 9, H2O-
DMF.
Sch em e 3a
Ch a r t 4
generation conjugates, is depicted in Scheme 3. The
starting key intermediate 13 was synthesized according
to a previously published procedure.26 Glycine and
6-aminohexanoic acid were chosen as spacers between
the polyaminopolycarboxylic moiety and the bile acid.
In this way, Z-glycine and N-Z-6-aminohexanoic acid
were activated by means of isobutyl chloroformate and
coupled to compound 13 to give, after hydrogenolysis
of the Z-group, derivatives 15c and 15f, respectively.
To achieve the whole series of bile acid derivatives (i.e.
with cholic acid, cholylglycine and cholyltaurine) the
side chain of compound 13 was modified. The cholyl-
glycine derivative 15d was obtained from 14 by hy-
drolysis of the methyl ester, coupling to glycine methyl
ester, and final hydrogenolysis of the Z-group. The
cholyltaurine derivative 15e was synthesized by cou-
pling Boc-glycine to 13, hydrolysis of the methyl ester,
coupling to taurine using 2-ethoxy-1-ethoxycarbonyl-1,2-
dihydroquinoline (EEDQ),27 and deprotection of the Boc
group with HCl in MeOH. The ligands 1c-f were
obtained as shown in Scheme 4. A four molar excess of
bis-anhydride 2128 (Chart 5) was suspended in DMF and
a
Reagents and conditions: (a) Z-Gly, IBCF, NMM, THF; (b) H2,
Pd/C, MeOH-H2O; (c) i: NaOH, H2O-MeOH; ii: HCl; (d)
GlyOMe, IBCF, Et3N, THF; (e) H2, Pd/C, MeOH; (f) Boc-Gly, IBCF,
Et3N, THF; (g) i: NH2CH2CH2SO3H, EEDQ, Et3N, DMF; ii: HCl,
MeOH; (h) ZNH(CH2)5COOH, IBCF, Et3N, THF; (i) H2, Pd/C,
EtOH.
Sch em e 4
partially hydrolyzed at 80°C by addition of water then
reacted with amino derivatives 15c-f. This convenient
procedure allows to increase the yield of the correspond-
ing DTPA monoamides, overcoming the problem faced
when using bis-anhydride 21 alone in DMF. In this case,
even with a large excess of 21, the formation of DTPA
bisamide derivatives is predominant.

3632 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Anelli et al.
Ch a r t 5
The synthesis of the third generation products 1k and
1l is depicted in Scheme 8. The key intermediate 32 was
prepared by activation of cholic acid by means of
isobutyl chloroformate, coupling to N6-Z-lysine, esteri-
fication with MeOH/PTSA, hydrogenolysis of the Z
group, and treatment with HCl/MeOH. Interestingly,
if the acidic treatment is not performed just before
evaporation, the only product obtained is the lactam 33
(Chart 6). Coupling of 32 with the monoacid 10 followed
by complete hydrolysis of the methyl esters with NaOH
gave ligand 1k . Ligand 1l was achieved by reaction of
32 with bis-anhydride 2128 (Chart 5) and successive
hydrolysis with NaOH, using the same procedure previ-
ously described for ligands 1c-f.
Ligands 1a -l were converted into the corresponding
gadolinium complexes 2a -l using two different proce-
dures. According to procedure A (see Experimental
Section), which is the most simple and straightforward,
Gd2O3 is added to a water solution of the ligand and a
suitable amount (according to the stoichiometry of the
final complex) of NaOH, and the suspension is heated
at 50 °C for several hours. The complex is then simply
recovered after filtration of the excess Gd2O3 and
evaporation of the aqueous solution.
After completion of the amidation reaction, NaOH
was added to hydrolyze the methyl ester moiety (when
present) and residual anhydride moieties. The crude
was purified by elution through an Amberlite XAD-16
resin column with a MeCN/H2O gradient. Elution with
only water enables elimination of the excess of DTPA
and the residual content of DMF, while the DTPA
monoamides 1c-f are usually eluted with 30-50%
MeCN in water.
The useful and versatile pro-ligand 2229 (Chart 5) was
employed for the synthesis of both DTPA ligands 1g and
1h . In particular, 22 was coupled to 3â-aminocholic acid
methyl ester 13 by means of DEPC to give the hexaester
23 which was fully deprotected to 1g with H2SO4 in
dioxane (90 °C). With the same strategy the coupling
of 22 to 3â-aminocholic acid benzyl ester 24 gave
derivative 25 which was hydrogenolysed, coupled to
taurine, and finally deprotected to ligand 1h (Scheme
5).
If the ligand is not stable to prolonged heating,
procedure B, which employs GdCl3 as a source of Gd(III)
ions, must be adopted. In this case a solution of GdCl3
is added dropwise to a solution of the ligand, while the
pH is constantly maintained at 7 by addition of a
suitable base, usually NaOH. A desalting step is then
necessary in order to eliminate the produced “byproduct”
salt (e.g. NaCl).
The ligand 1i was obtained by coupling the DOTA
tris-tert-butyl ester 2730 (Scheme 6) to the cholyltaurine
derivative 15e in the presence of benzotriazol-1-yloxy-
tris(dimethylamino)phosphonium hexafluorophosphate
(BOP) and deprotection of the tert-butyl esters in a
mixture of water, acetic acid, and 2-propanol at reflux.
The ligand 1j was the last second generation product
and its synthesis is shown in Scheme 7. Cholic acid tert-
butyl ester31 was reacted with an excess of epichloro-
hydrin under phase transfer conditions to give 29 as
the main product. Reaction of 1,4,7,10-tetraazacyclo-
dodecane-1,4,7-triaacetic acid tris(1,1-dimethylethyl)
Toler a bility. Animal tolerability tests were per-
formed in mice by the intravenous route, aimed at
assessing at least the low tolerability limit. When
available, survival at the dose of pharmacokinetic assay
in rats was used as toxicity estimate. Further investiga-
tions also assessed the toxicity by the intracerebro-
ventricular route on the most promising candidates, as
this test was recognized to be predictive for chemo-
toxicity of iodinated contrast agents.33 To allow for a
comparison, LD50 values for two of the extracellular
ionic gadolinium complexes which are used clinically,
ester (DO3A Bu ester)32 with the epoxy derivative 29
in refluxing ethanol gave compound 30 which was
hydrolyzed in 0.5 M sulfuric acid at 90 °C to the ligand
1j.
t
Sch em e 5a
a
Reagents and conditions: (a) 22, DEPC, Et3N, DMF; (b) aq H2SO4, dioxane, ∆; (c) i: H2, Pd/C, EtOH; ii: column chromatography
with CH2Cl2/MeOH/Et3N; (d) i: NH2CH2CH2SO3H, DEPC, Et3N, DMF; ii: aq H2SO4, dioxane, ∆.

Hepatocyte-Directed MRI Contrast Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3633
Sch em e 6a
Sch em e 8a
a
Reagents and conditions: (a) i: 15e, BOP, DIEA, DMF; ii:
a
Reagents and conditions: (a) i: N6-Z-Lys, IBCF, Et3N, THF;
H2O/AcOH/i-PrOH, ∆.
ii: PTSA, MeOH; (b) H2, Pd/C, MeOH‚HCl; (c) i: 10, BOP, DIEA,
DMF; ii: NaOH, H2O-MeOH; (d) i: 21, DMF-H2O; ii: 2 M
NaOH.
Sch em e 7a
Ch a r t 6
Ta ble 1. Tolerability of Complexes 2a -l
compound
iv LD50 (mmol/kg)a
ic LD50 (mmol/kg)a
2a
2b
2c
2d
2e
2f
2g
2h
2i
<2.0b
0.0512
<0.25b
n.p.c
>7.0d
0.30 (0.22-1.13)
>3.0d
n.p.c
9.5 (8.2-20)d
>0.25b
0.21 (0.16-0.27)
n.p.c
7.6 (5.9-16.4)d
>0.25b
n.p.c
a
n.p.c
Reagents and conditions: (a) epichlorohydrin, Bu4NHSO4, 50%
NaOH-CH2Cl2; (b) DO3A Bu ester,32 EtOH, ∆; (c) aq H2SO4, ∆.
t
>2.0d
<0.00125
2j
2k
2l
1.19 (0.93-1.45)d
>0.25b
0.003-0.006
namely Gd-DTPA-dimeg and Gd-DOTA-meg, are also
reported in Table 1.
n.p.c
>0.25b
n.p.c
First generation conjugates, i.e. 2a ,b, proved too toxic
to deserve further investigation. Differently, several
second generation conjugates showed more than prom-
ising preliminary results. Noticeably, all terms of the
homogeneous series 2c-e, i.e. featuring the same che-
late subunit coupled to cholic acid, cholylglycine, and
cholyltaurine, respectively, show iv LD50 > 3.0 mmol
kg-1. This seems to indicate that this class of conjugates
are characterized by good tolerability independently of
the nature of the bile acid. Furthermore, conjugates 2c
and 2e show a more than acceptable degree of neuro-
toxicity. Also, conjugate 2g, which differs from 2c
because all five carboxylate groups of DTPA are involved
in the complexation of the gadolinium ion, featured high
tolerability (i.e. iv LD50 7.6 mmol kg-1). The tolerability
data gathered for third generation conjugates, i.e., 2k ,l,
are only preliminary and do not allow us to draw any
definitive conclusion. Owing to their safety levels,
compounds 2c and 2g were selected for further safety
assessment and underwent cardiovascular safety and
mutagenicity studies.
Gd-DTPA-dimeg
Gd-DOTA-meg
6e
-
-
10.6f
a
LD50 ) median lethal dose; iv ) intravenous, ic ) intracere-
b
broventricular administration (95% CL). In rats, values express
mortality at the dose of pharmacokinetic assay (see Experimental
Section). c Not performed. In mice. e From ref 34. f From ref 35.
d
Neither 2c nor 2g showed significant activity on
cardiovascular system in anaesthetized rabbits. The
cardiovascular changes induced by these compounds
were short lasting: all the examined parameters recov-
ered the baseline values in 10-15 min after the injec-
tion. Moreover, the effects observed with both com-
pounds were not qualitatively different from those
observed after injection of saline and mannitol solutions
used as control articles.
When 2c and 2g underwent the Ames test, although
toxicity was observed with 2c at just 5000 µg/plate, no
evidence of mutagenic activity was seen for both com-
pounds in all tested strains, either in the presence or
in the absence of metabolic activation. The tolerability

3634 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Ta ble 2. Pharmacokinetic Evaluation of Complexes 1a -l.
Anelli et al.
bearing -3 global charge (2g vs 2h ). The third genera-
tion conjugates (2k and 2l) showed biliary elimination
of 34% and 7.5%, respectively. This could likely reflect
the importance of the benzyloxymethyl moiety in facili-
tating the biliary elimination of these conjugates.
Compound 2j, which contains a subunit of cholic acid
linked to a neutral macrocyclic chelating mojety and
features a global charge of -1, showed an high biliary
excretion. Moreover, for the compound 2j, the hepato-
biliary elimination was inhibited by coadministration
of sodium cholyltaurine (see Experimental Section) from
39% in control animals to 23% in animals with sodium
cholyltaurine coadministration (0-30 min). For other
conjugates (e.g. 2d , 2h ) no significant inhibition was
noticed. This seems to indicate that the latter conju-
gates do not use the Na+/taurocholate carrier to enter
hepatocytes, whereas that carrier is, at least in part,
involved in the uptake of 2j. This also suggests that the
stereochemical configuration has to be considered in the
design of contrast agents which may enter hepatocytes
using the Na+/taurocholate transporter.
Elimination in Rats, after 8 h (dose level 0.25 mmol kg-1
)
conjugate
biliary (%)
renal (%)
2a
2ba
2c
2d
2e
2f
2g
2h
2i
25.4 ( 3.6
71-77 (n ) 2)
45.6 ( 2.5
20.3 ( 2.9
19.9 ( 7.9
52 ( 11
76.1 ( 3.6
0-2.8 (n ) 2)
50 ( 13
71.32 ( 0.42
71 ( 18
36.6 ( 4.2
56.9 ( 1.0
89 ( 14
46.4 ( 2.7
12 ( 6.8
18.4 ( 2.0
63.5 ( 5.0
34.0 ( 3.5
7.5 ( 3.8
66 ( 10
2j
2k
2l
18.0 ( 6.7
57.4 ( 3.1
84 ( 12
a
Too toxic to be evaluated after administration at a dose level
of 0.25 mmol kg-1, tested at 0.1 mmol kg-1
.
data so far collected indicate that 2c and 2g are safe
enough candidates for possible development.
P h a r m a cok in etic Eva lu a tion . Conjugates 2a -l
underwent pharmacokinetic evaluation, and results, in
terms of biliary vs renal elimination, are reported in
Table 2 and deserve some comments. Among the first
generation conjugates, 2a showed a moderate biliary
elimination, whereas 2b, although more toxic than 2a ,
showed an higher biliary elimination. This difference
in the elimination pattern could be related to the global
charge of the conjugates (i.e. -2 for 2a and -1 for 2b)
and/or to the presence of the benzyloxymethyl residue
in 2b. More interesting are the results found for the
second generation conjugates. A comparison among
structurally analogous conjugates 2c-e indicates that
cholic acid (compound 2c) directs transport of gado-
linium conjugates from blood into bile two times more
effectively than cholylglycine and cholyltaurine. This
evidence is somehow unexpected. Indeed, after intra-
portal administration in rats of bile acids, it was
observed that cholyltaurine is the bile acid most ef-
ficiently transported into bile followed by cholylglicine
and cholic acid.36 These results were further confirmed
in rat liver perfusion experiments. Attempts to explain
this trend in terms of different extent of binding to
serum albumin failed since each of the three bile acids
have a similar fraction bound to albumin as determined
by equilibrium dialysis.37
Our observations with 2c-e can be explained on the
basis of two hypotheses: (i) 2d and 2e, possibly due to
their lower binding to rat albumin, are excreted more
quickly through the kidneys than 2c; (ii) conjugation
to gadolinium complexes of cholyltaurine and cholyl-
glycine influences their nature to such an extent that
the corresponding conjugates are less efficiently recog-
nized by the systems responsible for transport through
hepatocytes than the conjugate containing cholic acid.
The introduction of a spacer between cholic acid and
the chelating mojety, see 2f in comparison with 2c, did
not exert any change in the biliary elimination. Cholyl-
taurine conjugates 2e and 2i, which contain a subunit
of Gd-DTPA-monoamide and Gd-DOTA-monoamide,
respectively, show very similar elimination patterns.
This indicates that for second generation conjugates, a
variation of the global charge from -2 to -1 is without
major effects on the elimination route. The cholic mojety
is again more effective toward the biliary elimination
than the cholyltaurine moiety, also among conjugates
Con clu sion s
It has been shown that bile acids can be used as
carrier units for the preparation of MRI contrast agents
which are eliminated through the biliary route.38 The
extent to which this is achieved depends on several
structural features such as (i) nature of the bile acid,
(ii) site of conjugation of the Gd(III) complex to the bile
acid moiety, (iii) global charge of the conjugate. For
example cholic acid appears to be a much more effective
carrier than cholylglycine and cholyltaurine. Several
second generation conjugates (i.e. conjugates in which
the Gd(III) complex is linked to the position 3 of the
steroid moiety of the bile acid) show high biliary
elimination as well as good tolerabilities. Noticeably, for
such conjugates, the 3R stereochemical configuration on
the steroid moiety, such as in the case of conjugate 2j,
seems to play a peculiar role in addressing the Na+/
taurocholate transport system.
Exp er im en ta l Section
Ch em istr y. Gen er a l Meth od s. Melting points were de-
termined with a Bu¨chi 540 apparatus and are uncorrected.
TLC analyses were carried out using Merck KGaA silica gel
plates 60 F254. Detection: AcOH/concentrated H2SO4/p-anis-
aldeide, 100:2:1; 1% KMnO4 in 1 M NaOH. Flash chromatog-
raphy was performed using Merck KGaA silica gel 60 (230-
400 mesh ASTM). H and 13C NMR spectra were recorded on
1
a Bruker AC 200 spectrometer, equipped with a 5 mm dual
probe, using deuterium as lock signal in CDCl3 as the solvent
when not noted. The H NMR spectral data are not reported,
being useless for the assignment of the structure due to the
broadness and extreme overlapping of the signals. Elemental
analyses were performed by Redox laboratories, Monza, Italy.
Mass spectra were recorded on a TSQ 700 (Finnigan MAT)
spectrometer equipped with an ESI source.
1
Analytical HPLC was performed on a Merck KGaA ap-
paratus: Hitachi high-pressure gradient pump system (L6200
and L6000), Hitachi AS 2000 autosampler, T 6300 column
thermostat, Hitachi L 4250 UV detector (210 nm). Flow rate
of 1 mL min-1. Analytical HPLC methods: (A) stationary
phase: Lichrosorb Select B 5 µm, 250 × 4 mm column packed
by Merck KGaA; mobile phase: eluent A ) 0.01 M KH2PO4
and 0.017 M H3PO4 in H2O, eluent B ) MeCN, gradient
elution: t ) 0 min (5% B), t ) 30 min (80% B), t ) 45 min
(80% B); (B) stationary phase: Kromasil C4 5 µm, 250 × 4

Hepatocyte-Directed MRI Contrast Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3635
mm column packed by Bischoff; mobile phase: eluent A )
0.017 M H3PO4 in H2O, eluent B ) 2-propanol, gradient
elution: t ) 0 min (20% B), t ) 30 min (35% B), t ) 40 min
(35% B); (C) stationary phase: Inertsil C8 5 µm, 250 × 4 mm
column packed by GL Sciences; mobile phase: eluent A ) 0.01
M KH2PO4 and 0.017 M H3PO4 in H2O, eluent B ) MeCN,
gradient elution: t ) 0 min (5% B), t ) 30 min (80% B), t ) 45
min (80% B); (D) stationary phase: Lichrospher 100 RP-8 5
µm, 250 × 4 mm column packed by Merck KGaA; mobile
phase: isocratic elution with premixed mobile phase prepared
as follows: 1 g of n-octylamine is added to a solution of MeCN
and H2O (35:65; 1 L), and the resulting solution was buffered
to pH 6 with H3PO4; (E) stationary phase: Lichrospher 100
RP-18 5 µm, 250 × 4 mm column packed by Merck KGaA;
mobile phase: isocratic elution with premixed mobile phase,
same as method D. Retention times are in minutes.
Preparative HPLC was performed on a Merck KGaA Prep-
bar 100 apparatus. Preparative HPLC methods: (A) stationary
phase: Lichroprep RP-8; 45-63 µm; 250 × 50 mm column
packed by Merck KGaA; mobile phase: eluent A ) 0.01 M
KH2PO4 adjusted to pH 6.2 with 80:20 NaOH/MeCN, eluent
B ) 0.01 M KH2PO4 adjusted to pH 6.2 with 50:50 NaOH/
MeCN, gradient elution from 100/0 to 0/100; flow rate: 70 mL
min-1; detection (UV): 210 nm, 280 nm; injection: 100 mL;
sample concentration: 10 mg mL-1; (B) stationary phase:
Lichroprep RP-8; 25-40 µm; 250 × 50 mm column; mobile
phase: eluent A ) H2O, eluent B ) MeCN, gradient elution
from 95/5 to 0/100; flow rate: 70 mL min-1; detection (UV):
210 nm, 280 nm; injection: 200 mL; sample concentration: 15
mg mL-1. Nanofiltration was performed on a Unit P123C
(Celfa, Schwyz, Switzerland) instrument using DRA 4020
(Daicel, J apan) membranes.
was slowly added into a solution of 4 (11.0 g, 28.8 mmol) and
N,N-diisopropylethylamine (37.5 g, 290 mmol, 49.3 mL) in 1,2-
dichloroethane (50 mL) at 0-5 °C under nitrogen. The reaction
mixture was stirred at room temperature for 16 h, cooled to 0
°C, and filtered. After evaporation of the solvent, the oily
residue was taken up with EtOAc (100 mL) and washed with
brine and H2O. The organic layer was separated, dried
(Na2SO4), and evaporated. The oily residue was purified by
flash chromatography (eluent: n-hexane/EtOAc 7:3) to afford
5 as a pale yellow oil: yield 14.8 g, 61%; TLC Rf 0.5 (n-hexane/
EtOAc 7:3); 13C NMR δ 27.9, 28.0, 52.1, 52.7, 53.4, 54.3, 55.9,
54.6, 70.8, 71.7, 80.1, 80.3, 80.4, 80.9, 123.2, 127.3, 145.9, 147.0,
170.4, 170.7, 171.2; HPLC (Method A) retention time ) 33.5,
96% (area %).
5-[[(4-Am in op h en yl)m eth oxy]m eth yl]-6,9,12-tr is[2-(1,1-
d im et h ylet h oxy)-2-oxoet h yl]-2,2-d im et h yl-4-oxo-3-oxa -
6,9,12-tr ia za tetr a d eca n -14-oic Acid 1,1-Dim eth yleth yl
Ester (6). To a solution of 5 (13.7 g, 16.3 mmol) in EtOH (200
mL) at room temperature was added 10% Pd/C (1.37 g), and
the resulting suspension was stirred under hydrogen atmo-
sphere for 1 h. The reaction mixture was filtered, the solvent
evaporated, and the oily residue purified by flash chromatog-
raphy (eluent: n-hexane/Et2O/i-PrOH 70:25:5) to give 6 as a
pale yellow oil: yield 10.7 g, 81%; TLC Rf 0.15 (n-hexane/Et2O/
i-PrOH 70:25:5); 13C NMR δ 28.0, 28.1, 52.1, 52.4, 52.7, 53.2,
54.3, 55.8, 64.6, 70.4, 73.2, 80.0, 80.4, 80.6, 80.7, 114.6, 128.2,
129.1, 147.7, 170.5, 170.7, 170.9, 171.4; HPLC (Method A)
retention time ) 29.2, 99% (area %).
5-[[[4-[[[[(1,1-Dim eth yleth oxy)ca r bon yl]a m in o]a cetyl]-
am in o]ph en yl]m eth oxy]m eth yl]-6,9,12-tr is[2-(1,1-dim eth -
yleth oxy)-2-oxoeth yl]-2,2-d im eth yl-4-oxo-3-oxa -6,9,12-tr i-
a za t et r a d eca n -14-oic Acid 1,1-Dim et h ylet h yl E st er (7)
(CAUTION: see General Methods). Diethyl cyanophosphonate
(DEPC) (2.19 g, 13.4 mmol, 2.03 mL) was dropped over 15 min
into a stirred solution of 6 (4.95 g, 6.12 mmol), N-(t-butoxy-
carbonyl)glycine (2.14 g, 12.2 mmol), and Et3N (1.36 g, 13.4
mmol, 1.86 mL) in DMF (50 mL) at 0 °C under nitrogen. After
16 h, the reaction mixture was concentrated to one-fourth of
its initial volume, diluted with EtOAc (100 mL), and washed
with brine and H2O. The organic phase was separated, dried
(Na2SO4), and evaporated to an oily residue that was purified
twice by flash chromatography (eluent: n-hexane/Et2O/i-PrOH
50:45:5; second column eluent: n-hexane/Et2O/Et3N 30:70:2)
to yield 7 as a pale yellow oil: yield 3.02 g, 51%; TLC Rf 0.15
(n-hexane/Et2O/i-PrOH 70:25:5); 13C NMR δ 28.0, 28.2, 45.0,
52.2, 52.3, 52.7, 53.3, 53.5, 54.2, 56.0, 64.8, 70.2, 72.7, 80.0,
80.1, 80.4, 80.6, 80.7, 119.6, 129.0, 133.9, 137.0, 156.3, 167.9,
170.5, 170.7, 170.8, 171.4; MS m/z 967 (M + H)+; HPLC
(Method A) retention time ) 31.9, 98% (area %).
1-[4-[[Am in oa cet yl]a m in o]p h en yl]-4-ca r b oxy-5,8,11-
t r is(ca r b oxym et h yl)-2-oxa -5,8,11-t r ia za t r id eca n -13-oic
Acid (8). Trifluoroacetic acid (246 g, 2.16 mol, 167 mL) was
dropped over 2 h into a solution of 7 (35.1 g, 36.3 mmol) and
anisole (270 mL) in CH2Cl2 (270 mL) at 0 °C. The reaction
mixture was stirred at room temperature for 3 days. After
removal of the solvent, the residue was taken up with CH2Cl2
(350 mL) and the solution evaporated. This operation was
repeated twice. The residue was suspended in H2O (400 mL)
and the mixture neutralized with 25% NH4OH (19 mL) to give
a clear solution that was washed with Et2O and evaporated.
The oily residue was purified by flash chromatography (elu-
ent: EtOH/25% NH4OH 8:2) to give 8 as a white solid: yield
13.0 g (the compound is partially salified as the ammonium
salt); TLC Rf 0.19 (EtOH/25% NH4OH 8:2); 13C NMR (D2O) δ
43.7, 58.7, 59.8, 61.8, 63.5, 70.1, 75.0, 71.0, 123.5, 131.8, 136.8,
139.0, 166.0, 180.5, 182.3; MS m/z 586 (M + H)+; HPLC
(Method A) retention time ) 2.42, 98.9% (area %).
CAUTION: For the reactions in which diethyl cyanophos-
phonate (DEPC) was used, during the aqueous workup atten-
tion must be given to HCN evolution. All operations must be
carried out under a well ventilated fume cupboard, and the
aqueous washings must be treated with aqueous sodium
hypochlorite before disposal.
2-Bromo-3-[(4-nitrophenyl)methoxy]propionic acid 1,1-di-
methylethyl ester (3),21 cholic acid N-hydroxysuccinimidyl ester
(9),23 4-carboxy-5,8,11-tris(2-methoxy-2-oxoethyl)-1-phenyl-2-
oxa-5,8,11-triazatridecan-13-oic acid methyl ester (10),24 N-(2-
a m in oet h yl)(3R,5â,7R,12R)-3,7,12-t r ih ydr oxych ola n -24-
amide (11),25 (3â,5â,7R,12R)-3-amino-7,12-dihydroxycholan-24-
oic acid methyl ester (13),26 N,N-[2-(2,6-dioxo-4-morpholinyl)-
ethyl]glycine (21),28 N,N-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxo-
ethyl]amino]ethyl]-L-glutamic acid 1-(1,1-dimethylethyl) ester
(22),29 (3â,5â,7R,12R)-3-amino-7,12-dihydroxycholan-24-oic acid
phenylmethyl ester (24),26 1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid tris(1,1-dimethylethyl) ester (27),30
(3R,5â,7R,12R)-3,7,12-trihydroxycholan-24-oic acid 1,1-di-
methylethyl ester (28),31 and 1,4,7,10-tetraazacyclododecane-
t
1,4,7-triaacetic acid tris(1,1-dimethylethyl) ester (DO3A Bu
ester)32 were synthesized following the procedure reported in
the literature. Other reagents were commercially available and
used as purchased.
N-[2-[(2-Am in oeth yl)a m in o]eth yl]-O-[(4-n itr op h en yl)-
m eth yl]-D,L-ser in e 1,1-Dim eth yleth yl Ester (4). A solution
of 321 (14 g; 38.9 mmol) in MeCN (30 mL) was slowly added to
a solution of diethylenetriamine (20 g, 194 mmol, 20.1 mL) in
MeCN (20 mL) at 0-5 °C under nitrogen. The reaction mixture
was warmed to 35 °C and stirred for 4 h. After evaporation of
the solvent, brine (100 mL) was added to the oily residue and
the mixture was extracted with Et2O. The organic layer was
separated, washed with H2O, dried (Na2SO4), and evaporated
to give 4 as a pale yellow oil: yield 12 g, 81%; TLC Rf 0.5
(CHCl3/MeOH/25% NH4OH, 10:2:0.5); 13C NMR δ 27.7, 41.6,
47.6, 49.0, 51.8, 61.6, 71.6, 71.8, 80.9, 123.1, 127.2, 145.6, 146.9,
171.9; HPLC (Method A) retention time ) 12.2, 95.8% (area
%).
4-Ca r b oxy-5,8,11-t r is(ca r b oxym et h yl)-1-[4-[[[[(3r,5â,
7r,12r)-3,7,12-t r ih yd r oxy-24-oxoch ola n -24-yl]a m in o]-
a cetyl]a m in o]p h en yl]-2-oxa -5,8,11-tr ia za tr id eca n -13-oic
Acid (1a ). To a solution of 8 (13.0 g) in DMF/H2O (8:5, 104
mL) at room temperature was added portionwise 923 (21.9 g,
43.0 mmol). The reaction mixture was stirred at room tem-
perature for 30 h. Evaporation of the solvents gave a solid
6,9,12-Tr is[2-(1,1-d im et h ylet h oxy)-2-oxoet h yl]-2,2-d i-
m eth yl-5-[[(4-n itr op h en yl)m eth oxy]m eth yl]-4-oxo-3-oxa -
6,9,12-tr ia za tetr a d eca n -14-oic Acid 1,1-Dim eth yleth yl
Ester (5). tert-Butyl bromoacetate (28.3 g, 145 mmol, 21.4 mL)

3636 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Anelli et al.
residue which was purified by flash chromatography (eluent:
CH2Cl2/MeOH/25% NH4OH 6:3:0.7). The purified ligand was
dissolved in 1 M HCl (270 mL) and the solution loaded onto
an Amberlite XAD-16 resin column and eluted with MeOH/
H2O gradient to afford 1a as a white solid: yield 10.1 g, 28%
(from 7); mp 154-156 °C; TLC Rf 0.21 (CH2Cl2/MeOH/25%
NH4OH 6:3:0.7); 13C NMR (D2O) δ 15.0, 19.6, 25.0, 25.9, 29.0,
30.4, 30.6, 31.9, 34.2, 34.8, 36.3, 38.0, 37.1, 41.1, 42.3, 44.1,
45.8, 49.8, 51.5, 52.2, 54.2, 55.6, 57.8, 58.6, 59.8, 68.6, 75.3,
69.7, 70.7, 74.0, 75.3, 123.6, 131.9, 138.4, 139.6, 172.3, 172.9,
173.3, 173.6, 180.0, 180.6; MS m/z 976 (M + H)+; HPLC
(Method A) retention time ) 20.3, 98.0% (area %). Anal.
(C48H73N5O16) C, H, N.
3,6,9-Tr is(2-m et h oxy-2-oxoet h yl)-11-oxo-10-[(p h en yl-
m et h oxy)m et h yl]-14-[[(3r,5â,7r,12r)-3,7,12-t r ih yd r oxy-
24-oxoch ola n -24-yl]a m in o]-3,6,9,12-tetr a a za tetr a d eca n -
oic Acid Meth yl Ester (12) (CAUTION: see General Meth-
ods). This compound was synthesized according to the proce-
dure described for 7, using 1024 (39.2 g, 68.8 mmol), 1125 (33.1
g, 73.4 mmol), and DEPC (12.4 g, 76.0 mmol, 11.5 mL) in DMF
(400 mL) and Et3N (7.69 g, 76.0 mmol, 10.5 mL). The solid
residue was purified by flash chromatography (eluent: CH2Cl2/
MeOH/25% NH4OH 9:1:0.1) to give 12 as a pale yellow solid:
yield 25.2 g, 36%; TLC Rf 0.34 (CH2Cl2/MeOH/25% NH4OH
9:1:0.1); 13C NMR δ 12.3, 17.3, 22.3, 23.1, 26.1, 27.4, 28.1, 30.3,
31.5, 33.2, 34.6, 35.4, 38.8, 39.4, 39.9, 41.4, 46.2, 46.6, 50.3,
51.2, 51.3, 51.5, 51.8, 52.2, 52.4, 52.7, 54.3, 54.7, 64.6, 68.1,
68.2, 71.6, 72.8, 73.2, 127.4, 127.7, 128.2, 137.8, 171.4, 171.7,
172.5, 173.3, 174.1; MS m/z 570 (M + H)+; HPLC (Method A)
retention time ) 20.4, 91% (area %).
3,6,9-Tr is(car boxym eth yl)-11-oxo-10-[(ph en ylm eth oxy)-
m eth yl]-14-[[(3r,5â,7r,12r)-3,7,12-tr ih ydr oxy-24-oxoch olan -
24-yl]a m in o]-3,6,9,12-tetr a a za tetr a d eca n oic Acid (1b). To
a solution of 12 (11.5 g, 11.5 mmol) in MeOH/H2O (1:1; 300
mL) at room temperature was added dropwise 2 M NaOH (5.0
mL) until pH 12 was reached. The reaction mixture was stirred
at room temperature for 48 h, maintaining pH 12 by the
addition of 1 M NaOH (35 mL) through a pH-stat apparatus.
The solution was adjusted to pH 7 with 2 M HCl and
evaporated. The residue was dissolved with H2O/MeOH (7:3,
500 mL) and acidified with 6 M HCl (15 mL), and the solution
was loaded onto an Amberlite XAD-16 resin column and eluted
with MeOH/H2O gradient to afford a solid which was further
purified by reversed-phase preparative HPLC (Method A) to
give 1b as a white solid: yield 2.13 g, 18%; 13C NMR (D2O) δ
15.0, 19.5, 25.1, 25.8, 29.9, 30.1, 30.5, 32.0, 34.2, 35.0; 37.1,
38.0, 41.2, 41.5, 42.2, 43.9, 49.5, 49.7, 52.6, 54.2, 55.2, 57.3,
58.1, 60.5, 69.6, 70.6, 74.0, 75.3, 75.7, 130.6, 131.2, 140.4, 174.1,
175.4, 176.9, 179.5, 182.2; MS m/z 969 (M + Na)+; HPLC
(Method A) 96% (area %).
in MeOH/H2O (10:1, 11 mL) was added 10% Pd/C (0.2 g). The
resulting suspension was refluxed for 10 min. The reaction
mixture was allowed to cool to room temperature, filtered, and
evaporated. The residue was taken up with Et2O to give the
precipitation of a solid which was filtered to afford 15c as a
white solid: yield 0.67 g, 87%; mp 187-189 °C; TLC Rf 0.46
(CH2Cl2/MeOH/Et3N 5:5:0.1); 13C NMR δ 12.3, 17.1, 22.9, 23.2,
24.7, 25.7, 27.4, 28.4, 30.8, 31.0, 31.1, 33.6, 34.4, 35.1, 35.2,
37.3, 39.2, 41.5, 44.6, 44.7, 46.3, 47.0, 51.3, 68.0, 72.9, 76.4,
77.1, 77.7, 171.8, 174.7; MS m/z 479 (M + H)+; HPLC (Method
A) retention time ) 17, 96% (area %). Anal. (C27H46N2O5) H,
N; C: calcd, 67.75; found, 67.14.
(3â,5â,7r,12r)-3-[[13-Car boxy-6,9,12-tr is(car boxym eth yl)-
1,4-dioxo-3,6,9,12-tetr aazatr idecyl]am in o]-7,12-dih ydr oxy-
ch ola n -24-oic Acid (1c). A solution of H2O (6.1 mL, 0.34 mol)
and DMF (250 mL) was added dropwise over 75 min into a
suspension of 2128 (92.5 g, 0.26 mol) in DMF (1.2 L) at 80 °C.
After 2 h, a solution of 15c (25 g; 52.2 mmol) in DMF (400
mL) was added dropwise over 30 min. The reaction mixture
was cooled to 20 °C, and 2 M NaOH (650 mL) was added
dropwise, to afford the precipitation of a sticky solid. After 16
h at room temperature, the pH of the mixture was adjusted
to 7 with 12 M HCl (35 mL), and the solution was decanted
and evaporated to give a crude product which was taken up
with H2O/MeCN (85:15, 1.4 L) and 6 M HCl (100 mL). The
resulting suspension was filtered through paper and the
solution was loaded onto an Amberlite XAD-16 resin column
and eluted with MeCN/H2O gradient to give 1c as a white
solid: yield 26.0 g, 59%; mp 280 °C (dec); TLC Rf 0.13 (CH2Cl2/
MeOH/25% NH4OH 6:3:1); 13C NMR (D2O + KOD) δ 15.0, 19.6,
25.3, 25.7, 26.6, 28.7, 30.0, 30.7, 33.2, 35.0, 35.4, 36.2, 37.1,
37.4, 38.3, 39.3, 41.7, 44.3, 45.1, 48.6, 49.1, 53.3, 53.6, 54.4,
54.7, 58.0, 60.7, 60.9, 61.3, 71.1, 75.8, 173.0, 177.1, 177.5, 179.8,
181.5, 186.9; MS m/z 840 (M + H)+; HPLC (Method A)
retention time ) 17.6, 98.9% (area %). Anal. (C40H65N5O14) C,
H, N.
(3â,5â,7r,12r)-7,12-Dih yd r oxy-3-[[[[(p h en ylm et h oxy)-
ca r bon yl]a m in o]a cetyl] a m in o]ch ola n -24-oic Acid (16).
To a stirred solution of 14 (15.3 g, 25.0 mmol) in MeOH/H2O
(3:1, 240 mL) at room temperature was added 1 M NaOH (35
mL) dropwise over 1 h. After 36 h, the pH of the reaction
mixture was lowered to neutrality with 2 M HCl (3.5 mL) to
afford the precipitation of 16 as a white solid that was filtered
and dried: yield 13.0 g, 87%; mp 228-233 °C; TLC Rf 0.41
(CH2Cl2/i-PrOH/AcOH 85:15:2); 13C NMR (DMSO-d6) δ 12.3,
17.0, 22.5, 24.5, 25.6, 27.3, 28.7, 30.5, 30.7, 33.4, 34.3, 34.7,
35.1, 36.5, 39.5, 41.4, 43.4, 44.8, 45.8, 46.1, 65.4, 66.3, 71.1,
127.8, 129.0, 129.6, 136.2; MS m/z 599 (M + H)+; HPLC
(Method A) retention time ) 24.4, 96% (area %). Anal.
(C34H50N2O7) C, H, N.
(3â,5â,7r,12r)-7,12-Dih yd r oxy-3-[[[[(p h en ylm et h oxy)-
ca r bon yl]a m in o]a cetyl]a m in o]ch ola n -24-oic Acid Meth -
yl Ester (14). Isobutyl chloroformate (8.00 g, 58.5 mmol, 7.57
mL) was dropped over 10 min into a solution of Z-glycine (12.2
g, 58.5 mmol) and N-methylmorpholine (6.00 g, 59.3 mmol) in
THF (400 mL) at -4 °C under nitrogen. After 15 min, a
solution of 1326 (21.8 g, 51.7 mmol) in THF (100 mL) was
dropped over 1.5 h into the reaction mixture that was
maintained at -4 °C for additional 30 min and then allowed
to rise to room temperature and stirred overnight. After
evaporation of the solvent, the residue was taken up with Et2O
(500 mL) and washed with H2O. The organic layer was
separated, dried (Na2SO4), and evaporated, and the crude was
purified by flash chromatography (eluent: CH2Cl2/i-PrOH 9:1)
to afford 14 as a white solid: yield 27.9 g, 88%; mp 87-89 °C;
TLC Rf 0.20 (CH2Cl2/i-PrOH 9:1); 13C NMR δ 12.4, 17.2, 22.9,
23.2, 24.4, 25.7, 27.4, 28.4, 30.8, 31.0, 33.3, 34.4, 35.1, 35.2,
37.1, 39.2, 41.6, 44.8, 45.6, 46.4, 47.0, 51.4, 66.7, 68.1, 73.0,
127.8, 128.0, 129.4, 136.2, 156.8, 168.5, 174.8; MS m/z 613 (M
+ H)+; HPLC (Method A) retention time ) 28.5, 95.5% (area
%). Anal. (C35H52N2O7) C, H, N.
N-[(3â,5â,7r,12r)-7,12-Dih ydr oxy-3-[[[[(ph en ylm eth oxy)-
ca r bon yl]a m in o] a cetyl]a m in o]-24-oxoch ola n -24-yl]gly-
cin e Meth yl Ester (17). Isobutyl chloroformate (2.73 g, 20
mmol, 2.59 mL) was dropped over 20 min into a solution of 16
(10 g, 16.7 mmol) and Et3N (2.02 g, 20 mmol, 2.78 mL) in DMF
(150 mL) at - 5 °C under nitrogen. After 15 min, a suspension
of glycine methyl ester hydrochloride (2.31 g, 18.4 mmol) and
Et N (1.86 g, 18.4 mmol, 2.55 mL) in DMF (15 mL) was added
3
over 1.5 h into the reaction mixture that was maintained at
-4 °C for 30 min and then was allowed to rise to room
temperature and stirred overnight. The suspension was fil-
tered, and the solution was evaporated. The residue was taken
up with CH Cl 2 (100 mL) and washed with H O. The organic
2
2
layer was separated, dried (Na 2SO4), and evaporated to give
a crude product which was purified by flash chromatography
(eluent: EtOAc/MeOH 9:1) to afford 17 as a white solid: yield
6.56 g, 59%; mp 123-128 °C; TLC Rf 0.33 (EtOAc/MeOH 9:1);
13C NMR δ 12.5, 14.8, 17.6, 22.5, 22.7, 23.3, 24.4, 25.4, 27.5,
28.2, 30.9, 31.5, 32.7, 33.2, 34.5, 35.1, 37.0, 39.2, 41.0, 41.6,
44.7, 45.6, 48.3, 48.6, 52.0, 53.3, 60.2, 63.6, 66.8, 68.1, 72.9,
76.4, 77.0, 77.2, 77.7, 127.7, 128.0, 129.6, 136.1, 156.9, 168.8,
170.6, 171.8, 174.2; MS m/z 670 (M + H)+; HPLC (Method B)
retention time ) 28, 96% (area %). Anal. (C37H55N3O8) C, H,
N.
(3â,5â,7r,12r)-3-[(Am in oa cetyl)a m in o]-7,12-d ih yd r oxy-
ch ola n -24-oic Acid Meth yl Ester (15c). To a solution of 14
(1.0 g, 1.6 mmol) and ammonium formate (0.47 g; 7.4 mmol)

Hepatocyte-Directed MRI Contrast Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3637
N-[(3â,5â,7r,12r)-3-[(Am in oa cet yl)a m in o]-7,12-d ih y-
d r oxy-24-oxoch ola n -24-yl]glycin e Meth yl Ester (15d ). To
a solution of 17 (21.8 g, 32.5 mmol) in MeOH (100 mL) at room
temperature was added 5% Pd/C (2.2 g), and the resulting
suspension was stirred under hydrogen atmosphere for 1.5 h.
The reaction mixture was filtered, the solvent evaporated, and
the crude product purified by flash chromatography (eluent:
EtOAc/MeOH 1:1) to afford 15d as a white solid: yield 15.8 g,
91%; mp 168-172 °C; TLC Rf 0.27 (EtOAc/MeOH 1:1); 13C
NMR (CD3OD) δ 13.4, 18.1, 23.8, 24.5, 25.9, 27.5, 28.9, 30.0,
32.2, 33.2, 34.0, 34.8, 35.7, 36.5, 37.0, 38.7, 41.1, 42.1, 43.1,
45.4, 47.0, 47.7, 48.1, 48.2, 48.5, 48.9, 49.4, 49.9, 50.2, 50.8,
52.9, 69.2, 74.2, 172.1, 174.5, 177.3; MS m/z 536 (M + H)+;
HPLC (Method B) retention time ) 3.9, 96% (area %). Anal.
(C29H49N3O6) C, H, N.
43.2, 45.0, 47.2, 47.3, 47.7, 48.0, 48.2, 48.4, 48.8, 49.2, 49.7,
50.1, 50.5, 69.3, 74.2, 81.0, 159.5, 171.7, 178.3; MS m/z 565
(M + H)+; HPLC (Method A) retention time ) 24.1, 96% (area
%). Anal. (C31H52N2O7) C, N: H: calcd, 9.28; found, 9.98.
2-[[(3â,5â,7r,12r)-3-[(Am in oa cet yl)a m in o]-7,12-d ih y-
d r oxy-24-oxoch ola n -24-yl]a m in o]et h a n esu lfon ic Acid
(15e). Taurine (5.38 g, 43.1 mmol) and Et3N (5.16 g, 51.0
mmol, 7.07 mL) were added to a solution of 19 (22.1 g, 39.1
mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline
(EEDQ) (12.6 g, 51.0 mmol) in DMF (100 mL) under nitrogen.
The resulting suspension was heated to 90 °C for 70 min,
obtaining a clear solution which was then cooled to 25 °C. After
30 min, the reaction mixture was slowly poured into cold Et2O
(0 °C, 900 mL) to give the precipitation of a resinous product.
The suspension was kept at 4 °C overnight. The mixture was
decanted, and the resinous residue was dried under vacuum.
The resulting white solid was added to a stirred solution of
HCl in MeOH (0.85 M, 190 mL) at room temperature. The
initially milky reaction mixture slowly became thicker and
after 24 h was filtered. The solid was washed thoroughly with
Et2O/MeOH (1:1) and dried to give 15e as a white solid: yield
14.3 g, 64%; mp 200 °C (dec); TLC Rf 0.67 (MeOH/AcOH 95:
5); 13C NMR (D2O + KOD) δ 15.5, 19.7, 25.3, 25.9, 26.8, 28.7,
30.2, 31.0, 33.5, 34.3, 35.1, 35.5, 36.5, 37.5, 38.0, 38.4, 39.3,
42.2, 44.2, 46.7, 48.6, 48.8, 48.9, 52.6, 71.0, 75.7; MS m/z 572
(M + H)+; HPLC (Method A) retention time ) 14.3, 94% (area
%). Anal. (C28H49N3O7S) H, N, S; C: calcd, 52.17; found, 51.70.
(3â,5â,7r,12r)-3-[[N-[N-[N-[2-[2-[Bis(ca r b oxym et h yl)-
a m in o]eth yl](ca r boxym eth yl)a m in o]eth yl]-N-(ca r boxy-
m eth yl)glycyl]glycyl]a m in o]-7,12-d ih yd r oxy-N-(ca r boxy-
m eth yl)ch ola n -24-a m id e (1d ). A solution of H2O (5.8 mL,
322 mmol) and DMF (60 mL) was added dropwise over 75 min
into a suspension of 2128 (68.6 g, 0.19 mol) in DMF (1 L) at 80
°C. After 2 h, a solution of 15d (26.8 g, 50 mmol) in DMF (100
mL) was added dropwise over 30 min. The reaction mixture
was cooled to 20 °C, and 2 M NaOH (480 mL) was added
dropwise, to afford precipitation of a sticky solid. After 16 h,
at room temperature the pH of the mixture was adjusted to 7
with 12 M HCl (31 mL), and the solution was decanted and
evaporated to give a crude product which was taken up with
H2O/MeCN (9:1, 100 mL) and 2 M HCl (10 mL) The resulting
solution was loaded onto an Amberlite XAD-2 resin column
and eluted with MeCN/H2O gradient to give 1d as a white
solid: yield 21.9 g, 49%; mp 210 °C (dec); 13C NMR (D2O +
KOD) δ 15.8, 19.5, 25.3, 25.8, 26.8, 29.7, 30.1, 30.8, 33.3, 34.3,
34.9, 35.3, 36.3, 37.4, 39.0, 39.3, 42.0, 44.3, 45.0, 46.1. 48.7,
48.8, 53.9, 54.2, 54.4, 54.5, 59.7, 60.9, 61.1, 61.5, 70.9, 75.6,
175.2, 177.5, 179.3, 179.5, 180.8, 181.6; MS m/z 897 (M + H)+;
HPLC (Method A) retention time ) 15.1, 100% (area %). Anal.
(C42H68N6O15) C, H, N.
(3â,5â,7r,12r)-3-[[[[(1,1-Dim eth yleth oxy)ca r bon yl]a m i-
n o]acetyl]am in o]-7,12-dih ydr oxych olan -24-oic Acid Meth -
yl Ester (18). Isobutyl chloroformate (11.5 g, 84.0 mmol, 10.9
mL) was dropped over 15 min into a solution of N-(tert-
butoxycarbonyl)glycine (14.7 g, 84.0 mmol) and Et3N (8.5 g,
84.0 mmol, 11.6 mL) in THF (400 mL) at -5 °C under nitrogen.
After 15 min, a solution of 1326 (29.5 g, 70.0 mmol) in THF
(100 mL) was added dropwise over 1 h into the reaction
mixture that was maintained at -4 °C for 30 min and then
was allowed to rise to room temperature and stirred overnight.
The suspension was filtered, and the solution was evaporated.
The residue was taken up with Et2O and washed with
saturated aqueous NaHCO3 and with H2O. The organic layer
was separated, dried (Na2SO4), and evaporated to give a crude
which was purified by flash chromatography (eluent: EtOAc/
MeOH 1:1) to afford 18 as a white solid: yield 25.3 g, 62%;
mp 110-114 °C; TLC Rf 0.19 (EtOAc); 13C NMR (CD3OD) δ
13.1, 17.9, 23.6, 24.3, 25.8, 27.3, 29.0, 29.9, 32.1, 32.2, 32.4,
34.8, 35.7, 36.5, 36.9, 38.7, 41.1, 43.2, 45.0, 47.2, 47.3, 47.7,
48.0, 48.2, 48.4, 48.6, 49.2, 49.7, 50.1, 50.5, 52.3, 69.2, 74.5,
80.9, 159.6, 171.7, 171.8, 176.6; MS m/z 579 (M + H)+; HPLC
(Method A) retention time ) 27.7, 97% (area %). Anal.
(C32H54N2O7) H, N; C: calcd, 66.40; found, 65.47.
(3â,5â,7r,12r)-3-[[N-[N-[N-[2-[2-[b is(ca r b oxym et h yl)-
a m in o]eth yl] (ca r boxym eth yl)a m in o]eth yl]-N-(ca r boxy-
m eth yl)glycyl]glycyl]a m in o]-7,12-d ih yd r oxy-N-(2-su lfo-
eth yl)ch ola n -24-a m id e (1e). A solution of H2O (3.6 mL, 227
mmol) and DMF (150 mL) was added dropwise over 30 min
into a suspension of 2128 (54.3 g, 152 mmol) in DMF (700 mL)
at 80 °C. After 2 h, a solution of 15e (21.7 g, 37.9 mmol) and
Et3N (3.85 g, 38 mmol, 5.27 mL) in DMF (300 mL) was added
dropwise over 30 min. The reaction mixture was cooled to 20
°C, and 2 M NaOH (380 mL) was added dropwise, to afford
the precipitation of a sticky solid. After 16 h, at room
temperature the pH of the mixture was adjusted to 7 with 12
M HCl (25 mL), and the solution was decanted and evaporated
to give a crude product which was taken up with H2O (300
mL) and 12 M HCl (35 mL). The resulting solution was loaded
onto an Amberlite XAD-2 resin column and eluted with MeCN/
H2O gradient to give 1e as a white solid: yield 20.4 g, 57%;
mp 174-176 °C; 13C NMR (D2O + KOD) δ 15.1, 19.6, 25.4,
25.9, 26.7, 28.8, 32.2, 32.9, 33.4, 34.4, 35.1, 35.4, 36.6, 37.5,
38.0, 39.0, 39.3, 42.0, 44.3, 45.1, 49.9, 52.6, 53.2, 53.5, 54.4,
54.8, 58.6, 60.7, 61.0, 61.3, 70.9, 75.6, 173.0, 176.6, 177.3, 179.5,
179.6, 191.5; MS m/z 947 (M + H)+; HPLC (Method A)
retention time ) 16.6, 94% (area %); Anal. (C42H70N6O16S) C,
H, N, S.
(3â,5â,7r,12r)-7,12-Dih yd r oxy-3-[[1-oxo-6-[[(p h e n yl-
m eth oxy)car bon yl]am in o]h exyl]am in o]ch olan -24-oic Acid
Meth yl Ester (20). Isobutyl chloroformate (1.78 g, 13 mmol,
1.69 mL) was dropped over 5 min into a solution of 6-[[(phenyl-
methoxy)carbonyl]amino]hexanoic acid (3.45 g, 13.0 mmol) and
Et3N (1.31 g, 13.0 mmol, 1.8 mL) in THF (70 mL) at -5 °C
under nitrogen. After 15 min, a solution of 1326 (5 g, 11.9 mmol)
in THF (30 mL) was added dropwise over 1 h into the reaction
mixture that was maintained at -4 °C for 30 min and then
was allowed to rise to room temperature and stirred overnight.
The suspension was filtered, and the solution was evaporated.
The residue was taken up with CH2Cl2 and washed with
saturated aqueous NaHCO3 and with H2O. The organic layer
was separated, dried (Na2SO4), and evaporated. Crystallization
of the crude product from EtOAc gave 20 as a white solid: yield
5.60 g, 70%; mp 145-150 °C; TLC Rf 0.11 (EtOAc), Rf 0.41
(EtOAc/i-PrOH 95:5); 13C NMR δ 13.3, 17.9, 23.8, 24.5, 25.9,
27.1, 27.6, 28.9, 30.0, 30.9, 32.1, 32.2, 32.4, 34.9, 35.7, 36.5,
36.9, 37.1, 38.5, 41.1, 41.9, 43.1, 47.2, 47.7, 48.0, 48.2, 48.4,
48.9, 49.3, 49.7, 50.1, 50.6, 52.3, 67.5, 69.3, 74.2, 128.9, 129.2,
129.7, 139.7, 159.0, 175.7, 175.9, 176.6; MS m/z 669 (M + H)+;
HPLC (Method A) retention time ) 28.4, 98.5% (area %). Anal.
(C39H60N2O7) C, H, N.
(3â,5â,7r,12r)-3-[[[[(1,1-Dim eth yleth oxy)ca r bon yl]a m i-
n o]a cetyl]a m in o]-7,12-d ih yd r oxych ola n -24-oic Acid (19).
To a solution of 18 (24.5 g, 42.3 mmol) in MeOH/H2O (2:1, 160
mL) at room temperature was added 1 M NaOH (49.7 mL)
dropwise over 2 h. After 48 h, the reaction mixture was filtered
and evaporated. The glassy residue was taken up with EtOAc
(160 mL), and the solution was washed with 1 M HCl. After
separation, the aqueous phase was saturated with NaCl and
extracted with EtOAc. The organic layers were combined, dried
(Na2SO4), and evaporated to give 19 as a white solid: yield
22.2 g, 93%; mp 150-155 °C; TLC Rf 0.47 (EtOAc/i-PrOH/
AcOH 90:15:1); 13C NMR (CD3OD) δ 13.1, 17.3, 23.8, 24.4, 25.8,
27.5, 28.9, 29.9, 32.2, 32.5, 34.8, 35.5, 36.5, 37.0, 38.7, 41.1,

3638 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Anelli et al.
(3â,5â,7r,12r)-3-[(6-Am in o-1-oxoh exyl)a m in o]-7,12-d i-
h yd r oxych ola n -24-oic Acid Meth yl Ester (15f). To a solu-
tion of 20 (4.0 g; 5.99 mmol) in EtOH (50 mL) at room
temperature 5% Pd/C (0.8 g) was added, and the resulting
suspension was stirred under hydrogen atmosphere for 6 h.
The reaction mixture was filtered, and the solvent was
evaporated to afford 15f as a white solid: yield 2.70 g, 84%;
mp 70-75 °C; TLC Rf 0.33 (MeOH/Et3N 95:5), 13C NMR δ 12.4,
17.2, 23.0, 23.2, 24.5, 25.5, 25.8, 26.3, 27.4, 28.4, 30.8, 31.0,
31.2, 32.7, 33.4, 34.4, 35.1, 35.2, 36.7, 37.4, 39.3, 41.6, 45.2,
43.4, 47.1, 51.3, 67.9, 72.8, 76.4, 77.0, 77.2, 77.6, 172.4, 174.7;
MS m/z 535 (M + H)+; HPLC (Method A) retention time ) 18,
95% (area %). Anal. (C31H54N2O5) C,N; H: calcd, 10.18; found,
9.56.
18-[[(3â,5â,7r,12r)-23-Ca r boxy-7,12-d ih yd r oxy-24-n or -
ch olan -3-yl]am in o]-3,6,9-tr is(car boxym eth yl)-11,18-dioxo-
3,6,9,12-tetr a a za octa d eca n oic Acid (1f). A solution of H2O
(2.34 mL, 146 mmol) and DMF (190 mL) was added over 30
min into a suspension of 2128 (34.7 g, 97.2 mmol) in DMF (500
mL) at 80 °C. After 2 h, a solution of 15f (13 g, 24.3 mmol) in
DMF (195 mL) was added over 30 min. The reaction mixture
was cooled to 20 °C, and 2 M NaOH (245 mL) was dropped, to
afford the precipitation of a sticky solid. After 24 h at room
temperature, the pH of the mixture was adjusted to 7 with 12
M HCl (18 mL), and the solution was decanted and evaporated
to give a crude product which was taken up with H2O/MeCN
(9:1, 150 mL) and 12 M HCl (42 mL). The resulting solution
was loaded onto an Amberlite XAD-2 resin column and eluted
with MeCN/H2O gradient to give 1f as a white solid: yield 15
g, 69%; mp 172-174 °C; 13C NMR δ 15.0, 19.7, 25.5, 26.0, 26.5,
29.1, 29.5, 30.2, 31.1, 33.5, 35.0, 35.3, 36.4, 37.1, 37.5, 38.5,
39.4, 41.6, 42.2, 44.2, 48.9, 53.4, 53.5, 54.2, 54.4, 58.6, 60.5,
61.2, 61.6, 70.5, 75.5, 176.3, 177.5, 178.7, 180.0, 181.5, 186.6;
MS m/z 896 (M + H)+; HPLC (Method A) retention time ) 18,
93% (area %). Anal. (C45H75N5O14) C, H, N.
am in o]ch olan -3-yl]-L-glu tam in e (1h ). (CAUTION: see Gen-
eral Methods). Triethylamine (1.36 g; 13.4 mmol, 1.86 mL) was
added dropwise to a solution of 26 (12.5 g, 10.1 mmol), taurine
(1.5 g, 12 mmol), and DEPC (2.1 g, 12.8 mmol, 1.82 mL) in
DMF (50 mL) at 0 °C under nitrogen. After 3 h, the solvent
was evaporated, the residue was dissolved in dioxane (250 mL),
and 0.5 M aq H2SO4 (250 mL, 125 mmol) was added dropwise.
The resulting mixture was heated at 90 °C for 2 h. After the
solution was cooled to room temperature, the pH was adjusted
to neutrality with 2 N NaOH (30 mL) and the solvent
evaporated. The crude was purified by flash chromatography
(eluent CH2Cl2/MeOH/25% NH4OH, 5:4:1) and desalted by
elution through an Amberlite XAD-16.00 resin column with a
MeCN/H2O gradient and further purified by reverse-phase
preparative HPLC (Method B) to give 1h : yield 1.5 g, 14%;
mp >200 °C; TLC Rf 0.3 (CH2Cl2/MeOH/25% NH4OH 5:4:1);
13C NMR δ 15.0, 19.6, 25.2, 25.8, 26.7, 28.5, 28.8, 30.2, 30.9,
33.3, 34.3, 35.1, 35.9, 36.3, 37.4, 37.5, 37.9, 39.2, 41.9, 44.3,
48.6, 50.4, 52.6, 55.1, 60.6, 68.7, 70.9, 75.8, 177.6, 179.6, 180.4;
MS m/z 962 (M + H)+; HPLC (Method D) retention time )
5.61, 95% (area %). Anal. (C43H71N5O17S) C, H, N, S.
10-[2-[[2-[[(3â,5â,7r,12r)-7,12-Dih yd r oxy-24-oxo-24-[(2-
s u lfo e t h y l)a m i n o ]c h o la n -3-y l]a m i n o ]-2-o x o e t h y l]-
a m in o]-2-oxoeth yl]-1,4,7,10-tetr a a za cyclod od eca n e-1,4,7-
tr ia cetic Acid (1i). N,N-Diisopropylethylamine (7.5 g, 58
mmol, 9.9 mL) was added dropwise to a solution of 2730 (13.3
g, 20.3 mmol), 15e (12.1 g, 21.2 mmol), and benzotriazol-1-
yloxy-tris(dimethylamino)phosphonium hexafluorophosphate
(BOP) (9.4 g, 21.2 mmol) in DMF (250 mL) at room temper-
ature under nitrogen. After 3 h, the reaction mixture was
evaporated and the oily residue was treated twice with H2O
and filtered. The resulting solid was dissolved in H2O/AcOH/
n-PrOH (2:1:1, 200 mL) and refluxed for 8 h. The solvent was
evaporated, and the resulting viscous oil was dissolved in 0.1
M HCl (50 mL), loaded onto Amberlite XAD-16.00, and eluted
with a EtOH/H2O gradient to afford 1i as a white solid: yield
4.7 g, 24%; mp 200 °C (dec); TLC Rf 0.4 (CH2Cl2/MeOH/25%
NH4OH 6:3:1); 13C NMR (D2O) δ 15.1, 19.8, 25.7, 26.6, 28.5,
30.2, 30.9, 33.6, 34.2, 35.0, 35.4, 35.5, 37.5, 37.8, 38.0, 39.9,
42.1, 44.2, 45.5, 48.5, 48.6, 48.9, 52.6, 53.2, 59.7, 62.3, 70.9,
75.4, 175.4, 176.4, 179.4, 182.3, 182.4; MS m/z 958 (M + H)+;
HPLC (Method A) retention time ) 14.1, 98% (area %). Anal.
(C44H75N7O14S) H, N; C: calcd, 55.15; found, 55.60.
(3r,5â,7r,12r)-7,12-Dih yd r oxy-3-(oxyr a n ylm et h oxy)-
ch ola n -24-oic Acid 1,1-Dim eth yleth yl Ester (29). A solu-
tion of 2831 (45.5 g, 97.8 mmol) in CH2Cl2 (300 mL) was added
dropwise over 30 min to a vigorously stirred mixture of
1-chloro-2,3-epoxypropane (154 g, 1.66 mol, 130 mL) (CAU-
TION: cancer suspected agent), 50% aq NaOH (220 mL) and
Bu4NHSO4 (6.79 g, 20 mmol), keeping the temperature at 20
°C. After 24 h, the reaction mixture was diluted with H2O (100
mL), and the organic phase was separated, dried (Na2SO4),
and evaporated. The oily residue was purified by flash chro-
matography (eluent EtOAc/n-hexane, 1:1) to afford 29 as a
white solid: yield 23.6 g, 46%; mp 65-67 °C; TLC Rf 0.35
(EtOAc/n-hexane 6:4); 13C NMR δ 12.2, 17.2, 22.4, 23.1, 26.3,
26.8, 27.0, 27.3, 28.0, 28.1, 30.8, 32.4, 34.7, 34.9, 35.0, 36.0,
36.2, 39.4, 41.4, 41.6, 44.5, 46.4, 47.0, 51.1, 68.1, 68.3, 68.6,
72.6, 76.7, 77.0, 77.7, 79.7, 79.8, 173.6,; MS m/z 521(M + H)+;
Anal. (C31H52O6) H; C: calcd, 71.50; found, 70.66.
(3â(S),5â,7r,12r)-3-[4-Ca r b oxy-4-[b is[2-[b is(ca r b oxy-
m et h yl)a m in o]et h yl]a m in o]-1-oxob u t yl]a m in o]-7,12-d i-
h yd r oxych ola n -24-oic Acid (1 g). This product was synthe-
sized according to the procedure described in ref 29.
(3â(S),5â,7r,12r)-3-[[4-[Bis[2-[bis[2-(1,1-dim eth yleth oxy)-
2-oxoeth yl]a m in o]eth yl]a m in o]-5-(1,1-d im eth yleth oxy)-
1,5-dioxopen tyl]am in o]-7,12-dih ydr oxych olan -24-oic Acid
p h en ylm eth yl Ester (25). (CAUTION: see General Meth-
ods). This compound was synthesized according to the proce-
dure described for 7 using 2229 (37 g, 49.6 mmol), 2426 (27.4 g,
55 mmol), and DEPC (8.97 g, 55 mmol, 8.35 mL) in DMF (750
mL) and Et3N (5.3 g, 52.5 mmol, 7.3 mL). The solid residue
was purified by flash chromatography (eluent: n-hexane/
EtOAc 8:2) to give 25 as a white solid: yield 36 g, 59%; mp
54-56 °C; TLC Rf 0.3 (n-hexane/EtOAc 8:2); 13C NMR δ 12.3,
17.1, 22.8, 23.2, 24.6, 25.4, 25.7, 27.4, 28.0, 29.1, 30.9, 31.2,
32.8, 33.5, 34.6, 35.1, 37.0, 39.2, 41.5, 45.1, 46.4, 47.0, 49.4,
53.1, 55.7, 63.2, 65.9, 68.2, 72.9, 76.5, 77.1, 77.8, 80.6, 80.7,
170.5, 172.2, 172.3, 190.1; MS m/z 1226 (M + H)+; Anal.
(C68H112N4O15) C, H, N.
(3â(S),5â,7r,12r)-3-[[4-[Bis[2-[bis[2-(1,1-dim eth yleth oxy)-
2-oxoeth yl]a m in o]eth yl]a m in o]-5-(1,1-d im eth yleth oxy)-
1,5-dioxopen tyl]am in o]-7,12-dih ydr oxych olan -24-oic Acid
tr ieth yla m m on iu m sa lt (26). To a solution of 25 (36 g, 29.4
mmol) in EtOH (1.5 L) at room temperature was added 5%
Pd/C (3.6 g), and the resulting suspension was stirred under
hydrogen atmosphere for 3 h. The reaction mixture was
filtered, the solvent evaporated, and the oily residue purified
by flash chromatography (eluent CH2Cl2/MeOH/Et3N, 95:5:1)
to give 26 as a white solid: yield 22 g, 61%; mp 57-59 °C;
TLC Rf 0.33 (CH2Cl2/MeOH/Et3N 95:5:1); 13C NMR δ 9.0, 12.2,
17.2, 22.9, 23.2, 24.5, 25.4, 25.8, 27.5, 27.9, 28.0, 28.3, 31.0,
31.6, 32.8, 33.4, 34.5, 35.0, 35.6, 37.1, 39.2, 41.6, 44.8, 45.1,
46.3, 46.5, 48.7, 49.4, 49.9, 53.1, 55.7, 63.2, 67.6, 72.6, 76.5,
77.1, 77.8, 90.6, 90.7, 170.5, 172.2, 172.3, 190.1; MS m/z 1135
(M-Et3NH++H+)+; HPLC (Method C) retention time ) 28.8,
95% (area %). Anal. (C67H121N5O15S) C, H, N.
10-[3-[[(3r,5â,7r,12r)-24-(1,1-Dim eth yleth oxy)-7,12-d i-
h ydr oxy-24-oxoch olan -3-yl]oxy]-2-h ydr oxypr opyl]-1,4,7,10-
t et r a a za cyclod od eca n e-1,4,7-t r ia cet ic Acid Tr is(1,1-d i-
m eth yleth yl) Ester Ad d u ct w ith Na Cl (30). A solution of
29 (10.4 g, 20 mmol) in absol EtOH (60 mL) was added
dropwise over 20 min to a refluxing solution of DO3A tBu
ester32 (10.3 g, 20 mmol) in absol EtOH (100 mL). After 3 h of
reflux, the solvent was evaporated, and the residue was
dissolved in EtOAc (200 mL) and washed with brine. The
organic phase was separated, dried (Na2SO4), and evaporated.
The crude was purified by flash chromatography (eluent
CH2Cl2/MeOH 15:1) to give 30 as a white solid: yield 13 g,
60%; mp 117-119 °C; TLC Rf 0.36 (CH2Cl2/MeOH 15:1); 13C
NMR δ 12.3, 17.1, 22.4, 23.0, 26.4, 26.7, 27.1, 27.3, 27.7, 27.9,
N 2 ,N 2 -B is [2-[b is (c a r b o x y m e t h y l)a m in o ]e t h y l]-N -
[(3â,5â,7r,12r)-7,12-d ih yd r oxy-24-oxo-24-[(2-su lfoeth yl)-

Hepatocyte-Directed MRI Contrast Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3639
28.0, 28.1, 30.8, 32.4, 34.5, 34.8, 34.9, 35.8, 35.9, 39.3, 41.3,
41.6, 46.2, 46.9, 48.3, 49.2, 49.8, 50.4, 52.0, 52.3, 55.4, 55.8,
56.1, 66.6, 67.8, 70.1, 70.3, 72.7, 76.5, 77.1, 77.8, 79.6, 79.9,
81.8, 81.9, 82.1, 171.7, 172.2, 172.6, 173.5; MS m/z 1036 (M +
H)+; Anal. (C57H102ClN4NaO12) H, Cl, N, Na; C: calcd, 62.59;
found, 60.28.
5-10%) to obtain a yellow-brown solid that was dissolved in
MeOH/H2O (2:1, 100 mL), and the solution was basified to pH
12 with 1 M NaOH. The reaction mixture was stirred for 21 h
at room temperature, maintaining pH 12 by addition of 1 M
NaOH (35.5 mL) through a pH-stat apparatus. The resulting
solution was neutralized by addition of 1 M HCl and evapo-
rated. The yellow oily residue was dissolved in 1 M HCl/MeOH
(7:3, 200 mL), loaded onto an Amberlite XAD-16.00 resin
column, and eluted with a MeOH/H2O gradient. Ligand 1k
was recovered as a white solid: yield 4.5 g, 27%; mp 158-160
°C; TLC Rf 0.25 (CHCl3/MeOH/H2O/Et3N 80:30:5:5); 13C NMR
(CD3OD) δ 13.2, 19.4, 23.3, 24.4, 24.5, 29.2, 29.6, 29.8, 30.4,
30.6, 31.4, 33.5, 34.5, 36.0, 36.7, 37.2, 40.2, 40.3, 40.6, 41.2,
43.2, 43.4, 47.7, 47.9, 48.2, 48.6, 48.8, 49.2, 49.7, 50.1, 50.5,
53.1, 53.7, 55.6, 56.4, 58.5, 59.1, 59.7, 67.6, 69.0, 69.2, 69.5,
71.0, 73.0, 74.1, 74.5, 129.0, 129.1, 129.4, 129.7, 129.8, 139.6,
139.7, 173.3, 174.1, 174.7, 175.3, 176.1, 176.2, 177.5, 179.4,
179.5; MS m/z 1031 (M-H)- HPLC (Method A) retention time
) 19.8, 97% (area %). Anal. (C52H81N5O16) C,H,N.
10-[3-[[(3r,5â,7r,12r)-23-Ca r b oxy-7,12-d ih yd r oxy-24-
n or ch ola n -3-yl]oxy]-2-h yd r oxyp r op yl]-1,4,7,10-tetr a a za -
cyclod od eca n e-1,4,7-tr ia cetic Acid (1j). A suspension of 30
(35.1 g, 32.1 mmol) in H2O (130 mL) and 0.5 M H2SO4 (130
mL) was heated to 90 °C. After 2.5 h the homogeneous reaction
mixture was evaporated. The solid residue was purified by
flash chromatography (eluent CH2Cl2/MeOH/25% NH4OH, 6:3:
1). The product was dissolved in H2O (160 mL) and 6 N HCl
(40 mL), the solution was loaded onto an Amberlite XAD-16
resin column and eluted with a MeCN/H2O gradient. Ligand
1j was recovered as a white solid: yield 16 g, 61%, mp 220 °C
(dec); TLC Rf 0.5 (CH2Cl2/MeOH/25% NH4OH 6:3:1); 13C NMR
(D2O+KOD) δ 15.2, 19.7, 25.4, 25.9, 28.0, 30.3, 35.0, 36.1, 37.6,
39.6, 42.4, 44.1, 44.3, 48.5, 53.6, 60.8, 62.3, 69.7, 70.3, 74.1,
75.3, 83.2, 182.0, 182.4, 188.3; MS m/z 811 (M + H)+; HPLC
(Method E) retention time ) 11.2, 99% (area %). Anal.
(C41H70N4O12) C, H, N.
N6-[(P h en ylm eth oxy)car bon yl]-N2-[(3r,5â,7r,12r)-3,7,12-
tr ih ydr oxy-24-oxoch olan -24-yl]-L-lysin e Meth yl Ester (31).
Isobutyl chloroformate (6.56 g, 48 mmol, 6.22 mL) was dropped
over 10 min into a suspension of cholic acid (16.3 g, 39.9 mmol)
and Et3N (4.86 g, 48 mmol, 6.65 mL) in THF (350 mL) at 0 °C
under nitrogen. After 15 min, a solution of N6-(phenyl-
methoxy)carbonyl-L-lysine (11.2 g; 40 mmol) in 0.67 M NaOH
(60 mL) was added dropwise to the reaction mixture that was
then maintained at 0 °C for 1h and at room temperature for
5 h. After addition of 2 N HCl (30 mL) to the mixture, the
organic solvent was evaporated. The aqueous phase was
diluted with brine and extracted with EtOAc. The organic layer
was separated, dried (Na2SO4), and evaporated. The residue
was dissolved in MeOH (600 mL) and p-toluenesulfonic acid
monohydrate (1.56 g, 8.2 mmol) was added. After 20 h Et3N
(0.83 g, 8.2 mmol, 1.14 mL) was added, the solvent was
evaporated and the crude was purified by flash chromatogra-
phy (eluent: i-PrOH/EtOAc, 8-20%) to give 31 as a white
solid: yield 24.1 g, 88%; mp 80-83 °C; TLC Rf 0.22 (EtOAc/
i-PrOH 9:1); 13C NMR (CD3OD) δ 13.3, 18.0, 23.5, 24.3, 24.5,
25.5, 28.1, 29.0, 29.5, 30.6, 31.4, 32.3, 33.4, 33.9, 36.1, 36.7,
37.0, 40.7, 41.2, 41.7, 43.2, 43.4, 47.7, 48.0, 48.3, 48.4, 48.8,
49.3, 49.4, 49.7, 50.1, 50.5, 52.5, 53.5, 64.9, 67.5, 69.2, 73.2,
74.2, 128.9, 129.1, 129.7, 139.6, 159.1, 174.5, 177.1; MS m/z
707 (M + Na)+; HPLC (Method A) retention time ) 27.8, 99.5%
(area %). Anal. (C39H60N2O8) H,N; C: calcd, 68.39; found, 67.75.
N 6-[N -[2-[[2-[Bis(ca r b oxym e t h yl)a m in o]e t h yl](ca r -
boxym eth yl)a m in o]eth yl]-N-(ca r boxym eth yl)glycyl]-N2-
[(3r,5â,7r,12r)-3,7,12-t r ih yd r oxy-24-oxoch ola n -24-yl]-L-
lysin e (1l). A solution of H2O (3.76 mL, 240 mmol) and DMF
(155 mL) was added dropwise over 75 min into a suspension
of 2128 (57.2 g, 160 mmol) in DMF (700 mL) at 80 °C. After 3
h, a solution of 32 (23.5 g; 40 mmol) and Et3N (4.05 g, 40 mmol,
5.54 mL,) in DMF (330 mL) was added dropwise over 30 min.
The reaction mixture was cooled to 20 °C, and 2 M NaOH (400
mL) was added dropwise, to afford the precipitation of a sticky
solid. After 24 h at room temperature, the pH of the mixture
was adjusted to 7 with 12 M HCl (25 mL), and the solution
was decanted and evaporated to give a crude product which
was taken up with H2O/MeCN (85:15, 500 mL) and 6 M HCl
(25 mL). The resulting solution was loaded onto an Amberlite
XAD-2 resin column and eluted with MeCN/H2O gradient to
give 1l as a white solid: yield 20 g, 55%; 13C NMR δ 15.1, 19.5,
25.2, 25.5, 29.1, 30.1, 31.0, 32.0, 34.1, 34.6, 35.0, 36.6, 37.2,
39.2, 41.1, 41.6, 42.3, 44.1, 48.6, 48.9, 54.1, 54.2, 54.6, 57.6,
60.5, 61.2, 61.3, 61.8, 70.6, 74.1, 75.4, 176.7, 179.2, 180.9, 181.7,
181.9, 182.0; MS m/z 912 (M + H)+; HPLC (Method A)
retention time ) 15, 98.5% (area %). Anal. (C44H73N5O15) H,N;
C: calcd, 57.94; found, 58.52.
Gen er a l P r oced u r es for th e P r ep a r a tion of Gd (III)
Com p lexes 2a -l. P r oced u r e A. To a solution of the ligand
(10 mmol) and base [NaOH or 1-deoxy-1-(methylamino)-D-
glucitol (meglumine); 1 or 2 mol equiv according to the
stoichiometry of the complex] in H2O (300 mL) was added
Gd2O3 (5 mmol). The resulting suspension was heated at 50
°C for 5 h. The reaction mixture was filtered and the solvent
evaporated to give the complex as a solid. All yields obtained
with this procedure are at least 90%.
P r oced u r e B. The ligand (12 mmol) was suspended in H2O
(300 mL), and base (NaOH or meglumine; 3 mol equiv) was
added until complete solution. A solution of GdCl3‚H2O (12
mmol) in H2O (50 mL) was added dropwise to the reaction
mixture, maintaining pH 7 by addition of base (NaOH or
meglumine; 1 or 2 mol equiv according to the stoichiometry of
the complex). Desalting was performed by nanofiltration or
by preparative HPLC (Method B) or by elution through an
Amberlite XAD-16 resin column with a MeCN/H2O gradient.
Evaporation of the solvents gave the complex as a solid. All
yields obtained with this procedure are at least 70%.
N2-[(3r,5â,7r,12r)-3,7,12-Tr ih yd r oxy-24-oxoch ola n -24-
yl]-L-lysin e Meth yl Ester Mon oh yd r och lor id e (32) To a
solution of 31 (15 g, 21.9 mmol) in MeOH (150 mL) at room
temperature 5% Pd/C (1.5 g) was added, and the resulting
suspension was stirred under hydrogen atmosphere for 1.5 h.
After filtration, the solution was cooled to 0 °C and HCl in
MeOH (1.1 M, 20.5 mL) was added. Evaporation of the solvent
gave 32 as a white solid: yield 12.4 g, 96%, mp 108-110 °C;
TLC Rf 0.33 (MeOH/Et3N 95:5); 13C NMR δ 13.3, 18.1, 23.5,
24.2, 28.2, 28.3, 32.2, 33.4, 36.2, 37.2, 40.8, 41.3, 43.3, 43.4,
48.3, 49.1, 49.3, 49.5, 53.1, 53.8, 69.3, 73.1, 74.3, 174.3, 177.3;
MS m/z 551 (M + H)+; HPLC (Method A) retention time )
17.1, 92% (area %). Anal. (C31H55ClN2O6) C,H,N,Cl.
2a : procedure B with meglumine; mp 178-180 °C; MS m/z
564 (M - 2Meglu+)2-; Anal. (C62H104GdN7O26) C,H,Gd,N.
2b: procedure B with meglumine; mp > 250 °C; MS m/z
1099 (M - Meglu+)-; Anal. (C55H89GdN6O19) C,H,Gd,N.
2c: procedure A with NaOH; mp > 250 °C; MS m/z 496
(M - 2Na+)2-; Anal. (C40H60GdN5Na2O14) C,H,Gd,N,Na.
2d : procedure A with NaOH; mp > 250 °C; MS m/z 525
(M - 2Na+)2-; Anal. (C42H63GdN6Na2O15) C,H,Gd,N,Na.
2e: procedure A with NaOH; mp > 250 °C; MS m/z 549
(M - 2Na+)2-; Anal. (C42H65GdN6Na2O16S) C, H,Gd,N,Na,S.
2f: procedure B with NaOH; mp >250 °C; MS m/z 532
(M - 2Na+)2-; Anal. (C44H68GdN5Na2O14) C,H,Gd,N,Na.
N 6-[N -[2-[[2-[Bis(ca r b oxym e t h yl)a m in o]e t h yl](ca r -
boxym eth yl)a m in o]eth yl]-N-(ca r boxym eth yl)-O-(p h en yl-
m eth yl)-DL-ser yl]-N2-[(3r,5â,7r,12r)-3,7,12-tr ih yd r oxy-24-
oxoch ola n -24-yl]-L-lysin e (1k ). N,N-Diisopropylethylamine
(6.48 g, 50.1 mmol, 8.52 mL) was added dropwise to a solution
of 32 (9.35 g, 15.9 mmol), 10 (9.06 g, 15.9 mmol), and BOP
(6.3 g, 15.9 mmol) in DMF (140 mL) at room temperature
under nitrogen. After 6 h, the solvent was evaporated, and
the residue was dissolved in EtOAc and washed with saturated
aqueous NH4Cl then with H2O. The organic phase was
separated, dried (Na2SO4), and evaporated. The crude was
purified by flash chromatography (eluent: MeOH/CH2Cl2

3640 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Anelli et al.
2g: procedure A with NaOH; mp > 250 °C; MS m/z 335
(M - 3Na+)3-; Anal. (C41H60GdN4Na3O15) C,H,Gd,N,Na.
2h : procedure A with NaOH; mp > 250 °C; MS m/z 371
(M - 3Na+)3-; Anal. (C43H65GdN5Na3O17S) H,Gd,N,Na,S; C:
calcd, 43.78; found, 43.10.
2i: procedure A with NaOH; mp >250 °C; MS m/z 1111
(M - Na+)-; Anal. (C44H71GdN7NaO14S) H,Gd,N,Na,S; C:
calcd, 46.59; found, 47.09.
2j: procedure A with NaOH; mp > 250 °C; MS m/z 984
(M - Na+)-; Anal. (C41H66GdN4NaO12) H,Gd,N,Na; C: calcd,
49.88; found 50.34.
2k : procedure A with meglumine; mp 171-174 °C; MS m/z
592 (M - 2Meglu+)2-; Anal. (C66H112GdN7O26) H,Gd,N; C:
calcd, 50.27; found, 50.86.
rificed at the end of the experiment by injecting an overdose
of sodium pentobarbital.
Mu ta gen icity. In in vitro assessment of the mutagenic
potential, histidine-dependent auxotrophic mutants of Salmo-
nella typhimurium (strains TA1535, TA1537, TA98, and
TA100) and a tryptophan-dependent mutant of Escherichia coli
(strain CM891) were exposed to the test substance diluted in
water, which was also used as a negative control. Two
independent mutation tests were performed in the presence
and absence of liver preparations from Aroclor 1254-induced
rats (S9 mix). The first was a standard plate incorporation
assay, and the second involved a preincubation stage. Dose
levels of up to 5000 µg/plate were tested in the mutation tests.
This is the standard limit dose recommended in the regulatory
guidelines this assay follows. Other dose levels used were a
series of ca. half-log dilutions of the highest concentration.
Bilia r y a n d Ur in a r y Excr etion in Nor m a l Ra ts. Su r -
gica l P r oced u r es. Animals were anesthetized by intraperi-
2l: procedure B with NaOH; MS m/z 532 (M - 2Na+)2-
Anal. (C44H68GdN5Na2O15) C, H, N,Gd; C: calcd, 47.60; found,
48.25.
Toxicity. Crl-CD-1(ICR)BR mice of both sexes (five males
and five females per dose group) were used in the body weight
range 18-25 g at treatment. Animals were housed under
controlled environmental conditions according to EEC guide-
lines. For intravenous administration, aqueous solutions of the
compounds were injected via lateral tail vein at 1 mL/min
injection speed. For intracerebral administration, aqueous
solutions of the compounds were injected in the left cerebral
ventricle according to the method of Haley and Mc Cormick.40
Animals were treated by single administration at escalating
doses and mortality and onset of clinical signs were recorded
throughout the following 7- or 14-day observation period.
Terminal sacrifice was then performed, followed by gross
necropsy. The median lethal dose (LD50) and its 95% confidence
limits (95% CL) were calculated by probit analysis using
specialized software.
toneal injection of sodium pentobarbital at 30 mg kg-1
.
Laparatomy was performed, and the common bile duct and
the urinary bladder were cannulated with a PE 50 polyeth-
ylene catheter. The abdominal cavity was closed with sutures
and the animal placed on a surgical table warmed to 37°C to
keep the body temperature within physiological limits. The
right femoral vein was exposed for the contrast medium
injection.
Dosin g a n d Sa m p lin g P r oced u r es. The compounds (as
aqueous solutions: 0.1 M for 2a , b, d , i; 0.25 M for 2f-h , k ,
l; 0.5 M for 2c, e, j) were injected at a dose level of 0.25 mmol
kg-1 into a femoral vein at a rate of 6 mL min-1. Bile and urine
were collected for 30 min before test article administration and
every 30 min starting from 0 up to 480 min.
Assa y of Ga d olin iu m in Bile a n d Ur in e. Bile and urine
contents of the compounds were calculated in terms of gado-
linium concentration. Gadolinium was assayed in bile and
urine by X-ray fluorescence, XRF. Quantitation was performed
by interpolation from standard curves.
Sod iu m Ch olylta u r in e (CT) Com p etition . Surgical pro-
cedures were the same as for the biliary and urinary excretion
experiments. Moreover, jugular vein was isolated and cannu-
lated for CT solution infusion.
Dosin g a n d Sa m p lin g P r oced u r es. Bolus injection (7.5
µmol kg-1) of the CT solution (3% w/v in physiological saline)
was followed by infusion at a rate of 7.5 µmol kg-1 via jugular
vein (total time of infusion 120 min). After 30 min from the
start of the infusion, the compounds were injected (0.25 mmol
kg-1) into a femoral vein. Bile and urine were collected every
30 min starting from the end of test article administration up
to 90 min after injection. Assay of gadolinium in bile and urine
was performed as reported above.
Car diovascu lar Safety Assessm en t. Cardiovascular safety
assessment on conjugates 2c and 2g was performed in NZW
rabbits (n ) 3) in the body weight range 2 to 3 kg. Each rabbit
(fasted overnight) was anaesthetized with about 30 mg/kg iv
of sodium pentobarbital. The dose of anaesthetic was supple-
mented when appropriate to maintain a surgical plan of
anaesthesia. Animals were then placed on an operating table
for surgical preparation. Tracheostomy was then performed,
followed by intubation to allow artificial ventilation when the
chest was opened. Artificial respiration was achieved using a
respiratory pump. A 21G needle was inserted into the marginal
ear vein for compounds administration; afterward, a 2F Millar
transducer (Millar, Texas), with two probes connected to Gould
pressure amplifiers (Gould, Ohio), was introduced into the left
cardiac ventricle by the right carotid artery to measure blood
pressure in both ascending aorta and left ventricle. Sternotomy
was then performed. The pericardium was opened and seg-
ment of ascending aorta was dissected free from the other
tissues. Electromagnetic flowmeter was placed around the
aorta for aortic flow determination (flowmeter Biotronex
Laboratory Inc., Kensington). Another electromagnetic probe
was placed around the left carotid artery for determination of
carotid flow. Then, subcutaneouos needle electrodes were
inserted into the legs and connected to an ECG amplifier
(Gould) to record the ECG (normally II lead) and to monitor
the heart rate. Compounds 2c and 2g were administered as
0.25 M aqueous solutions at a dose of 1 mmol/kg (4 mL/kg)
with an injection rate of about 0.1 mL/s. The cardiovascular
parameters were then monitored for 30 min after the injection
and recorded at the following times: end administration, 1,
5, 10, 15, and 30 min after the administration. The following
parameters were examined: heart rate (HR) in beats/min,
mean arterial blood pressure (mABP) in mmHg, systolic left
ventricular pressure (sLVP) in mmHg, cardiac output (CO) in
mL/min, stroke volume (SV) in mL/beat, dP/dt in mmHg/s2,
systemic vascular resistance (SVR) in 103 dyne‚s cm-5. All the
cardiovascular parameters were recorded using a Gould
polygraph and stored magnetically on an on-line data acquisi-
tion system (Morra Ing., Cuneo, Italy). The same software
elaborated the data in order to give values relative to the
examined cardiovascular parameters. Each animal was sac-
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