An evaluation of a C-glucuronide as a liver targeting group: Conjugate of a glucocorticoid antagonist

Article abstract of DOI:10.1016/S0960-894X(03)00431-1

A β-C-glucuronide conjugate of the glucocorticoid receptor antagonist, Mifepristone 1, was prepared which maintained binding affinity, had modest in vitro activity, and was metabolically more stable than the parent. Pharmacokinetic studies suggest that the conjugate is recognized by the liver like O-glucuronides and may undergo a portion of the enterohepatic recirculation loop.

Full text of DOI:10.1016/S0960-894X(03)00431-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2307–2310
An Evaluation of a C-Glucuronide as a Liver Targeting Group:
Conjugate of a Glucocorticoid Antagonist
Bryan K. Sorensen,a,* J. T. Link,a Thomas von Geldern,a Maurice Emery,a
Jiahong Wang,a Bach Hickman,a Marlena Grynfarb,b Annika Goos-Nilssonb
and Sherry Carrolla
aMetabolic Disease Research, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA
bKaro Bio AB Novum SE-141 57 Huddinge, Sweden
Received 20 February 2003; revised 22 April 2003; accepted 23 April 2003
Abstract—A b-C-glucuronide conjugate ofthe glucocorticoid receptor antagonist, Mifepristone 1, was prepared which maintained
binding affinity, had modest in vitro activity, and was metabolically more stable than the parent. Pharmacokinetic studies suggest
that the conjugate is recognized by the liver like O-glucuronides and may undergo a portion ofthe enterohepatic recirculation loop.
# 2003 Elsevier Science Ltd. All rights reserved.
Cortisol is a hormone synthesized in the adrenal gland
which has anti-inflammatory effects and influences
hepatic glucose production (HGP).1 Other glucocorti-
coids are widely employed as anti-inflammatory agents
which unfortunately can aggravate established diabetes.
The underlying mechanism is glucocorticoid-induced
upregulation ofhepatic enzymes required for gluconeo-
genesis mediated by the glucocorticoid receptor (GR).2
The primary modulated enzymes are phosphoenolpyr-
uvate carboxykinase and glucose-6-phosphatase which
catalyze two key transformations required to convert
oxaloacetate into glucose.3 GR is a nuclear hormone
receptor that resides in the cytoplasm in an inactive
state until bound to a ligand.4 Upon binding, the ligand
bound GR complex translocates to the cell nucleus
where it can either initiate, or repress, specific gene
transcription. We sought to identify a GR antagonist
that could be used to prevent upregulation ofgluco-
neogenesis by cortisol as a treatment for diabetes.5
other steroid receptors, such as the progesterone and
mineralcorticoid receptor, and by systemic glucocorti-
coid antagonism. Systemic antagonism could potentially
lead to problems ofglucocorticoid insufficiency, such as
hypotension, inflammation, and symptoms ofAddi-
son’s disease.7 In addition, systemic blockade ofthe
glucocorticoid receptor by mifepristone 1 produces
compensatory responses mediated through the hypo-
thalamic-pituitary-adrenal axis.8 Activation ofthis axis
leads to secretion ofadrenocorticotrophic hormone,
which stimulates production ofendogenous glucocorti-
coid, potentially reducing the effectiveness ofa GR
antagonist (Fig. 1).
Selectively targeting a GR antagonist to the liver should
lower HGP while avoiding the effects resulting from
systemic exposure. To achieve liver targeting, we sought
to design compounds that undergo enterohepatic recir-
culation.9 Glucuronidation is one potential strategy to
induce a GR antagonst to undergo enterohepatic recir-
culation. After oral administration, absorption, and
passage into the liver via the portal vein an agent can
undergo glucuronidation. Glucuronidation is a con-
jugation reaction ofan activated ofrm ofglucuronic
The glucocorticoid receptor antagonist mifepristone
(RU-486) 1 reduces levels ofPEPCK and glucose-6-
phosphatase and causes a 50% reduction ofplasma
glucose levels in obese diabetic db/db mice.6 However,
the utility ofthis agent is limited by cross-reactivity with
acid 2 that takes place in the liver, and is an important
10
pathway ofdrug metabolism and clearance.
These
reactions, catalyzed by glucuronyl transferases, result in
the formation of structurally varied glucuronide con-
jugates 3. Polar, high molecular weight (>300),
*Corresponding author. Tel.: +1-847-9388-176; fax: +1-847-9381-
674; e-mail: bryan.k.sorensen@abbott.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00431-1

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B. K. Sorensen et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2307–2310
Figure 1. Steroidal glucocorticoid receptor antagonist, mifepristone 1,
glucuronic acid 2, b-glucuronide conjugate 3, and C-glucuronide GR
antagonist conjugate 4.
glucuronides often pass from the hepatocyte to the bili-
ary fluid en route to the intestine. Once in the intestine,
the conjugate may be reabsorbed, pass through the colon
due to low membrane permeability and be excreted, or,
as is the case with O-linked glucuronide conjugates, be
enzymatically hydrolyzed by b-glucuronidases. This
liberates the parent molecule for reabsorption. Through
enterohepatic recirculation a drug may have multiple
passes through the liver and have low systemic exposure.
Alternatively, low molecular weight (<300) glucuronides
are frequently exported from the hepatocyte into the
bloodstream where they are filtered by the kidney and
undergo urinary excretion.
13,14
Scheme 1. Formation of b-C-glucuronide precursors. (a) See refs
(6 steps-22%); (b) dimethyldioxirane, CH2Cl2, rt; (c) allyl tri-n-butyl-
stannane, n-Bu3SnOTf, CH2Cl2, À78 ꢀC, 57%; (d) TBSCl, imidazole,
DMF, 120 ꢀC, 54%; (e) 9-BBN, THF; NaBO4, H2O, rt, 89%; (f) TsCl,
pyridine, CHCl3, rt, 76%.
With these considerations in mind, we sought to deter-
mine whether glucuronic acid GR antagonist conjugates
4 are liver targeted and undergo enterohepatic cirucula-
tion. Curley and co-workers reported b-C-glucuronic
acid conjugates ofretinoids that are stable to acid and
b-glucuronidase hydrolysis.11 Furthermore, they show
in vitro and in vivo (oral administration) effects in can-
cer models.12 These results suggested C-glucuronic acid
conjugates as a starting point.
The synthesis ofa C-glucuronide is outlined in Scheme
1. We sought to build b-C-glucuronide precursors cap-
able ofcoupling to GR antagonists. The synthesis star-
ted with d-glucuronolactone 5, and through known
procedures, produced methyl (3,4-di-O-t-butyldi-
methylsilyl-d-glucuronate) glycal 6.13,14 Treatment of
glycal 6 with dimethyldioxirane provided epoxide 7
diastereoselectively.13 The crude epoxide underwent
Lewis acid mediated reaction with allyl tri-n-butyl-
stannane to provide b-C-glycoside 8.15 Protection ofthe
alcohol with a TBS group provided the fully protected
allyl b-C-glycoside 9. Hydroboration ofthe alkene using
9-BBN followed by oxidation with sodium borate yielded
alcohol 10 which was converted to the tosylate 11. Alkene
9 and tosylate 10 could be coupled to GR antagonists by
Suzuki and alkylation reactions, respectively.
Scheme 2. Formation of b-C-glucuronide mifepristone conjugate. (a)
(i-Pr)2NEt, NaI, CH3CN, 90 ꢀC, 61%; (b) TBAF, THF, H2O, rt, 20%.
tone 12 was alkylated with the C-glycoside tosylate 11
to produce the protected mifepristone C-glucuronide 13.
The protecting groups, including the methyl ester, were
removed using tetrabutylammonium fluoride (TBAF) to
provide the desired conjugate 14. The deprotection
conditions gave a clean conversion. However, separaton
ofthe polar conjugate 14 from the TBAF by reverse
phase HPLC was ofmodest efficiency (unoptimized). It
The formation of the C-glucuronide mifepristone con-
jugate is outlined in Scheme 2. N-desmethyl-mifepris-

B. K. Sorensen et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2307–2310
2309
was hoped conjugate 14 would maintain the potency of
mifepristone 1 since groups at this position project out-
16
side the ligand binding domain ofthe GR.
Table 1 compares the biological activity ofmifepristone
1 and conjugate 14. In a competitive binding assay using
the radiolabeled agonist dexamethasone and human
GR, conjugate 14 gave an IC50 of16 nM which was
weaker then mifepristone 1.17 The second assay in col-
umn 3 uses a glucocorticoid hormone reporter CHO cell
line genetically engineered to express the glucocorticoid
response element and a reporter gene encoding a secre-
ted form of alkaline phosphatase (ALP). Compounds
are evaluated for antagonism of dexamethasone induced
ALP expression. The conjugate 14 has diminished in
vitro potency. Low cell permeability may account for
the weaker activity. The assay in column 4 measures the
inhibition ofthe tyrosine aminotranesrfase (TAT)
activity induced by a dose ofthe GR agonist pre-
dnisolone by a GR antagonist in rat hepatocytes. Given
the difference in potency in the binding assay, the com-
pound shows excellent functional potency in hepato-
cytes, suggesting the possibility ofactive uptake by
hepatocytes.
Figure 2. Metabolism ofmifepristone 1 and the conjugate 14 both at
10 mM in rat liver microsomes (0.25 mg microsomal protein/mL) at
37 ꢀC.
One desirable property ofa liver targeting group is
protection from metabolism. Mifepristone 1 is known to
undergo N-demethylation, alkyne metabolism, and its
metabolites inhibit its own metabolism.18 Figure 2
shows the results from a metabolism study using rat
liver microsomes incubated with 1 and 14. The C-glu-
curonide conjugate exhibits greater stability and does
not undergo N-dealkylation, which is the major meta-
bolic pathway for mifepristone 1. Thus the C-glucuro-
nide group protects the GR antagonist 14 from
metabolism.
Figure 3. Pharmacokinetic profile ofthe conjugate 14 at 5 mg/kg iv
and oral.
rats were iv dosed (5 mg/kg) and, as before, plasma drug
levels were low (1–2 mg/mL). This time, there were low,
but measureable, liver levels (0.22 mg/g). Most impor-
tantly, no parent drug was found in the urine or kidney.
These results suggest active transport from the liver to
the bile, indicating the conjugate may be undergoing a
portion ofthe enterohepatic recirculation loop. A final
study incubating efflux pump substrates 17b-estradiol-
d-glucuronide, a cMOAT/MRP2 inhibitor, and ver-
apamil, a MRP2 inhibitor, with the conjugate in the rat
hepatocyte/TAT assay was conducted.19,20 The appar-
ent efficacy ofthe conjugate was increased by as much
as 10-fold using these efflux pump inhibitors, supporting
the hypothesis.
The oral and iv pharmacokenetic profile ofconjugate 14
at 5 mg/kg in Sprague–Dawley rats is shown in Figure
3. The conjugate has a high plasma clearance (5.3 L/h
kg) and a short halflief ( t1/2=1.8 h). Plasma con-
centrations at 7 h after iv dosing are low (0.003 mg/mL).
No parent drug was detected in the plasma after oral
dosing. No trace ofthe C-glucuronide 14 was detected
in the livers regardless ofthe mode ofdosing (7 h).
The oral results indicate the conjugate 14 is not absor-
bed. The iv results are consistent with several elimina-
tion possibilities. Once in the liver, the conjugate may
reenter the bloodstream where it is filtered by the kidney
and excreted in the urine. The conjugate may also be
actively transported from the liver to the biliary fluid.
From there, the conjugate could enter the intestine and
be eliminated in the feces. To check on excretion, three
b-C-glucuronides were successfully prepared and con-
jugated to the GR antagonist, mifepristone 1. The con-
jugate 14 maintained GR binding and had modest in
vitro effects. Furthermore, it was metabolically more
stable then the parent. Pharmacokinetic studies suggest
that the conjugate may be treated by the liver like
O-glucuronides and may undergo part ofthe enter-
ohepatic recirculation loop. Unfortunately, the conjugate
had low bioavailability. Decreasing the hydrophilicity
ofthis compound may increase membrane permeability
and absorption. Such modifications may realize the goal
ofthese studies: the discovery ofa liver targeting group.
Table 1. Comparison ofmiefpristone
binding and functional assays
1 and the conjugate 14 in
Compd
GR binding
IC50 (nM)
GR-ALP
(nM)
TATa inhibition
IC50 (mM)
1
14
1.1
16.7
5.0
550
0.41
2.8
aLiver enzyme upregulated by GR agonists.

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B. K. Sorensen et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2307–2310
Walker, J. R.; Alshafie, G.; Abou-Issa, H.; Curley, R. W.
Bioorg. Med. Chem. Lett. 2002, 12, 2447.
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