Synthesis and biological evaluation of novel steroidal pyrazoles as substrates for bile acid transporters

Article abstract of DOI:10.1016/j.bmcl.2004.10.027

A series of novel C2-C3 annulated steroidal pyrazoles was synthesized as substrates for bile acid transporters. The pyrazoles were conjugated with drugs and drug surrogates. Their in vitro transport activities were evaluated in human ileal bile acid transporter (hIBAT) and human liver bile acid transporter (hLBAT) systems. The results of synthetic and transporter assay studies are described. A series of novel steroidal pyrazoles was synthesized as substrates for bile acid transporters to explore their potential as drug carriers. The selected pyrazole fused bile acids were further conjugated with drugs and drug surrogates. Their in vitro transport activities were evaluated in human ileal bile acid transporter (hIBAT) and human liver bile acid transporter (hLBAT) expressing Chinese hamster ovary (CHO)-cells and Xenopus laevis oocytes. The results of synthetic efforts and transporter assays studies are described herein.

Full text of DOI:10.1016/j.bmcl.2004.10.027

Bioorganic & Medicinal Chemistry Letters 15 (2005) 85–87
Synthesis and biological evaluation of novel steroidal pyrazoles as
substrates for bile acid transporters
Laxminarayan Bhat,* Bernd Jandeleit, Tracy M. Dias, Tristen L. Moors and
Mark A. Gallop
XenoPort Inc., 3410 Central Expressway, Santa Clara, CA 95051, USA
Received 18 August 2004; revised 5 October 2004; accepted 9 October 2004
Abstract—A series of novel steroidal pyrazoles was synthesized as substrates for bile acid transporters to explore their potential as
drug carriers. The selected pyrazole fused bile acids were further conjugated with drugs and drug surrogates. Their in vitro transport
activities were evaluated in human ileal bile acid transporter (hIBAT) and human liver bile acid transporter (hLBAT) expressing
Chinese hamster ovary (CHO)-cells and Xenopus laevis oocytes. The results of synthetic efforts and transporter assays studies are
described herein.
Ó 2004 Elsevier Ltd. All rights reserved.
The Na+/taurocholate co-transporting polypeptide
(NTCP; SLC10A1), also termed the liver bile acid trans-
porter (LBAT), and the apical sodium-dependent bile
salt transporter (ASBT; SLC10A2), alternatively named
the ileal (or intestinal) bile acid transporter (IBAT), are
solute carrier transporters regulating the enterohepatic
circulation of bile salts.1 LBAT and IBAT are mem-
brane co-transporters that mediate sodium dependent,
electrogenic uptake of bile acids into hepatocytes and
ileal enterocytes, respectively. In recent years interest
in developing ligands for the human intestinal bile acid
transporter (hIBAT) has been motivated by two distinct
applications. First, inhibitors of hIBAT block the up-
take of endogenous bile acids from the intestinal lumen
resulting in increased fecal bile salt excretion and a com-
pensatory up-regulation of de novo bile acid synthesis
from cholesterol in the liver.2 Enhanced bile acid biosyn-
thesis via activation of cholesterol 7a-hydroxylase
(CYP7A1) results in net reduction in plasma cholesterol,
and hIBAT inhibitors are currently under clinical inves-
tigation as novel antihypercholesterolemic agents.3 Sec-
ond, the realization that the intestinal bile acid transport
system represents a high capacity solute uptake pathway
has prompted several groups to investigate chemical
conjugation of bile acid moieties to drugs and diagnostic
agents either via non-cleavable or enzymatically labile
(i.e., prodrug) linkages. While the use of non-labile bile
acid conjugates has been promoted as a method for
achieving liver-selective targeting of drugs,4 the latter
approach has been proposed as an attractive strategy
for improving systemic exposure to otherwise poorly
orally bioavailable drugs.5
As part of our interest in developing novel ligands for
the ileal and hepatic bile acid transporters, we have ex-
plored the synthesis of heterosteroids based on the chol-
anoic acid skeleton.6,7 In this report we describe the
synthesis of steroidal pyrazoles8 and their interaction
with bile acid transporters heterologously expressed in
mammalian cells and Xenopus oocytes.
The bile acid building blocks, 3-ketocholanoic acid
methyl esters 1a–c were prepared in 70–92% overall
yields following reported protocols.9 The 3-keto bile
acid esters 1a–c were subsequently condensed with ethyl
formate in the presence of sodium hydride in toluene to
afford the 2-hydroxymethylene substituted intermediates
2a–c in 81–94% yields. The bile acid derivatives 2a–c
were treated with hydrazines in ethanol at reflux temper-
ature to yield the corresponding pyrazole fused bile acid
methyl esters, which after saponification under standard
conditions, afforded the steroidal pyrazoles 3a–g in 63–
95% overall yields. The glycine conjugated steroidal pyr-
azoles 4a–g were prepared by coupling glycine tert-butyl
Keywords: Steroidal pyrazoles; Bile acids; Bile acid transporters; Oral
bioavailability.
*
Corresponding author at present address: ARYx Therapeutics, Inc.,
2255 Martin Avenue, Suite F, Santa Clara, CA 95050, USA. Tel.: +1
408 869 2761x226; fax: +1 408 869 2773; e-mail: lbhat@aryx.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.027

86
L. Bhat et al. / Bioorg. Med. Chem. Lett. 15 (2005) 85–87
O
O
R2
R2
OMe
H
O
OMe
i
HO
(81-94%)
R1
O
R1
2a-c
1a, R1 = R2 = H
b, R1 = OH; R2 = H
c, R1 = R2 = OH
(63-95%)
ii, iii
R2
O
O
R2
OH
N
OR
iv
H
O
R3
N
N
R3
N
N
R1
(71-97%)
R1
4a-g (R = tBu)
5a-g (R = H)
3a-g
v (87-98%)
Scheme 1. Reagents and conditions: (i) NaH/HCO2Et/cat. EtOH/toluene, rt, 12–24h; (ii) H2NNHR3/EtOH, reflux, 8–12h; (iii) 2N NaOH/THF, rt,
5–8h; (iv) EDAC/H–Gly–OButÆHCl/DIEA/DCM, rt, 12–18h; (v) 4M HCl in dioxane, rt, 5–8h.
ester with 3a–g under standard coupling conditions in
71–97% overall yields. Finally, the pyrazole annulated
glycocholanoic acids 5a–g were synthesized by treatment
4a–g with 4M hydrogen chloride (HCl) solution in diox-
ane in excellent yields (Scheme 1).
(Table 1). Their affinity for the bile acid transporters
was assessed from the inhibition of radio-labeled tauro-
cholate uptake in recombinant CHO-cells. The func-
tional transport activity was determined from an
electrophysiology assay using transfected Xenopus laevis
oocytes. The glycodeoxycholic acid (GDC) derivatives
5b, 5d, and 5f showed good binding affinity in both
hIBAT and hLBAT assays. In contrast, the glycolitho-
cholic acid derivatives 5a and 5c and the glycocholic
acid derivative 5g showed high binding affinity for
hLBAT but only weak affinity for hIBAT. The steroidal
pyrazoles 5a–g are weak to moderate substrates in
hIBAT expressing oocytes (inducing 7–44% of the max-
imal current stimulated by the control substrate GDC).
The Naproxen conjugated steroidal pyrazole derived
from glycolithocholic acid 6a showed lower binding
affinity for hLBAT than that derived from glycodeoxy-
cholic acid 6b but both had very weak affinity for
hIBAT. The drug conjugate 6b showed weak substrate
activity (12% Imax) whereas 6a did not show any specific
activity in the hIBAT expressing oocytes. The drug sur-
rogate conjugated pyrazole 7 showed good binding
affinity for hLBAT and reasonable affinity for hIBAT
in radio-labeled competition uptake assays. Interest-
To investigate the possibility of using the steroidal pyr-
azole template as a drug carrier various drugs and drug
surrogates were conjugated to functionalized substitu-
ents or linkers on the pyrazole moiety via an enzymati-
cally cleavable covalent bond. Selected drugs and/or
drug surrogate conjugates are described in Scheme 2.
Naproxen conjugated steroidal pyrazoles 6a–b were pre-
pared by coupling naproxen with 4e–f under standard
conditions using trichlorobenzoyl chloride (TCBC) as
coupling agent followed by treatment with 4M HCl in
dioxane in good yields. Similarly, the carbamate deriva-
tive 7 was synthesized in moderate yield by coupling
pyrazole derivative 4c with 3,4-dimethoxyquinoline
using 1,10-carbonyldiimidazole (CDI) as coupling agent.
The membrane transport activity of the steroidal pyraz-
oles and their drug conjugates10 was evaluated in both
human IBAT and LBAT in vitro transporter assays11
O
N
H
OH
O
O
N
i, ii
4e, f
4c
N
O
O
R
O
6a, R = H
b, R = OH
O
iii
N
O
O
H
OH
O
N
O
N
N
7
Scheme 2. Reagents and conditions: (i) (a) Naproxen/TCBC/pyridine/DCM, 0°C to rt, 5h; (b) 4e–f/pyridine/DCM, 0°C to rt, 8h; (ii) 4M HCl in
dioxane, rt, 12h; (iii) (a) 4c/CDI/pyridine/DCM, 0°C, 1h; (b) 3,4-dimethoxyquinoline/pyridine/DCM, 0°C to rt, 12h.

L. Bhat et al. / Bioorg. Med. Chem. Lett. 15 (2005) 85–87
87
Table 1. In vitro transport results of the steroidal pyrazoles 5a–g and their drug conjugates 6a–b, 7 in hIBAT and hLBAT expressing CHO-cells and
Xenopus laevis oocytes
Entry
Steroidal pyrazoles and their drug conjugates
Competition assaya
Oocyte assayb % max GDC
IC50 (lM)
Number
R1
R2
R3
hIBAT
LBAT
hIBAT
1
2
3
4
5
6
7
8
9
10
5a
5b
5c
5d
5e
5f
H
H
H
H
H
H
H
OH
H
H
H
H
60
1.6
1.6
0.3
0.7
0.2
0.3
0.3
8.6
63
26
21
17
7
OH
H
H
b-Hydroxyethyl
b-Hydroxyethyl
m-Hydroxybenzyl
m-Hydroxybenzyl
m-Hydroxybenzyl
—
>300
22
OH
H
0.9
1.5
44
7
OH
OH
H
5g
6a
6b
7
>300
>300
>300
32
22
0
OH
H
—
—
3.4
0.22
12
59
a IC50 data are generated from radio-labeled taurocholate competition assays in transporter expressing CHO cells at a highest test sample con-
centration of 300lM.
b % Max values are relative to glycodeoxycholic acid (GDC) in transporter expressing Xenopus laevis oocytes at a test sample concentration of
200lM.
4. For recent examples, see: von Geldern, T. W.; Tu, N.;
Kym, P. R.; Link, J. T.; Jae, H.-S.; Lai, C.; Apelqvist, T.;
Rhonnstad, P.; Hagberg, L.; Koehler, K.; Grynfarb, M.;
ingly, conjugate 7 was well transported by hIBAT (59%
of Imax), perhaps reflecting the influence of the b-
hydroxyethyl linker moiety in permitting productive
interactions with the transporter protein.
¨
Goos-Nilsson, A.; Sandberg, J.; Osterlund, M.; Barkhem,
T.; Ho¨glund, M.; Wang, J.; Fung, S.; Wilcox, D.; Nguyen,
˚
P.; Jakob, C.; Hutchins, C.; Fa¨rnegardh, M.; Kauppi, B.;
Ohman, L.; Jacobson, P. B. J Med. Chem. 2004, ASAP
Web Release Date: 13-Jul-2004; Anelli, P. L.; Lattuada,
L.; Lorusso, V.; Lux, G.; Morisetti, A.; Morosini, P.;
Serleti, M.; Uggeri, F. J. Med. Chem. 2004, 47, 3629–
3641.
In summary, we have synthesized novel steroidal pyraz-
oles as substrates for bile acid transporters (hIBAT and
hLBAT). The selected pyrazole fused bile acids were
further conjugated with drugs and drug surrogates.
The annulated pyrazoles and their drug conjugates
showed good affinity for hLBAT as compared to
hIBAT, and weak to moderate transport activity in
hIBAT expressing oocytes. The results of this investiga-
tion demonstrated that the bile acid analogs derived
from heteroannulation onto bile acid ring A at the
C2–C3 positions can be substrates for bile acid trans-
porters and be potentially used as shuttles for drug
targeting.
¨
5. Tolle-Sander, S.; Lentz, K. A.; Maeda, D. Y.; Coop, A.;
Polli, J. E. Mol. Pharm. 2004, 1, 40–48.
6. Bhat, L.; Gallop, M. A.; Jandeleit, B. PCT Intl. Patent
Appl. WO 03/020214 A2, 2003.
7. Other heterosteroid analogs of cholic acid are described in:
Venepalli, B. R. et al. J. Med. Chem. 1992, 35, 374–378;
Kovar, K. A.; Weidemann, M. Arch. Pharm. (Weinheim,
Ger.) 1984, 317, 977–983; Bellini, A. M.; Rocchi, R.;
Fornasini, G.; Benassi, C. A. Farmaco, Ed. Sci. 1970, 25,
226–233; Bellini, A. M.; Rocchi, R.; Fornasini, G.;
Benassi, C. A. Farmaco, Ed. Sci. 1970, 25, 125–132;;
Bellini, A. M.; Rocchi, R.; Benassi, C. A. Gazz. Chim. Ital.
1969, 99, 1243–1251.
Acknowledgements
8. Other steroidal pyrazoles are described in: Christiansen,
R. G.; Bell, M. R.; DꢀAmbra, T. E.; Mallamo, J. P.;
Herrmann, J. L.; Ackerman, J. H.; Opalka, C. J.; Kullnig,
R. K.; Winneker, R. C.; Snyder, B. W.; Batzold, F. H.;
Schane, H. P. J. Med. Chem. 1990, 33, 2094–2100.
9. Tserng, K.-Y. J. Lipid Res. 1978, 19, 501–504.
10. Detailed experimental procedures for the synthesis of
intermediates and the final steroidal pyrazoles, and the
corresponding drug conjugates are described in Ref. 6. All
compounds gave satisfactory NMR spectroscopic and
mass spectral data consistent with their structural
assignments.
The authors thank Quincey Wu and Hui Yan for excel-
lent analytical support.
References and notes
1. Hagenbuch, B.; Dawson, P. Pflugers Arch. 2004, 447, 566–
570.
2. Kurata, H.; Suzuki, S.; Ohhata, Y.; Ikeda, T.; Hasegawa,
T.; Kitayama, K.; Inaba, T.; Kono, K.; Kohama, T.
Bioorg. Med. Chem. Lett. 2004, 14, 1183–1186, and
references cited therein.
11. The experimental procedures for the in vitro compound
transport assays with hIBAT and hLBAT expressing cell
lines and oocytes are described in Ref. 6.
3. Booker, M. L. Curr. Opin. Invest. Drugs 2001, 2, 393–
395.