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Table 2
References and notes
Nuclear receptor selectivity of FXR agonist 1n
1. Forman, B. M.; Goode, E.; Chen, J.; Oro, A. E.; Bradley, D. J.; Perlmann, T.;
Noonan, D. J.; Burka, L. T.; McMorris, T.; Lamph, W. W.; Evans, R. M.;
Weinberger, C. Cell 1995, 81, 687.
2. Higashiyama, H.; Kinoshita, M.; Asano, S. Acta Histochem. 2008, 110, 86.
3. Sinal, C. J.; Tohkin, M.; Miyata, M.; Ward, J. M.; Lambert, G.; Gonzalez, F. J. Cell
2000, 102, 731.
4. Rizzo, G.; Renga, B.; Mencarelli, A.; Pellicciari, R.; Fiorucci, S. Curr. Drug Targets:
Immune, Endocr. Metab. Disord. 2005, 5, 289.
5. Zhang, Y.; Lee, F. Y.; Barrera, G.; Lee, H.; Vales, C.; Gonzalez, F. J.; Willson, T. M.;
Edwards, P. A. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 1006.
6. Liu, Y.; Binz, J.; Numerick, M. J.; Dennis, S.; Luo, G.; Desai, B.; MacKenzie, K. I.;
Mansfield, T. A.; Kliewer, S. A.; Goodwin, B.; Jones, S. A. J. Clin. Invest. 2003, 112,
1678.
7. Fiorucci, S.; Clerici, C.; Antonelli, E.; Orlandi, S.; Goodwin, B.; Sadeghpour, B. M.;
Sabatino, G.; Russo, G.; Castellani, D.; Willson, T. M.; Pruzanski, M.; Pellicciari,
R.; Morelli, A. J. Pharmacol. Exp. Ther. 2005, 313, 604.
NR
Assay typea
1n XC50 (nM)
1n %Maxb
LRH
LXR
LXRb
PXR
ROR
FRET
FRET
SPA
SPA
FRET
4200
1400
2500
960
46
33
101
100
—
a
a
>10,000
a
FRET, ligand seeking assay measuring ligand-mediated interaction of the NR
LBD with a coactivator peptide; SPA, scintillation proximity assay. The XC50 values
are the mean of at least two assays.
b
Maximum percent efficacy of the test compound relative to activation via a
standard.
8. Stedman, C.; Liddle, C.; Coulter, S.; Sonoda, J.; Alvarez, J. G.; Evans, R. M.;
Downes, M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 11323.
Table 3
Pharmacokinetics of FXR agonists in rat
9. Fiorucci, S.; Antonelli, E.; Rizzo, G.; Renga, B.; Mencarelli, A.; Riccardi, L.;
Orlandi, S.; Pellicciari, R.; Morelli, A. Gastroenterology 2004, 127, 1497.
10. Fiorucci, S.; Rizzo, G.; Antonelli, E.; Renga, B.; Mencarelli, A.; Riccardi, L.;
Morelli, A.; Pruzanski, M.; Pellicciari, R. J. Pharmacol. Exp. Ther. 2005, 315, 58.
11. Fiorucci, S.; Rizzo, G.; Antonelli, E.; Renga, B.; Mencarelli, A.; Riccardi, L.;
Orlandi, S.; Pruzanski, M.; Morelli, A.; Pellicciari, R. J. Pharmacol. Exp. Ther. 2005,
314, 584.
12. Maloney, P. R.; Parks, D. J.; Haffner, C. D.; Fivush, A. M.; Chandra, G.; Plunket, K.
D.; Creech, K. L.; Moore, L. B.; Wilson, J. G.; Lewis, M. C.; Jones, S. A.; Willson, T.
M. J. Med. Chem. 2000, 43, 2971.
13. Akwabi-Ameyaw, A.; Bass, J. Y.; Caldwell, R. D.; Caravella, J. A.; Chen, L.; Creech,
K. L.; Deaton, D. N.; Jones, S. A.; Kaldor, I.; Liu, Y.; Madauss, K. P.; Marr, H. B.;
McFadyen, R. B.; Miller, A. B.; Navas, F., III; Parks, D. J.; Spearing, P. K.; Todd, D.;
Williams, S. P.; Wisely, G. B. Bioorg. Med. Chem. Lett. 2008, 18, 4339.
14. The alcohol 2h was prepared from commercially available (E)-2,6-
dichlorocinnamic acid in three synthetic steps. First, the acid was esterified
employing the Fischer method with catalytic sulfuric acid in methanol (94%
yield). Then, the resulting unsaturated methyl ester was converted to the
methyl 2-(2,6-dichlorophenyl)cyclopropanecarboxylate via its addition to the
in situ generated sulfur ylide prepared by adding sodium hydride to
trimethylsulfoxonium iodide in dimethyl sulfoxide (26% yield). Finally,
reduction of the ester with di-iso-butylaluminum hydride in tetrahydrofuran
afforded the desired [2-(2,6-dichlorophenyl)cyclopropyl]methanol 2h (78%
yield).
Fd (%)
a
c
#
t1/2 (min)
Clb (mL/min/kg)
VSS (mL/kg)
1b
1j
1k
1ab
52
48
20
16
15
1100
1200
2100
1400
7.8
9.2
2.6
1.9
130
140
140
a
t1/2 is the iv terminal half-life dosed as a solution. All in vivo pharmacokinetic
values are the mean of two experiments.
b
Cl is the iv total clearance.
VSS is the iv steady state volume of distribution.
F is the oral bioavailability.
c
d
exhibited medium volumes of distribution (VSS = 1100–2100 mL/
kg), with the three two atom tether analogs (1j, 1k, and 1ab) hav-
ing moderate clearances (Cl = 15–20 mL/min/kg) of less than one
third of hepatic blood flow in the rat, translating into terminal
half-lives (t1/2 = 130–140 min) of greater than 2 h. Unfortunately,
all of the analogs had poor oral exposure (F = 1.9–9.2%). These
clearances were significantly lower than the starting lead GW
4064 1a (Cl = 36 mL/min/kg). Furthermore, despite similar clear-
ances between phenol 1j and aniline 1k, the potentially more sol-
uble aniline 1k (F = 2.6%) had a lower oral bioavailability than the
phenol 1j (F = 9.2%). Possibly, the stilbene moiety that is present in
all of the analogs is detrimental to oral exposure and overrides any
changes elsewhere in the agonists.
In summary, a series of analogs of GW 4064 1a were synthe-
sized as potential modulators of FXR. Incorporation of a two atom
tether between the isoxazole and the 2,6-dichlorophenyl ring pro-
duced a number of highly efficacious FXR agonists with compara-
ble efficacy and potency to the original lead 1a. Introduction of a
heteroatom into the linker allowed the rapid production of termi-
nal ring analogs. Several of the compounds with a two atom tether
had lower in vivo clearances than GW 4064 1a. Structure–activity
relationships identified from this work should prove useful in the
design of improved FXR modulators based on GW 4064 1a.
15. Miller, W. H.; Dessert, A. M.; Anderson, G. W. J. Am. Chem. Soc. 1948, 70, 500.
16. Bass, J. Y., III; Deaton, D. N.; Caravella, J.; McFadyen, R. B.; Navas, F., III;
Spearing, P. K. PCT Int. Appl. WO/08 051942 A1 20071023.
17. Bell, M. G.; Doti, R. A.; Dowling, M. S.; Genin, M. J.; Lander, P. A.; Ma, T.; Mantlo,
N. B.; Ochoada, J. M.; Stelzer, L. S.; Stites, R. E.; Warshawsky, A. M. PCT Int. Appl.
WO 140174, 2007; Chem. Abstr. 2007, 148, 55040.
18. Mitsunobu reactions in this Letter were performed under the following
conditions: a mixture of 1.0 equiv each of the alcohol, the phenol, di-iso-
propyl diazodicarboxylate, and triphenylphosphine were placed in
a
microwave reaction vessel and toluene was added to make a 0.1 M mixture.
Then, the vessel was sealed and heated in a microwave reactor between 85 and
100 °C for between 600 and 1000 s. The resulting mixture was then
concentrated and the residue was purified by silica gel chromatography with
gradients of ethyl acetate in hexanes.
19. Saponification reactions in this Letter were performed under the following
conditions: The ester was placed in a microwave reaction vessel and a 2:1
mixture of tetrahydrofuran and methanol was added, followed by 1.5 equiv of
1 M sodium hydroxide. Then, the vessel was sealed and heated in a microwave
reactor between 100 and 120 °C for 500 s. The resulting mixture was
neutralized with 1 M hydrochloric acid and extracted with ethyl acetate. The
organic layer was dried over magnesium sulfate, filtered, and concentrated.
20. The stereoisomers of the sec-butyl group were separated at the alcohol 6l stage
and carried through the rest of the synthesis separately to give analogs 1am
and 1an.