Addressing PXR liabilities of phthalazine-based hedgehog/smoothened antagonists using novel pyridopyridazines

Article abstract of DOI:10.1016/j.bmcl.2010.06.006

Pyridopyridazine antagonists of the hedgehog signaling pathway are described. Designed to optimize our previously described phthalazine smoothened antagonists, a representative compound eliminates a PXR liability while retaining potency and in vitro metab

Full text of DOI:10.1016/j.bmcl.2010.06.006

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4607–4610
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Addressing PXR liabilities of phthalazine-based hedgehog/smoothened
antagonists using novel pyridopyridazines
Jacob A. Kaizerman, Wade Aaron, Songzhu An, Richard Austin, Matt Brown, Angela Chong, Tom Huang,
Randall Hungate, Ben Jiang, Michael G. Johnson, Gary Lee, Brian S. Lucas, Jessica Orf, Minqing Rong,
*
Maria M. Toteva, Dineli Wickramasinghe, Guifen Xu, Qiuping Ye, Wendy Zhong, Dustin L. McMinn
Amgen, Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyridopyridazine antagonists of the hedgehog signaling pathway are described. Designed to optimize our
previously described phthalazine smoothened antagonists, a representative compound eliminates a PXR
liability while retaining potency and in vitro metabolic stability. Moreover, the compound has improved
efficacy in a hedgehog/smoothened signaling mouse pharmacodynamic model.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 27 April 2010
Accepted 1 June 2010
Available online 8 June 2010
Keywords:
Hedgehog
Smoothened
Hh
SMO
Cancer
Oncology
The hedgehog (Hh) signaling pathway regulates cell growth and
differentiation in developing tissues.1 Deregulation of this pathway
has been linked to tumorigenesis in a variety of cancers2,3 and is
clearly implicated in development of basal cell carcinoma4 and
medulloblastoma.5 Smoothened (Smo), a seven-transmembrane
cell surface receptor, transduces Hh signaling and is responsible
for activation of the Gli family of zinc finger transcription factors.
Once activated, Gli transcription factors drive the expression of
genes involved with cell proliferation and, when deregulated, can-
cer (cyclins and CDK’s, BCL2 (survival) and Snail (metastasis), as
well as Gli1). In normal tissues, Smo is prevented from activating
Gli transcription factors by the action of the 12-transmembrane
protein Patched (Ptch). Upon binding to Hh or when plagued by
inactivating mutations, Ptch no longer inhibits Smo, thus allowing
the effector Smo to drive unregulated expression of Hh target
genes. The promise of treating cancer via antagonism of Smo and
disruption of aberrant Hh signaling has driven considerable recent
efforts in our labs6 and on the part of the wider pharmaceutical
industry.7
over, 1 showed efficacy in established pharmacodynamic and tu-
mor models representative of Hh pathway disruption.11 Further
evaluation of 1 and related compounds revealed a pregnane X
receptor (PXR) activation liability, thus presenting for this series
of compounds the potential for transactivation of a variety of drug
metabolizing enzymes and subsequent drug–drug interactions.
The cancer indication presents an ever-increasing polypharmic
population and thus encourages the development of oncology
drugs known to avoid erratic drug metabolism and the associated
potential for therapeutic failure.12 We describe here our efforts to
eradicate PXR liabilities from our Smo antagonists while preserv-
ing potency and pharmacodynamic efficacy.
From our previously submitted work, we disclosed that appro-
priate substitution of the 4-phenyl group was generally well toler-
ated and in some cases led to improved in vitro and in vivo
metabolic stability. For this reason, we initially sought to evaluate
PXR activation as it relates to modification at this region of the
molecule. Synthesis of compounds 1–7, 12, 14–16, 21 and 22 have
We have recently described a series of 4-(4-phenylphthalazine-
1-yl)piperazine amide-based inhibitors of Smo.8,9 Structural opti-
mization led to 1, a compound which proved highly potent against
mouse (mSMO) and human (hSMO) forms of Smo (Fig. 1).10 More-
* Corresponding author.
E-mail address: dmcminn@amgen.com (D.L. McMinn).
Figure 1. Smoothened antagonist.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.006

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J. A. Kaizerman et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4607–4610
quite potent, showed compromised in vitro metabolic stability in
rat or human liver microsome homogenates. Compounds 12 and
13 exhibited potency and in vitro metabolic stability comparable
to the parent compound 1. For these compounds, though, there
was a disconnect between in vitro metabolic stability in rat liver
microsomal homogenates and their observed moderate to high
clearance in vivo (Table 3). Substituting the phenyl group with het-
eroaryl functionality containing one heteroatom resulted in com-
pounds with significantly compromised potency (14–16) or
decreased in vitro metabolic stability (17 and 18). Support for a
hydrophobicity-driven PXR transactivation hypothesis in this
structural class was corroborated by comparison of PXR activation
by the N-linked pyrrole 17 to the more hydrophobic N-linked in-
dole 18 (12% vs 74%, respectively). Heteroaryl groups at the 4-posi-
tion containing more than two heteroatoms showed low PXR
potential and minimal in vitro metabolic turnover, albeit at the
cost of target potency (19–22).
Having shown that increasing polarity of pendant features
could potentially mitigate PXR transactivation, we hypothesized
that increased polarity in the upper ring of the phthalazine core
would also provide compounds with low PXR potential. Moreover,
such new scaffolds may provide an opportunity to address the PXR
liability while preserving potency and metabolic stability. We
chose to test this hypothesis by incorporating single nitrogen at
each position of the benzo-portion of the phthalazine core, thus
rendering pyridopyridazines 23–26 (Table 2).
O
O
a, b, or c
Cl
N
N
R1
N
N
N N
N N
R2
R2
8, R1 = 4-(CH3)2N-phenyl
9, R1 = 4-N-morphilinophenyl
10, R1 = 4-(CH3OOC)-phenyl
11, R1 = 4-HOOC-phenyl
12, R1 = 4-HOCH2-phenyl
13, R1 = 4-H2NCOCH2-phenyl
17, R1 = N-pyrrole
28
d
18, R1 = N-indole
19, R1 = 2-oxazole
20, R1 = N-imidazole
Scheme 1. (a) R1-B(OH)2, Pd(PPh3)4, Na2CO3, toluene, 100 °C, 71–97%; (b) pyrrole,
indole, or imidazole, CuI, K3PO4, trans-1,2-diaminocyclohexane, dioxane, 110 °C,
14–50%; (c) 2-(tri-N-butylstannyl)oxazole, Pd[P(t-Bu)3]2, CsF, dioxane, 25%; (d)
2,2,2-trichloroacetyl isocyanate, neutral alumina (Brockmann II), chloroform, 99%.
been described.8,13 Compounds 8–12 and 17–20 were derived from
our previously described intermediate 28 (Scheme 1) utilizing
Suzuki (8–12), Buchwald copper mediated N-heteroarylation15
(17, 18, and 20) or Stille couplings (19). Conversion of the benzyl
alcohol 12 to the primary carbamate 13 was achieved using 2,2,2-
trichloroacetyl isocyanate and subsequent alumina hydrolysis.
With the exception of 11, substitutions on the 4-phenyl ring
rendering increased calculated log D coefficients led to increased
PXR activation (1 vs 2, 3 vs 4, and 5 vs 6, Table 1). In contrast, com-
pounds with less lipophilic groups at the para-position of the
4-phenyl ring exhibited reduced PXR activation (7–13). Com-
pounds 7–9 failed to combine minimized PXR potential with suffi-
cient biochemical potency, while compounds 10 and 11, although
Syntheses of pyridopyridazines are outlined in Schemes 2–4.
Preparation of 5-((2R)-2-methyl-4-(phenylcarbonyl)-1-piperazi-
nyl)-8-phenylpyrido[3,2-d]pyridazine 23 went though the 5,8-di-
chloropyrido[2,3-d]pyridazine intermediate 31 which was prepared
by sequential treatment of 2,3-pyridinedicarboxylic anhydride with
hydrazine and POCl3 (Scheme 2). Regioselective nucleophilic
Table 1
Phthalazine-based Smoothened inhibitors
Compd
R1
R2
hSMO IC50
(l
M)a
PXR, % activ. at 2
l
Mb
log D (pH 7.4)c
%TO RLMd
%TO HLMd
1
2
3
4
5
6
7
8
4-(Trifluoromethyl)phenyl
Phenyl
3,4-Dichlorophenyl
Phenyl
F-phenyl
Phenyl
4-NH2CO-phenyl
4-N-Morpholinophenyl
4-HOOC-phenyl
4-(CH3)2N-phenyl
4-(CH3OCO)-phenyl
4-HOCH2-phenyl
4-H2NCOCH2-phenyl
4-Pyridyl
3-R-methyl
3-R-methyl
H
0.0028
0.0022
0.017
0.019
0.012
0.0053
0.019
0.021
>1
0.0053
0.0033
0.0027
0.0048
0.025
0.079
0.057
0.0087
0.0080
>1
81
30
48
12
61
32
5.9
6.6
2.7
17
6.1
3
2.7
25
23
16
12
74
7.8
3.7
5.4
7.1
3.74
2.29
3.34
1.8
2.49
2.29
1.12
0.95
0.08
1.97
2.59
1.3
1.52
1.46
1.49
1.56
2.02
2.99
1.61
0.72
0.04
0.52
11
12
13
39
21
<10
<10
28
<10
82
>95
15
10
36
16
17
63
72
22
16
<10
<10
13
44
31
H
19
2-S-methyl
2-S-methyl
3-R-methyl
3-R-methyl
3-R-methyl
3-R-methyl
3-R-methyl
3-R-methyl
3-R-methyl
3-R-methyl
3-R-methyl
3-R-methyl
3-R-methyl
3-R-methyl
3-R-methyl
3-R-methyl
3-R-methyl
3-R-methyl
<10
<10
<10
19
<10
59
>95
14
10
11
19
12
54
9
10
11
12
13
14
15
16
17
18
19
20
21
22
3-Pyridyl
2-Pyridyl
N-Pyrrolyl
N-Indolyl
55
2-Oxazolyl
<10
<10
<10
<10
N-Imidazolyl
2-Pyridazinyl
5-Pyrimidinyl
>1
>1
>1
a
b
c
Values are the average of a minimum of three measurements.
Rifampin used as control.14
ACD based calculation.
d
Percent turnover within 30 min in liver microsome homogenates.

J. A. Kaizerman et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4607–4610
4609
Table 2
Pyridopyridazine-based smoothened inhibitors
O
N
N
N
N
Compd Ring
hSMO IC50
0.066
(l
M) PXR, % activ. at 2
4.1
l
Ma %TO HLMb
N
23
13
N
24
0.016
0.012
5.7
7.5
56
30
N
Scheme 3. (a) Hydrazine, ethanol, reflux, 71%; acetic acid, hydrazine, 100 °C, 68%;
(b) POCl3, pyridine, 110 °C, 80%; (c) (R)-tert-butyl 3-methylpiperazine-l-carboxyl-
ate, 130 °C melt, 52%; (d) TFA, CH2C12, 77%; (e) benzoyl chloride, TEA, CH2C12, 84%.
25
N
26
0.0058
4.9
13
c
Percent turnover within 30 min in liver microsome homogenates.
a
Values are the average of a minimum of three measurements.
b
Rifampin used as control.
heteroaromatic substitution with (R)-1-benzoyl-3-methylpipera-
zine provided 32 which served as the substrate for a final Suzuki
coupling. Alternatively, 8-((2R)-2-methyl-4-(phenylcarbonyl)-
1-piperazinyl)-5-phenylpyrido[2,3-d]pyridazine synthesis com-
menced with conversion of 3-benzoylpicolinic acid 33 to the
8(7H)-pyrido[2,3-d]pyridazinone 34 (Scheme 3). Chloride synthesis,
substitution, deprotection of intermediate 36, and benzoylation pro-
vided 24. Synthesis of the common intermediate 39 for preparation
of 1-((2R)-2-methyl-4-(phenylcarbonyl)-1-piperazinyl)-4-phenyl-
pyrido[4,3-d]pyridazine and 4-((2R)-2-methyl-4-(phenylcarbon-
yl)-1-piperazinyl)-1-phenylpyrido[3,4-d]pyridazine followed that
of 31 (Scheme 4). Predictive electronics of the 1,4-dichloropyri-
do[3,4-d]pyridazine system afforded significantly higher isolated
substitution yields for 41 than for 40. Suzuki coupling with the
appropriate boronic acid was followed by removal of Boc protection
and benzoylation to provide 25 and 26. Compounds 23–26 success-
fully reduced PXR activation to well below the levels of 1. The 4-
((2R)-2-methyl-4-(phenylcarbonyl)-1-piperazin-yl)-1-phenyl-pyr-
ido[3,4-d]pyridazine compound, 26, proved to be the optimal regio-
isomer addressing in concert potency, in vitro metabolic stability,
and PXR activation. To assess the capacity for this scaffold to reduce
PXR activation, we reinstalled the trifluoromethyl feature at the
para-position of the 1-phenyl group (27, Fig. 2). To our satisfaction,
Scheme 4. (a) NH2NH2, ethanol, H2O, reflux, 99%; (b) POCl3, Hünig’s base, reflux,
67%; (c) (R)-tert-butyl 3-methylpiperazine-l-carboxylate, Hünig’s base, NMP,
100 °C, 2% isolated yield for 40, 62% isolated yield for 41; (d) PhB(OH)2, Pd(PPh3)4,
Na2CO3, toluene, 100 °C, 99%; (e) TFA, CH2C12, 99%; (f) benzoyl chloride, TEA,
CH2C12, 55–77%; (g) 4-CF3PhB(OH)2, Pd(PPh3)4, Na2CO3, toluene, 100 °C, 99%.
N
N
N
a
b
HO
OH
Cl
Cl
O
O
O
N N
N N
29
30
31
c
d
N
23
O
Cl
N
N
N
N
32
Scheme 2. (a) Acetic acid, hydrazine, 100 °C, 68%; (b) POCl3, pyridine, 110 °C, 38%;
(c) (R)-(3-methylpiperazin-l-yl)(phenyl)methanone, melt, 44%; (d) PhB(OH)2,
Pd(PPh3)4, Na2CO3, toluene, 100 °C, 77%.
Figure 2. Compounds 26 and 27.

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3. Lum, L.; Beachy, P. A. Science 2004, 304, 1755.
Table 3
4. Epstein, E. H. Nat. Rev. Cancer 2008, 8, 743.
Rat and mouse pharmacokinetics for selected phthalazine and pyridopyridazine-
based smoothened inhibitorsa
5. Berman, D. M.; Karhadkar, S. S.; Hallahan, A. R.; Pritchard, J. I.; Eberhart, C. G.;
Watkins, D. N.; Chen, J. K.; Cooper, M. K.; Taipale, J.; Olson, J. M.; Beachy, P. A.
Science 2002, 297, 1559.
Compd
Species
CL (iv, L/h/kg)
Vdss (L/kg)
AUCpo
(lg h/L)
%F
6. (a) Austin, R. J.; Kaizerman, J.; Lucas, B.; McMinn, D. L.; Powers, J. PCT Int. Appl.
WO 2009/002469 A1, 2009.; (b) Kaizerman, J.; Lucas, B.; McMinn, D. L.;
Zamboni, R. PCT Int. Appl. WO 2009/035568 A1, 2009.
1
2
Rat
Rat
Rat
Rat
Rat
Rat
Mouse
0.41
0.28
5.7
5.1
1.7
1.8
1.4
1.4
3.5
2
1200
1800
BQL
39
15
nd
22
35
73
46
12
13
26
27
27
7. (a) Miller-Moslin, K.; Peukert, S.; Jain, R. K.; McEwan, M. A.; Karki, R.; Llamas, L.;
Yusuff, N.; He, F.; Li, Y.; Sun, Y.; Dai, M.; Perez, L.; Michael, W.; Sheng, T.; Lei, H.;
Zhang, R.; Williams, J.; Bourret, A.; Ramamurthy, A.; Yuan, J.; Guo, R.;
Matsumoto, M.; Vattay, A.; Maniara, W.; Amaral, A.; Dorsch, M.; Kelleher, J. F.
J. Med. Chem. 2009, 52, 3954; Review of recent efforts in the field of Hh
signaling regulation: (b) Mahindroo, N.; Punchihewa, C.; Fujii, N. J. Med. Chem.
2009, 52, 3829; (c) Peukert, S.; Jain, R. K.; Geisser, A.; Sun, Y.; Zhang, R.; Bourret,
A.; Carlson, A.; DaSilva, J.; Ramamurthy, A.; Kelleher, J. F. Bioorg. Med. Chem.
Lett. 2009, 19, 328; (d) Tremblay, M. R.; Nevalainen, M.; Nair, S. J.; Porter, J. R.;
Castro, A. C.; Behnke, M. L.; Yu, L.-C.; Hagel, M.; White, K.; Faia, K.; Grenier, L.;
Campbell, M. J.; Cushing, J.; Woodward, C. N.; Hoyt, J.; Foley, M. A.; Read, M. A.;
Sydor, J.; Tong, J. K.; Palombella, V. J.; McGovern, K.; Adams, J. J. Med. Chem.
2008, 51, 6646; (e) Feldmann, G.; Fendrich, V.; McGovern, K.; Bedja, D.; Bisht,
S.; Alvarez, H.; Koorstra, J.-B. M.; Habbe, N.; Karikari, C.; Mullendore, M.;
Gabrielson, K. L.; Sharma, R. Mol. Cancer Ther. 2008, 7, 2725; (f) Brunton, S. A.;
Stibbard, J. H. A.; Rubin, L. L.; Kruse, L. I.; Guichert, O. M.; Boyd, E. A.; Price, S. J.
Med. Chem. 2008, 51, 1108; (g) Remsberg, J. R.; Lou, H.; Tarasov, S. G.; Dean, M.;
Tarasova, N. I. J. Med. Chem. 2007, 50, 4534; (h) Rudin, C. M.; Hann, C. L.; Laterra,
J.; Yauch, R. L.; Callahan, C. A.; Fu, L.; Holcomb, T.; Stinson, J.; Gould, S. E.;
Coleman, B.; LoRusso, P. M.; Von Hoff, D. D.; de Sauvage, M. J.; Low, J. A. NEJM
2009, 361, 1173.
1.1
460
0.45
0.46
0.64
1600
3300
3500
a
Dosed iv 0.5 mg/kg, po 2 mg/kg.
200
1600
Ave Gli/RGS
180
1400
1200
1000
800
600
400
200
0
Mean plasma concentration of 27(ug/L)
160
140
120
100
80
60
8. Lucas, B. S.; Aaron, W.; An, S.; Austin, R. J.; Brown, M.; Chan, H.; Chong, A.;
Hungate, R.; Huang, T.; Jiang, B.; Johnson, M. G.; Kaizerman, J. A.; Lee, G.;
McMinn, D. L.; Orf, J.; Powers, J. P.; Rong, M.; Toteva, M. M.; Uyeda, C.;
Wickramasinghe, D.; Xu, G.; Ye, Q.; Zhong, W. Bioorg. Med. Chem. Lett. 2010, 20,
3618.
40
20
0
vehicle
1 mpk
5 mpk
50 mpk
9. Inhibitors of the Hh pathway featuring a piperazine-substituted phthalazines
have been described, Ref. 7a.
Figure 3. Reduction of Gli1 expression in skins of mice treated with 27. Four
animals per group, two samples per animal; 1 mg/kg, = 0.355; 5 mg/kg,
= 0.0011; 50 mg/kg, <0.0001 (Dunnet’s method).
10. Biological methods: IC50 assay for mouse Smo: mouse smoothened activity was
measured in vitro using a modified version of the method described.11d An
oligonucleotide cassette with five consensus Gli1 binding sites was ligated into
the luciferase reporter plasmid pGL4.16 (Promega, Madison, WI, USA). A stable
clone of NIH-3T3 cells stably transfected with the Gli1-binding-site plasmid
was used for the reporter assay. Compounds were incubated with cells for 15 h
in the presence of Optimem medium (Invitrogen, Carlsbad, CA, USA)
supplemented with 0.5% charcoal–dextran treated fetal bovine serum
(HyClone) and 10 mM myristoylated mouse Shh protein (Williams et al.,
1999). Luciferase activity was measured by addition of Bright-Glo (Promega)
and reading on a luminometer. Compounds were tested in quadruplicate using
a threefold dilution series.
q
q
q
not only was PXR activation suppressed, but potency in the human
biochemical assay was improved sixfold relative to 26. These
qualities were augmented by impressive in vivo pharmacokinetic
profiles in potential representative pharmacodynamic model spe-
cies (Table 3). Compound 27 provided superior oral exposure rela-
tive to 26 in rats and exhibited low clearance and good oral
bioavailability in the mouse. Finally, compounds 26 and 27, as rep-
resentatives of this class, showed excellent potency in the mouse,
our species of choice for pharmacodynamic efficacy assessment.
Our pharmacodynamic model relies upon the known overex-
pression of Hh target genes in the depilated skin of mice.10 The Hh
signaling pathway is activated during the growth stage of the hair
cycle resulting in up-regulation of target genes including Gli1. Skins
of mice were depilated and, after 5 days, were harvested 6 h post-
oral dosing with 27. Gli1 expression was measured against reference
genes. Figure 3 shows a dose dependent reduction of Gli1 expression
IC50 assay for human SMO: HEPM cells were used to measure human SMO
activity in vitro using a modified version of the method described by others [US
Patent 6,613,798]. In 96 well tissue culture plates, compounds were incubated
with HEPM cells in the presence of MEM media supplemented with 0.5%
charcoal–dextran treated fetal bovine serum (HyClone) and 50 mM
myristoylated mouse Shh protein (Williams et al. 1999). Twenty-four hours
after the addition of compound and Shh, GLI expression was measured using a
Quantigene assay (Affymetrix, Santa Clara, CA, USA).
Mouse skin pharmacodynamic model: Female NOD-Scid mice (Taconic, Hudson,
NY, USA) aged 5–8 weeks were shaved on one side of the hind flank. Four days
after shaving, mice were anesthetized and regrown hair was removed using
hair removal wax strips. After 5 days, mice were dosed orally with compounds
or vehicle. At specified time points after dosing, animals were sacrificed and
the waxed regions of skin were excised and stored in RNALater solution
(Ambion, Austin, TX, USA) at 4 °C. Skin samples were homogenized in Lysis
buffer (Ambion) using a Tomtec Autogizer (Hamden, CT, USA); RNA was
purified using an mirVana miRNA Isolation Kit (Ambion). RNA was converted
cDNA using reverse transcriptase prior to quantitative, real time PCR analysis
(Applied Biosystems, Foster City, CA, USA). Gli1 expression was normalized to
Rgs3 expression using the standard curve method.
was observed (approximate EC50 = 0.46
an improvement over the performance of our previously reported
compound 1 in this model (EC50 = 2.0
M).16,8
lM). This result constitutes
l
In conclusion, pyridopyridazines described in this work consti-
tute a novel class of SMO inhibitors capable of suppressing PXR
transactivation, a potential liability seen for our previously reported
potent phthalazine-based inhibitors. Compounds 26 and 27 of the 4-
((2R)-2-methyl-4-(phenylcarbonyl)-1-piperazin-yl)-1-phenylpyri-
do[3,4-d]pyridazine regio-isomeric sub-class highlight our ability to
redress PXR liabilities while maintaining potency and metabolic sta-
bility in vitro. Finally, compound 27 proved efficacious at disrupting
Hh signaling in an in vivo pharmacodynamic model. These results
merit our continued investigation of this structural class.
11. (a) Paladini, S.; Qian, X.; Rubin J. Invest. Dermatol. 2005, 125, 638; (b) Williams,
K. P.; Rayhorn, P.; Chi-Rosso, G.; Garber, E. A.; Strauch, K. L.; Horan, G. S. B.;
Reilly, J. O.; Baker, D. P.; Taylor, F. R.; Koteliansky, V.; Pepinsky, R. B. J. Cell Sci.
1999, 112, 4405; (c) U.S. Patent 6,613,798.; (d) Chen, J. K.; Taipale, J.; Young, K.
E.; Maiti, T.; Beachy, P. A. PNAS 2002, 99, 14071.
12. Sinz, M. W. Ann. Rep. Med. Chem. 2008, 43, 405.
13. The purity of final compounds was determined by HPLC. The final structures
are consistent with their 1H NMR and LCMS spectral data. The enantiomeric
purity was determined by HPLC and found to be >98% ee.
14. PXR activation relative to percent of control (rifampin) was determined using
HepG2 cells transfected with a luciferase reporter construct driven by human
PXR cDNA with luciferase activity determined by chemiluminescence.
15. Klapars, A.; Antilla, J. C.; Huang, X.; Buchwald, S. L. J. Am. Chem. Soc. 2001, 123,
7727.
References and notes
16. Fraction unbound 1 in mouse plasma = 0.06; fraction unbound 27 in mouse
plasma = 0.035 (ultracentrifugation).
1. Ingham, P. W.; McMahon, A. P. Genes Dev. 2001, 15, 3059.
2. Rubin, L. L.; de Sauvage, F. J. Nat. Rev. Drug Discov. 2006, 5, 1026.