Synthesis of novel ketoconazole derivatives as inhibitors of the human Pregnane X Receptor (PXR; NR1I2; also termed SXR, PAR)

Article abstract of DOI:10.1016/j.bmcl.2008.06.018

PXR, pregnane X receptor, in its activated state, is a validated target for controlling certain drug-drug interactions in humans. In this context, there is a paucity of inhibitors directed toward activated PXR. Using prior observations with ketoconazole as a PXR inhibitor, the target compound 3 was synthesized from (s)-glycidol with overall 56% yield. (+)-Glycidol was reacted with 4-bromophenol and potassium carbonate in DMF to yield the ring opened compound 6. This was then heated to reflux in benzene along with 2′, 4′-difluoroacetophenone and catalytic amount of para-toluene sulfonic acid to yield 8. The resultant acetal 8 was then functionalized using Palladium chemistry to yield the target compound 3. The activity of the compound was compared with ketoconazole and UCL2158H. However, in contrast with ketoconazole (IC₅₀ ～ 0.020 μM; ～100% inhibition), 3 has negligible effects on inhibition of microsomal CYP450 (maximum ～20% inhibition) at concentrations >40 μM. In vitro, micromolar concentration of ketoconazole is toxic to passaged human cell lines, while 3 does not exhibit cytotoxicity up to concentrations ～100 μM (viability >85%). This is the first demonstration of a chemical analog of a PXR inhibitor that retains activity against activated PXR. Furthermore, in contrast with ketoconazole, 3 is less toxic in human cell lines and has negligible CYP450 activity.

Full text of DOI:10.1016/j.bmcl.2008.06.018

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3974–3977
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel ketoconazole derivatives as inhibitors of the human
Pregnane X Receptor (PXR; NR1I2; also termed SXR, PAR)
b
a
c
c
Bhaskar C. Das a, , Ankanahlli V. Madhukumar , Jaime Anguiano , Sean Kim , Michael Sinz ,
*
Tatyana A. Zvyaga c, Eoin C. Power d, C. Robin Ganellin e, Sridhar Mani b,
*
a Albert Einstein Cancer Center, Albert Einstein College of Medicine, Departments of Developmental and Molecular Biology and Nuclear Medicine, Bronx, NY 10463, USA
b Albert Einstein Cancer Center, Albert Einstein College of Medicine, Broux, NY 10461, USA
c Bristol-Myers Squibb Co., Wallingford, CT 06492, USA
d Sienabiotech S.P.A., Via Fiorentina, 53100 Siena, Italy
e Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
PXR, pregnane X receptor, in its activated state, is a validated target for controlling certain drug–drug
interactions in humans. In this context, there is a paucity of inhibitors directed toward activated PXR.
Using prior observations with ketoconazole as a PXR inhibitor, the target compound 3 was synthesized
from (s)-glycidol with overall 56% yield. (+)-Glycidol was reacted with 4-bromophenol and potassium
carbonate in DMF to yield the ring opened compound 6. This was then heated to reflux in benzene along
with 20, 40-difluoroacetophenone and catalytic amount of para-toluene sulfonic acid to yield 8. The resul-
tant acetal 8 was then functionalized using Palladium chemistry to yield the target compound 3. The
activity of the compound was compared with ketoconazole and UCL2158H. However, in contrast with
Received 30 April 2008
Revised 2 June 2008
Accepted 4 June 2008
Available online 10 June 2008
Keywords:
Pregnane X receptor
PXR
Ketoconazole
Azole
Analogs
ketoconazole (IC50 ꢀ 0.020
CYP450 (maximum ꢀ20% inhibition) at concentrations >40
ketoconazole is toxic to passaged human cell lines, while 3 does not exhibit cytotoxicity up to concentra-
M (viability >85%). This is the first demonstration of a chemical analog of a PXR inhibitor that
l
M; ꢀ100% inhibition), 3 has negligible effects on inhibition of microsomal
l
M. In vitro, micromolar concentration of
Nuclear receptors
tions ꢀ100
l
retains activity against activated PXR. Furthermore, in contrast with ketoconazole, 3 is less toxic in
human cell lines and has negligible CYP450 activity.
Ó 2008 Elsevier Ltd. All rights reserved.
Pregnane X receptor or PXR (NR1I2; also termed SXR, PAR), a
member of the adopted orphan nuclear receptor family, is the pri-
mary xenobiotic sensor in human and mammalian tissues.1 It
responds to a wide range of structurally and chemically distinct li-
gands that range from small lipophilic drugs (e.g., rifampicin) to
potentially toxic bile acids as well as cholesterol metabolites.1–10
This promiscuous activation of PXR by natural substances and xeno-
biotics has been shown as a mechanism that accelerates the metab-
olism of affected drugs, which leads to unanticipated adverse drug
interactions and loss of efficacy.8 Recent data also support a major
role for PXR activation in controlling (decreasing) the transport of
drugs across the blood brain barrier, underscoring PXR’s role in drug
delivery to the brain in disease state (e.g., brain metastases).11
Furthermore, there is increasing evidence that PXR activation
leads to increased cancer cell growth and drug resistance.12
Together, in the context of cancer therapeutics, controlling PXR
activation is expected to provide many therapeutic benefits by
improving both drug metabolism and delivery. Therefore, the
development of novel and non-toxic inhibitors of PXR activation
is warranted.
In addition to its therapeutic utility, PXR inhibitor would be use-
ful to study the molecular basis of receptor function. To date, only
few PXR inhibitors have been described: ketoconazole (and related
azoles13), suphoraphane,14 and ecteinascidin-743 (ET-743).15 Keto-
conazole was first described as a PXR antagonist by Takeshita et
al., and was subsequently shown to disrupt the binding of co-regula-
tors (including both coactivators and corepressors) to the surface of
PXR in an agonist-dependent fashion.16,17 In addition, in the pres-
ence of the established PXR activator rifampicin, ketoconazole, and
related azoles were shown to prevent the activation of the receptor
both in cell-based assays and in a humanized PXR mouse model.18
Scintillation proximity assays using PXR ligand binding domain
co-purified with SRC-1 peptide show that ketoconazole does
indeed bind to PXR over a concentration range (10–100
l
M) but
is concentration-dependent.17 Since this assay is
a ligand
(3H-SR12813) displacement assay, the concentration dependence
relates to ligand displacement (by binding to the ligand binding
pocket) and not to allosteric binding. However, our laboratory has
shown that ketoconazole binds to at least a region outside the
* Corresponding authors. Tel.: +1 17184302422; fax: +1 17184308853.
E-mail address: bdas@aecom.yu.edu (B.C. Das).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.018

B. C. Das et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3974–3977
3975
ligand binding pocket. We also have shown that the revertant AF-2
region double mutant of PXR (T248E/K277Q) activates with rifam-
picin but is not inhibited by ketoconazole.13,19 These data establish
that small molecule modulators such as ketoconazole can antago-
nize PXR, and that the surface AF-2 site of the receptor appears to
be a target of such compounds. Based on these data and the knowl-
edge of the PXR:SRC-1 crystal structure, we embarked on develop-
ing novel ketoconazole analogs 3 that would retain its antagonist
potential on activated PXR but also have minimal cytotoxicity as
well as CYP3A inhibition potential (Scheme 1).
from GIBCO/Invitrogen (Carlsbad, CA). Charcoal/dextran-treated
FBS was purchased from Hyclone (Logan, UT). HepG2 cells were
obtained from ATCC (Manassas, VA). Steady-GloÒ Luciferase
Assay System was purchased from Promega (Madison, WI). Ala-
mar-Blue reagent was purchased from Trek Diagnostics (Cleve-
land, OH). Rifampicin, ketoconazole, and midazolam were
purchased from Sigma (St. Louis, MO). Human liver microsome
and 1-hydroxymidazolam were obtained from BD Biosciences
(San Jose, CA). Human PXR-pcDNA3 and luciferase reporter
containing CYP3A4 promoter, CYP3A-Luc, were generated at
Bristol-Myers Squibb.
The discoveryof novel ketoconazole analogs thatinhibit PXR only
in its ligand-activated state would serve as tools to better under-
stand receptor function. These novel analogs would be more selec-
tive to PXR antagonism (i.e., lack cytochrome P450 inhibition
effects, idiosyncratic organ toxicity, and cellular cytotoxicity in
vitro). Ideally, the IC50 for antagonism of the lead analogs would be
lower and more selective to PXR. Finally, non-toxic lead compounds
could be further developed for clinical use in the setting of cancer
chemotherapy. Focusing on the above objectives, we synthesized
ketoconazole analogs (FL-B # 12, FL-B # 15, FL-B # 24, FL-B # 31
and FL-B # 45). From our preliminary study we found compound 3
(FL-B-12) is our lead compound shown in Scheme 2. Hence onwards,
our lead compound 3 will be read as FL-B-12 in all figures and tables.
Cell culture, PXR transactivation and cytotoxicity assays: HepG2
cells were cultured in DMEM containing 10% FBS. For transient
transfection,
pcDNA3 plasmid DNA, 20
90
l of LipofectamineTM 2000 was prepared in 1 mL of serum-free
a
transfection mixture containing
1 lg of PXR-
lg of Cyp3A-Luc plasmid DNA and
l
DMEM. The transfection mixture was then applied to the cells in
fresh medium (20 mL per flask) and was incubated at 37 °C (5%
CO2) overnight. The transfected cells were trypsinized and cryopre-
served for long-term storage. On the day of experiment, vials of
cryopreserved cells were thawed and then resuspended in fresh
Media II (DMEM containing 5% charcoal/dextran-treated FBS, 1%
penicillin/streptomycin,100
sodium pyruvate, and 2 mM
cells were added to wells of 384-well plates containing either test
compound alone or a mixture of test compound and rifampicin
lM non-essential amino acids, 1 mM
L
The synthesis of compound
3
was as follows.20–22 First,
-glutamine). Approximately 8 ꢁ 103
(+)-glycidol was reacted with 4-bromophenol and potassium car-
bonate in DMF to yield the ring opened compound 6. This was
then heated to reflux in benzene along with 20,40-difluoroacetoph-
enone and catalytic toluene sulfonic acid to yield 8 as racemic
mixture. The resultant acetal 8 was then functionalized using
Buchwald chemistry to yield 3, overall 56%. The general proce-
dure for synthesis of compound 3 is briefly described in Ref. 22.
After synthesizing the compounds, we tested the biological
activity of analogs and compared with parent ketoconazole and
UCl2158H.
(10
l
M) dissolved in DMSO (0.1% in final incubations). Compounds
M–2.5 mM, 1:3 serial dilu-
were tested at ten concentrations (50
l
tion). The plates then were incubated at 37 °C for 24 h and subse-
quently Alamar Blue reagent was added to each well. Plates were
incubated for 2 h at 37 °C, 5% CO2 and then 1 h at room tempera-
ture. Fluorescence was read at Ex525/Em598 to measure cytotox-
icity of the test articles. Subsequently, luciferase substrate
(Steady-GloÒ) was added to each well. The plates were incubated
for 15 min at room temperature, after which the luminescence
was read on a Viewlux (Perkin-Elmer, Waltham, MA) plate reader.
In addition, drug induced cytotoxicity was assessed by the MTT
Materials: Dulbecco’s modified Eagle medium (DMEM), Lipo-
fectamineTM 2000, heat-inactivated fetal bovine serum (FBS), tryp-
sin–EDTA (0.25%), and penicillin–streptomycin were purchased
N
O
N
O
Cl
O
Cl
O
H3C
N
N
O
H3C
N
N
O
O
O
UCL2158H (2)
Cl
Ketoconazole (1)
Cl
O
O
H3C
N
N
O
O
F
(3)
B-12
F
Scheme 1. Structures of ketoconazole, UCL2158H, and 3.
O
O
Br
OH
(5)
O
(7)
O
Br
O
F
F
OH
Br
O
K2CO3, DMF, 88%
PTSA, Benzene,
52%
(8)
OH
(4)
(6)
OH
F
F
O
O
HN
N
O
O
(9)
H3C
N
N
Pd(dba)2,NaOtBu,
P(tBu)3, toluene, 80 ºC,
56%
O
F
(3)
S
F
1-(4-(4-(((2
R
,4
)-2-(2,4-difluorophenyl)
-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)ethanone
Scheme 2. Synthesis of 3 and derivates.

3976
B. C. Das et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3974–3977
assay17 in cancer cell lines (LS174T and SKOV3) as well as a fibro-
blast cell line (CRL). Cells were exposed to a concentration range of
the drug(s) for 48 h. These assays were repeated three separate
times each in triplicate.
Table 1
Maximum activation and inhibition of PXR transactivation by ketoconazole and its
derivatives
Substance (Ref.)
Maximum % activation
observed
Maximum % inhibition
observed
Data analysis: Rifampicin (10 lM), a well known agonist of PXR,
Ketoconazole
UCL2112H (28)
UCL2134D (27)
UCL2135 (23)
UCL2158H (28)
UCL2202D (28)
UCL2245 (28)
UCL2238 (27)
FL-B-12
FL-B-15
FL-B-24
FL-B-31
FL-B-45
20
0
2
93
0
is included in each plate as an internal standard and positive con-
trol. The data were then expressed as percent activation (% Act),
where the total signal is the signal from the 10 lM rifampicin
and the blank signal is that from the DMSO vehicle.
82
32
109
108
58
1
103
90
75
59
48
18
10
10
43
2
91
10
36
50
33
72
107
Compound signal ꢂ Blank signal
%Act ¼
ꢁ 100%
Total signal ꢂ Blank signal
For PXR activation, a plot of concentration versus % Act was gener-
ated for each compound tested and from the plot, EC50 and the
highest percent activation observed for a particular compound
(Emax). For PXR inhibition, where the cells were incubated with a
mixture of 10 M rifampicin and the test compound, a percent inhi-
bition (%Inh) was calculated. From the concentration–%Inh plot, IC50
and the highest percent inhibition observed for that compound
(Imax) were reported.
Rifampicin
iated activation of the receptor whereas UCL2138H {cpd 17}28 was
a potent activator of PXR but was not an inhibitor. However, other
five compounds behaved like ketoconazole, in that it is a weak acti-
vator of PXR and a moderate antagonist of activated PXR and
UCL2158H and UCL2202D {cpd 2}28 appeared to be the most sig-
nificant inhibitors of activated PXR. UCL2158H appeared to be a
better PXR antagonist than UCL2202D as UCL2203 moderately
activated PXR (43%) while UCL2158H did not induce significant
PXR activation (10%) (Supplementary Table 2 and 3). The new com-
pounds are fluorinated derivatives of ketoconazole lacking the
imidazole group, synthesized by our laboratory. Five new com-
pounds were tested and all showed a varying degree of PXR antag-
onism. However, except for FL-B-12 (our lead compound 3 in
Scheme 2), all showed moderate potent activation of PXR in addi-
tion to their activities against rifampicin-mediated PXR activation
(Table 1). FL-B-12 (should read as 3) did not significantly activate
PXR (10%) but inhibit PXR activation by rifampicin to a full extent
(103%) (Supplementary Fig. 1 and Table 2). These results suggested
that the removal of the imidazole group from ketoconazole or its
analogs did not significantly alter their ability to inhibit PXR.
Subsequently, we examined whether the loss of imidazole
group improved or eliminated CYP inhibition associated with the
parent compound, ketoconazole in human liver microsome using
midazolam as the CYP3A4-specific substrate. In addition, a time-
dependency of a potential CYP inhibition was tested to see if the
loss of the imidazole group brings about new liabilities previously
not associated with ketoconazole by preincubating the compounds
with microsome. Two compounds, UCL2158H and 3, were chosen
for this study along with ketoconazole as the positive control. Ta-
ble 3 illustrates the IC50 values with and without pre-incubation
with the test compounds. Ketoconazole, a prototypical reversible
inhibitor of CYP3A4 (and thus midazolam 1-hydroxylase activity),
completely inhibits conversion of midazolam to its metabolite
Reversible and time-dependent CYP3A4 inhibition assay in human
liver microsomes: The CYP3A4 inhibition potential of ketoconazole
and its two analogs, 3 and UCL3158H, were tested in human liver
microsomes. Briefly, test articles were incubated in a reaction
mixture containing 5 M midazolam, 0.1 mg/mL pooled human
liver microsomes, and 1 mM NADPH for 5 min. The enzyme reac-
tion was stopped by adding an equal volume of quench solution
(H2O/acetonitrile/formic-acid = 94:5:1) containing 13C-1-hydrox-
ymidazolam (0.1 M final) as the internal standard. In order to test
time-dependency of CYP3A4 inhibition, an identical reaction was
started without midazolam and the reaction mixture was incu-
bated for 30 min. After the pre-incubation, 5 M midazolam was
added and incubated for 5 min before the quench solution was
added. The samples were centrifuged at 1500 RPM for 15 min to
precipitate denatured microsomal proteins. An ultra high-
throughput RapdiFireTM mass-spectrometer analysis (Biotrove,
Inc., Woburn, MA) was performed to measure the quantity of
1-hydroxymidazolam present in the reaction by monitoring mass
transition of 342.0 ? 203.1 and 345.0 ? 206.1 for 1-hydroxymi-
dazolam and 13C-1-hydroxymidazolam, respectively. A percent
CYP3A4 inhibition (%Inh) was calculated by the following
equation:
ꢀ
ꢁ
CDMSO ꢂ Ccompound
%Inh ¼
ꢁ 100
CDMSO
DMSO and compounds are concentrations of 1-hydroxymidazolam
in the vehicle control and compound tested, respectively. A plot
of concentration–%Inh was then created and from the plot, IC50
and maximum % inhibition were reported.
Results: The basis for structure–function analysis of inhibitors of
activated PXR comes from the initial observations that ketocona-
zole is a weak activator of PXR but an inhibitor of activated PXR
(103% inhibition with IC50 of 0.020 lM) with no indication of
(IC50 = 18.73
l
M) (Table 1). Based on previous modeling studies
time-dependency (no IC50 change). In the same assay performed
from our laboratory, we believe that the site of inhibition may in-
clude the AF-2 surface (estimated area and volume by castP using
atoms detected by GOLD ꢀ 88 Å2 and 117.6 Å3).13,16,17,19,23,24 Keto-
conazole induces toxicity at high doses in humans. This may be
partly due to its inhibitory activity against heme-iron containing
enzymes, which is attributed to the imidazole group.25–27 Thus,
we first designed tested analogs of ketoconazole that lacked the
imidazole group to see if this modification improved on CYP inhib-
itory activity but still retained PXR activity. These compounds,
developed at University College London, were tested in the PXR
transactivation assay. Among 7 compounds tested, UCL2112H
{cpd 12}28 did not transactivate PXR or inhibit the rifampicin-med-
simultaneously using the same pool of microsomes, UCL2158H
was shown to be a weak inhibitor (IC50 of 10.8 or 19.6
CYP3A4, which demonstrated no time-dependency. FL-B-12 (com-
pound 3) appears to be very weak activator of CYP3A4
(IC50 > 40 M), again without a time-dependency of its CYP3A4
lM) of
a
l
inhibition. Hence, elimination of the imidazole group in these ana-
logs markedly improved CYP3A4 inhibition compared to ketocon-
azole without introducing new liability (time-dependent
inhibition).
In a cytotoxicity screen that was performed simultaneously
with PXR transactivation assay, some of the tested compounds
including ketoconazole were toxic in HepG2 cells with 24 h incu-

B. C. Das et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3974–3977
3977
bation especially at the highest concentration tested (50 lM) (data
References and notes
not shown). Thus the cytotoxic potential for the most potent PXR
inhibitors (FL-B-12 and UCL-2158H) was tested in three cancer cell
lines (Caco-2, LS174T and SKOV3) as well as in a transformed fibro-
blast cell line (CRL).17,29 We show that these compounds
(UCL2158H and FL-B-12) are less cytotoxic than ketoconazole in
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these epithelial cells across a concentration range up to 100 lM
(Fig. 3). FL-B-12 was the least cytotoxic (ꢀ98% viability across all
cell lines tested) in the effective concentration range for PXR inhi-
bition (e.g., two times the IC50 ꢀ 30
lM). However, in the same
concentration range, ketoconazole is cytotoxic (viability is ꢀ65–
75% across all cell lines tested). These results together showed that
FL-B-12 (lead compound 3) and UCL2158H have better cytotoxic
profiles than ketoconazole and also demonstrated that the PXR
inhibition exhibited by these compounds was not a result of cell
death induction.
We have shown that it is possible to modify the structure of
ketoconazole to compounds while preserving PXR antagonist
activity. Furthermore, we have demonstrated that these modifica-
tions (loss of the imidazole group) can markedly reduce CYP3A4
inhibition activity associated with ketoconazole. We conclude that
it is feasible to identify PXR specific antagonists with reduced lia-
bilities using a rationally designed structure–activity relationship.
Finally, we speculate that the mechanism of action of FL-B-12 is
similar to that described for ketoconazole,19 in that FL-B-12 may
partially or wholly fit into the AF-2 site in a docking mode that
may be similar or dissimilar to ketoconazole. This allosteric inter-
action on PXR may inhibit SRC-1 co-activator binding and receptor
activation when engaged by an agonist ligand.
In summary, we have shown that it is possible to modify the
structure of ketoconazole to compounds while preserving PXR
antagonist activity. Furthermore, we have demonstrated that these
modifications (loss of the imidazole group) can markedly reduce
CYP3A4 inhibition activity associated with ketoconazole without
reduction of PXR activity. The IC50s obtained are identical which
again suggests that the imidazole group does not contribute to
binding. Also the similarities in structure between UCL 2158 and
FL-B-12 (Scheme 2, compound 3) are reflected in there IC50s. Given
the relatively small number of analogues we conclude that it may
be feasible to identify more potent PXR specific antagonists with
the design and synthesis of analogs. After obtaining this prelimin-
ary hit, we are developing a diversified library of compounds using
SAR analysis on PXR.
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D.; Venkatesh, M. K.; Wang, H.; Kim, S.; Sinz, M.; Goldberg, G. L.;
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22. Synthesis of compound 3: A mixture of compound 8, 1-acetylpiperazine, tert-
BuONa, P(tBu)3, and Pd(dba)2 in dry toluent was gently refluxed and stirred
under a nitrogen atmosphere for 18 h. To the mixture was added 10 mL water,
extracted with ethyl acetate (20 mL 3ꢁ), washed with water (10 mL), brine
(15 mL). The extracts were dried over Na2SO4 and evaporated off. The residue
was purified by column chromatography on silica gel to give 3 as thick liquid 3.
1H NMR (300 MHz,CDCl3): d 7.52 (m, 1H), 6.87–6.71 (m, 6H), 4.58 (m, 1H), 4.28
(m, 1H), 3.95 (m,1H), 3.76 (m,4H), 3.58 (m,2H), 3.00 (m, 4H), 2.11 (s, 3H), and
1.72 (s,3H). 13C NMR CDCl3: d 169.8, 162.7,160.7, 153.7, 144.5, 127.9, 126.8,
115.3, 115.2, 110.5, 108.01, 74.7, 68.2, 67.3, 51.4, 45.8, 41.2, 26.5 and 21.2. ESI-
MS: calculated for C23H26F2N2O4 ([M+H]+) 433:0046; found: 433.0067 Rostein,
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.06.018.