2-(3-Methoxyphenyl)quinazoline Derivatives: A New Class of Direct Constitutive Androstane Receptor (CAR) Agonists

Article abstract of DOI:10.1021/acs.jmedchem.5b01891

Constitutive androstane receptor (CAR) is a key regulator of xenobiotic and endobiotic metabolism. Together with pregnane X (PXR) and aryl hydrocarbon (AHR) receptors, it is referred to as xenobiotic receptor. The unique properties of human CAR, such as i

Full text of DOI:10.1021/acs.jmedchem.5b01891

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Article
2-(3-Methoxyphenyl)quinazoline Derivatives: A New Class
of Direct Constitutive Androstane Receptor (CAR) Agonists
Tomáš Smutný, Alice Nova, Marcela Drechslerová, Alejandro Carazo Fernandez, Lucie
Hyrsova, Zuzana Rania Hrušková, Jirí Kunes, Milan Pour, Marcel Spulák, and Petr Pavek
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01891 • Publication Date (Web): 04 May 2016
Downloaded from http://pubs.acs.org on May 6, 2016
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2ꢀ(3ꢀMethoxyphenyl)quinazoline Derivatives: A
New Class of Direct Constitutive Androstane
Receptor (CAR) Agonists
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Tomas Smutny,1 Alice Nova,1,2 Marcela Drechslerová,3 Alejandro Carazo,1 Lucie Hyrsova,1
Zuzana Rania Hrušková,3 Jiří Kuneš,3 Milan Pour,3 Marcel Špulák3* and Petr Pavek.1*
1Department of Pharmacology and Toxicology, 3Department of Inorganic and Organic
Chemistry, Faculty of Pharmacy, Heyrovského 1203, Hradec Kralove, Czech Republic, CZꢀ500
05, 2Institute of Molecular and Translation Medicine, Faculty of Medicine, Palacky University in
Olomouc, Hnevotinska 5, Olomouc, CZꢀ779 00, Czech Republic.
KEYWORDS: nuclear receptor, constitutive androstane receptor, inverse agonist, cellꢀbased
assays, quinazoline.
ABSTRACT
Constitutive androstane receptor (CAR) is a key regulator of xenobiotic and endobiotic
metabolism. Together with Pregnane X (PXR) and Aryl hydrocarbon (AHR) receptors, it is
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referred to as “xenobiotic receptor”. The unique properties of human CAR, such as its high
constitutive activity, both direct (ligandꢀbinding domainꢀdependent) and indirect activation have
hindered the discovery of direct selective human CAR ligands. Herein, we report a novel class of
direct human CAR agonists in a group of 2ꢀ(3ꢀmethoxyphenyl)quinazoline derivatives. The
compounds are even more potent activators of human CAR than is prototype 6ꢀ(4ꢀ
chlorophenyl)imidazo[2,1ꢀb][1,3]thiazoleꢀ5ꢀcarbaldehyde Oꢀ(3,4ꢀdichlorobenzyl)oxime
(CITCO). The three most potent ligands are at the same time extremely potent activators of the
other xenobiotic or hormonal receptors, namely PXR, AHR and Vitamin D receptor, which
regulate major xenobioticꢀmetabolizing enzymes and efflux transporters. Thus, the novel CAR
ligands can be also considered as constituting the first class of potent panꢀxenobiotic receptor
ligands that can serve as potential antidotes boosting overall metabolic elimination of xenobiotic
or toxic compounds.
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INTRODUCTION
Constitutive androstane receptor (CAR, NR1I3) together with pregnane X receptor (PXR,
NR1I2) and aryl hydrocarbon receptor (AHR) are ligandꢀactivated transcription factors that play
pivotal roles in xenobiotic clearance. These transcription factors control gene expression across a
broad spectrum of target genes that encode key phase I and phase II drug metabolizing enzymes
(DMEs) and some drug transporters.1 They are activated by a variety of exogenous ligands that
include drugs, environmental toxicants, industrial chemicals, and herbal compounds. They are
therefore sometimes referred to as “xenosensors” or “xenobiotic receptors.”2
Although CAR was originally identified as a “xenosensor” of environmental, dietary, natural,
and synthetic ligands,3, 4 recent findings suggest that CAR also plays important roles in energy
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metabolism of fatty acids, bile acids, lipids, and glucose; in thyroid hormone metabolism; in cellꢀ
cycle regulation; and in cell–cell interaction.5, 6 CAR is composed of three domains: a highly
conserved DNAꢀbinding domain, a hinge region, and a divergent ligand
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binding/dimerization/transcriptional activation domain.7 In contrast to other nuclear receptors,
activation of this receptor is complex. Both ligandꢀbinding domain (LBD)ꢀdependent and
independent activation of human CAR have been shown. These release CAR from its
cytoplasmic tethering complex and translocate the receptor into the nucleus, where CAR
transactivates CARꢀinducible genes as a heterodimer with retinoid X receptor α (RXRα) nuclear
receptor. Dephosphorylationꢀinduced translocation of CAR to the nucleus is a key step for
indirect activation.8
Numerous ligands, agonists, antagonists, as well as inverse agonists have been reported for
human CAR. There is discrepancy in assigning a CARꢀinteracting compound, since validated
methods are not widely used for direct human CAR ligand identification or distinguishing
between indirect activators (such as acetaminophen, bilirubin, and phenobarbital) and ligands
interacting within its ligandꢀbinding domain. 3, 4 Moreover, CAR is constitutively active and it is
localized in the nucleus in model tumor cell lines as opposed to its cytoplasmic localization in
normal hepatocytes. CAR activation by ligands is determined by their effect on the position of
the Cꢀterminal helix12 that binds coactivators of corepressors. In addition, there are significant
species differences in the ligand specificity of CAR ligands.4 Currently, there is no specific highꢀ
affinity agonist for human CAR that would help decipher the diverse physiological functions of
CAR. Known to date is 6ꢀ(4ꢀchlorophenyl)imidazo[2,1ꢀb]thiazoleꢀ5ꢀcarbaldehydeꢀOꢀ(3,4ꢀ
dichlorobenzyl)oxime (CITCO, 1), which is a potent human – but not mouse – CAR agonist.9
However, this unstable compound also activates PXR.9 The potent mouse CAR ligand 1,4ꢀ
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bis[(3,5ꢀdichloropyridineꢀ2ꢀyl)oxy]benzene (TCPOBOP, 2) does not activate human or rat
CAR.10 Reported activators of human CAR identified to date include pesticides such as
pyrethroids (permethrin, cypermethrin), the carbamate benfuracarb, and organochlorines (e.g.,
methoxychlor); synthetic compounds such as CITCO, FL81, octicizer, thiazolidinꢀ4ꢀones, 6ꢀ
arylpyrrolo[2,1ꢀd][1,5]benzothiazepine derivatives, and sulfonamides; and natural flavonoids
such as chrysin and drugs (e.g., artemisinin derivatives, nevirapine, nicardipine, efavirenz,
carbamazepine).4, 11ꢀ17
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In this work, a library of compounds previously prepared as potential antituberculotics was
subjected to random screening. The screening revealed that 2ꢀ(3ꢀmethoxyphenyl)ꢀ3,4ꢀ
dihydroquinazolinꢀ4ꢀone (3) displayed promising activation of the CAR receptor comparable to
that of CITCO in reporter gene assay. We therefore synthesized other derivatives of 2ꢀ(3ꢀ
methoxyphenyl)quinazoline and analyzed their interaction with the human CAR receptor.
Interaction of the compounds with the additional “xenoreceptors” PXR and AHR as well as with
other nuclear receptors controlling expression of certain drug metabolism genes, such as vitamin
D receptor (VDR) and glucocorticoid receptors (GR)18 was analyzed simultaneously.
Herein we report that three 2ꢀ(3ꢀmethoxyphenyl)quinazoline derivatives are robust ligands of
CAR as well as of the other “xenobiotic receptors” PXR and AHR and even exceed their
prototype ligands in their potencies to activate the receptors in cellꢀbased reporter gene assays.
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RESULTS
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In a random screening of a library previously prepared as potential antituberculotics, we
identified a hit compound 2ꢀ(3ꢀmethoxyphenyl)ꢀ3,4ꢀdihydroquinazolinꢀ4ꢀone (3) that displayed
promising activation of the CAR receptor comparable to that of CITCO. Quinazolinone 3
(Scheme 1) was therefore prepared19 in order to confirm its activity in relationship to the CAR
receptor. Thus, 3ꢀmethoxybenzoic acid (4) was converted to its chloride 5 which was
subsequently treated with 2ꢀaminobenzonitrile to yield amide 6. Compound 6 was then cyclized
to afford the title quinazoline 3 in 55 % overall yield. The repeated evaluation of 2ꢀ(3ꢀ
methoxyphenyl)ꢀ3,4ꢀdihydroquinazolinꢀ4ꢀone (3) in reporter gene assays fully confirmed the
ability to activate the CAR receptor.
Scheme 1. Synthesis of 2ꢀ(3ꢀmethoxyphenyl)ꢀ3,4ꢀdihydroquinazolinꢀ4ꢀone
A library of six 2ꢀ(3ꢀmethoxyphenyl)quinazolines was subsequently prepared via simple
alkylation conditions using RX/NaI/K2CO3 at reflux in acetone (Scheme 2). In contrast to the
alkylation of 3,4ꢀdihydroquinazolinꢀ4ꢀone,20 we observed no N3ꢀalkylated products. The
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reactions afforded only Oꢀalkylated species, the structures of which were clearly confirmed by
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13C NMR shifts of ꢀOꢀCHnꢀ carbon atom (Table 1).20
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Scheme 2. Alkylation of quinazoline 3 with various primary halides
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Table 1. Isolated yields and 13C NMR chemical shifts in CDCl3 of C(1') group of compounds 3a-
f
Compound
Yield (%) δC (ppm)
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54.0
62.7
68.3
72.8
67.3
68.2
3a
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Quinazoline derivative 7a bearing methylsulfanyl moiety in position 4 was further synthesized.
The starting 2ꢀ(3ꢀmethoxyphenyl)ꢀ3,4ꢀdihydroquinazolinꢀ4ꢀone 3 was first converted into its
sulfur analog 7, followed by the same alkylation protocol (Scheme 3).
Scheme 3. Preparation of 4ꢀmethylsulfanyl derivative 7a
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Library screening and ligands identification
We randomly screened a library of compounds previously prepared as potential antituberculotics
for activation of the human CAR receptor using a reporter gene assay with p(ER6)3ꢀluc construct
transfected together with human CAR expression vector into HepG2 cells. This assay showed
itself to be a sensitive test for selecting several potential activators of CAR. 2ꢀ(3ꢀ
methoxyphenyl)ꢀ3,4ꢀdihydroquinazolinꢀ4ꢀone (3) displayed the most promising activation of
CAR in the assay and was used as the “lead” compound.
In the next series of experiments, we tested the interactions of the newly synthesized compounds
3a–f, 7 and 7a with CAR using the same assay (Fig. 1A). Interestingly, we found even more
robust activation of the CARꢀresponsive assay with derivatives 3a, 7 and 7a. The activation was
even more potent than that produced by the prototype CAR activator/ligand CITCO in equimolar
concentrations (Fig. 1A).
Next, the compounds 3, 3a, 7 and 7a were tested using luciferase gene reporter vectors with
promoter responsive sequences of CAR target genes CYP3A4 and CYP2B6. We found that 7, 3a
and 7a compounds significantly activated CYP3A4ꢀluc and CYP2B6ꢀluc reporter constructs in a
doseꢀdependent manner through both wildꢀtype CAR (CAR) characterized by high constitutive
activity as well as its variant 3 (CAR3) with low constitutive activity21 (Fig. 1B, C). All tested
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compounds appeared as lower affinity but high potency activators of human CAR and CAR3
receptors in the assays, as indicated by the EC50 and Imax parameters (Fig. 1C). No effects of
compounds 7, 3a and 7a on HepG2 cell viability have been observed (Fig. 1 Supporting
information).
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In next set of experiments, we employed mammalian two hybrid assay with GAL4ꢀCAR LBD
and VP16ꢀSRC1 fusion constructs. All tested compounds 7, 3a and 7a significantly stimulated
interaction of CAR ligand binding domain with steroid receptor coactivator 1 (SRC1)ꢀVP16
fusion protein (Fig. 1D).
Finally, we used the in vitro LanthaScreen timeꢀresolved (TR)ꢀFRET Constitutive Androstane
Receptor (CAR) Coactivator Assay that monitors ligandꢀdependent, but not indirect
phenobarbital activatorꢀdependent, interaction with CAR ligand binding domain (LBD) as we
have shown in our previous report22. We observed that the tested compounds 7, 3a and 7a all
activated the assay, thus suggesting direct interaction with human CAR LBD (Fig. 1E). These
activations were, however, weaker than that of CITCO, therefore indicating potential indirect
cellular signalingꢀdependent effects of the quinazoline compounds on CAR activation or a ligand
specific interactions of CAR with its coactivators SRCꢀ1 (Fig. 1D) and PGC1α (Fig. 1E) or
corepressors based on position of helix 12 determined by the ligands.23, 24
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Figure 1. Compounds 7, 3a and 7a activate human wildꢀtype CAR and its CAR variant 3 in
transient transfection reporter gene assays and in TRꢀFRET CAR coactivation assays
Compounds 7, 3a and 7a activate PXR, AHR and VDR receptors
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In the next set of experiments, we analyzed whether 7, 3a and 7a compounds activate other
xenobiotic receptors, such as PXR, AHR and VDR, which control expression of many xenobiotic
metabolizing enzymes in the liver and intestine.25 We found that compounds 7, and, to a lesser
extent 3a and 7a significantly activated both PXR and AHR receptors, as indicated by low
micromolar EC50 and high Imax (Fig. 2A and B). In addition, compound 7 significantly activated
the Vitamin D receptor (VDR) in transient transfection reporter gene assay in low micromolar
concentrations (EC50=0.51 ꢀM) (Fig. 2C). Glucocorticoid receptor was not significantly
activated by the compounds (data not shown).
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Figure 2. Compounds 7, 3a and 7a activate PXR, AHR and VDR receptors in transient
transfection reporter gene assays
Compounds 7, 3a and 7a induce prototype CAR target gene CYP2B6 mRNA in HepG2
cells and in primary human hepatocytes
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We analyzed whether compounds 7, 3a and 7a induce CYP2B6 mRNA expression in HepG2
cells expressing exogenous human CAR or in three primary human hepatocyte preparations.
CYP2B6 is the most responsive target gene of the CAR receptor. We found statistically
significant induction of CYP2B6 mRNA expression both in CARꢀexpressing HepG2 cells and in
human hepatocytes after treatment with compounds 7, 3a and CITCO (Fig. 3A and B).
Compound 7a most likely undergoes fast metabolism in metabolically competent primary human
hepatocytes since we did not observed CYP2B6 mRNA induction (Fig. 3B).
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Compounds 7 and 3a up-regulate other major inducible xenobiotic-metabolizing enzymes
and P-glycoprotein transporter mRNAs
Compounds 7, 3a and 7a also significantly induced CYP3A4, CYP3A5, CYP2C9, MDR1/Pꢀ
glycoprotein genes controlled by CAR, PXR and VDR as well as AHR target genes CYP1A1,
CYP1A2 (also regulated by CAR), CYP1B1 and UGT1A1 (also regulated by PXR) (Fig. 3C).
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Figure 3. Compounds 7 and 3a induce target genes expression in CARꢀexpressing HepG2,
HepaRG cells or in primary human hepatocytes
Compounds 7, 3a and 7a do not significantly inhibit major cytochrome P450 enzymes
CYP3A4 and CYP2C9
Finally, we analyzed if compounds 7, 3a and 7a interfere enzymatic activities of the most
abundant liver CYP enzymes induced by CAR, PXR, VDR and AHR receptors. We found that
tested compound do not significantly affect CYP3A4 and CYP2B6 enzyme activities (Fig. 4A,
B). On the other hand, compounds 7, 3a and 7a inhibit enzymatic activity of CYP1A2 enzyme,
which activates numerous procarcinogens to active carcinogens in the liver (Fig. 4C). In case of
CYP2C9, the IC50 were at concentrations leading to CAR, PXR or VDR nuclear receptors
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activation (Fig. 4D). We observed no significant interaction of tested compounds with CYP2D6
enzyme (Fig. 4E).
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These data thus indicate that enzymatic activities of the major induced CYP liver enzymes via
CAR, PXR and VDR are not significantly affected with the compounds and that compound 7
least interferes with the enzymes.
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Figure 4. Inhibition of recombinant human CYP3A4, CYP2B6, CYP1A2, CYP2C9 and
CYP2D6 enzymes with compounds 7, 3a and 7a
DISCUSSION
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CAR is one of the key regulators of xenobiotic and endobiotic metabolism. Recently, the
therapeutic potential for treatment of such metabolic disorders as obesity, type 2 diabetes
mellitus, insulin resistance, dyslipidemia, and atherosclerosis has been indicated in animal
models using a ligand of rodent CAR.26 However, there exists no potent, specific, and nontoxic
agonist of human CAR receptor that can serve as a chemical tool to address various biological
functions of CAR or to consider CAR as a therapeutic target. The unique properties of human
CAR, such as its high constitutive activity and the complexity of the related signaling, make
discovery of specific ligands difficult. In addition, the lack of robust and validated cellꢀbased
assays to study direct interactions of compounds with the CAR ligandꢀbinding pocket (LBP)
hinders the discovery of selective human CAR ligands. Therefore, the ligand specificity of
human CAR as well as of highꢀaffinity endogenous ligands remains obscure.11
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Our aim was to discover novel compounds that would interact with the human CAR receptor as a
direct agonist with high potency. We randomly screened a library comprising about 400
compounds for the activation of human CAR receptor using a reporter gene assay with pER6ꢀluc
construct transfected together with human CAR expression vector into HepG2 cells. This assay
provided to be a sensitive test for selecting the lead compound based on 2ꢀ(3ꢀmethoxyphenyl)ꢀ
3,4ꢀdihydroquinazolinꢀ4ꢀone (3) structure (Fig. 1A). The synthesized compounds 7, 3a and 7a
displayed even more potent activation of CAR in transient transfection reporter gene assays (Fig.
1A and B), in two hybrid CAR LBD/SRCꢀ1 interaction assay (Fig.1D), and in induction
experiments in HepG2 cells transfected with exogenous CAR (Fig. 3A). At the same time, we
observed activation of variant 3 of the human CAR gene with compounds 7, 3a and 7a (Fig. 1C),
which was found to be a ligandꢀactivated lowꢀconstitutive activity receptor variant. CAR3 differs
from CAR1 by a 5 amino acid insertion outside of the ligand binding pocket.21, 27 Interestingly,
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in TRꢀFRETꢀbased coactivator LBD competitive assay, CITCO activated the system more
significantly at equimolar concentrations than did compounds 7, 3a and 7a (Fig. 1E). This
phenomenon might reflect additional cellular or epigenetic effects of compounds 7, 3a and 7a
that stimulate CAR transactivation23 or indicate ligand dependent coactivation with PGC1α.
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Unlike other NRs that have evolved as receptors for specific highꢀaffinity endogenous ligands,
CAR and PXR are highly promiscuous. Only two human CAR crystal structures with agonist
bound have been reported to date.28 The CAR LBD sequence creates a ligandꢀbinding pocket
with volume ranging from 525 to 675 Å.5, 28 The LBD cavity has flexible and hydrophobic
character, which correlates with the structural promiscuity of CAR ligands.4, 7 The apoꢀPXR
binding cavity volume is even double (approximately1150 Å3) and can extend in the presence of
ligands to 1290–1540 Å3. Cavity of VDR is larger but narrower than that of CAR (870 Å3) (Fig.
2 Supporting information). A single residue (T350M) difference in the Cꢀterminal region of the
mouse versus human CAR might account for the extensive species selectivity for some agonists.7
The coꢀactivator recruitment appears to play a central role in fixing ligands in the correct
arrangement in large CAR and PXR cavities. In addition, it was clearly shown that ligands
determine helix12 position, which is critical for coactivator or corepressor binding and
subsequent CAR activation.24
The ligand pockets of CAR, PXR as well as VDR are lined by mostly hydrophobic residues. The
27 residues of the murine CAR pocket create a highly hydrophobic environment, with only a
quarter of them being polar. Similarly, the cavity of PXR is lined by 28 amino acids, of which
eight have polar or charged side chains.5, 29 Therefore van der Waals forces play crucial roles in
the interactions of the ligand with CAR and hydrogen bonding is rare. For example, CITCO form
no hydrogen bonds and makes weak electrostatic interactions with His203, Asn165, and Tyr326.
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Another CAR agonist 5βꢀpregnanedione forms a single hydrogen bond with Hisꢀ2035, 30, but
display hydrophobic interactions with Phe161, Ile164, Leu206, Phe217, Tyr224, Phe234, and
Leu242 in human CAR cavity.28 In our docking study, we found that compound 7 and 3a form
hydrophobic bonding with three amino acids Phe161, Phe234, and Leu242 that also interact with
CITCO and 5βꢀpregnanedione (Fig. 2 Supporting information).
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CAR appears to crossꢀtalk with other two members of subfamily NR1I PXR (NR1I2) and VDR
(NR1I1) since these receptors recognize similar response elements, coactivators and corepressors
and share a number of similar target genes involved in xenobiotic detoxification.31 CAR, PXR
and VDR mainly control inducible expression of such major xenobioticꢀmetabolizing enzymes
of cytochrome P450 as CYP3A4, CYP2C9, CYP2B6 or Pꢀglycoprotein efflux transporter in the
hepatocytes or in enterocytes.32
Aryl hydrocarbon receptor (AHR) is another key ligandꢀactivated "xenosensor", although it
belongs to the basic helixꢀloopꢀhelix/PerꢀSimꢀARNT (bHLH/PAS) family of transcription
factors. It controls another set of genes involved in the metabolism and transport of xenobiotics
including CYP1A1, CYP1A2, CYP1B1, GST1, UGT1A1, UGT1A6, NQO1, ALDH3A1, and
BCRP transporter.33, 34 The AHR ligands comprise a wide variety of known toxic and highly
hydrophobic environmental contaminants, such as halogenated aromatic hydrocarbons including
2,3,7,8ꢀtetrachlorodibenzoꢀpꢀdioxin (TCDD), polychlorinated biphenyls (PCBs), and the
polycyclic aromatic hydrocarbons (PAHs) benzopyrene and 3ꢀmethylcholanthrene (MC).35 Many
AHR agonists represent planar aromatic chemicals; however, the SAR analysis showed that
absolute planarity is not necessary for ligand binding of polychlorinated biphenyls. It was
estimated that an AhR ligand are hydrophobic, electronegative compounds with
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hydrogenbonding properties between 12 Åꢀ14 Å in length, less than 12 Å in width and no more
than 5 Å deep.35
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Due to enormous substrate ligand variability of xenosensors PXR, CAR and AHR, the receptors
are sometimes supposed not to strictly discriminate between molecules on the basis of size or
chemical structure. The only one 3D QSAR model has been published for human CAR.24
Considering our tested compounds structural features, we can see that methylation of oxygen or
sulfur in position 4 of the quinazoline ring significantly increases activation of CAR (Fig. 1A).
On the contrary, alkylation of quinazoline 3 employing longerꢀchain alkyl halides (b-f) abolish
activity toward CAR activation. This correlates with our docking experiment showing reasonable
positioning of compounds 7 and 3a in CAR LBD (Fig. 2 Supporting information).
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CONCLUSION
In summary, we have shown that compounds 7, 3a and 7a are potent ligands of human CAR
receptor. We also observed in the study that compounds 7, 3a and 7a are at the same time highly
potent ligands of the xenobiotic receptors PXR and AHR. We can therefore suppose that the
compounds boost overall xenobiotic metabolism via induction of their target genes. To our
knowledge, this is the first group of such xenobiotic metabolism “boosters” and panꢀxenobiotic
receptor agonists with potential application as antidotes to toxic compounds dominantly cleared
from the body through metabolism. We assume that such antidotes could be seriously considered
for treatment of intoxication by some natural compounds (such as mycotoxins) or synthetic toxic
compounds, or, in cases of drugꢀinduced liver injury (DILI),36 under the assumption that the
toxicants are mainly eliminated by biotransformation and are not significantly bioactivated to
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reactive intermediates by the xenobiotic detoxification system. Secondly, the new class of
metabolism activators might be considered in some cases for controlled and stimulated activation
of prodrugs.
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Additional experiments should further characterize the toxicological properties of the compounds
and consider their applications as antidotes in several intoxications or as prodrug activators. In
addition, the compound could help us to more precisely characterize the limits of xenobiotic
metabolism in different tissues or in the organism as a whole. We are currently synthesizing
further set of quinazoline derivatives with a view to identify either more potent human CAR
ligands or more selective human CAR agonists.
EXPERIMENTAL SECTION
Biology
Activation of human CAR in transient transfection reporter gene assays (Fig. 1). HepG2
(A,B) or COSꢀ1 (C) cells were transiently transfected with either p(ER6)3ꢀluc (A), CYP3A4ꢀluc
(B) or CYP2B6ꢀluc (C) luciferase gene reporter constructs together with wildꢀtype CAR
expression vector (A, B) or its lowꢀconstitutive activity variant CAR3 (C) and pRLꢀTK control
plasmid for transfection normalization. After 24 h of stabilization, the cells were treated with the
tested compounds 7, 3a and 7a, CITCO and artemisinin (CAR agonists), clotrimazole an inverse
agonist) in indicated concentrations or with vehicle (DMSO; 0.1%, v/v) for an additional 48 h
(A) or 24 h (B, C). HepG2 cells transfected with CYP3A4ꢀluc and wildꢀtype CAR have been in
parallel treated with androstenol (5 µM) to suppress high basal activity (Fig. 1B). In experiments
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with CAR3, RXRα has been cotransfected into COSꢀ1 cells (100 ng/well). After treatment, the
cells were lysed and assayed for both firefly and Renilla luciferase activities. The data are
presented as the means ± SD from triplicate measurements of a representative experiment (A) or
as means of three independent experiments (n=3). The results are expressed as the foldꢀchange in
inducing firefly luciferase activities relative to vehicleꢀtreated cells (normalized to 1) *p <0.05 ꢀ
statistically significant. (D) Compounds 7, 3a and 7a activate interaction of CAR ligand binding
domain with SRCꢀ1 coactivator in two hybrid assay. HepG2 cells have been transfected with
GAL4ꢀCAR LBD construct together with VP16ꢀSRC1 construct and pGL5ꢀluc reporter vector.
After 24 h of stabilization, cells were treated with tested compounds, CITCO and clotrimazole
(known ligands of CAR, 10 µM) or vehicle (0.1%, control) for 24 h. Cells were lysed and
analyzed for firefly and Renilla luciferase activities. The results are expressed as the foldꢀchange
in inducing firefly luciferase activities relative to vehicleꢀtreated cells (normalized to 1). (E)
Effect of compounds 7, 3a and 7a on CAR activation in the TRꢀFRET LanthaScreen CAR
Coactivator Assay. Compounds were tested in a serial dilution together with the prototype CAR
agonist CITCO. Data are presented as the relative activation to background activity (no CAR
LBD in the reaction mixture, set to 0%) and to the effect of CITCO (1 ꢀM) set as 100%
activation. The dotted line represents the constitutive activity of CAR LBD (vehicleꢀtreated
samples). Data are presented as the means and S.D. from three independent experiments (n=3).
EC50 indicates the xenobiotic concentration required to achieve halfꢀmaximum activation and
relative Imax represents the overall maximal calculated activation produced by the tested
compound (i.e., maximal efficacy).
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Activation of PXR, AHR and VDR receptors in transient transfection reporter gene assays
(Fig. 2). HepG2 (A, B) or Huhꢀ7 (C) cells were transiently transfected with either p3A4ꢀluc (A),
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pXREꢀluc (B), or pDR3ꢀluc (C) luciferase gene reporter constructs together with appropriate
expression vectors pSG5ꢀPXR (A) or pSG5ꢀVDR (C) and pRLꢀTK control plasmid for
transfection normalization. After 24 h of stabilization, the cells were treated with the tested
compounds 7, 3a and 7a, 3ꢀmethylcholantrene (an AHR ligand), rifampicin (a PXR ligand),
1α,25ꢀdihydroxyvitamin D3 (1,25OHvit D3, a VDR ligand) at the concentration range indicated
for an additional 24 h. After treatment, the cells were lysed and assayed for both firefly and
Renilla luciferase activities. The results are expressed as the relative change in induction of
firefly luciferase activities versus vehicleꢀtreated cells (normalized to 1), when activation with
maximal tested concentration of 3ꢀmethylcholantrene (10 µM), rifampicin (25 µM), and
1,25OHvitD3 (100 nM) was set to 100%. The data are presented as the relative means ± SD from
triplicate measurements of three independent experiments (n = 3). EC50 (xenobiotic
concentration required to achieve halfꢀmaximum promoter activation) and relative Imax
(representing the overall maximal calculated induction produced by the tested compound ꢀ i.e.,
maximal efficacy) values were determined using GraphPad Prism Software.
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Induction of target genes mRNA expression in CAR-expressing HepG2 cells, HepaRG cells
or in primary human hepatocytes (Fig. 3). (A) HepG2 cells were transfected with CAR
expression plasmid and 24 h later treated with the tested compounds 7, 3a and 7a (1, 5 or 10 µM)
or CITCO (1 µM) or vehicle (DMSO; 0.1%, v/v) for 24 h. (B) Three primary human hepatocyte
preparation have been treated with tested compounds 7, 3a and 7a (10 µM) or CITCO (10 µM)
or vehicle (DMSO; 0.1%, v/v) for 24 h. (C) Primary human hepatocytes (Batch No. 2220900,
Biopredic) or HepaRG cells (for CYP1A1 and CYP1B1) have been treated with 3ꢀ
methylcholanthrene (3ꢀMC, an AHR prototype ligand, 10 µM), rifampicin (rif, a prototype PXR
ligand, 10 µM), CITCO (CAR ligand, 10 µM) and tested compounds 7, 3a and 7a (10 µM).
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Total RNA was isolated and CYP2B6 (the gene dominantly controlled by CAR) or CYP1A1,
CYP1A2, CYP1B1, CYP2C9, CYP3A4, CYP3A5, MDR1/ABCB1 and UGT1A1 genes mRNAs
were assessed via qRTꢀPCR. The data in Fig. 1A are presented as the means ± SD from three
experiments (n = 3) performed in triplicates and are expressed as the foldꢀchange in CYP2B6
mRNA induction relative to vehicleꢀtreated cells (normalized to 1). In Fig. 1B and 1C,
experiments were performed with three samples for each treatment. The values were normalized
to HPRT mRNA as a reference gene. *p < 0.05 indicates statistically significant difference from
vehicleꢀtreated cells transfected with appropriate reporter construct (ANOVA with Dunnett’s
post hoc test).
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Inhibition of recombinant human CYP3A4, CYP2B6, CYP1A2, CYP2C9 and CYP2D6
enzymes with compounds 7, 3a and 7a (Fig. 4). Assays with recombinant human enzymes
CYP3A4, CYP2B6, CYP1A2, CYP2C9 and CYP2D6 have been performed according to
manufacturer’s protocols (Promega). IC50 (xenobiotic concentration required to achieve halfꢀ
maximum enzyme inhibition) was determined using GraphPad Prism Software fitting for each
compound.
Transient transfection and luciferase reporter gene assays
All transient transfection assays were carried out using TransFectin transfection reagent
purchased from BioꢀRad (Hercules, CA, USA) in HepG2, HuH7 or COSꢀ1 cells, as described
previously.37 DNA constructs are described in Supplementary data. Briefly, cells were seeded
into 48ꢀwell plates and transfected with a luciferase reporter construct (150 ng/well), an
expression plasmid (100 ng/well), and the Renilla reniformis luciferase transfection control
plasmid (pRLꢀTK) (30 ng/well) 24 h later. In case of mammalian two hybrid assay, HepG2 cells
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have been transfected with GAL4ꢀCAR LBD (100 ng/well), VPꢀ16ꢀSRCꢀ1 (100 ng/well) and
pGL5ꢀluc (150 ng/well) constructs in 48ꢀwell plate format. Cells were maintained in a phenol
redꢀfree medium (220 µl) supplemented with 10% charcoal/dextranꢀstripped FBS and treated
with the tested compounds (at indicated concentrations in a range from 0.1 up to 30 µM) or
reference compounds CITCO, artemisinin38 and clotrimazole (CAR ligands and agonists, 10
µM), 1,25OHvitD3 (10 nM, a VDR receptor ligand), rifampicin (10 µM, PXR ligand), 3ꢀ
methylcholantrene (10 µM, an AHR ligand) for 24 h. Reference compound have been purchased
from SigmaꢀAldrich. Luminescence activity in the cell lysate was measured using a
commercially available luciferase detection system (Dual Luciferase Reporter Assay Kit,
Promega). The data are expressed as the foldꢀchange of firefly luciferase activity normalized to
Renilla luciferase activity in each sample and relative to the vehicle (DMSO 0.1%)ꢀtreated
controls, which were set equal to 1. Activity of a reference agonist at the maximum tested
concentration has been set equal to 100% activation.
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Primary cultures of human hepatocytes and HepaRG cells
Preparation of primary human hepatocytes and information about donors is described in
Supplementary data. HepaRG cells were cultivated as we described recently.39
TR-FRET Constitutive Androstane Receptor (CAR) Coactivator Assay
LanthaScreen timeꢀresolved (TR)ꢀFRET Constitutive Androstane Receptor (CAR) Coactivator
Assay was performed according to the recently published protocol.22 Data are presented as
means and SD from three independent experiments, and the curve fitting was performed using a
sigmoidal dose response (variable slope) algorithm in GraphPad software ver. 6. Activity of
CITCO at the maximum tested concentration has been set equal to 100% activation.
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qRT-PCR analysis
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Total RNA was isolated from HepG2 or HepaRG cells and primary human hepatocytes treated
with the tested compounds or with CITCO (1 µM), rifampicin (10 µM), and 3ꢀMC (10 µM) for
24 h. qRTꢀPCR expression analyses were performed as previously described.37 In expression
experiments with CARꢀexpressing HepG2 cells, 2 × 105 cells were seeded into 12ꢀwell plates
and transiently transfected with the pCR3ꢀhCAR expression construct. After 24 h of
stabilization, the cells were treated with the tested compounds for 24 h in the indicated
concentrations. The data are presented as the foldꢀchanges in gene expression relative to the
vehicleꢀtreated control (DMSO 0.1%) samples (set equal to 1). Statistical analyses were
performed using the means of at least three independent experiments (n = 3) performed in
triplicate.
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Chemistry
General. The structural identities of the prepared compounds were confirmed by 1H NMR and
13C NMR spectroscopy (Varian MercuryꢀVx BB 300 and Varian VNMR S500 spectrometers).
Each of the reported compounds had ≥95% purity, as determined by combustion analysis
(CHNSꢀOCE FISONS EA1110CE automatic microanalyzer instrument).
Experimental procedure for the preparation of compound 3: A mixture of 3ꢀmethoxybenzoic
acid 4 (9.700 g, 63.8 mmol) and thionyl chloride (30 mL) was heated under reflux for 3 h. The
resultant carboxylic acid chloride 5 was purified by distillation with 95 % yield and immediately
used for the next reaction step.
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Chloride 5 (7.165 g, 42.0 mmol) was slowly added to a cooled (0 °C) solution of 2ꢀ
aminobenzonitrile (4.962 g, 42.0 mmol) and dry pyridine (17 mL). The reaction mixture was
stirred at 0 °C for 4 h, then pyridine was removed under reduced pressure and crude product was
recrystallized from an ethanol–water (2:1) mixture with quantitative yield.
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A mixture of the resultant amide 6 (7.291 g, 28.9 mmol), sodium hydroxide (32 g) and aqueous
hydrogen peroxide solution (30 %, 48 mL) in water (200 mL) was heated at 100 °C for 1 h. After
cooling to room temperature, the reaction mixture was acidified with concentrated aqueous
hydrogen chloride solution (36 %) to pH ≅ 4. The product crystallized out of the solution, after
which it was filtered, washed with water and dried to afford 2ꢀ(3ꢀmethoxyphenyl)ꢀ3,4ꢀ
dihydroquinazolinꢀ4ꢀone (3) in 58 % yield. White crystalline compound, mp. 214 °C, mp. lit.40
209–210 °C. 1H NMR: (300 MHz, DMSOꢀd6) δ 12.52 (1H, bs, OH), 8.14–8.18 (1H, m, Ar),
7.85–7.71 (4H, m, Ar), 7.54–7.21 (2H, m, Ar), 7.15–7.11 (1H, m, Ar), 3.83 (3H,s,OCH3); 13C
NMR: (75 MHz, DMSOꢀd6) δ 162.5, 159.5, 152.3, 148.8, 134.8, 134.2, 129.9, 127.7, 126.8,
126.1, 121.2, 120.3, 117.8, 112.7, 55.6; IR: 1047, 1148, 1223, 1251, 1267, 1286, 1310, 1445,
1471, 1482, 1586, 1612, 1670, 3038 cm−1; LRMS (APCI): m/z (relative intensity) 253.6 [M+H]+
(100), 130.6 (3), 88.6 (1.5), 87.6 (6), 73.6 (8); Anal. Calcd for C15H12N2O2: C, 71.42; H, 4.79; N,
11.10. Found: C, 71.38; H, 5.00; N, 11.21.
General experimental procedure for the preparation of compounds 3a–f: A mixture of 2ꢀ(3ꢀ
methoxyphenyl)ꢀ3,4ꢀdihydroquinazolinꢀ4ꢀone (3, 0.5 mmol), sodium iodide (0.05 mmol),
potassium carbonate (2.5 mmol) and an appropriate alkylating agent (2.5 mmol) in acetone (5
mL) was heated under reflux for 24 h. The resultant mixture was diluted with ethyl acetate (10
mL), washed with brine (10 mL), and the organic phase was dried with sodium sulfate. Crude
products were purified by column chromatography (hexane – ethyl acetate 9:1).
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4-Methoxy-2-(3-methoxyphenyl)quinazoline (3a). Yield: 85 %. Yellowish crystalline
compound, mp. 92–94 °C. 1H NMR: (500 MHz, CDCl3) δ 8.22–8.15 (3H, m, Ar), 8.03–7.99
(1H, m, Ar), 7.86–7.8 (1H, m, Ar), 7.55–7.41 (2H, m, Ar), 7.08–7.03 (1H, m, Ar), 4.30 (3H, s,
OCH3), 3.96 (3H, s, OCH3); 13C NMR: (125 MHz, CDCl3) δ 167.0, 159.8, 159.8, 151.8, 139.7,
133.4, 129.4, 128.0, 126.4, 123.4, 121.1, 116.6, 115.3, 113.4, 55.4, 54.0. IR: 1047, 1108, 1184,
1195, 1282, 1327, 1355, 1380, 1443, 1453, 1509, 1561, 1574, 1620, 2966 cm−1; LRMS (APCI):
m/z (relative intensity) 267.6 [M+H]+ (100), 252.6 (75), 224.6 (7), 223.6 (25), 148.6 (7); Anal.
Calcd for C16H14N2O2: C, 72.17; H, 5.30; N, 10.52. Found: C, 72.26; H, 5.37; N, 10.43.
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4-Ethoxy-2-(3-methoxyphenyl)quinazoline (3b). Yield: 91 %. Yellowish crystalline
compound, mp. 93 °C, mp. lit.41 not mentioned. 1H NMR: (300 MHz, CDCl3) δ 8.24–8.14 (3H,
m, Ar), 8.02–7.97 (1H, m, Ar), 7.84–7.77 (1H, m, Ar), 7.5–7.41 (2H, m, Ar), 7.08–7.03 (1H, m,
Ar), 4.8–4.72 (2H, q, J = 7.2 Hz, OCH2), 3.94 (3H, s, OCH3), 1.56–1.51 (3H, t, J = 7.2 Hz, CH);
13C NMR: (75 MHz, CDCl3) δ 166.5, 159.7, 159.7, 151.6, 139.6, 133.2, 129.3, 127.8, 126.2,
123.4, 120.9, 116.4, 115.3, 113.3, 62.7, 55.3, 14.3; IR: 1023, 1050, 1105, 1161, 1282, 1322,
1346, 1356, 1380, 1426, 1458, 1507, 1561, 1575, 2989 cm−1; LRMS (APCI): m/z (relative
intensity) 281.5 [M+H]+ (24), 255.5 (2), 254.4 (13), 253.6 (100), 238.5 (4), 210.5 (6), 91.5 (5);
Anal. Calcd for C17H16N2O2: C, 72.84; H, 5.75; N, 9.99. Found: C, 72.81; H, 5.64; N, 10.08.
2-(3-Methoxyphenyl)-4-propoxyquinazoline (3c). Yield: 88 %. Yellowish amorphous
compound. 1H NMR: (300 MHz, CDCl3) δ 8.24–8.17 (3H, m, Ar), 8.02–7.98 (1H, m, Ar), 7.83–
7.78 (1H, m, Ar), 7.53–7.41 (2H, m, Ar), 7.07–7.03 (1H, m, Ar), 4.65 (2H, t, J = 7.0 Hz, OCH2),
3.93 (3H, s, OCH3), 2.06–1.93 (2H, m, CH2), 1.15 (3H, t, J = 7.0 Hz, CH3); 13C NMR: (75
MHz, CDCl3) δ 166.7, 159.7, 151.7, 139.6, 133.3, 129.3, 127.8, 126.2, 123.4, 121.0, 116.4,
115.3, 113.3, 68.3, 55.3, 22.1, 10.6; IR: 1046, 1105, 1162, 1252, 1279, 1346, 1363, 1421, 1499,
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1561, 1575, 1965 cm−1; LMRS (APCI): m/z (relative intensity) 295.6 [M+H]+ (16), 254.4 (12.5),
253.6 (100), 238.6 (3), 210.6 (3); Anal. Calcd for C18H18N2O2: C, 73.45; H, 6.16; N, 9.52.
Found: C, 73.12; H, 6.00; N, 9.31.
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4-Isobutyloxy-2-(3-methoxyphenyl)quinazoline (3d). Yield: 78 %. Yellowish amorphous
compound. 1H NMR: (300 MHz, CDCl3) δ 8.23–8.15 (3H, m, Ar), 8.01–7.97 (1H, m, Ar), 7.82–
7.77 (1H, m, Ar), 7.52–7.41 (2H, m, Ar), 7.07–7.03 (1H, m, Ar), 4.49 (2H, d, J = 6.8 Hz,
OCH2), 3.95 (3H, s, OCH3), 2.35–2.22 (1H, m, CH), 1.14 (6H, d, J = 6.8 Hz, CH3); 13C NMR:
(75 MHz, CDCl3) δ 166.7, 159.8, 159.7, 151.7, 139.7, 133.3, 129.3, 127.9, 126.2, 123.4, 121.0,
116.4, 115.4, 113.3, 72.8, 55.3, 27.9, 19.3; IR: 1047, 1107, 1162, 1253, 1280, 1344, 1385, 1421,
1461, 1499, 1561, 1575, 1620, 2959 cm−1; LRMS (APCI): m/z (relative intensity) 309.6 [M+H]+
(7.5), 255.5 (2), 254.4 (16), 253.6 (100), 238.6 (2.5), 210.6 (3); Anal. Calcd for C19H20N2O2: C,
74.00; H, 6.54; N, 9.08. Found: C, 73.87; H, 6.44; N, 9.21.
4-Allyloxy-2-(3-methoxyphenyl)quinazoline (3e). Yield: 58 %. Yellowish crystalline
compound, mp. 50–52 °C. 1H NMR: (300 MHz, CDCl3) δ 8.22 (3H, m, Ar), 8.02–7.98 (1H, m,
Ar), 7.82–7.77 (1H, m, Ar), 7.52–7.4 (2H, m, Ar), 7.07–7.03 (1H, m, Ar), 6.31–6.18 (1H, m,
CH), 5.56 (1H, d, J = 17.2 Hz, =CH2 A), 5.37 (1H, d, J = 10.4 Hz, =CH2 B), 5.21 (2H, d, J = 5.5
Hz, OCH2), 3.95 (3H, s, OCH3); 13C NMR: (75 MHz, CDCl3) δ 166.1, 159.7, 159.5, 151.7,
139.5, 133.4, 132.6, 129.3, 127.9, 126.3, 123.4, 120.9, 118.3, 116.5, 115.2, 113.3, 67.3, 55.3; IR:
1001, 1052, 1109, 1217, 1280, 1317, 1339, 1355, 1408, 1457, 1500, 1561, 1574, 1619, 2939
cm−1; LRMS (APCI): m/z (relative intensity) 293.6 [M+H]+ (100), 291.7 (2.5), 265.6 (3), 264,6
(11), 253.6 (50), 250.7 (9), 210.6 (2). Anal. Calcd for C18H16N2O2: C, 73.95; H, 5.52; N, 9.58.
Found: C, 74.16; H, 5.65; N, 9.42.
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4-Benzyloxy-2-(3-methoxyphenyl)quinazoline (3f). Yield: 78 %. White crystalline compound,
mp. 109–111 °C. 1H NMR: (300 MHz, CDCl3) δ 8.32–8.17 (3H, m, Ar), 8.07–8.03 (1H, m, Ar),
7.84–7.78 (1H, m, Ar), 7.64–7.6 (2H, m, Ar), 7.52–7.37 (5H, m, Ar), 7.13–7.08 (1H, m, Ar),
5.76 (2H, s, OCH2), 3.97 (3H, s, OCH3); 13C NMR: (75 MHz, CDCl3) δ 166.2, 159.7, 159.5,
151.7, 139.4, 136.4, 133.3, 129.3, 128.4, 128.1, 128.0, 127.8, 126.3, 123.3, 120.9, 116.5, 115.1,
113.2, 68.2, 55.2; IR: 1041, 1091, 1222, 1255, 1280, 1331, 1342, 1356, 1416, 1459, 1500, 1563,
1575, 1600, 1619, 2940 cm−1; LRMS (APCI): m/z (relative intensity) 343.6 [M+H]+ (65), 253.5
(1), 92.5 (7.5), 91.5 (100); Anal. Calcd for C22H18N2O2: C, 77.17; H, 5.30; N, 8.18. Found: C,
77.01; H, 5.32; N, 8.34.
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Experimental procedure for the preparation of compound 7: A mixture of 2ꢀ(3ꢀ
methoxyphenyl)ꢀ3,4ꢀdihydroquinazolinꢀ4ꢀone (3, 1.000 g, 4.0 mmol) and phosphorus
pentasulfide (0.880 g, 4.0 mmol) in toluene (10 mL) was heated under reflux for 4 h. The
reaction mixture was poured onto ice and washed with chloroform (3x20 mL). Collected organic
layers were dried with sodium sulfate. Crude product was purified by column chromatography
(hexane – ethyl acetate 1:1) to afford thiol 7 in 83 % yield. Yellow crystalline compound, mp.
222–225 °C, mp. lit.42 not mentioned. 1H NMR: (500 MHz, DMSOꢀd6) δ 13.84 (1H, bs, SH),
8.64–8.57 (1H, m, Ar), 7.92–7.86 (1H, m, Ar), 7.8–7.63 (3H, m, Ar), 7.6–7.55 (1H, m, Ar),
7.48–7.43 (1H, m, Ar), 7.16–7.12 (1H, m, Ar), 3.87 (3H, s, OCH3); 13C NMR: (125 MHz,
DMSOꢀd6) δ 187.9, 159.4, 151.4, 144.5, 135.6, 133.5, 129.8, 129.5, 128.5, 128.2, 127.8, 121.0,
118.0, 113.3, 55.6; IR: 1029, 1040, 1154, 1189, 1222, 1252, 1263, 1339, 1492, 1508, 1570, 2985
cm−1; LRMS (APCI): m/z (relative intensity) 269.9 [M+H]+ (100), 253.6 (9), 235.5 (19), 226.5
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(6), 210.6 (3); Anal. Calcd for C15H12N2OS: C, 67.14; H, 4.51; N, 10.44; S, 11.95. Found: C,
67.01; H, 4.42; N, 10.28; S, 11.90.
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Experimental procedure for the preparation of compound 7a: A mixture of 2ꢀ(3ꢀ
methoxyphenyl)ꢀ3,4ꢀdihydroquinazolinꢀ4ꢀthione (7, 0.5 mmol), potassium carbonate (2.5 mmol)
and an methyl iodide (2.5 mmol) in acetone (5 mL) was heated under reflux for 24 h. The
resultant mixture was diluted with ethyl acetate (10 mL), washed with brine (10 mL), and the
organic phase was dried with sodium sulfate. The crude product was purified by column
chromatography (hexane – ethyl acetate 9:1) to afford 2ꢀ(3ꢀmethoxyphenyl)ꢀ4ꢀ
methylsulfanylquinazoline (7a) in 79 % yield. Yellowish crystalline compound, mp. 89–91 °C.
1H NMR: (300 MHz, CDCl3) δ 8.28–8.21 (2H, m, Ar), 8.11ꢀ8.01 (2H, m, Ar), 7.86–7.8 (1H, m,
Ar), 7.56–7.41 (2H, m, Ar), 7.09–7.4 (1H, m, Ar), 3.95 (3H, s, OCH3), 2.83 (3H, s, SCH3); 13C
NMR: (75 MHz, CDCl3) δ 171.4, 159.8, 158.6, 148.6, 139.5, 133.6, 129.5, 129.0, 126.7, 123.7,
122.6, 121.1, 116.7, 113.4, 55.4, 12.6; IR: 1039, 1217, 1249, 1328, 1348, 1429, 1483, 1494,
1559, 1600, 2924 cm−1; LMRS (APCI): m/z (relative intensity) 283.6 [M+H]+ (100), 268.5 (7),
250.6 (3.5), 236.5 (5.5), 235.6 (38), 192.6 (1); Anal. Calcd for C16H14N2OS: C, 68.06; H, 5.00;
N, 9.92; S, 11.35. Found: C, 68.31; H, 4.89; N, 10.11; S, 11.17.
ASSOCIATED CONTENT
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Supporting Information. General experimental procedures, supplementary data and copies of
1H and 13C NMR spectra of compounds 3, 3a-f, 7, 7a. This material is available free of charge
via the Internet at http://pubs.acs.org.
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*Corresponding Author (biology): Petr Pavek, Ph.D., Department of Pharmacology and
Toxicology, Faculty of Pharmacy, Heyrovského 1203, Hradec Kralove, Czech Republic, CZ500
05; Tel.: +420 495 067 334; Fax: +420 495 067 170; Eꢀmail address: petr.pavek@faf.cuni.cz
*Corresponding Author (chemistry): Marcel Špulák, Ph.D., Department of Inorganic and
Organic Chemistry, Faculty of Pharmacy, Heyrovského 1203, Hradec Kralove, Czech Republic,
CZ500 05; Tel.: +420 495 067 348; Fax: +420 495 067 166; Eꢀmail address: spulak@faf.cuni.cz
ACKNOWLEDGMENT
This work was supported by Projects Nos. 303/12/0472 (biology) and 15ꢀ07332S (Chemistry) of
the Czech Science Foundation. Postgraduate student (Z. R. H.) acknowledges Charles University
for financial support (Project No. 398315).
ABBREVIATIONS USED
AhR, aryl hydrocarbon receptor; CAR, constitutive androstane receptor; CYP450, cytochrome
P450; GR, glucocorticoid receptor; LBD, ligand binding domain; MRP2, multidrug resistanceꢀ
associated protein; NR, nuclear receptor; PGC1α, peroxisome proliferatorꢀactivated receptor
gamma, coactivator 1 α (PPARGC1A); PXR, pregnane X receptor; SLC, solute carrier family;
SRCꢀ1, steroid receptor coactivator 1 (NCOA1); TCDD, 2,3,7,8ꢀtetrachlorodibenzoꢀpꢀdioxin;
XME, xenobioticꢀmetabolizing enzyme.
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