Stereoisomerization of human constitutive androstane receptor agonist CITCO

Article abstract of DOI:10.1016/j.tet.2020.131886

CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl) oxime) is a widely used agonist of the human constitutive androstane receptor (hCAR), a key hepatic xenobiotic sensor protein with emerging therapeutic indications.

Full text of DOI:10.1016/j.tet.2020.131886

Tetrahedron 79 (2021) 131886
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Tetrahedron
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Stereoisomerization of human constitutive androstane receptor
agonist CITCO
Benjamin Diethelm-Varela a, Anmol Kumar a, Caitlin Lynch b, Gregory H. Imler c,
Jeffrey R. Deschamps c, Yue Li d, Menghang Xia b, Alexander D. MacKerell Jr. a,
,
*
Fengtian Xue a
a Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 Penn Street, Baltimore, MD, 21201, United States
b National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, 20892-3375, United States
c Naval Research Laboratory, Code 6930, 4555 Overlook Avenue, Washington, D.C., 20375, United States
d Department of Chemistry and Biochemistry, University of Maryland College Park, College Park, MD, 20740, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl) oxime) is
a widely used agonist of the human constitutive androstane receptor (hCAR), a key hepatic xenobiotic
sensor protein with emerging therapeutic indications. To address the insufficient stability of CITCO,
which limits its therapeutic potential, the E- and Z-isomers of CITCO were synthesized and characterized
by X-ray crystallography, one- and two-dimensional NMR spectroscopy. The two isomers were found to
Received 30 October 2020
Accepted 11 December 2020
Available online 16 December 2020
undergo E/Z isomerizations in solution likely via
a protonation-rotation mechanism, time- and
concentration-dependently. Our molecular modeling studies suggests both stereoisomers can bind to
hCAR.
© 2020 Elsevier Ltd. All rights reserved.
1. Introduction
Upon xenobiotic activation, hCAR regulates the expression of
various hepatic genes that encode important enzymes involved in
phase I oxidation, phase II conjugation, and phase III drug trans-
CITCO
(6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-
carbaldehyde-O-(3,4-dichlorobenzyl) oxime) was first discovered
by Maglich and coworkers in 2003 [1]. Since its introduction, it has
been widely used as a selective agonist for the human constitutive
androstane receptor (hCAR), a key hepatic xenobiotic sensor that
plays important regulatory roles in drug metabolism and activa-
tion, glucose and lipid homeostasis, and cell fate [2e5]. Recently Lin
and coworkers reported that CITCO also directly activates the hu-
man pregnane X receptor (hPXR), another important xenobiotic
receptor [6]. As a small molecule tool, CITCO has been involved in
numerous studies regarding biological regulation of hCAR by
different proteins (e.g., PP2A, [7] ATF5, [8] DAX1, [9] MPR410, and
EGFR [11]) and protein complexes (e.g., proteasome [12] and WNT/
porters [20,21]. Therefore, selective modulation of hCAR has
become a pharmacological strategy to enhance the metabolic effi-
ciency of drugs [22]. This strategy was highlighted by the devel-
opment of hCAR ligands as metabolic enhancers of the
chemotherapy cyclophosphamide [23]. As a positive control, CITCO
was implicated in the development of various biological assays of
hCAR [24e28] and in the discovery of hCAR ligands including ag-
onists, [29e35] antagonists, [36,37] and inverse agonists [36,38].
Recently using the chemical scaffold of CITCO as a parent, efforts
have emerged in the development of novel potent and selective
agonists of hCAR [39,40].
The binding of CITCO to the ligand binding domain (LBD) of
hCAR was confirmed by direct-binding assays including fluores-
cence resonance energy transfer (FRET) and surface plasmon
resonance (SPR) [1]. The crystal structure of CITCO in complex with
the LBD of hCAR has also been solved [41]. The chemical structure
of CITCO contains an oxime functionality connecting the imidazo
[2,1-b]thiazole and dichlorobenzyl moieties (Fig. 1). Interestingly,
while both E- and Z-isomers around the oxime double bond are
possible, in the literature CITCO has been displayed exclusively as
b
-catenin [13]). It has also been used to elucidate the role of hCAR in
regulating its target genes, [14e17] and other molecules such as P-
glycoprotein, multidrug resistance protein, [18] and steroid hor-
mones [19].
* Corresponding author.
E-mail address: fxue@rx.umaryland.edu (F. Xue).
https://doi.org/10.1016/j.tet.2020.131886
0040-4020/© 2020 Elsevier Ltd. All rights reserved.

B. Diethelm-Varela, A. Kumar, C. Lynch et al.
Tetrahedron 79 (2021) 131886
Fig. 1. A. Single crystal structure of E-CITCO. B. Schematized through-space interactions detected using NOESY spectroscopy. NOESY crosspeaks that link protons are represented as
red arrows. C. Potential energy scan of the dihedral "C8eC15eC16eN17" in the range of ꢁ180ꢀ degree to 180ꢀ degree at HF/6-31G(d) model chemistry using Psi4 package [43]. This
potential energy scan spans through conversion of cis-to trans-conformation of E-CITCO, where dihedral value of 180ꢀ (or ꢁ180ꢀ) implies trans-rotamer of two chlorinated moieties
and 0ꢀ implies cis-rotamer. Four different conformations with "C8eC15eC16eN17" dihedral values ꢁ90ꢀ, 0ꢀ, 90ꢀ and 180ꢀ are projected on the plot.
the E-isomer, although no experimental evidence was documented
[1,23,41].
yields, however reactions employing other solvents such as
dioxane and toluene showed decreased yields. Interestingly, we
found that under all tested conditions, CITCO was generated
exclusively as the E-isomer, and this stereoselectivity remained
during the formation of other oximes and the corresponding imine
analog (Table S1). Therefore, the condensation reaction for the
formation of CITCO probably has a kinetic drive that favors the
formation of E-CITCO.
2. Results and discussion
We synthesized CITCO via a condensation reaction of 6-(4-
chlorophenyl)imidazo[2,1-b]thiazole-3-carbaldehyde and O-(3,4-
dichlorobenzyl)hydroxylamine in the presence of AcOH at 70 ꢀC
(Scheme S1, Table 1) [39,42]. The 1H-NMR data indicated the for-
mation of a single product with signals compatible to that of CITCO
(Fig. S1). This product was successfully crystallized and confirmed
by X-ray diffraction studies as E-CITCO, with a trans-configuration
at the oxime double bond C16]N17 (Fig. 1A). The molecule adopts
In the 1H NMR spectrum of E-CITCO in CDCl3, measured one day
after dissolution, two sets of signals with different relative in-
tensities were observed. A time-course of the NMR spectra showed
the original set of signals decreased over time while the newly
formed signal set increased (Fig. S4A). Conversely, the high-
resolution mass spectra of the same NMR sample showed the
presence of the peak at m/z 435.9477, corresponding to CITCO,
while no other peaks were observed (Fig. S4B). The new compound
was purified and successfully crystallized. The X-ray single crys-
tallographic result revealed that the new compound was Z-CITCO,
with the oxime double bond C16]N17 adopting a cis-configuration
(Fig. 2A). While the overall structure of Z-CITCO employed a similar
configuration to that of E-CITCO, the distance between the imidazo
[2,1-b]thiazole and dichlorobenzyl groups was shorter in Z-CITCO
compared to that in the E-isomer. This result was also confirmed by
NOESY experiments (Fig. 2B, S20, and S21), in which new through-
space interactions between hydrogen pairs H12/H21 and H13/H27
were observed in addition to those detected in E-CITCO.
In an attempt to elucidate the agonistic activity of both CITCO
stereoisomers for hCAR, we performed luciferase reporter-based
activation assays using purified E- and Z-isomers along with the
commercially available CITCO (E-isomer). No obvious difference
was detected among the three samples, with the EC50 and Emax
values falling within the same order of magnitude (Fig. 3). While
this result might suggest both stereoisomers are equipotent, the
aforementioned stereoisomerization could also play an important
role. To better understand this reaction, a study of the isomeriza-
tion kinetics was undertaken using 1H-NMR spectroscopy.
an overall t-shaped geometry, with the hydrogen atoms on C1 (H1)
and C16 (H16) close to each other, and the dichlorobenzyl moiety
pointing toward the imidazothiozole core. It is worth noting that
the configuration of E-CITCO in the single crystal was significantly
different to the one reported in the cocrystal structure of E-CITCO in
hCAR, in which the dichlorobenzyl group was in close proximity to
the p-chlorophenyl ring [41]. The crystallographic result was sup-
ported by NOESY spectroscopy in either aqueous environment (10%
D2O in DMSO, Fig. 1B and S19) or organic solvent (CDCl3) (Fig. S21),
which indicated through-space interactions between hydrogen
pairs H1/H16 and H13/H19A, while no interactions were detected
between the imidazothiazole and dichlorophenyl rings. It was
interesting that the same trans rotamer, in which a through-space
interaction observed between H1 and H16, was present in both
solid and solution environments. These experimental observations
were further confirmed by our quantum mechanics (QM) simula-
tion studies. As shown in Fig.1C, the calculated energy for the trans-
rotamer was ꢁ6.5 kcal/mol lower than that of the cis-rotamer.
Next we sought to screen conditions for the synthesis of CITCO
(Table 1). Removal of AcOH from the reaction condition caused a
significant decrease of yields, while a reaction performed at room
temperature only indicated a slight decrease of yields. Changing the
solvent from EtOH to MeOH showed no obvious impact on reaction
The kinetic and mechanistic studies of oxime isomerizations are
known [44e47]. Two distinct mechanisms, nucleophilic catalysis
and protonation-rotation, are proposed for the isomerization re-
actions (Scheme S3). As shown in Fig. 4, at the concentration of
16 mM in CDCl3, continuous isomerization reactions proceeded
until equilibriums were reached with an approximate 20/80 (E/Z)
ratio of the two isomers. The reaction half-lives t1/2 were 17 and 8.1
days for E- and Z-CITCO, respectively (Table 2). The Gibbs free en-
Table 1
Conditions for the synthesis of CITCO.
solvent
Additives
temp (ꢀC)
yielda (%)
E or Zb
EtOH
EtOH
EtOH
MeOH
dioxane
toluene
AcOH
e
70
70
rt
70
70
70
40
26
35
35
22
29
E
E
E
E
E
E
AcOH
AcOH
AcOH
AcOH
ergy
DGZ-E, as determined from the corresponding equilibrium
constant K, were 3.4 and 3.8 kJ/mol, indicating the Z-isomer was
favored under this condition.
Changing the solvent to DMSO-d6 caused no significant differ-
ence in the kinetics of isomerization (Table 2 and Fig. S5). The
addition of D2O as a co-solvent indicated no detectable effect in
a
The low yield was due to a common deformylation reaction of the starting
material aldehyde, yielding p-chlorophenyl imidazothiazole as a byproduct (Scheme
S2, and Figs. S2 and S3).
b
Stereoselectivity determined by analysis of the 1H-NMR spectra.
2

B. Diethelm-Varela, A. Kumar, C. Lynch et al.
Tetrahedron 79 (2021) 131886
Fig. 2. A. Single crystal structure of Z-CITCO. B. Schematized through-space interactions detected using NOESY spectroscopy. NOESY cross peaks that link protons are represented as
red arrows.
Table 2
a
Kinetics of the isomerization of E-CITCO (16 mM) in CDCl3
.
6
4
2
0
additive
t1/2 (days)
k (dayꢁ1
)
K ([E]/[Z])
DGZ-E (KJ/mol)
17
0.041
0.25
3.4
e
b
-
8.1
16
20
19
17
6.2
0.086
0.044
0.036
0.038
0.042
0.11
0.21
0.23
0.25
0.26
0.33
1.9
3.8
3.7
3.4
3.4
2.7
ꢁ1.6
c
DMSO-d6
D2O, 2%
D2O, 4%
Et3N
TFA
a
All experiments were conducted at room temperature.
Z-CITCO was used.
The experiment was performed in DMSO-d6.
b
c
-8
-7
-6
-5
-4
reaction dramatically changed kinetics of the isomerization reac-
tion. In the presence of 1 equiv of TFA, the reaction proceeded much
faster with the t1/2 value of 6.2 days, indicating that the isomeri-
zation likely went through an acid-catalyzed protonation-rotation
mechanism. Meanwhile at equilibrium, the E-CITCO predominated
log CITCO
M
Fig. 3. Dose-dependent activation of hCAR by CITCO samples in an hCAR agonist
luciferase gene reporter assay. Green: commercial CITCO; blue; Z-CITCO: red: E-CITCO.
The incomplete dose response curves were due to the limited solubility of the samples.
the mixture with the K and
DGZ-E values of 1.9 and ꢁ1.6 kJ/mol,
respectively.
Next, a concentration-dependency study was performed (Fig. 5
and Table 3). The t1/2 values of the isomerization reaction were
significantly dependent on the concentration, ranging between 0.1
and over 50 days. For reactions beginning with a concentration of
2.5 mM or higher, at equilibrium the Z-isomer predominated
(K < 0.5). The fraction of E-isomer increases when the concentra-
tion decreases. Meanwhile, shorter reaction times were required to
reach equilibrium when starting with less concentrated samples
(see Fig. S6 for full time courses). The concentration dependency
was pronounced; the sample with a concentration of 2.5 mM
showed t1/2 ¼ 1.1 days compared to t1/2 ¼ 17 days of 16 mM, which
accounts for an over 6-fold increase in time necessary to reach half
the equilibrium concentration. When the concentration was near
the solubility of CITCO (110 mM) in CDCl3, no obvious isomerization
was evidenced by NMR spectroscopy over a 40-day period (data not
shown). Conversely, at the concentration of 100 mM in either CDCl3
or aqueous DMSO-d6 that broadly mimics conditions used for
biological tests, rapid isomerizations were observed with equilib-
riums being reached in 0.1 and 0.3 days, respectively. These results
can explain the apparently identical agonistic activity of both E- and
Z-isomers determined by the luciferase reporter assays, which
involve low to medium micromolar concentrations of the testing
compounds, and require an incubation period of 24 h [48].
These experiments also revealed that the equilibrium position is
concentration-dependent. At higher concentrations, the Z-isomer
seems to be favored in solution, as the equilibrium leads to over 80%
Fig. 4. Isomerization kinetics of CITCO (16 mM) in CDCl3 at room temperature. The 1H-
NMR spectra were recorded over time at room temperature. The proportion of each
isomer was estimated by NMR.
isomerization kinetics, suggesting that the isomerization rate is
probably not dependent on nucleophilic catalysis. While inclusion
of the base triethylamine (Et3N) showed no impact on t1/2, the
isomerization reached a new equilibrium containing more E-iso-
mer with the K and
DGZ-E values of 0.33 and 2.7 kJ/mol, respec-
of this isomer. At lower concentrations, such as 2.5 mM and 100
mM,
tively. Interestingly, the addition of trifluoroacetic acid (TFA) to the
the percentage of Z-isomer in the mixture decreased to
3

B. Diethelm-Varela, A. Kumar, C. Lynch et al.
Tetrahedron 79 (2021) 131886
following a protonation-rotation mechanism.
E
Z
The results from the kinetic studies prompted us to revisit the
binding mode of CITCO by docking both isomers into the reported
electron density map (Fig. 6) [41]. Compared to the binding mode of
E-CITCO proposed in the original cocrystal structure (Fig. 6A), our
docking studies predicted E-CITCO to adopt a highly similar binding
mode (Fig. 6B), with the dichlorobenzyl and p-chlorophenyl groups
close to each other. Interestingly, the Z-isomer was also predicted to
bind favorably to the LBD of hCAR, although with a different
inversed binding mode (Fig. 6C), in which the p-chlorophenyl and
imidazo[2,1-b]thiazole groups bound in opposite positions to those
shown in E-CITCO.
In summary, we have shown that the widely-used hCAR acti-
vator CITCO was synthesized exclusively as the E-stereoisomer.
Both E- and Z-isomers undergo time- and concentration-dependent
stereoisomerization through a protonation-rotation mechanism,
leading to an equilibrium position. Given the rapid rate of isomer-
ization at low concentration, it must be noted that every biological
measurement done with CITCO thus far likely stems from a mixture
of isomers, with the activity of each still being unknown. Our
computational simulations suggest the existence of unique binding
poses for each isomer, both of which match the experimental
electron-density of the published crystal structure. In order to
properly understand the biological properties of this compound,
and to arrive at a three-dimensional pharmacophore, future studies
will involve the synthesis of stable CITCO-like stereoisomers.
d
100
80
60
40
20
0
0
5
10
15
Time days
Fig. 5. Isomerization kinetics of E- and Z-CITCO at different concentrations.
Table 3
Kinetics studies of E- or Z-CITCO at different concentrations in CDCl3
a
.
conc. (mM)
t1/2 (days)
k (dayꢁ1
)
K ([E]/[Z])
DGZ-E (KJ/mol)
69 (E)b
2.5 (E)
0.1 (E)
0.1 (E)c
82 (Z)b
2.5 (Z)
>50
1.1
0.11
0.33
>50
1.9
e
e
e
0.65
6.6
2.1
e
0.41
0.63
0.71
e
2.2
1.2
0.83
e
3. Experimental section
3.1. General procedures
0.36
0.37
2.5
a
All experiments were conducted at room temperature.
Experiments were stopped at day 50 and data points were not enough to pro-
All reagents and solvents were of analytical grade and used
without further purification. Reactions were monitored using thin-
layer chromatography (TLC) on commercial silica-gel plates
(GF254). Flash column chromatography was performed on silica gel
(200e300 mesh). NMR spectra were obtained on a Varian INOVA
400 MHz NMR spectrometer at 25 ꢀC. Chemical shifts are reported
b
duce accurate fits.
c
The NMR sample was dissolved in 10% D2O in DMSO-d6.
approximately 70% and 60%, respectively. Thus, an interplay be-
tween the thermodynamic (equilibrium position) and kinetic
(isomerization rate) parameters is evident in this process, where
the higher the concentration, the more favored the Z-isomer is.
Taken together our X-ray crystallographic and NMR results
provide direct experimental evidence for the formation and isom-
erization of CITCO (Scheme 1). CITCO is generated, via a conden-
sation reaction of 6-(4-chlorophenyl)imidazo[2,1-b]thiazole-3-
carbaldehyde and O-(3,4-dichlorobenzyl)hydroxylamine, as the E-
isomer, which was a kinetic product with the least steric strain.
After formation, both E- and Z-CITCO can undergo a time- and
concentration-dependent isomerization in solution, likely
as
internal reference. Chemical shifts (
referenced to the CDCl3 residual peak (
d
values (parts per million) using the residual solvent peak as an
) were reported in ppm
7.264) for 1H NMR.
d
d
Chemical shifts of 13C NMR were reported relative to CDCl3
(d
77.04). Data for 1H NMR were reported in the following order:
chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; sext,
sextet; m, multiplet), coupling constant (Hz), number of protons.
Data for 13C NMR were reported as
d values (parts per million).
High-resolution mass spectra (HRMS) were obtained on a JEOL
AccuTOF with ESI/APCI ion sources coupled to an Agilent 1100 HPLC
system. HPLC analysis was performed on a Shimadzu HPLC fitted
Scheme 1. Proposed Mechanism for E-CITCO Formation and Stereoisomerization.
4

B. Diethelm-Varela, A. Kumar, C. Lynch et al.
Tetrahedron 79 (2021) 131886
Fig. 6. Comparison of published binding mode of CITCO (A) to docking results of E-CITCO (B) and Z-CITCO (C) using the software AutoDock Vina, overlaid on the electron-density
map of hCAR (yellow mesh).
with a C-18 reversed-phase column (Phenomenex, luna 5
m
M
anhydrous Na2SO4 and concentrated to 100 mL under vacuum. To
the resulting solution, HCl in EtOH (5 mol/L, 8.5 mL) was added
dropwise. The precipitate was filtered under and the filtrate was
washed with diethyl ether to provide the desired product as white
crystals. Yield: 6.2 g (75%).
C18(2) 4.6 mm ꢂ 100 mm) with a flow rate of 0.8 mL/min using
CH3CNeH2O 8:2 mobile phase. The purity of final products is >95%.
3.2. Synthesis
3.2.1. 6-(4-Chlorophenyl)imidazo[2,1-b]thiazole
3.2.4. 6-(4-Chlorophenyl)imidazo[2,1-b]thiazole-3-carbaldehyde
This compound was synthesized by following a procedure that
has been reported [39]. To dimethyl formamide (179 mmol) in an
ice-bath was added phosphorus (V) oxychloride (29.8 mmol)
dropwise. The mixture was stirred for 5 min before the addition of
6-(4-chlorophenyl)imidazo[2,1-b]thiazole (12.4 mmol) as a solu-
tion in dimethyl formamide. The reaction mixture was warmed to
room temperature, and vigorously stirred for 3 h, then potassium
hydroxide (120 mmol) was added as a solution in water (3.8 M). The
mixture was heated under reflux overnight, cooled, and poured
over an ice bath. The resulting precipitate was collected by filtration
under vacuum and washed generously with water to give the
desired product as a white solid that was used in the next step
without further purification. Yield: 3.1 g (95%).
This compound was synthesized by following a procedure that
has been reported [39]. To
a
solution of 4-chloro-2-
bromoacetophenone (15 mmol) in EtOH (15 mL) was added 2-
aminothiazole (15 mmol). The reaction mixture was heated under
reflux for 24 h and cooled down. The solvent was removed under
vacuum, and the resulting material was partitioned between
saturated NaHCO3 (20 mL) and EtOAc (20 mL). The aqueous layer
was extracted by EtOAc (2 ꢂ 20 mL) and the combined organic
layers were dried over anhydrous Na2SO4, and concentrated. The
crude product was purified by flash chromatography using EtOAc/
hexanes (1:4) to yield the titled product as a yellow solid. Yield:
2.8 g (80%).
3.2.2. 6-(4-Chlorophenyl)imidazo[2,1-b]oxazole
This compound was synthesized by following a procedure that
has been reported [39]. A solution of oxazol-2-amine (5 mmol) and
bromomethyl ketone (5 mmol) in THF (20 mL) and CH3CN (30 mL)
was stirred at room temperature for 24 h. The precipitate from the
reaction mixture was collected by filtration and the filtrate was
washed using CH3CN. To a solution of the resulting solid in toluene
(50 mL) at 0 ꢀC was added titanium (IV) chloride (5 mmol) as a
solution in toluene (5 mL) dropwise over a period of 30 min. The
reaction mixture was heated at 100 ꢀC for an additional 3 h, and
cooled. The solvent was removed by rotary evaporation, and ice was
added to the residue. The pH value of the resulting mixture was
adjusted to 9 using Na2CO3, and the resulting solution was
extracted using EtOAc (30 mL ꢂ 3). The organic layers were com-
bined and dried over anhydrous Na2SO4. The concentrated crude
product was dried overnight under vacuum to get the crude
product, which was purified by flash chromatography using EtOAc/
hexanes (1:4) to yield the desired product as a white solid. Yield:
612 mg (56%).
3.2.5. 6-(4-Chlorophenyl)imidazo[2,1-b]oxazole-3-carbaldehyde
This compound was synthesized by following a procedure that
has been reported [39]. To dimethyl formamide (15 mmol) in an
ice-bath was added phosphorus (V) oxychloride (2.5 mmol) drop-
wise. The mixture was stirred for 5 min before the addition of 6-(4-
chlorophenyl)imidazo[2,1-b]thiazole (1 mmol) as a solution in
dimethyl formamide. The reaction mixture was warmed to room
temperature, and vigorously stirred for 3 h, then potassium hy-
droxide (10 mmol) was added as a solution in water (3.8 M). The
mixture was heated under reflux overnight, cooled, and poured
over an ice bath. The resulting precipitate was collected by filtration
under vacuum and washed generously by water to give the desired
product as a white solid that was used in the next step without
further purification. Yield: 234 mg (95%).
3.2.6. General procedure A: synthesis of Aryl-3-carbaldehyde O-
Oximes
Procedure adapted from Liang and coworkers [39]. 6-(4-
3.2.3. O-(3,4-Dichlorobenzyl)hydroxylamine
Chlorophenyl)imidazo[2,1-b]thiazole-3-carbaldehyde
or
This compound was synthesized by following a procedure that
has been reported [49]. To a solution of sodium ethoxide (43 mmol)
in EtOH (70 mL) was added N-hydroxyurethane (43 mmol) drop-
wise, followed by 3,4-dichlorobenzyl chloride (43 mmol). The re-
action mixture was allowed to stir at room temperature for 10 h. A
solution of sodium hydroxide (86 mmol) in water (70 mL) was
added and the resulting suspension was heated under reflux for an
additional 2 h. The EtOH was removed under vacuum and the
resulting aqueous mixture was extracted by diethyl ether
(3 ꢂ 100 mL). The combined organic layers were dried over
(0.38 mmol) was dissolved in ethanol (5 mL). The corresponding
hydroxylamine or amine (0.38 mmol) was added, followed by
acetic acid (1.9 mmol). The mixture was heated at 70 ꢀC for 16 h.
The crude was partitioned between saturated NaHCO3 and EtOAc
and extracted with EtOAc three times. The combined organic layers
were washed with brine and dried over anhydrous Na2SO4. The
crude product was filtered and concentrated under vacuum. The
resulting solid was purified by flash chromatography using 20%
ethyl acetate in hexanes as the solvent system to yield the desired
product as a white solid.
5

B. Diethelm-Varela, A. Kumar, C. Lynch et al.
Tetrahedron 79 (2021) 131886
3.2.7. (E)-6-(4-Chlorophenyl)imidazo[2,1-b]thiazole-3-
carbaldehyde O-(3,4-dichlorobenzyl) oxime (E-CITCO)
(d, J ¼ 4.2 Hz, 1H), 4.14 (t, J ¼ 6.7 Hz, 2H), 1.76 (sext, J ¼ 7.0 Hz, 2H),
1.00 (t, J ¼ 7.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6):
d .152.3,
Synthesized through general procedure
chlorophenyl)imidazo[2,1-b]thiazole-3-carbaldehyde and O-(3,4-
dichlorobenzyl)hydroxylamine. 1H NMR (400 MHz, CDCl3):
8.39
A
using 6-(4-
148.4, 138.5, 134.2, 132.1, 129.5, 128.9, 121.8, 116.3, 112.7, 76.2, 22.4,
10.4. Exact mass calcd for C15H14ClN3OS [M þ Hþ] 319.0546, found
319.0620. Yield: 60 mg (57%). Yield: 69 mg (57%).
d
(s, 1H), 7.99 (d, J ¼ 4.5 Hz, 1H), 7.59 (d, J ¼ 8.3 Hz, 2H), 7.51 (s, 1H),
7.46e7.42 (m, 3H), 7.24 (d, J ¼ 6.3 Hz, 1H), 6.92 (d, J ¼ 4.2 Hz, 1H),
3.2.12. 6-(4-Chlorophenyl)imidazo[2,1-b]oxazole-3-carbaldehyde
O-(3,4-dichlorobenzyl) oxime
5.14 (s, 2H); 13C NMR (100 MHz, CDCl3):
d 152.7, 149.3, 139.9, 137.7,
134.5, 132.6, 132.1, 131.9, 130.5, 130.1, 129.5, 129.0, 127.4, 121.6, 115.7,
113.0, 75.0. HRMS (ESI): Exact mass calcd for C19H12Cl3N3OS
[M þ Hþ] 434.9767, found 434.9477. Yield: 66 mg (40%).
Synthesized through a procedure adapted from Liang and co-
workers
[39].
6-(4-Chlorophenyl)imidazo[2,1-b]oxazole-3-
carbaldehyde (0.38 mmol) was dissolved in ethanol. O-(3,4-
dichlorobenzyl)hydroxylamine (0.38 mmol) was added, followed
by acetic acid (1.9 mmol). The mixture was heated at 70 ꢀC for 16 h.
The crude was parti-tioned between saturated NaHCO3 and EtOAc
and extract-ed with EtOAc three times. The combined organic
layers were washed with brine and dried over anhydrous Na2SO4.
The crude product was filtered and concentrated under vacuum.
The resulting solid was purified by flash chromatography using 20%
ethyl acetate in hexanes to yield the desired product as a white
solid. Yield: 42 mg (26%).
As part of our synthetic study on the stereoselectivity in the
synthesis of CITCO, the general procedure conditions were modi-
fied by substituting the solvent for either methanol, dioxane, or
toluene, by omitting the acid additive, and by assaying the pro-
cedure at room temperature. The results in terms of yield and
stereoselectivity are summarized on Table 1.
3.2.8. (Z)-6-(4-Chlorophenyl)imidazo[2,1-b]thiazole-3-
carbaldehyde O-(3,4-dichlorobenzyl) oxime (Z-CITCO)
Synthesized through general procedure
A
using 6-(4-
chlorophenyl)imidazo[2,1-b]thiazole-3-carbaldehyde and O-(3,4-
dichlorobenzyl)hydroxylamine. The pure E stereoisomer was dis-
solved in acetone to a final concentration of 2.5 mM and left to
isomerize for several days. Z stereoisomer was resolved by flash
3.3. Isomerization time-course
Pure E- or Z-CITCO was weighted on an analytical scale and
dissolved on an appropriate solvent. Catalysts were added as
required, and additional solvent was added to achieve the desired
concentration. Solution was transferred to an NMR tube, and an 1H-
NMR spectrum was immediately acquired employing a d1 ¼ 5s and
16 total scans. The proportion of each stereoisomer was estimated
by normalizing the methylene protons signal integral of the E iso-
chromatography (36 mg). 1H NMR (400 MHz, CDCl3):
d 7.66 (d,
J ¼ 4.1 Hz, 1H), 7.59 (d, J ¼ 8.3 Hz, 2H), 7.49e7.40 (m, 5H), 7.21 (d,
J ¼ 8.3 Hz, 1H), 6.81 (d, J ¼ 4.5 Hz, 1H), 5.20 (s, 2H); 13C NMR
(100 MHz, CDCl3):
d 152.5, 149.9, 137.1, 136.3, 134.6, 132.7, 132.4,
131.9, 130.6, 130.3, 129.9, 128.8, 127.6, 123.1, 115.0, 111.8, 75.3. HRMS
(ESI): Exact mass calcd for C19H12Cl3N3OS [M þ Hþ] 434.9767, found
434.9477.
mer (1H NMR (400 MHz, CDCl3):
calculating the ratio of the relative integral of the methylene pro-
tons in the Z isomer (1H NMR (400 MHz, CDCl3):
5.20 (s, 2H)) to
this value. Samples were kept at room temperature, and spectra
were re-acquired at regular intervals. The proportion of each iso-
mer was calculated with each acquisition as described.
d 5.14 (s, 2H)) to a value of 1, and
3.2.9. 6-(4-Chlorophenyl)imidazo[2,1-b]thiazol-5-yl)-N-(3,4-
dichlorophenethyl)methanimine
d
Synthesized through a procedure adapted from Liang and co-
workers
[39].
6-(4-Chlorophenyl)imidazo[2,1-b]thiazole-3-
carbaldehyde (0.38 mmol) was dissolved in ethanol. 2-(3,4-
dichlorophenyl)ethanamine (0.38 mmol) was added, followed by
acetic acid (1.9 mmol). The mixture was heated at 70 ꢀC for 16 h.
The crude was partitioned between saturated NaHCO3 and EtOAc
and extracted with EtOAc three times. The combined organic layers
were washed with brine and dried over anhydrous Na2SO4. The
crude product was filtered and concentrated under vacuum. The
resulting solid was purified by flash chromatography using 20%
ethyl acetate in hexanes as the solvent system to yield the desired
product as a white solid. Yield: 119 mg (72%).
3.4. X-ray crystallography
Single-crystal X-ray diffraction data on compounds were
collected using Cu Ka radiation and a Bruker Photon II CPAD area
detector. The crystals were prepared for data collection by coating
with high viscosity microscope oil. The oil-coated crystals were
mounted on a micro-mesh mount (MiteGen, Inc.) and transferred
to the diffractometer and a data set collected at 150 K for E-CITCO
and 293 K for Z-CITCO. The structures were solved by direct
methods and refined by full-matrix least squares on F [2] values
using the programs found in the SHELXL suite (Bruker, SHELXL
v2014.7, 2014, Bruker AXS Inc., Madison, WI). Corrections were
applied for Lorentz, polarization, and absorption effects. Parame-
ters refined included atomic coordinates and anisotropic thermal
parameters for all non-hydrogen atoms. The H atoms were included
using a riding model. Complete information on data collection and
refinement is available in the supplemental material.
3.2.10. 6-(4-Chlorophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde
oxime
Synthesized through general procedure
chlorophenyl)imidazo[2,1-b]thiazole-3-carbaldehyde and hydrox-
ylamine hydrochloride. 1H NMR (400 MHz, DMSO-d6):
11.43 (s,
A
using 6-(4-
d
1H), 8.36 (s, 1H), 8.08 (d, J ¼ 4.6 Hz, 1H), 7.71 (d, J ¼ 7.9 Hz, 2H), 7.53
(d, J ¼ 8.3 Hz, 2H), 7.46 (d, J ¼ 4.2 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6):
d 151.4, 146.0, 138.6, 132.7, 132.4, 129.6, 128.8, 126.4,
121.0, 115.0. HRMS (ESI): Exact mass calcd for C12H8ClN3OS
[M þ Hþ] 277.0077, found 277.0154. Yield: 60 mg (57%).
3.4.1. E-CITCO
The 0.070 ꢂ 0.027 ꢂ 0.010 mm3 crystal was monoclinic in space
group C2/c, with unit cell dimensions a ¼ 35.774(11) Å, b ¼ 6.935(3)
3.2.11. 6-(4-Chlorophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde
O-propyl oxime
Å, c ¼ 20.419(4) Å,
a
¼ 90ꢀ,
b
¼ 121.277(14)ꢀ, and
g
¼ 90ꢀ. Data was
Synthesized through general procedure
chlorophenyl)imidazo[2,1-b]thiazole-3-carbaldehyde and O-pro-
pylhydroxylamine. 1H NMR (400 MHz, CDCl3):
8.35 (s, 1H), 8.16 (d,
J ¼ 4.0 Hz, 1H), 7.61 (d, J ¼ 8.4 Hz, 2H), 7.42 (d, J ¼ 8.4 Hz, 2H), 6.92
A
using 6-(4-
95.5% complete to 67.679ꢀ
q with an average redundancy of 2.91.
The final anisotropic full matrix least-squares refinement on F [2]
with 372 variables converged at R1 ¼ 7.27%, for the observed data
and wR2 ¼ 19.38% for all data.
d
6

B. Diethelm-Varela, A. Kumar, C. Lynch et al.
Tetrahedron 79 (2021) 131886
3.4.2. Z-CITCO
results was performed in PyMOL.
Associated content
The 0.078 ꢂ 0.021 ꢂ 0.020 mm3 crystal was monoclinic in space
group P21/c, with unit cell dimensions
a
¼
17.0194(12) Å,
b ¼ 7.1790(5) Å, c ¼ 32.056(2) Å,
a
¼ 90ꢀ,
b
¼ 100.064(4)ꢀ, and
g
¼ 90ꢀ. Data was 80.1% complete to 67.679ꢀ
q with an average
Supporting information
redundancy of 2.31. The final anisotropic full matrix least-squares
refinement on F [2] with 536 variables converged at R1 ¼ 9.34%,
for the observed data and wR2 ¼ 32.91% for all data.
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
General experimental procedures, supplementary data, and
copies of 1H and 13C NMR spectra (PDF).
3.5. hCAR agonist luciferase reporter gene assay
Author contributions
HepG2-CYP2B6-hCAR [48] cells were cultured in DMEM (Invi-
trogen, Carlsbad, CA) supplemented with 5
(Invitrogen), 0.5 mg/mL geneticin (Invitrogen), 10% Hyclone™ FBS
(GE Healthcare Life Sciences, Logan, UT), and 100 U/mL penicillin
mg/mL blasticidin
The manuscript was written through contributions of all au-
thors. All authors have given approval to the final version of the
manuscript.
and 100
mg/mL streptomycin (Invitrogen). For the assay, the HepG2-
CYP2B6-hCAR cells were dispensed at 2500 cells/4
m
L/well in tissue
Declaration of competing interest
cultureetreated 1536-well white assay plates (Greiner Bio-One
North America, Monroe, NC) using a Thermo Scientific Multidrop
Combi (Thermo Fisher Scientific Inc., Waltham, MA). The media
used for plating was DMEM supplemented with 10% Hyclone™ FBS
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
and 100 U/mL penicillin and 100
mg/mL streptomycin. After the
assay plates were incubated at 37 ꢀC/5% CO2 for 5 h, 23 nL of
compounds dissolved in dimethyl sulfoxide (DMSO), CITCO (Sigma-
Aldrich Corp., St. Louis, MO), or DMSO were transferred to the assay
plates by a Wako Pintool station (Wako Automation, San Diego, CA).
Acknowledgement
We thank the Samuel Waxman Cancer Research Foundation for
their funding support to FX. We are grateful to Dr. Kellie Hom of the
Department of Pharmaceutical Sciences, University of Maryland
School of Pharmacy, for her technical advice on our NMR experi-
ments. The X-ray crystallographic work was supported by NIDA
through an Interagency Agreement #Y1-DA1101 with the Naval
Research Laboratory (NRL).
One
centration of 0.75
(FRD, Aurora Discovery, Carlsbad, CA). The final test compound
concentrations in the 5 L assay volume ranged from 6.41 pM to
92 M in 16 different concentrations at a 1:3 dilution. The final
mL of PK11195 (Sigma-Aldrich Corp.) was added (final con-
mM PK11195) using a Flying Reagent Dispenser
m
m
concentration of DMSO (used for the negative control) was 0.46%.
The plate format of the positive control is as follows: Column 1:
concentration-response titration of CITCO from 2.81 nM to 92
a 1:2 dilution with DMSO; Column 2 top half: 60 M of CITCO;
Column 2 bottom half: 48 M of CITCO; Column 3 top half and
Column 4: DMSO only; Column 3 bottom half: 92 M of tetraoctyl
ammonium bromide. After 23 h of incubation at 37 ꢀC/5% CO2, one
L of CellTiter-Fluor™ (Promega, Madison, WI) was added, using
mM at
Appendix A. Supplementary data
m
m
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tet.2020.131886.
m
m
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