Human constitutive androstane receptor agonist DL5016: A novel sensitizer for cyclophosphamide-based chemotherapies

Article abstract of DOI:10.1016/j.ejmech.2019.06.031

The DNA alkylating prodrug cyclophosphamide (CPA), alone or in combination with other agents, is one of the most commonly used anti-cancer agents. As a prodrug, CPA is activated by cytochrome P450 2B6 (CYP2B6), which is transcriptionally regulated by the

Full text of DOI:10.1016/j.ejmech.2019.06.031

Accepted Manuscript
Human constitutive androstane receptor agonist DL5016: A novel sensitizer for
cyclophosphamide-based chemotherapies
Dongdong Liang, Linhao Li, Caitlin Lynch, Bryan Mackowiak, William D. Hedrich,
Yong Ai, Yue Yin, Scott Heyward, Menghang Xia, Hongbing Wang, Fengtian Xue
PII:
S0223-5234(19)30559-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.031
EJMECH 11434
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 April 2019
Revised Date: 10 June 2019
Accepted Date: 11 June 2019
Please cite this article as: D. Liang, L. Li, C. Lynch, B. Mackowiak, W.D. Hedrich, Y. Ai, Y. Yin, S.
Heyward, M. Xia, H. Wang, F. Xue, Human constitutive androstane receptor agonist DL5016: A novel
sensitizer for cyclophosphamide-based chemotherapies, European Journal of Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.ejmech.2019.06.031.
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ACCEPTED MANUSCRIPT
Human constitutive androstane receptor agonist DL5016: a novel sensitizer for
cyclophosphamide-based chemotherapies
a,1
a,1
b
a
a,d
Dongdong Liang, Linhao Li, Caitlin Lynch, Bryan Mackowiak, William D. Hedrich,
a
a
c
b
a,
,
a,
Yong Ai, Yue Yin, Scott Heyward, Menghang Xia, Hongbing Wang, ** and Fengtian Xue *
a
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy,
Baltimore, Maryland 21201, United States
b
9
800 Medical Center Drive, National Center for Advancing Translational Sciences, National
Institutes of Health, Bethesda, Maryland 20892, United States
c
BioIVT, 1450 S Rolling Rd, Halethorpe, Maryland 21227, United States
d
Current address: Bristol-Myers Squibb, Wallingford, Connecticut
Corresponding authors
*
*H.W.: phone, 410-706-1280; fax, 410-706-5017;
E-mail, hongbing.wang@rx.umaryland.edu
F.X.: phone, 410-706-8512; fax, 410-706-0886;
*
E-mail, fxue@ rx.umaryland.edu
1
D.L. and L.L. contributed equally to this work.
KEYWORDS: Human constitutive androstane receptor, cyclophosphamide, structure-activity
relationship, co-culture, chemotherapy
1

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ABBREVIATIONS
CAR, constitutive androstane receptor; CPA, Cyclophosphamide; CYP2B6, cytochrome P450
2
B6; CYP3A4, cytochrome P450 3A4; CLL, chronic lymphocytic leukemia; ALL,
lymphoblastic leukemia; PXR, pregnane X receptor; TCPOBOP, 1,4-bis(3,5-dichloro-2-
pyridinyl oxy)benzene; HTS, high throughput screening; CITCO, 6-(4-chlorophenyl)imidazo-
[2,1-b]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime; LBD, ligand binding domain;
TLC, thin-layer chromatography; RIF, rifampicin; PB, phenobarbital; HPH, human primary
hepatocytes.
2

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GRAPHIC ABSTRACT
3

ACCEPTED MANUSCRIPT
ABSTRACT
The DNA alkylating prodrug cyclophosphamide (CPA), alone or in combination with other
agents, is one of the most commonly used anti-cancer agents. As a prodrug, CPA is activated by
cytochrome P450 2B6 (CYP2B6), which is transcriptionally regulated by the human constitutive
androstane receptor (hCAR). Therefore, hCAR agonists represent novel sensitizers for CPA-
based therapies. Among known hCAR agonists, compound 6-(4-chlorophenyl)imidazo-[2,1-
b
]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) is the most potent and broadly
utilized in biological studies. Through structural modification of CITCO, we have developed a
novel compound DL5016 (32), which has an EC50 value of 0.66 µM and EMAX value of 4.9 when
activating hCAR. DL5016 robustly induced the expression of hCAR target genes CYP2B6, at
both the mRNA and protein levels, and caused translocation of hCAR from the cytoplasm to the
nucleus in human primary hepatocytes. The effects of DL5016 were highlighted by dramatically
enhancing the efficacy of CPA-based cytotoxicity to non-Hodgkin lymphoma cells.
4

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1
. Introduction
Cyclophosphamide (CPA), an alkylating prodrug of the oxazaphosphorine family, remains
one of the most widely prescribed anti-cancer drugs [1]. CPA is used, often in combination with
other agents, for the treatment of numerous malignant diseases including Hodgkin and non-
Hodgkin lymphomas, multiple myeloma, chronic lymphocytic leukemia (CLL), acute
lymphoblastic leukemia (ALL), and solid tumors such as ovarian cancer, breast cancer, small cell
lung cancer, and sarcoma [1]. Although CPA and its combination regimens have successfully
improved the survival rates of patients with early stage cancer, their effects for those with late
stage diseases are often limited. Thus, there is still an urgent need for new CPA-based
treatments with an enhanced efficacy and better controlled toxicity. One of the potential
solutions is to increase the metabolic conversion of CPA to its pharmacologically active
metabolite [2].
CPA is biotransformed predominantly in the liver to the active metabolite 4-hydroxy
cyclophosphamide by cytochrome P450 isozyme 2B6 (CYP2B6) [3,4]. In addition to this
desirable metabolic pathway, CPA can also be converted into a pharmacologically inactive
metabolite dechloroethyl-CPA, along with another toxic metabolite, 2-chloro acetaldehyde,
almost exclusively by CYP3A4 [5]. It is well documented that in the human body, the
expression of CYP2B6 and CYP3A4 are positively regulated by nuclear receptors constitutive
androstane receptor (CAR) and pregnane X receptor (PXR), respectively, with certain levels of
cross-talk [6-9]. While activation of PXR has been shown to induce expression of CYP3A4 and
CYP2B6 in concert, selective activation of CAR has shown preferential induction of CYP2B6
over CYP3A4 [6-9]. Therefore, development of potent and selective human CAR (hCAR)
agonists represents a promising approach to sensitize CPA-based chemotherapies [2].
5

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As a part of its xenobiotic response mechanism, CAR is activated by a wide variety of
ligands including environmental chemicals and drugs [10-12]. For instance, Tzameli and
coworkers showed that the cytochrome P450 inducing agent 1,4-bis(3,5-dichloro-2-pyridinyl
oxy)benzene (TCPOBOP) [13] functions by selective activation of mouse CAR [14]. Sueyoshi
and coworkers reported that the polybrominated diphenyl ether, BDE-47, was a non-selective
activator for both CAR and PXR [15]. Various high-throughput screening (HTS) efforts have
been devoted to the identification of novel and selective hCAR agonists [16-19]. Among others,
a
screening campaign from GlaxoSmithKline led to the identification of 6-(4-
chlorophenyl)imidazo-[2,1- ]thiazole-5-carbaldehyde- -(3,4-dichlorobenzyl)oxime (CITCO,
b
O
Figure 1), which binds to the ligand binding domain (LBD) of hCAR and activates the receptor
with a high potency [20,21]. Since its discovery, CITCO has been widely used as a biological
tool in numerous studies investigating the functions of hCAR [22-25]. Although CITCO can
activate human PXR at higher concentrations, its high affinity for hCAR leads to the potent and
preferred induction of CYP2B6 over CYP3A4 in human primary hepatocytes (HPH) [20].
Recently, synthetic efforts have started to emerge in developing novel hCAR agonists. Smutny
and coworkers disclosed their efforts in the synthesis of 2-(3-methoxyphenyl) quinazoline-based
hCAR agonists, although the top compounds of the series failed to significantly improve the
expression levels of hCAR regulated genes [26].
Herein, we disclose the development of a novel hCAR agonist, DL5016, from a series of new
compounds 1-32 (Figure 1). The compounds have been designed by modifying the chemical
structure of CITCO. Specifically, the oxime linker of CITCO, which might cause chemical
instability [26,27], was replaced by functional groups such as amine, amide, imine, and ether. In
addition, the three hydrophobic ring systems of CITCO, imidazothiazole, 3,4-diCl-phenyl and 4-
6

ACCEPTED MANUSCRIPT
Cl-phenyl, were optimized using a variety of substituted arene moieties, for their hCAR potency
and selectivity over hPXR. Our lead compound of the series, DL5016, was stable in an aqueous
environment and, activated hCAR with good potency and selectivity over hPXR. DL5016
caused hCAR translocation from the cytoplasm into the nucleus of HPH and induced the
expression of CYP2B6 at both the mRNA and protein levels. In co-culture of HPH and human
non-Hodgkin lymphoma cells, DL5016 also potentiated the cytotoxicity of the CPA-based
combination regimen CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) against
the targeted lymphoma cells.
Figure 1. Design of compounds 1-32 by modifying the structure of CITCO.
2
. Results and discussion
2
.1. Chemistry
The syntheses of compounds 1-32 are summarized in Schemes 1-5. To optimize the linker of
the agonist, compounds 1-5 (Scheme 1) were prepared using a common precursor 33 (Scheme
S1). Condensation of aldehyde 33 with 2-(3,4-dichloro phenyl)ethan-1-amine in the presence of
acetic acid (AcOH) generated imine 1 in modest yields. Reduction of compound 1 using NaBH4
gave secondary amine 2 in modest yields. Methylation of the amino group of compound 2 using
MeI provided tertiary amine 3 in modest yields [28]. The amide analog 4 was synthesized via a
7

ACCEPTED MANUSCRIPT
2
two-step procedure from 33. NaClO mediated oxidation of aldehyde 33 generated carboxylic
acid 34, which was coupled to 2-(3,4-dichlorophenyl)ethan-1-amine using EDC and DMAP to
generate amide 4 [29]. To get the ether analog 5, aldehyde 33 was first reduced to primary
alcohol 35 using NaBH in good yields. Then, the resulting hydroxy group of 35 was activated
4
using MsCl to give compound 36 [30]. Finally, nucleophilic substitution of mesylate 36 using 2-
(3,4-dichlorophenyl)ethan-1-ol as a nucleophile provided ether analog 5 [31].
Scheme 1. Synthesis of compounds 1-5. Reagents and conditions: (a) 2-(3,4-
o
dichlorophenyl)ethan-1-amine, AcOH, EtOH, 90 C, overnight, 50%; (b) NaBH , MeOH, rt, 6 h,
4
o
6
4%; (c) MeI, NaH, DMF, 60 C, overnight, 61%, (d) NaClO
2
, NaH
2
PO
4
, 2-methyl-2-butene, t-
2
BuOH/H O/THF, rt, overnight, 85%; (e) 2-(3,4-dichlorophenyl)ethan-1-amine, EDCI, DMAP,
DCM, rt, overnight, 65%; (f) NaBH
4
3
, MeOH, rt, 2 h, 75%; (g) MsCl, Et N, DCM, 2 h; (h) 2-
o
(
3,4-dichlorophenyl) ethan-1-ol, K CO , DMF, 60 C, overnight, 28% for two steps.
2
3
To optimize the imidazothiazole core of CITCO, three compounds (6-8) were synthesized
using imidazole derivatives 37-39 as the starting materials (Scheme 2). Vilsmeier reaction of
compounds 37-39 using DMF and POCl
3
gave aldehydes 40-42 in modest to good yields [32].
8

ACCEPTED MANUSCRIPT
Reductive amination of aldehydes 40-42 using 2-(3,4-dichloro phenyl)ethan-1-amine give final
compounds 6-8 in modest yields [33].
o
Scheme 2. Synthesis of compounds 6-8. Reagents and conditions: (a) DMF, POCl
3
, CHCl
3
, 0 C
to reflux, 6 h, 65-87%; (b) i) 2-(3,4-dichlorophenyl)ethan-1-amine, AcOH, EtOH, reflux,
overnight; ii) NaBH , MeOH, rt, overnight, 22-57% for two steps.
4
To optimize B-ring, compounds 9-18 were synthesized (Scheme 3). Condensation of various
amines with aldehyde 33 generated the corresponding imines, which were then submitted to a
4
NaBH mediated reduction to give the final compounds 9-18 in modest overall yields [33].
Scheme 3. Synthesis of compounds 9-18. Reagents and conditions: (a) i) R-NH , AcOH, EtOH,
2
4
reflux, overnight; ii) NaBH , MeOH, rt, overnight, 15-49% for two steps.
To optimize A-ring, compounds 19-29 were synthesized using a collection of bromomethyl
ketones 43a-k (Scheme 4). Treatment of compounds 43a-k with thiazol-2-amine in refluxing
EtOH provided imidazothiazoles 44a-k [34], which were submitted to Vilsmeier reaction using
9

ACCEPTED MANUSCRIPT
DMF and POCl
3
to give aldehydes 45a-k in good yields [32]. Reductive amination of aldehydes
4
5a-k with 2-(3,4-dichlorophenyl)ethan-1- amine generated compounds 19-29 in good overall
yields [33].
Scheme 4. Synthesis of compounds 19-29. Reagents and conditions: (a) thiazol-2-amine, EtOH,
o
reflux, overnight; (b) DMF, POCl
3
, CHCl
3
, 0 C to reflux, 6 h, 51-69% for two steps; (c) i) 2-
(3,4-dichlorophenyl)ethan-1- amine, AcOH, EtOH, reflux, overnight; ii) NaBH
4
, MeOH, rt,
overnight, 12-56% for two steps.
The syntheses of compounds 30-32 started with bromomethyl ketone 43k (Scheme 5).
Condensation of compound 43k with either 2-amino-thiazole or 2-amino-oxazole gave
compounds 44k and 46, respectively [34,35]. Vilsmeier reaction of compounds 44k and 46
3
using DMF and POCl yielded the corresponding aldehydes 45k and 47 [32], which underwent
reductive amination with either 2-phenylethan-1-amine or 2,3-dihydro-1H- inden-2-amine to
provide the final products 30-32 [33].
10

ACCEPTED MANUSCRIPT
Scheme 5. Synthesis of compounds 30-32. Reagents and conditions: (a) 2-amino-thiazole, EtOH,
o
reflux, overnight; (b) i) 2-amino-oxazole, CH CN/THF, rt, 24 h; ii) TiCl , toluene; 0-100 C, 4 h;
3
4
o
(
c) DMF, POCl , CHCl , 0 C to reflux, 6 h, 51-79% for two steps; (d) i) amine, AcOH, EtOH,
3 3
4
reflux, overnight; ii) NaBH , MeOH, 0-rt, overnight, 26-42% for two steps.
2
.2. Structure-activity relationship (SAR)
To modify the chemical structure of CITCO, we first replaced the unstable oxime linker with
various functional groups (1-5, Figure 2A, blue). The EC and E values of new compounds
5
0
MAX
were determined using an hCAR luciferase gene reporter assay [18]. Compared to the parent
CITCO, the imine analog 1 was a much weaker agonist for hCAR (EC50 = 54 µM vs 3.1 µM for
CITCO, Figure 2A). The poor potency of this compound was probably due to the instability of
the imine group, which hydrolyzes readily in aqueous solutions to generate the inactive
intermediate 33. The secondary amine 2, on the other hand, provided a 1.4-fold increase in
potency and a slightly decreased EMAX value compared to CITCO. The additional methyl group
of the tertiary amine analog 3 turned out to be detrimental to both the EC and E values of
MAX
5
0
the compound. Decreased activities were also observed for compound 4 that employed a more
rigid amide linker. Interestingly, the ether analog 5 indicated an excellent EC50 value of 0.19 µM
while having a slightly decreased EMAX value. These results showed that flexible small linkers,
such as secondary amine and ether, are preferred for hCAR agonist activity.
11

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Using secondary amine 2 as a template, three compounds 6-8 were synthesized by modifying
the imidazothiazole core (Figure 2A, red). Introduction of a methyl group at the 2-position of the
thiazole ring caused a 5.5-fold (EC = 2.2 µM vs 0.4 µM for 2) drop of the potency. This result
5
0
is consistent with the fact that the imidazothiazole moiety of CITCO binds to the bottom of a
hydrophobic pocket in the LBD of hCAR (Figure 6) [21]. Replacement of the sulfur atom with
an oxygen yielded compound 7 with a 3.2-fold decrease in the EC value. However, compared
5
0
to compound 2, the resulting compound also demonstrated a small increase in the EMAX value.
These results showed that the imidazo[2,1-b]oxazole could be a good substitution of the
imidazothiazole core, which forms toxic metabolites in vivo [36]. Expansion of the core to an
imidazo[1,2-a]pyrimidine ring system resulted in a new compound (8) with a 2-fold decrease in
potency and a slight decrease of the EMAX value.
Compounds 2, 5, 7 and 8 were selected for further evaluation based on their effects in
activating hCAR and hPXR using the HepG2 cell reporter assays (Figure 2B) [37]. At a
concentration of 1 µM, compounds 2 and 5 demonstrated similar efficacies in activating hCAR,
although the more potent compound 5 in the hCAR agonist assay also significantly activated
hPXR. These results together indicated that the secondary amino linker is key for both potency
and specificity of hCAR agonists. Note that the imidazo[2,1-b]oxazole analog 7, although
indicated a slight decrease in hCAR activation potency, still indicated good specificity for
hCAR. The imidazo[1,2-a]pyrimidine analog 8, at the same tested concentration (1 µM), was
less potent compared to CITCO and the other three compounds.
12

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Figure 2. Activation of hCAR and hPXR by CITCO and compounds 1-8. (A) Concentration
dependent activation curves along with EC and E values of CITCO and compounds 1-8 in
MAX
5
0
the hCAR agonist luciferase gene reporter assays. EC50 values obtained from at least three
independent experiments in quadruplicate were expressed as mean ± SEM. EMAX values were
calculated by nonlinear regression. (B) Activation of hCAR and hPXR by CITCO and
compounds 2, 5, 7 and 8 in the HepG2 cell reporter assays. Data obtained from three
independent experiments were expressed as mean ± SD normalized against positive control
CITCO for hCAR activities or rifampicin (RIF) for hPXR activities. *, P < 0.05; **, P < 0.01.
Next, substitutions of the 3,4-diCl-phenyl ring of CITCO were studied (Figure 3). When one
of the two Cl atoms was removed, the resulting compounds 9 and 10 demonstrated 1.6- and 7-
fold decreases in potency, respectively (Figure 3A). These results indicated that the 4-Cl group
contributes more significantly to hCAR agonist activity. When the 4-Cl was changed to a
smaller F, the resulting compound 11 indicated a 3-fold decrease of potency while keeping a
similar EMAX value. When a larger Br (12) was introduced to the same position, although the
EC50 was further decreased, the EMAX value of the resulting compound increased significantly.
13

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Inclusion of a polar OH group led to a new compound (13) with a significantly decreased EC50
,
indicating that polar substitutions were not preferred on the bottom ring. Interestingly, the
analog with an unsubstituted phenyl group (14) turned out to be a potent hCAR agonist, and its
analog with one CH shorter (15) indicated an over 11-fold decrease in potency. These results
2
highlighted the importance of linker length in maintaining the agonistic potency of hCAR
ligands. Three new compounds (16-18) with fused ring systems were also tested. While the
indole analog 16 generated modest potency (EC50 = 2.7 µM) with a significant increase in EMAX
,
it was exciting to see the 2,3-dihydro-1H-inden-2-yl analog 17 indicated good EC50 and EMAX
values. The large naphthyl compound (18) was inactive towards hCAR.
Compounds 9, 14, and 17 were selected for further evaluation of their activities in activating
hCAR and hPXR using the HepG2 cell reporter assays (Figure 3B) [37]. All three compounds,
at 1 µM, had similar activation effects compared to CITCO, while also maintaining selectivity
for hCAR against hPXR.
14

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Figure 3. Activation of hCAR and hPXR by CITCO and compounds 9-18. (A) Concentration
dependent activation curves alongside EC50 and EMAX values of compounds 9-18 in high
throughput hCAR agonist luciferase gene reporter assays. EC50 values obtained from at least
three independent experiments in quadruplicate were expressed as mean ± SEM. EMAX values
were calculated by nonlinear regression. (B) Activation of hCAR and hPXR by CITCO and
compounds 9, 14, and 17 in the HepG2 cell reporter assays. Data obtained from three
independent experiments were expressed as mean ± SD normalized against the positive control,
CITCO for hCAR activities or RIF for hPXR activities. **, P < 0.01.
We then optimized the 4-Cl-phenyl ring of CITCO using a collection of compounds 19-29
(
Figure 4). Replacement of the phenyl group with a small aliphatic ethyl group (19) caused a
dramatic drop in hCAR activity (Figure 4A). This result indicated that an aromatic system at this
15

ACCEPTED MANUSCRIPT
position is important for the potency of hCAR agonists. Moving the Cl from the 4-position to 3-
position (20) caused a 2.3-fold decrease in the EC50 (0.93 µM vs 0.40 µM) for compound 2.
However, the new compound (20) also had an increased EMAX (from 2.8 to 3.2). These results
suggested that the 3-substitution might also be beneficial for new agonists. Replacement of the
Cl with a similar-sized Me group led to the formation of compound 21 that had similar EC and
5
0
EMAX values, while with a more polar CF group, new compound 22 was a much weaker agonist.
3
Substitution of the Cl with smaller F or H atoms led to new compounds 23-24 with excellent
potencies, although their EMAX values were decreased. The interesting profiles of these
compounds (23-24) make them good candidates for hCAR activation at low concentrations. In
addition, large substitution at the 4-position, such as Br (25), OMe (26), CN (27) and t-Bu (28),
were also tested. While compounds 25 and 26 had comparable EC and E values to the
MAX
5
0
parent 2, compounds 27 and 28 were significantly less active. Interestingly, the naphthyl analog
9, although with a 3-fold decrease in potency, had a large EMAX value of 3.6. These results
2
indicated that the naphthyl group at this position is also preferred for hCAR agonists.
The promising compounds 23, 24, and 29 were then selected and further evaluated for their
activation effects, of hCAR and hPXR, using the HepG2 cell reporter assays (Figure 4B) [37].
While compounds 23 and 24 indicated slightly decreased activation potency, compared with
CITCO, the naphthyl analog 29 showed a slight increase in activation of hCAR. At the same
time, compound 29 retained selectivity for hCAR over hPXR.
16

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Figure 4. Activation of hCAR and hPXR by CITCO and compounds 19-29. (A) Concentration
dependent activation curves alongside EC50 and EMAX values of compounds 19-29 in high-
throughput hCAR agonist luciferase gene reporter assays. EC50 values obtained from at least
three independent experiments in quadruplicate were expressed as mean ± SEM. EMAX values
were calculated by nonlinear regression. (B) Activation of hCAR and hPXR by CITCO and
compounds 23, 24, and 29 in the HepG2 cell reporter assays. Data obtained from three
independent experiments were expressed as mean ± SD normalized against the positive control,
CITCO for hCAR activities or RIF for hPXR activities. **, P < 0.01.
Finally, we combined the desirable features from the above compounds 1-29 and synthesized
compounds 30-32 (Figure 5). While compounds 30 and 31 had similar activities, compound 32
(
DL5016) showed excellent EC and E
values (Figure 5A). These three compounds were
5
0
MAX
also evaluated in the HepG2 cell reporter assays and compared to CITCO (Figure 5B) [37].
17

ACCEPTED MANUSCRIPT
Compound 30 caused similar hCAR activation with respect to CITCO; however, it also
significantly activated hPXR. Compound 31 demonstrated enhanced hCAR activation over
CITCO, while hPXR activation was also slightly increased. It was exciting to see that compound
DL5016 demonstrated significant improvement in hCAR activation over CITCO while
maintaining selectivity over hPXR.
Figure 5. Activation of hCAR and hPXR by CITCO and compounds 30-32. (A) Concentration
dependent activation curves alongside EC50 and EMAX values of compounds 30-32 in high-
throughput hCAR agonist luciferase gene reporter assays. EC50 values obtained from at least
three independent experiments in quadruplicate were expressed as mean ± SEM. EMAX values
were calculated by nonlinear regression. (B) Activation of hCAR and hPXR by CITCO and
18

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compounds 30-32 in the HepG2 cell reporter assays. Data obtained from three independent
experiments were expressed as mean ± SD normalized against the positive control, CITCO for
hCAR activities or RIF for hPXR activities. *, P < 0.05; **, P < 0.01.
2
.3. Binding mode of DL5016 in the LBD of hCAR
Compound DL5016 indicated an overall binding mode similar to that of CITCO (Figure 6)
[21]. Specifically, the 2,3-dihydro-1H-inden-2-yl fragment of DL5016 occupied the same
hydrophobic pocket (Tyr224, Phe132, and Phe217) as the dichlorophenyl group of CITCO.
Because the length of 2,3-dihydro-1H-inden-2-yl is slightly shorter than the dichlorophenyl
group, it fits deeper into this hydrophobic pocket than dichlorophenyl, which might explain why
the 2,3-dihydro-1H-inden-2-yl analog demonstrated improved agonistic activity compared to the
corresponding dichlorophenyl analog (17 vs 2). Similar to the imidazothiazole core of CITCO,
the imidazo[2,1-b]oxazole core of DL5016, located at the junction of helixes 3 and 5 in close
proximity to Met168 (H3) and Cys202 (H5). This explains why an additional methyl group on
the imidazothiazole core was not tolerated (6 vs 2). The S atom in the imidazothiazole core of
CITCO is 3.0 Å away from the side chain hydroxyl group of Tyr326, implying a possible H-
bonding interaction. Considering the fact that Tyr326 belongs to helix 10 which is adjacent to
helix X and AF2, this interaction might contribute to the agonistic activity of CITCO with
respect to hCAR. The distance between the imidazothiazole oxygen atom of DL5016 to the
same Tyr326 residue was 4.4 Å, which could explain the slightly decreased EC50 value of
DL5016 compared to CITCO. The naphthyl group of compound DL5016, similar to the p-Cl-
phenyl group of CITCO, pointed directly to the residue Gly229 of helix 6 and Leu239 in helix 7.
This result explains why large substitutions could not be tolerated on the A-ring in our SAR
studies. It is worth noting that the naphthyl group formed a favorable π-π stacking interaction
19

ACCEPTED MANUSCRIPT
with the side chain of Phe234. The same interaction was not observed in the crystal structure of
CITCO in hCAR.
Figure 6. Docking result of compound DL5016 (cyan) in the LBD of hCAR (grey) in
comparison with CITCO (magenta, PDB: 1XVP). Key residues of hCAR that interact with
CITCO and DL5016 are highlighted in green, the helix X (H-X) is shown in light pink, and the
activation function 2 helix (AF2) is orange.
2
.4. Physiochemical properties of DL5016
Compound DL5016 demonstrated similar hCAR agonist potency and an improved EMAX
value compared to CITCO (Table 1). Unlike CITCO, which gave unstable results from the
hCAR luciferase gene reporter assay [26,27], compound DL5016 indicated excellent stability. In
addition, the potential toxicophores of CITCO, the thiazole and oxime groups, were replaced in
compound DL5016 by oxazole and amino groups, respectively. Moreover, CITCO is a highly
hydrophobic (logP 7.2) structure without an ionizable functional group, while compound
20

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a
DL5016 indicated a significantly decreased logP value and an ionizable amino group (pK 7.85).
The solubilities of compound DL5016 in various solvent systems have also been tested. DL5016
indicated reasonable aqueous solubility in both H O and PBS buffer.
2
Table 1
Comparison of properties for CITCO and DL5016.
CITCO a
DL5016
0.66 ± 0.10
4.9
EC50
0.57 ± 0.11
a
E
MAX
3.1
E
MAX / EC50
5.4
thiazole, oxime
7.26
7.2
none
4.62
Potential toxicophore [38]
logP
pKa
NA
7.85
18 ± 5
17 ± 5
b
Solubility
µg/mL)
H
2
O
NDb
(
PBS buffer
ND
a
Number shown here is an average of six different batches of samples.
b
The solubility was not determined.
2
.5. Activation of hCAR induces hepatic expression of CYP2B6 and CYP3A4
Numerous examples have shown that compounds identified using hCAR gene reporter assays
might not be able to induce the expression of the CYP2B6 gene, the rate-determining enzyme for
CPA bioactivation [26,39]. To study the effects of compound DL5016 on the expression of CPA-
metabolizing enzymes, HPH were treated with CITCO, DL5016, and the known hPXR activator
Rifampin (RIF, Figure 7). For HPH from two liver donors (HL-#131 and HL-#133), the
treatment of compound DL5016, similar to CITCO [2], concentration-dependently induced the
expression of CYP2B6 mRNA (Figure 7A). This was not achieved by the amide analogue 1
(figure S1), implying that different gene induction effects can happen even within the same
scaffold. Similar to the effect of CITCO, the induction of CYP3A4 by DL5016 was significantly
lower compared to CYP2B6. The same trend of results was also observed at the protein level in
HPH prepared from the same liver donors (Figure 7B). As expected, the hPXR activator RIF
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preferentially induced CYP3A4 over CYP2B6 at both mRNA and protein levels in the same liver
donors.
Figure 7. hCAR regulated induction of CYP2B6 and CYP3A4 in HPH. (A) CYP2B6 and
CYP3A4 mRNA levels were measured in HPH prepared from human liver donors (HL-#131 and
HL-#133). The HPH were treated with CITCO, RIF, DL5016 or the vehicle control (0.1%
DMSO). (B) Representative immunoblots of CYP2B6 and CYP3A4 proteins in HPH from liver
donors #131 and #133. RT-PCR data obtained from three independent experiments were
expressed as mean ± SD normalized against vehicle control. *, P < 0.05; **, P < 0.01.
2
.6. DL5016 causes hCAR nuclear translocation in HPH
It has been well-documented that chemically-stimulated activation causes hCAR to
translocate from the cytoplasm to the nucleus in HPH as well as in vivo [40]. As shown in
Figure 8, compound DL5016 triggered hCAR nuclear translocation using Ad-EYEP-hCAR-
22

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infected HPH treated with vehicle control (0.1% DMSO), CITCO, or DL5016. While in the
vehicle control-treated HPH, Ad-EYFP-hCAR mostly remains in the cytoplasm, it is efficiently
translocated into the nucleus upon treatment of the positive control CITCO and compound
DL5016. These results confirmed that compound DL5016 mediates the first, essential step in
hCAR activation.
Figure 8. DL5016 promotes translocation of hCAR from cytoplasm to nucleus. HPH were
infected with Ad-EYFP-hCAR overnight and then treated with DMSO (0.1%), phenobarbital
(PB, 1 mM), CITCO (1 µM), or DL5016 (1 µM) for 8 h. Cells were fixed, stained with DAPI to
represent the nucleus, and imaged. In representative images from HPH139, hCAR is sequestered
in the cytoplasm of HPH without treatment, while both CITCO and DL5016 promote significant
nuclear accumulation of hCAR, denoted with a red mask (A). Quantitation of hCAR
translocation reveals DL5016 translocates hCAR similar to agonists PB and CITCO, where CAR
translocation (%) is calculated based on the percent of cells that expressing hCAR where over
5
0% of the nucleus overlaps with EYFP-hCAR (B). **, P < 0.01.
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2
.7.
DL5016 promotes CHOP-mediated cytotoxicity of SU-DHL-4 cells in the HPH/SU-
DHL-4 co-culture system.
The HPH/SU-DHL-4 cell co-culture model has been proved to be an excellent in vitro
compound evaluation assay that mimics the in vivo conditions of human body and allows
simultaneous studies of hepatic metabolism and extrahepatic anti-cancer effects [2]. To evaluate
the anti-cancer effects of DL5016, different concentrations of the CPA-based regimen CHOP
were added to the co-cultures with the presence of vehicle (0.1% DMSO) or DL5016 (1 µM)
(
Figure 9A). The results indicate that co-treatment with DL5016 significantly enhanced CHOP-
induced cytotoxicity in SU-DHL-4 cells. Specifically, in the presence of DL5016, the
cytotoxicity of 100 µM CHOP was similar or even stronger than that of 500 µM CHOP alone.
We also studied the temporal effects of CHOP in the presence of DL5016 (Figure 9B). CHOP
caused cytotoxicity in SU-DHL-4 cells time-dependently, and addition of DL5016 dramatically
enhanced CHOP-induced cytotoxicity in 24 h after co-treatment. A control experiment
demonstrated that compound DL5016, by itself, did not show cytotoxicity in the co-cultured SU-
DHL-4 cells.
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Figure 9. Activation of hCAR enhanced cytotoxicity against SU-DHL-4 cells by CPA-based
regimen CHOP in the HPH/SU-DHL-4 co-culture system. (A) Effects of hCAR activation on the
concentration-dependent anti-cancer effects of CHOP in SU-DHL-4 cells. The HPH/SU-DHL-4
co-cultures were treated with vehicle control (0.1% DMSO) or CHOP (5, 100 and 500 µM) in
the presence and absence of DL5016 (1 µM). Cell viability was analyzed 24 h after the co-
treatment. (B) Effects of hCAR activation on the time-dependent cytotoxicity of CHOP in SU-
DHL-4 cells. The HPH/SU-DHL-4 co-cultures were treated with vehicle control (0.1% DMSO)
or CHOP (100 µM) in the presence and absence of DL5016 (1 µM). Data obtained from three
independent measurements were expressed as mean ± SD normalized as the percentage of
viability of vehicle control. Statistical significance between the treatment groups CHOP and
DL5016/CHOP were analyzed; **, P < 0.01.
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3
. Conclusion
In summary, the development of potent and selective hCAR agonists represents a promising
approach to sensitize CPA-based chemotherapies by improving the metabolic efficiency of CPA.
As the most well-known hCAR agonist, CITCO is widely utilized in various biological studies.
However, CITCO is not stable, which undergoes an isomerization reaction in solution. Through
structural modification of CITCO, we have developed a novel compound, DL5016. DL5016 is
chemically stable while also potently and selectively activates hCAR over hPXR. DL5016
induces the expression of hCAR target genes on both the mRNA and protein levels. It causes
translocation of hCAR from cytoplasm to the nucleus. Addition of DL5016 dramatically
decreases the efficacious dose of the CPA-based combination regimen CHOP.
4
. Experimental section
4
.1. General information
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). UV spectra were obtained on a Nanodrop 2000c spectrophotometer. Flash
column chromatography was performed on silica gel (200−300 mesh). NMR spectra were
obtained on a Varian INOVA 400 MHz NMR spectrometer at 25 °C. Chemical shifts were
reported as
δ values (parts per million) using the residual solvent peak as an internal reference.
1
Data for H NMR were reported in the following order: chemical shift, multiplicity (s, singlet; d,
doublet; t, triplet; sept, septuplet; dd, double doublet; dt, double triplet; m, multiplet), coupling
1
3
constant J (Hz), number of protons. Data for C NMR were reported as
δ values (parts per
million). High-resolution mass spectra (HRMS) were obtained on a JEOL AccuTOF with
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ESI/APCI ion sources coupled to an Agilent 1100 HPLC system. HPLC analysis was performed
on a Shimadzu HPLC fitted with a C18 reversed-phase column (Phenomenex, luna 5 µM C18(2)
4
.6 mm × 100 mm) with a flow rate of 0.8 mL/min using CH OH/H O 75:25 with 0.1%
3 2
NH OAc or CH CN/H O 80:20 mobile phase. The purity of final products was > 95%.
4
3
2
General Procedure A: A solution of 2-aminothiazole (5 mmol, 1 equiv) and bromomethyl
ketone (5 mmol, 1 equiv) in EtOH (30 mL) was heated under reflux for 16 h. The solvent was
removed under reduced pressure, and saturated NaHCO
solid. The mixture was extracted using EtOAc (30 mL × 3), and the organic layers were
combined, and dried over Na SO . The concentrated crude product was dried overnight under
3
(30 mL) was added to the remaining
2
4
vacuum to get the crude imidazothiazole that was used directly in the next step. The Vilsmeier
reagent was prepared by dropping POCl (16.5 mmol, 3.3 equiv) into a solution of DMF (5
3
mmol, 1.0 equiv) in CHCl (5 mL) at 0 °C. To the resulting mixture at 0-5 °C was added a
3
solution of imidazothiazole (5 mmol) in CHCl (30 mL) dropwise. The reaction was warmed to
3
the room temperature over 1 h, and then heated under reflux for an additional 5 h. The solvent
was removed under reduced pressure and the resulting residue was poured onto ice. The crude
aldehyde was collected by filtration and further purified using flash chromatography.
General Procedure B: A solution of oxazol-2-amine (5 mmol, 1 equiv) and bromomethyl
ketone (5 mmol, 1 equiv) in THF (30 mL) and CH CN (30 mL) was allowed to stir at room
3
temperature for 24 h. The precipitation from the reaction mixture was collected by filtration and
o
then washed using CH CN. To a mixture of the resulting solid in toluene (50 mL) at 0 C was
3
added titanium (IV) chloride (0.94 g, 5 mmol) as a solution in toluene (5 mL) over 30 min. The
o
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
27

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mixture was adjusted to 9 using Na
2 3
CO , and the resulting solution was extracted using EtOAc
(
30 mL × 3). The organic layers were combined, and dried over Na SO . The concentrated
2
4
crude product was dried overnight under vacuum to get the imidazooxazole that was used
without further purification. The Vilsmeier reagent was prepared by dropping POCl (16.5
3
mmol, 3.3 equiv) into a solution of DMF (5 mmol, 1.0 equiv) in CHCl (5 mL) at 0 °C. To the
3
resulting mixture at 0-5 °C was added a solution of imidazothiazole (5 mmol) in CHCl (30 mL)
3
dropwise. The reaction was warmed to the room temperature over 1 h, and then heated under
reflux for an additional 5 h. The solvent was removed under reduced pressure and the resulting
residue was poured onto ice. The crude aldehyde was collected by filtration and further purified
using flash chromatography.
General Procedure C: To a solution of aldehyde (1 mmol) in EtOH (5 mL) was added amine
(
1 mmol) followed by AcOH (5 mmol, 5 equiv). The reaction mixture was heated under reflux
overnight, and then concentrated. To a solution of the resulting residue in MeOH at 0 °C was
added NaBH (5 mmol, 5 equiv). The reaction mixture was allowed to stir at room temperature
overnight. The reaction mixture was partitioned between saturated NH Cl (30 mL) and EtOAc
30 mL). The aqueous layer was extracted using EtOAc (30 mL × 2) and the combined organics
were washed with brine (45 mL), dried (Na SO ) and concentrated. The crude product was
4
4
(
2
4
purified by flash chromatography (1-5% MeOH in CH Cl ) to give the desired product.
2
2
4
.1.1. 6-(4-Chlorophenyl)-N-(3,4-dichlorophenethyl)imidazo[2,1-b]thiazole-5-carboxamide (1)
Compound 1 was synthesized starting with 33 and 2-(3,4-dichlorophenyl)ethan-1- amine
1
using the first half of General Procedure C (50%): H NMR (400 MHz, CDCl ):
δ 8.36 (s, 1H),
3
8
.28 (s, 1H), 8.51 (d, J = 7.2 Hz, 2H), 7.43-7.36 (m, 4H), 7.07 (d, J = 7.6 Hz, 1H), 6.91 (s, 1H),
28

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1
3
3
1
3
3
.85 (t, J = 6.8 Hz, 2H), 2.96 (t, J = 6.8 Hz, 2H); C NMR (100 MHz, CDCl ): δ 152.8, 150.0,
40.3, 134.4, 132.2, 132.1, 131.0, 130.24, 130.17, 129.8, 128.9, 128.6, 121.8, 120.7, 112.8, 62.4,
+
6.9; HRMS (ESI): Exact mass calcd for C H Cl N S [M+H] 434.0052, found 434.0037;
2
0
15
3
3
HPLC analysis: retention time = 12.1 min, peak area 98.1%, 80:20 CH CN/H O
3
2
4
.1.2. N-((6-(4-Chlorophenyl)imidazo[2,1-b]thiazol-5-yl)methyl)-2-(3,4-dichlorophenyl)ethan-1-
amine (
2
)
1
Compound 2 was synthesized using the second half of General Procedure C (64%): H NMR
400 MHz, CDCl3): 7.57 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 4.4 Hz, 1H), 7.38 (d, J = 8.4 Hz, 2H),
.32 (d, J = 8.0 Hz, 1H), 7.25 (d, J = 6.8 Hz, 1H), 6.96 (d, J = 8.8 Hz, 1H), 6.75 (d, J = 4.8 Hz,
(
δ
7
1
1
3
H), 4.12 (s, 2H), 2.87 (t, J = 6.0 Hz, 2H), 2.71 (t, J = 6.4 Hz, 2H); C NMR (100 MHz,
CDCl3):
δ
149.1, 143.7, 140.1, 133.1, 132.3, 130.6, 130.3, 130.2, 129.0, 128.7, 128.1, 120.3,
+
1
4
3 3
18.7, 111.9, 49.6, 43.7, 35.3; HRMS (ESI): Exact mass calcd for C20H17Cl N S [M+H]
36.0209, found 436.0207; HPLC analysis: retention time = 18.5 min, peak area 97.4%, 75:25
CH OH/H O with 0.1% NH OAc.
3
2
4
4
.1.3.
N-((6-(4-Chlorophenyl)imidazo[2,1-b]thiazol-5-yl)methyl)-2-(3,4-dichlorophenyl)-N-
methylethan-1-amine (3)
To a solution of compound 2 (130 mg, 0.3 mmol) and K
2
CO
3
(50 mg, 0.36 mmol) in DMF
o
was added MeI (47 mg, 0.33 mmol). The reaction mixture was stirred at 60 C overnight and
then cooled to room temperature. The resulting residue was dissolved in H O (50 mL) to give a
2
yellow solution. The resulting solution was extracted using CH Cl (30 mL × 3), and the
2
2
combined organic layers were dried over Na SO . The solvent was removed by rotary
2
4
evaporation. The crude product was purified by flash chromatography to give compound 3 (83
29