Identification of new aryl hydrocarbon receptor (AhR) antagonists using a zebrafish model

Article abstract of DOI:10.1016/j.bmc.2019.07.030

A new series of 1,3-diketone, heterocyclic and α,β-unsaturated derivatives were synthesized and evaluated for their AhR antagonist activity using zebrafish and mammalian cells. Compounds 1b, 2c, 3b and 5b showed significant AhR antagonist activity in a transgenic zebrafish model. Among them, compound 3b, and 5b were found to have excellent AhR antagonist activity with IC₅₀ of 3.36 nM and 8.3 nM in a luciferase reporter gene assay. In stem cell proliferation assay, compound 5b elicited marked HSC expansion.

Full text of DOI:10.1016/j.bmc.2019.07.030

Accepted Manuscript
Identification of new aryl hydrocarbon receptor (AhR) antagonists using live
zebrafish model
Jieun Jeong, Kun-Hee Kim, Dong-Young Kim, Gopalakrishnan
Chandrasekaran, Minhee Kim, Suvarna H. Pagire, Mahesh Dighe, Eun Young
Choi, Su-Min Bak, Eun-Young Kim, Myung-Geun Shin, Seok-Yong Choi, Jin
Hee Ahn
PII:
S0968-0896(19)30794-1
https://doi.org/10.1016/j.bmc.2019.07.030
BMC 15014
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 May 2019
11 July 2019
17 July 2019
Please cite this article as: Jeong, J., Kim, K-H., Kim, D-Y., Chandrasekaran, G., Kim, M., Pagire, S.H., Dighe, M.,
Young Choi, E., Bak, S-M., Kim, E-Y., Shin, M-G., Choi, S-Y., Hee Ahn, J., Identification of new aryl hydrocarbon
receptor (AhR) antagonists using live zebrafish model, Bioorganic & Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.bmc.2019.07.030
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Identification of new aryl hydrocarbon receptor (AhR) antagonists using live
zebrafish model
Jieun Jeong,1,6 Kun-Hee Kim,2,6 Dong-Young Kim,3,6 Gopalakrishnan Chandrasekaran,2 Minhee
Kim,1 Suvarna H Pagire,1 Mahesh Dighe,1 Eun Young Choi,3 Su-Min Bak4, Eun-Young Kim4,
Myung-Geun Shin,5,* Seok-Yong Choi,2,* and Jin Hee Ahn1,*
1
Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju 61005,
Republic of Korea
2 Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju,
Republic of Korea,
3 Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul, Republic
of Korea
4 Department of Life and Nanopharmaceutical Science and Department of Biology, Kyung Hee
University, Seoul, Republic of Korea
5 Department of Laboratory Medicine, Chonnam National University Medical School, Gwangju,
Republic of Korea
*Corresponding authors: e-mail address: jhahn@gist.ac.kr; address: Department of Chemistry,
Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea, e-mail address:
zebrafish@chonnam.ac.kr; address: Department of Biomedical Sciences, Chonnam National University
Medical School, Gwangju, Republic of Korea, & e-mail address: mgshin@chonnam.ac.kr; address:
Department of Laboratory Medicine, Chonnam National University Medical School, Gwangju, Republic
of Korea.
6 Authors contributed equally
Abstract
A new series of 1,3-diketone, heterocyclic and -unsaturated derivatives were synthesized
and evaluated for their AhR antagonist activity using zebrafish and mammalian cells.
Compounds 1b, 2c, 3b and 5b showed significant AhR antagonist activity in a transgenic
zebrafish model. Among them, compound 3b, and 5b found to have excellent AhR antagonist
activity with IC50 of 3.36 nM and 8.3 nM in luciferase reporter assay. In stem cell proliferation
assay, compound 5b elicited marked HSC expansion.
Keywords: Aryl hydrocarbon receptor (AhR), antagonist, zebrafish, stem cell
1

1. INTRODUCTION
The aryl hydrocarbon receptor (AhR) is a transcription factor that belongs to a family of
basic helix-loop-helix/Per-Arnt-Sim (b-HLH/PAS) proteins1,2. AhR consists of b-HLH and PAS
domains. The b-HLH domain is located in the N-terminal region and involved in DNA binding3.
The PAS domain has two subdomains. The PAS-A subdomain is responsible for protein
dimerization and the PAS-B is implicated in ligand binding4. In the absence of its ligand, AhR
forms an inactive complex with a heat shock protein 90 (HSP90) dimer and a co-chaperone
protein X-associated protein 2 (XAP2) and thus remains in the cellular cytoplasm. The HSP90
dimer protects inactive AhR complex from proteolysis and AhR Nuclear Translocator (ARNT)
binding. Ligand binding changes inactive AhR to its active form, resulting in exposure of a
nuclear localization signal (NLS) in the PAS domain. Subsequently, the active AhR complex
translocates to the nucleus where ARNT displaces HSP90 and XAP2 from the AhR complex by
forming an AhR-ARNT heterodimer2,5,6. Further, this heterodimer binds to a dioxin-responsive
element (DRE) in the promoters of various genes, thus inducing transcription of these genes that
regulate immune, hepatic, cardiovascular and reproductive systems as well as lipid and
xenobiotic metabolisms7.
The activation of AhR by the ligand including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
and benzo[a]pyrene causes dysfunction of cholesterol pathway that leads to formation of
atheroma due to accumulation of fats in the blood vessels, thereby resulting atherosclerosis8,9.
Also, AhR regulates stem cell proliferation10. In 2010, Cooke et al reported that StemRegenin 1
(SR1), an AhR antagonist, expanded human hematopoietic stem cells (HSCs) ex vivo11.
Therefore, the development of AhR antagonists would be of great help with the treatment of
atherosclerosis and HSC expansion.
2

Several AhR antagonists including flavones, resveratrol, kaempferol, and pyrazole-5-
carboxamide derivatives (Fig. 1) exhibited anti-atherosclerosis effect. However, most of them
have not developed further, which prompted us to identify a new series of AhR antagonists.
OCH3
NO2
H3CO
O
OCH3
OH
O
O
HO
H3CO
O
OH
6,2'4'-trimethoxyflavone
3'-Methoxy-4'-nitroflavone
Resveratrol
OH
OH
HN
N
H
N
N
HO
O
N
N
N
N
O
OH
N
N
OH
O
S
Kaempferol
CH-223191
StemReginin-1
(SR-1)
Fig. 1. Reported AhR antagonists.
To identify novel AhR antagonists, we screened a chemical library (6,400 compounds) of
Korea Chemical Bank (KCB) using cyp1a reporter zebrafish (Tg(cyp1a:egfp))12. We found
compound 1a having 1, 3-diketone scaffold as a hit compound (Fig. 2). It exhibited moderate
AhR inhibitory activity in a zebrafish in vivo model. Initial outcome encouraged us to perform
scaffold modification for further pharmacophoric exploration.
O
O
O
F3C
O
1a
Fig. 2. Structure of hit compound 1a (GM-90203).
3

2. Chemistry
Diketone derivatives were synthesized according to Scheme 1. Initially, various methyl
alkoxy-benzoate were prepared from different hydroxy benzoic acids through esterification
followed by Mitsunobu reaction with various alcohols. Similarly, on other side, different alkoxy-
acetophenones were synthesized by carrying out Mitsunobu coupling between various hydroxy
acetophenones and alcohols. Subsequently, 1,3-Diketone derivatives (1-3) were synthesized by
cross Claisen condensation between various substituted methyl benzoates (B) and acetophenones
(C) under basic condition.
Scheme 1. Synthetic route for series 1-3 compounds.
O
O
O
i
ii
O
OH
O
R1O
HO
HO
O
O
iii
A
B
R1O
OR2
R
1 = -CF3, -CH3, -CH2CH3
3'-hydroxy
4'-hydroxy
3'-hydroxy
4'-hydroxy
-CH(CH3)2, -CH2CH(CH3)2, -Bn
1a-f
2a-e
3a-d
O
O
ii
R2O
HO
2'-hydroxy
3'-hydroxy
4'-hydroxy
C
R
2 = -CH3, -CF3, -CH2CH3,
Reagents and condition: i) H2SO4, MeOH, reflux. ii) R1-OH or R2-OH, PPh3, DIAD, THF, 0 °C,
RT. iii) NaH, THF, reflux.
Subsequently, heterocyclic compounds and alkylated diketones were synthesized as shown
in Scheme 2. 1,3-Diketone derivatives (1b and 2c) reacted with hydroxylamine hydrochloride
under reflux to afford respective isoxazole derivatives (4a and 4e). Similarly, 1b and 2c reacted
with hydrazine hydrate to give pyrazole derivatives (4b and 4c), respectively. Compound 2c
4

underwent cyclocondensation with formamidine to give pyrimidine derivative 4d. Moreover,
Compound 2c underwent methylation to afford dimethyl compound 4f.
Scheme 2. Synthetic route for series 4 compounds.
N
N
O
4d
O
ii
X, Y = N, O
X
Y
O
O
O
O
N
NH
O
i
iii
R3
R3
R3
1d R3 =OCF3
2c R3 =OCH(CH3)2
4b R3 =OCF3
4c R3 =OCH(CH3)2
4a R3 =OCF3
4e R3 =OCH(CH3)2
iv
O
O
O
O
4f
o
Reagents and conditions: i) NH2OH.HCl, EtOH, reflux. ii) formamide, 180 C, overnight. iii)
NH2NH2, EtOH, reflux. iv) CH3I, NaH, acetone, reflux.
Compounds 5a and 5b were synthesized according to scheme 3. They were synthesized by
Aldol condensation between respective benzaldehydes and 2-methoxy acetophenones in the
presence of sodium hydroxide as a catalyst.
Scheme 3. Synthetic route for series 5 compounds.
5

O
O
O
O
O
i
H
+
R4
R4
5a R3 =OCF3
5b R3 =OCH(CH3)2
Reagents and conditions: i) NaOH, EtOH, 0 oC, room temperature.
3. RESULTS AND DISCUSSION
In the last few decades, zebrafish has emerged as a screening model in drug discovery owing
to its significant genomic similarity to human and its transparency during organogenesis that
allows for in vivo labeling and imaging. Recently, we developed transgenic zebrafish
Tg(cyp1a:egfp) by incorporating into the zebrafish genome a cyp1a fosmid where enhanced
green fluorescence protein (EGFP) gene replaced the start codon of cyp1a. As cyp1a is a target
gene of AhR, EGFP fluorescence in Tg(cyp1a:egfp) zebrafish can become readout for AhR
transcriptional activity. TCDD, an AhR ligand with the highest efficacy, induced EGFP
expression in blood vessels, cloaca, gut, lateral line, liver, pectoral fin bud, pronephros, pancreas
and swimming bladder of Tg(cyp1a:egfp) zebrafish in a dose-dependent manner13.
6

Fig. 3. Fluorescence based screening (A) and optimization (B) for AhR antagonists using
Tg(cyp1a:egfp) zebrafish.
Compound 1a was identified as a hit through a chemical library screening using the
Tg(cyp1a:egfp) zebrafish (Fig. 3). CH223191, a known AhR antagonist12, was used as the
reference. Optimization of the compound 1a was performed and the results of the zebrafish study
are summarized in Table 1. Compound 1b, which has p-OCF3 (on left phenyl group) and o-OCH3
(on right phenyl group) substitutions on the phenyl group, showed better antagonistic activity
than that of the hit 1a. However, mono-substituted 1c and 1d were not active. Compound 1e
7

having p-OCF3 (on left phenyl group) and p-OCH3 (on right phenyl group) substitution showed
decreased AhR antagonistic activity. These seemed to suggest that both substituents are
necessary for activity. From the above results, we fixed o-methoxy group at right phenyl and
further modified left phenyl group with diverse substituents. p-Methoxy (2a), p-ethoxy (2b), p-
isopropoxy (2c), p-isobutyloxy (2d) and p-benzyloxy (2e) derivatives were synthesized and
evaluated. Compound 2c exhibited improved AhR antagonistic activity, whereas methoxy (2a),
isobutyloxy (2d) or benzyloxy (2e) derivatives were not active. From the results, we fixed p-
isopropoxy group at left phenyl and further modified right phenyl group with diverse substituents.
Compound 3b exhibited good AhR antagonist activity. However, di and tri-substituted
derivatives (3c and 3d) were not effective.
Table 1. AhR antagonist activity of 1,3-diketone derivatives in a zebrafish model.
AhR Antagonist activity* based on
fluorescence in Tg(cyp1a:egfp)
Compound
Structure
zebrafish
O
O
O
1a
++
F3C
O
O
O
O
1b
1c
++++
F3C
O
O
O
O
-
8

O
O
1d
1e
1f
-
-
F3C
O
O
O
F3C
O
O
O
O
O
O
+
F3C
O
O
O
2a
2b
-
O
O
O
O
O
O
+++
O
O
2c
++++
O
O
O
O
2d
-
O
O
O
O
2e
3a
-
-
BnO
CF3
O
O
O
O
9

O
O
O
3b
++++
O
O
O
O
O
3c
-
-
O
O
O
O
3d
O
HO
O
*Excellent (++++), Good (+++), Moderate (++), Low (+), No (-),
In the preceding study, compounds 1b, 2c and 3b were most active. As they have diketone
moiety, we sought more druggable derivatives, which are more rigid with heterocycles and less
reactive than diketone moiety, by replacing diketo moiety. Modification of flexible 1,3-diketone
to ring closure isoxazole, pyrazole and pyrimidine diminished the AhR antagonist activity
compared to 2c as shown in Table 2. Unfortunately, alkylated diketone derivative (4f) was not
active either.
Table 2. AhR antagonist activity of heterocyclic and alkylated derivatives in a zebrafish model.
AhR Antagonist activity* based on
fluorescence in Tg(cyp1a:egfp)
Compound
Structure
zebrafish
X, Y = N, O
X
Y
O
4a
-
F3C
O
10

HN
HN
N
N
N
N
O
O
O
-
4b
4c
4d
F3C
(Embryotoxicity )
O
O
O
-
(Embryotoxicity )
++
+
X, Y = N, O
X
Y
O
4e
O
O
O
O
O
4f
-
*Moderate (++), Low (+), No (-).
To identify another scaffold, -unsaturated derivatives were synthesized and evaluated for
their AhR inhibition. As shown in Table 3, 5b showed potent AhR inhibition.
Table 3. AhR antagonist activity of -unsaturated derivatives in a zebrafish model.
AhR Antagonist activity* based on
Compound
Structure
fluorescence in Tg(cyp1a:egfp)
zebrafish
O
O
O
-
5a
F3C
(Embryotoxicity)
O
O
O
5b
++++
11

*Excellent (++++),No (-).
From the compounds screening in a zebrafish model, we found that compounds 1b, 2c, 3b
and 5b exerted significant AhR antagonistic activity in a dose-dependent manner (Fig. 4). This
finding was validated by Western blotting with anti-GFP antibody showing that compounds 1b,
2c, 3b and 5b inhibit AhR activity in a dose-dependent manner (Fig. 5).
Fig. 4. Assessment of anti-AhR activity of compounds using GFP fluorescence in
Tg(cyp1a:egfp) zebrafish embryos. Tg(cyp1a:nls-egfp) embryos at 30 hours post-fertilization
(hpf) were treated for 48 h with TCDD (10 nM) and indicated compound at various
concentration and imaged. Upper and lower panels are bright field and fluorescence images,
respectively. DMSO was used as vehicle control. Scale bar = 300 μm.
12

Fig. 5. Assessment of anti-AhR activity of compounds using GFP protein in Tg(cyp1a:egfp)
zebrafish embryos. The embryos in Fig. 4 were subjected to Western blotting with anti-GFP
antibody. Anti--actin antibody was used for a loading control. * P < 0.05; ** P < 0.01; *** P <
0.001 by the two-tailed Student’s t-test.
Selected compounds were further evaluated in luciferase reporter gene AhR assay. The
luciferase reporter assay indirectly measures AhR transcriptional activity by quantification of the
luciferase expression in the mammalian cells induced by promoter of an AhR target gene. All
investigational compounds showed better AhR antagonistic activity than the reference compound
CH223191. Moreover, compounds 3b and 5b exhibited excellent in vitro activity with IC50
values of 3.36 nM and 8.3 nM, respectively (Table 4).
13

Table 4. IC50 of AhR antagonists.
AhR antagonist
activity
Compound
Structure
(IC50)
O
O
O
1b
15.93 nM
10.98 nM
3.36 nM
F3C
O
O
O
O
2c
O
O
O
O
3b
O
O
O
5b
8.34 nM
O
H
N
N
N
CH223191
98.15 nM
N
O
N
14

Finally, we assessed the effect of the compounds on the expansion of HSCs. After 35 days
of HSC culture with individual compound, CD34+ cells were counted. Compound 5b exhibited
more than 4 fold enhancements in cell number compared to media alone (Table 5). However, 1b,
2c, 3b and CH223191 did not elicit significant change in cell number (data not shown).
Table 5. Haematopoietic stem cell expansion at 35 days after treatment with AhR antagonists.
Day 35
Compound
CD34+ number (x106 cells)
DMSO (0.01 %)
1.75
5b
9.82
*** P < 0.001 by the two-tailed unpaired Student’s t-test.
4. Conclusions
A new series of 1,3-diketone and -unsaturated derivatives were identified and evaluated
for their AhR antagonist activity using zebrafish and mammalian cells. Here, compounds 1b, 2c,
3b and 5b showed significant AhR antagonist activity in a transgenic zebrafish model. Among
them, compound 3b and 5b were found to have excellent AhR antagonist activity with IC50 of
3.36 nM and 8.3 nM in luciferase reporter assay, respectively. In HSC proliferation assay,
compound 5b elicited marked HSC expansion.
15

5. Experimental section
5.1 Chemistry: general procedure
The reagents used in this study were purchased from commercial suppliers and used without
further purification. Thin layer chromatography for analysis was performed using pre-coated
silica gel glass plate (silica gel 60, F-254, 0.25 nm) of Merck. For the identification of the
material separated on TLC, it was confirmed by UV lamp (254 nm and 365 nm). Merck silica gel
grade 9385 (230-400 mesh) was used as a filler for the column for the purification of the
reactants. For the determination of the structure of the synthesized compound, 1H NMR spectra
were run on was JEOL JNMECS400 spectrometers at 400MHz. Chemical shift (δ) are expressed
in ppm downfield from tetramethylsilane (TMS) as an internal standard, and CDCl3, DMSO-d6
were used as a solvent. The letters s, d, t, q and m are used to indicate singlet, doublet, triplet,
quartet and multiplet. HRMS (EI) measurements were conducted at the Korea Basic Science
Institute (KBSI).
5.2 Synthesis
5.2.1 Synthesis of methyl hydroxy benzoate (A).
Hydroxy benzoic acid and H2SO4 were in 50 ml MeOH. The resulting mixture was refluxed
during 4 h, then cooled down to room temperature and concentrated in vacuo. The resulting
residue was carefully quenched by NaHCO3 saturated water solution and extracted with DCM.
The combined organic layers were washed with brine, dried over Na2SO4 and the solvent were
evaporated in vacuo.
5.2.2 Synthesis of methyl alkoxy benzoate (B).
To a solution of methyl hydroxy benzoate (1 eq) in dry THF were added alcohol (1 eq) and
triphenylphosphine (PPh3) (1.2eq). The reaction mixture was allowed to stir at 0 ℃ (ice bath) for
15 minutes. Diisopropyl azodicarboxylate (DIAD) (1.2 eq) was added dropwise into the cold
solution and then stirred at room temperature for overnight under N2. The mixture was diluted
with water and extracted with ethyl acetate twice, and the combined organic phase was washed
with brine solution, dried with Na2SO4, filtrated, and concentrated under reduced pressure. The
residue was purified on a silica gel column.
16

5.2.3 Synthesis of alkoxy-acetophenone (C).
To a solution of hydroxyacetophenone (1eq) in dry THF were added alcohol (1 eq) and
triphenylphosphine (PPh3) (1.2 eq). The reaction mixture was allowed to stir at 0 ℃ (ice bath) for
15 minutes. Diisopropyl azodicarboxylate (DIAD) (1.2 eq) was added dropwise into the cold
solution then stirred at room temperature for overnight under N2. The mixture was diluted with
water and extracted with ethyl acetate twice, and the combined organic phase was washed with
brine solution, dried with Na2SO4, filtrated, and concentrated under reduced pressure. The
residue was purified on a silica gel column.
5.2.4 Synthesis of 1,3-diketone derivatives.
To a suspension of sodium hydride (60% in mineral oil, 5 eq) in dry THF was added a solution
of substituted methyl benzoate (2 eq) in dry THF. After mixture refluxed, substituted
acetophenone (1 eq) in dry THF was added dropwise. The mixture was refluxed overnight, then
quenched with 1N HCl. The mixture was extracted with ethyl acetate twice, and the combined
organic phase was washed with brine solution, dried with anhydrous Na2SO4, filtrated, and
concentrated under reduced pressure. The residue was purified on a silica gel column.
1-(2-Methoxyphenyl)-3-(4-(trifluoromethoxy)phenyl)propane-1,3-dione. (1b)
Using the procedure for 5.2.4 with methyl 2-methoxybenzoate and 4'-
trifluoromethoxyacetophenone provided the title compound in 20.5% yield. 1H-NMR (400 MHz,
CDCl3) δ 8.00 (dt, J = 9.3, 2.4 Hz, 2H), 7.95 (dd, J = 7.9, 1.8 Hz, 1H), 7.49 (ddd, J = 8.7, 6.9, 1.4
Hz, 1H), 7.34-7.28 (m, 2H), 7.12 (s, 1H), 7.11-7.05 (m, 1H), 7.02 (d, J = 8.2 Hz, 1H), 3.97 (s,
3H). 13C NMR (100 MHz, DMSO-d6): δ 184.73 (C) ,184.34 (C), 159.00 (C), 151.79 (C), 134.59
(CH), 134.42(C), 132.20 (C), 130.21 (C), 130.05(CH, CH), 123.93 (CH), 121.57(CH, CH),
121.16 (CH), 113.04(CH), 98.74(CH), 56.50 (CH3). HRMS for C17H13F3O4 calcd, 338.0766;
found, 338.0769 [M+].
1-(4-Isopropoxyphenyl)-3-(2-methoxyphenyl)propane-1,3-dione. (2c)
Using the procedure for 5.2.4 with methyl 4-methoxybenzoate and 2'-methoxyacetophenone
1
provided the title compound in 66.1% yield. H-NMR (400 MHz, CDCl3) δ 7.97-7.90 (m, 3H),
7.49-7.42 (m, 1H), 7.11-7.04 (m, 2H), 7.00 (d, J = 8.5 Hz, 1H), 6.98-6.91 (m, 2H), 4.71-4.60 (m,
17

1H), 3.96 (s, 3H), 1.38 (d, J = 6.1 Hz, 6H). 13C NMR (100 MHz, DMSO-d6): δ 186.61 (C)
,182.18(C), 162.03 (C), 158.79 (C), 134.01 (CH), 131.07(CH), 130.01(CH), 129.98(CH),
127.55(C), 124.06(C), 121.10(CH), 115.99 (CH, CH), 112.93(CH), 97.71(CH), 70.24(CH3),
56.46(CH2), 22.18(CH3, CH3). HRMS for C19H20O4 calcd, 312.1362; found, 312.1364 [M+].
1-(2-Ethoxyphenyl)-3-(4-isopropoxyphenyl)propane-1,3-dione. (3b)
Using the procedure for 5.2.4 with methyl 4-isopropoxybenzoate and 2'-ethoxyacetophenone
1
provided the title compound in 10.0% yield. H-NMR (400 MHz, CDCl3) δ 8.01-7.97 (m, 1H),
7.93 (dt, J = 9.6, 2.4 Hz, 2H), 7.43 (ddd, J = 8.6, 6.9, 1.4 Hz, 1H), 7.26 (d, J = 1.2 Hz, 1H), 7.08-
7.02 (m, 1H), 7.00-6.90 (m, 3H), 4.71-4.60 (m, 1H), 4.18 (q, J = 6.9 Hz, 2H), 1.55 (d, J = 6.1 Hz,
13
3H), 1.40-1.35 (m, 6H). C NMR (100 MHz, DMSO-d6): δ 186.70 (C), 181.50(C), 162.03(C),
158.29(C), 134.08 (CH), 131.06(CH), 129.99 (CH, CH), 129.74 (C), 127.71(C), 123.63(CH),
121.00 (CH), 116.02 (CH),113.62 (CH), 97.79 (CH), 70.24(CH2), 64.59(CH2), 22.19 (CH3,
CH3), 15.19 (CH3). HRMS for C20H22O4 calcd, 326.1518; found, 326.1517 [M+].
5.2.5. Synthesis of chalcone derivatives.
To a substituted benzaldehyde in EtOH added NaOH at 0 ℃ (ice bath) then stir for 10 min. 2'-
methoxyacetophenone was added slowly dropwise and the mixture was allowed to warm to room
temperature overnight. The solvent was concentrated under reduced pressure. The residue was
diluted with water and extracted with ethyl acetate twice, and the combined organic phase was
washed with brine solution, dried with anhydrous Na2SO4, filtrated, and concentrated under
reduced pressure. The residue was purified on a silica gel column.
(E)-3-(4-Isopropoxyphenyl)-1-(2-methoxyphenyl)prop-2-en-1-one. (5b)
Using the procedure for 5.2.5 with methyl 4-isopropoxybenzaldehyde and 2'-
methoxyacetophenone provided the title compound in 90.0% yield. 1H-NMR (400 MHz, DMSO-
D6) δ 7.66 (d, J = 8.9 Hz, 2H), 7.56-7.49 (m, 1H), 7.49-7.42 (m, 2H), 7.25 (d, J = 15.9 Hz, 1H),
7.18 (d, J = 8.5 Hz, 1H), 7.05 (t, J = 7.3 Hz, 1H), 6.96 (d, J = 8.9 Hz, 2H), 4.75-4.64 (m, 1H),
13
3.85 (s, 3H), 1.27 (d, J = 6.1 Hz, 6H). C NMR (100 MHz, DMSO-d6): δ 192.67 (C), 160.09
(C), 158.07(C), 143.32 (CH), 133.23 (CH), 130.97 (CH, CH), 129.92 (C), 129.69(C), 127.28
(CH), 125.03(CH), 121.03 (CH), 116.32 (CH, CH), 112.79 (CH), 69.92(CH), 56.29 (CH3), 22.24
(CH3, CH3). HRMS for C19H20O3 calcd, 296.1412; found, 296.1414 [M+].
18

5.3. Zebrafish
Tg(cyp1a:egfp) zebrafish were maintained using standard procedures14 and staged in hours post-
fertilization (hpf) as per standard criteria15.
5.3.1. A small molecule library screen using Tg(cyp1a:egfp) zebrafish
Tg(cyp1a:nls-egfp) embryos13 at 30 hpf were placed in 96 well-plates (three embryos per well;
SPL Life Sciences) harboring E3 media (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2·2H2O and
0.4 mM MgCl2·6H2O, pH7.2) with 10 nM TCDD (catalog number: 48599; Sigma Aldrich)
dissolved in DMSO. Each well contained three embryos and 200 µL of the media. Subsequently,
5 µM of 6,400 small molecules (Korea Chemical Bank) dissolved in DMSO were individually
added to the well and incubated for 48 h. EGFP expression in the embryos were assessed under a
fluorescent stereomicroscope (MZ16 FA, Leica). Efficacy of hit compounds and their derivatives
dissolved in DMSO were tested in 2-ml glass vials (Suju Science) containing 10 embryos and 1-
ml E3 media with 6 nM TCDD. CH-223191 (C8124, Sigma Aldrich) and StemRegenin1 (72342,
STEMCELL Technologies), both of which are AhR antagonists, were used as a positive control.
5.4 Western blotting
Embryos were lysed with RIPA Lysis and Extraction Buffer (89900, Thermo Fisher
Scientific), and the resulting lysates were separated by 10% sodium dodecyl sulfate
polyacrylamide gel electrophoresis and then transferred to PVDF membranes (IPVH00010,
Millipore). The membranes were incubated with anti-EGFP antibody (1:2,000; sc-9996, Santa
Cruz Biotechnology) for 16 h at 4°C, washed with TBST (0.2 M Tris, 1.37 M NaCl and 0.1%
Tween-20, pH 7.6) three times and probed with IRDye 800CW goat anti-mouse antibody
(1:5,000; 925-32210, LI-COR Biosciences) for 1 h at room temperature. After washing with
TBST, the membranes were imaged with an Infrared Imaging System (Odyssey CLx, LI-COR
Biosciences).
5.5 DNA manipulation
To generate expression plasmids encoding zebrafish AhR, N- and C-terminal halves of
AhR2 (GenBank Accession number NM_131264.1) were PCR-amplified from the cDNA with
19

specific primers (forward: 5’-AAG CTT ACC ATG TCG GCG GGT ATC GGT AC-3’, reverse:
5’-CCT GCT TTA TAA ACA AGC CTA GCA TTG-3’; forward: 5’-GCT TGT TTA TAA AGC
AGG AAG AC-3’, reverse: 5’-ATC GAT GCG TAG TCA CAG CAG TTG CTT TG-3’) and
then simultaneously cloned into the HindIII/ ClaI sites of pCS4+-3xFLAG plasmid19. To
construct expression plasmids encoding zebrafish AhR nuclear translocator (ARNT)-1c
(NM_001045271.1), ARNT-1c was PCR-amplified from the cDNA with specific primers
(forward: 5’-GCT AGC GGA TCC ACC ATG ACA TCA GCT AAT CCG-3’; reverse: 5’-GCT
AGC CTC GAG TCA CTT GAC GTT GGT GTT GG-3’) and cloned into the BamHI/XhoI sites
of pCS4+ plasmid16. To generate zebrafish cyp1a reporter plasmids, two cis-regulatory regions
(p-2608/-2100 and p-580/+71) within the cyp1a promoter17 were PCR-amplified from the
genomic DNA with specific primers (forward: 5’-GCT AGC ACG CGT TAG AGC AGA ACT
AGT GAA-3’, reverse: 5’-GCT AGC CTC GAG TGT GAT GGT TTA GGC AAT-3’; forward:
5’-GCT AGC CTC GAG CTT TAA TCA GGG GTC GCT-3’, reverse: 5’-ACT ATT AAG CTT
CCG GAG CGA TCG AGT GGA-3’) and then simultaneously cloned into the MluI/HindIII
sites of pGL3-Basic vector (Promega).
5.6 Cell culture
COS-7 cells were cultured in RPMI1640 media (Welgene) supplemented with 10% fetal
bovine serum (Welgene) and 1x Antibiotic-Antimycotic (Thermo Fisher Scientific) at 37°C and
5% CO2 atmosphere.
5.7 Determination of the half maximal inhibitory concentration (IC50) of compounds
IC50 of compounds was determined as described previously18 with some modification. In
brief, COS-7 cells were seeded onto 24-well plate at a density of 5 x 104 cells/well. When
reaching approximately 65% confluency, the cells were transfected with ARNT-1c expression
plasmid (50 ng), AhR2 expression plasmid (5 ng), Cyp1a-firefly luciferase reporter plasmid (20
ng) and pRL-TK Renilla luciferase plasmid (Promega, 1 ng) using Lipofectamine 3000 (Thermo
Fisher Scientific) according to the manufacturer’s protocol. The pEGFP-C1 plasmid (Clontech)
was used to assess the transfection efficiency. At 5 hr post transfection, compounds were added
to the cells at various concentrations (1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 µM and
10 µM). In parallel, TCDD (10 nM) or the compounds (10 µM) were added as a control. At 1 hr
20

post compound treatment, TCDD (10 nM) was added to all of the wells except the control wells.
At 18 hr post TCDD treatment, the media was removed, and the cells were rinsed thoroughly
with 500 µl of Dulbecco's phosphate buffered saline (DPBS), treated with 100 µl of 1x Passive
Lysis Buffer (Promega) and rocked at room temperature for 15 min. The dual luciferase assay
was carried out using a Dual-Luciferase Reporter Assay System (Promega) and an Orion L
Microplate Luminometer (Titertek Berthold). The cell lysate (20 µl) was mixed with the
Luciferase Assay Reagent II (100 µl), and the firefly luciferase activity was measured for 10 sec
after a 3-sec delay. Subsequently, Stop & Glo Buffer plus Stop & Glo Buffer Substrate (100 µl)
was added and the Renilla luciferase activity was measured for 10 sec after a 3-sec delay. The
ratio of firefly luciferase activity to Renilla luciferase activity was expressed as relative light unit
(RLU) and RLU values were normalized and expressed as toxic induced ratio (TIR). IC50 was
determined using Prism (GraphPad Software).
5.8 Assessment of expansion of human CD34+ cells
Expansion of human CD34+ cells was assessed as described previously11 with some
modification. In brief, human CD34+ progenitor cells from cord blood (C-12921, PromoCell)
were cultured in hematopoietic progenitor expansion medium DXF (C-28021, PromoCell)
supplemented with 1x antibiotics (15240062, Gibco), Supplement Mix (C-39821, PromoCell)
and Cytokine Mix E (C-39891, Promocell) containing recombinant human thrombopoietin, stem
cell factor (SCF), Fms like tyrosine kinase 3 (flt-3) ligand and IL-3. The cells (5 x 104 cells in 0.5
ml culture media) were seeded into 6-well plates (3471, Corning), and then immediately treated
with compounds. Subsequently, the cells were transferred to 75-cm2 flask (3814, Corning) and
cultured at 37°C in 5% CO2 for 5 weeks. The cell density was maintained between 5 x 105 and 2
× 106 cells/ml by adding fresh media. To count CD34+ cells, the cells were stained in staining
media (PBS supplemented with 2% FBS and 2 mM EDTA) at 4°C for 1 h with anti-hCD34-APC
(dilution ratio 1:50, 343608, BioLegend,) and anti-hCD45RA-FITC (1:50, 347723, BD
Biosciences) or anti-hLin1-FITC (1:50, 348801, BioLegend) antibodies, washed with staining
media and analyzed with flow cytometry (Accuri C6 instrument, BD Biosciences) at weeks 1
and 5. To perform a colony forming assay, cells were seeded into methylcellulose containing
media (MethoCult SF H4436, 04436, StemCell Technologies) and cultured for 14 days. Colony
forming units (CFUs) were scored under an inverted microscope (CKX41SF, Olympus). Colony
21

forming content of the expansion culture was calculated as follows: the average number of
scored colonies from three dishes × total mononuclear cell number/input cell number. Total
mononuclear cells were determined by multiplying the number of cells per ml by the culture
volume.
Acknowledgements
This work was supported by grants from the Ministry of Trade, Industry and Energy, Korea
(2017-10063396), the Ministry of Science, ICT & Future Planning(MSIP)/National Research
Foundation of Korea (NRF) (2016M3A9B6902868) and the Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded by the Ministry of Education
(NRF-2013R1A1A2008587). The chemical library used in this study was kindly provided by
Korea Chemical Bank (http://www.chembank.org/) of Korea Research Institute of Chemical
Technology. Kun-Hee Kim thanks Health Fellowship Foundation for a graduate fellowship.
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