Development of benzochalcone derivatives as selective CYP1B1 inhibitors and anticancer agents

Article abstract of DOI:10.1039/c9md00258h

A series of benzochalcone derivatives have been synthesized and evaluated for CYP1 inhibitory activity and cytotoxic properties against wild type cell lines (MCF-7 and MDA-MB-231) and drug resistant cell lines (LCC6/P-gp and MCF-7/1B1). All of these compounds were found to have selective inhibition towards CYP1B1 and the most potent two possessed single-digit nanomolar CYP1B1 potency. In addition, some of them showed promising cytotoxic activities not only against wild type cells, but also against drug resistant cells at low micromolar concentrations. More importantly, these multi-functional compounds may surmount drug-drug interactions that frequently occur during the combination of CYP1B1/P-gp inhibitors and anticancer drugs to overcome drug resistance. This study may provide a good starting point for the further development of more potent multi-functional agents with CYP1B1 inhibitory activity and cytotoxic potency in cancer prevention and treatment.

Full text of DOI:10.1039/c9md00258h

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Development of benzochalcone derivatives as selective CYP1B1
inhibitors and anticancer agents
Received 00th January 20xx,
Accepted 00th January 20xx
Jinyun Dong, Guang Huang, Qijing Zhang, Zengtao Wang, Jiahua Cui, Yan Wu, Qingqing Meng*and
Shaoshun Li*
DOI: 10.1039/x0xx00000x
A series of benzochalcone derivatives has been synthesized and evaluated for CYP1 inhibitory activity and
cytotoxic property against wild type cell lines (MCF-7 and MDA-MB-231) and drug resistant cell lines (LCC6/P-
gp and MCF-7/1B1). All of these compounds were found to be selective inhibition towards CYP1B1 and the
most potent two possessed single-digit nanomolar CYP1B1 potency. In addition, part of them showed
promising cytotoxic activities not only against wild type cells, but also against drug resistant cells at low
micromolar concentrations. More importantly, these multi-functional compounds may surmount drug-drug
interactions that frequently occur during the combination of CYP1B1/P-gp inhibitors and anticancer drugs to
overcome drug resistance. This study may provide a good starting point for the further development of more
potent multi-functional agents with CYP1B1 inhibitory activity and cytotoxic potency in cancer prevention and
treatment.
contrary, CYP1A1 and CYP1A2 are prone to producing 2-
hydroxyestradiol (2-OHE2), while its further-oxidized product E2-2,3-
quinone is found to be non-mutagenic.4
1. Introduction
Cancer remains one of the most serious threats to human
health, for its incidence and mortality are rapidly growing worldwide.
Many environmental factors have been proposed to promote
carcinogenesis, including ultraviolet radiation, chemical carcinogens
and infection. Chemical carcinogens are frequently found in
everyday life. For example, benzo[a]pyrene, found in tobacco smoke
and charred foods, induces tumorigenesis after being subject to
oxidation by cytochrome P450 family 1 enzymes (CYP1s).1, 2 CYP1
family consist of three members, namely CYP1A1, CYP1A2, and
CYP1B1. Among them, CYP1B1 is the most extensively studied one.
Beyond its role in metabolizing xenobiotics into carcinogenic
products, CYP1B1 can catalyze the 4-hydroxylation of 17β-estradiol
(E2) subsequently leading to the formation of E2-3,4-quinone, a
mutagenic compound able to bind covalently to DNA.3 On the
To date, although a variety of drugs have been approved for
cancer treatment, most patients will eventually develop drug
resistance, leading to therapy failure.5 Thus there is still an urgent
need for new agents that are effective against this disease. Various
mechanisms have been attributed to drug resistance, such as
enhanced drug efflux (overexpression of P-glycoprotein (P-gp) etc.),
altered drug metabolism and reduced apoptosis.6 CYP1B1 has also
been reported to be associated with drug resistance, for its role in
the metabolism of certain anticancer agents such as docetaxel,
paclitaxel, and cisplatin.7 More studies have given evidence that
CYP1B1 is overexpressed in various cancers and is specially localized
to tumors.8 Therefore, development of selective CYP1B1 inhibitors
could be a promising strategy for cancer prevention and overcoming
drug resistance.9
School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road,
Shanghai, China
*Corresponding author. E-mail, ssli@sjtu.edu.cn; Fax, +86 21 34204775 ; Tel, +8621
34204775.

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Flavonoids have long been recognized for their broad spectrum Synthesis of compounds 14, 17 and 18 required the corresponding
View Article Online
DOI: 10.1039/C9MD00258H
11
of pharmacological activities.10, α-Naphthoflavone (ANF), a 7,8- intermediates of 1,4,5,8-tetramethoxy-2-naphthaldehyde (4e), 1-(1-
benzoflavone derivative, is a potent CYP1 inhibitor with no obvious aminonaphthalen-2-yl)ethan-1-one (5c) and 1-(1-hydroxy-4,5,8-
selectivity for CYP1B1 and CYP1A2.12 In the study of reversing trimethoxynaphthalen-2-yl)ethan-1-one (4g) that were prepared
CYP1B1-mediated drug resistance, owing to its low cytotoxicity, ANF according to our reported procedures.18,19. Compound 19 was
only acts as a modulator to increase the anticancer efficacy of obtained by selective reduction of carbon-carbon double bond of α,
cytotoxic drugs. As a result, the safety issues caused by drug-drug β-unsaturated ketone of 1.20 Treatment of compound 15 with
interactions should be taken into consideration when combining guanidine hydrochloride in the presence of KOH and H2O2 generated
modulators and anticancer drugs. Notably, chalcones, as an compound 20 in good yield.21
important member of flavonoid family, also display a wide range
Scheme 1. Reagents and conditions, a) KOH, EtOH, RT.; b) Zn; NH4Cl, RT.; c)
of biological activities including anticancer and suppression of CYP1
guanidine hydrochloride, KOH, H2O2, EtOH, Reflux; d) (CH3)2SO4, NaOH, THF/H2O,
enzymes as well as P-glycoprotein.13-17 Inspired by the biological
O
CHO
O
(i)
properties of chalcones, we herein describe our efforts to identify
benzochalcone-based bi-functional compounds with both cancer
preventive and therapeutic potential that were designed from lead
compound ANF (shown in Fig.1). In this way, their inhibitory potency
on CYP1B1 enzyme and P-gp, not only can block the metabolic
activation of chemically diverse procarcinogens, but also may
increase their efficacy in killing CYP1B1 or P-gp overexpressed cells.
More importantly, these multi-functional agents with CYP1B1 and P-
gp inhibitory activity as well as cytotoxic potency may be of
advantage, especially in terms of surmounting drug-drug interactions
that frequently occur during the combined administration of drugs.
a
R1
R
R
+
R1
1-18
X
X
X= C or N
R1=1- OH, H, 1-OCH3
1-NH2, 1-OH-4,5,8-triOCH3
(ii)
OH
O
OH
O
b
19
NH2
N
1
(iii)
(iv)
O
N
c
15
20
O
O
Br
O
OH
O
O
d
e
f
n
Linker
O
D
B
Br
OH
O
O
o
i
t
O
C
a
c
i
C
f
i
R
4c
4b
A
4a
r
d
i
n
o
g
4d
-
o
m
p
r
e
e
n
g
i
n
k
n
g
i
O
L
ANF
OH
A
O
O
O
OH
O
O
O
O
OH
O
O
O
O
O
OHC
h
B
D
n
o
i
t
a
c
i
f
i
d
j
i
o
m
A
,
D
g
r
n
i
O
O
i
n
g
r
O
O
O
O
B
m
o
4f
d
i
O
4g
4e
f
4g
OH
O
i
c
a
t
i
o
n
R
Ar
NO2
NH2
O
(v)
NH2
l
k
CN
Fig.1 Design and structural modification of benzochalcones
5b
5c
5a
2. Results and discussion
2.1. Chemistry
RT.; e) NBS, CH3CN, -20 OC; f) CH3ONa, CuI, DMF/MeOH, reflux; g) DMF, POCl3, DCM,
reflux; h) CH3I, Mg, Et2O, N2, HCl; i) MnO2, DCM, reflux; j) AlCl3, CH3CN; k) KCN,
NCCH2COOEt, KOH, DMF, 60 OC, NaOH-H2O (5%), reflux ; l) Mg, CH3I, Et2O, N2, HCl.
These different benzochalcones with varying substituents on
rings A and B could be divided into three different subtypes, B ring
modified benzochalcones (1-14), A/D ring modified benzochalcones
(15-18) and Linker modified benzochalcones (19-20). The synthesis
of compounds 1-20 followed the general pathway outlined in
Scheme 1. Compounds 1-18 were synthesized by Claisen-Schmidt
condensation of different substituted acetophenones and
benzaldehydes in the presence of ethanolic KOH. Particularly,
2.2. CYP1 inhibitory activity
These synthesized benzochalcones were evaluated for their
capacity to suppress recombinant human CYP1 enzymes using EROD
(7-ethoxyresorufin O-deethylation) assay. The results were
summarized in Tables 1 and 2. As a general trend, these
2 | J. Name., 2012, 00, 1-3
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ARTICLE
benzochalcone derivatives displayed
a significantly increased substituent at the para position of B ring generally provided superior
selectivity toward CYP1B1 over CYP1A2 when compared to ANF, activity. To investigate the effect of the type of B ring on CYP1B1
because the CYP1A2 inhibitory activity of all compounds were inhibitory ability, we changed the benzene ring with some aromatic
sharply decreased after the cleavage of ring-C of ANF, while their heterocyclic rings. Among compounds 10-13, 3-pyridine ring
CYP1B1 inhibitory activity showed a slower decline, and two of them substituted derivative 11 showed a remarkable CYP1B1 inhibitory
possessed similar levels of CYP1B1 inhibition as compared with ANF. effect (IC50 = 16.7 nM). With an aim of enhancing the hydrophobic
Therefore, we can conclude that CYP1A2 is much more susceptible interaction with heme pocket, we extended the benzene ring with
to the planarity of molecules. It was observed that compound 1 methoxyl substituted naphthalene ring resulting in a significant loss
showed mild inhibitory potency on CYP1B1 enzyme (IC50 = 157.7 nM), of its CYP1B1 inhibitory activity (IC50 >1000 nM), as seen with
which was 26-fold less potent than the lead compound ANF (IC50
=
compound 14, suggesting that larger functional groups were not
5.9 nM). However, its CYP1B1 inhibitory activity was slightly tolerated for B ring.
increased by introduction of substituent to B ring (2-9), and
Table 1 Inhibitory potency of B ring modified benzochalcones against CYP1 enzymes.
OH
O
Ar
IC50 values (nM)
1A1
80.3 ± 10.8
IC50 ratio
1A2/1B1
No
ANF
Ar
-
1B1
5.9 ± 1.3
1A2
18.0 ± 3.6
1A1/1B1
13.6
3.1
1
2
phenyl
157.7 ± 18.5
90.2 ± 11.4
81.9 ± 14.7
48.31 ± 8.2
55.4 ± 7.8
46.6 ± 8.7
40.2 ± 6.3
76.9 ± 3.9
37.8 ± 5.8
135.0 ± 14.8
16.7 ± 3.7
90.2 ± 6.2
51.8 ± 7.4
> 1000
> 1000
> 6.3
3.7
> 6.3
o- fluorophenyl
m- fluorophenyl
p-fluorophenyl
o- chlorophenyl
m- chlorophenyl
p- chlorophenyl
o- hydroxyphenyl
o- methoxyphenyl
2- pyridyl
330.0 ± 30.2
499.0 ± 18.9
> 1000
> 1000
> 11.1
> 12.2
> 20.7
> 18.1
> 21.5
> 24.9
> 13.0
> 26.5
> 7.4
3
> 1000
6.1
4
> 1000
> 20.7
9.5
5
529.0 ± 28.2
434.9 ± 25.8
401.2 ± 33.1
364.7 ± 17.2
556.6 ± 28.7
> 1000
> 1000
6
> 1000
9.3
7
> 1000
10.0
4.7
8
> 1000
9
> 1000
14.7
> 7.4
7.0
10
11
12
13
14
> 1000
3- pyridyl
117.1 ± 18.9
300.9 ± 21.7
250.0 ± 25.9
227.0 ± 25.2
> 1000
13.6
4- pyridyl
3.3
> 11.1
> 19.3
2-pyrrolyl
> 1000
4.8
1,4,5,8-tetrameth-
oxynaphthalen-2-yl
> 1000
532.6 ± 37.4
> 1000
< 0.5
> 1.0

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DOI: 10.1039/C9MD00258H
In order to get more information about the relationship ANF (IC50 value of 5.9 nM against CYP1B1). Further confirmation of
between the structure and CYP1 inhibitory effects, we therefore the importance of α, β-unsaturated ketone (Michael reaction
turned our attention to the naphthalene and linker moieties. As acceptor) was also conducted. Compound 19, obtained by selective
shown in Table 2, methylation of the hydroxyl group of compound 1 reduction of the carbon-carbon double bond of compound 1,
led to 16, which exhibited an increased inhibitory effect on CYP1B1 retained its inhibitory activity against CYP1B1 despite lacking of
(IC50 values of 51.4 vs 157.7 nM). In addition, the unsubstituted Michael reaction acceptor. However, a significantly increased effect
analogue 15 was also more potent than the hydrophilic group was observed from substitution with aminopyrimidine (20), resulting
substituted compounds 1 and 17 in blocking CYP1B1 enzyme. We in 20-fold increase in enzymatic potency of CYP1B1 as compared with
hypothesized that hydrophobic group would be beneficial for compound 15 (IC50 values of 4.8 and 95.5 nM, respectively). The
inhibiting CYP1B1. To test this hypothesis, we introduced three results suggested that α, β-unsaturated ketone is not a necessary
methoxyl groups to the naphthalene moiety (18). As expected, this structure for CYP1 inhibitory potency and a rigid functional group is
compound exhibited a dramatic increase in the CYP1B1 inhibitory more favorable.
activity with an IC50 value of 4.9 nM, which is equipotent to that of
Table 2 Inhibitory potency of naphthalene ring and linker modified benzochalcones against CYP1 enzymes.
Linker
8
1
4
R
5
IC50 values (nM)
1A1
IC50 ratio
1A2/1B1
Linker
No
15
R
1B1
1A2
1A1/1B1
> 10.5
O
H
95.5 ± 7.4
> 1000
> 1000
> 10.5
> 19.5
> 8.9
O
O
O
16
17
18
19
20
1-OCH3
51.4 ± 8.9
220.5 ± 32.3
285.8 ± 25.7
> 1000
> 1000
4.3
2.5
1-NH2
112.4 ± 7.3
4.9 ± 0.6
1-OH-4,5,8-
161.3 ± 17.4 734.7 ± 31.2
32.9
2.0
149.9
> 6.6
triOCH3
O
1-OH
H
151.3 ± 25.8 306.4 ± 11.3
> 1000
> 1000
NH2
4.8 ± 0.5
51.8 ± 9.7
10.8
> 208.3
N
N
2.3. Cytotoxic activity on cancer cells
To explore the potential anticancer activity of these synthesized LCC6/P-gp and MCF-7/1B1. Among them, LCC6/P-gp cells were
compounds, we evaluated their cytotoxic activity against four obtained by transfection of the mdr1 gene that can encode P-
selected cancer cell lines, MCF-7 (estrogen receptor positive tumor glycoprotein (P-gp), while the CYP1B1 overexpressed MCF-7 cell line
cell line), MDA-MB-231(estrogen receptor negative tumor cell line), (referred to as MCF-7/1B1) was obtained by the induction of TCDD
4 | J. Name., 2012, 00, 1-3
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(2,3,7,8-tetrachlorodibenzo-p-dioxin) in MCF-7 cells. It should be benzochalcones 10-12 generally showed higher cytotoxic activity
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DOI: 10.1039/C9MD00258H
noted that LCC6/P-gp and MCF-7/1B1are drug resistant cancer cell against the tested cells than the other compounds. In particular, the
lines, which were characterized by MTT (3-(4, 5-dimethylthiazol-2- 2-pyridyl substituted chalcone derivative 10, the most vigorous one
yl)-2, 5-diphenyltetrazolium bromide) assay (data not shown).
in this series, was found to have a broad spectrum of anticancer
The cytotoxic effect of these benzochalcones on selected activity that showed strong growth inhibitory effect not only on wild
tumour cells was determined by a standard MTT cell viability assay. type cells (MCF-7 and MDA-MB-231, IC50 values of 1.8 and 14.3 μM,
5-Fluorouracil (5-FU) and ANF were used as positive control drugs. As respectively), but also on drug resistant cells (LCC6/P-gp and MCF-
the data presented in Table 3, these compounds exhibited varying 7/1B1, IC50 values of 7.7 and 11.3 μM, respectively). To our surprise,
degree of activities, and most of them were more potent than 5-FU replacement of the benzene ring with tetramethoxy substituted
and ANF. In terms of selectivity, they were found to be superior in its naphthalene ring (14) suffered a dramatic drop of its CYP1B1 enzyme
activity against MCF-7 cells. As illustrated by compounds 1 through activity, while increasing cytotoxic activities against tumor cells MCF-
9, variations of the substituents on the phenyl ring did not result in 7 and LCC6/P-gp with IC50 values of 1.9 and 3.9 μM, respectively. It
any obvious change in activity. However, a substantial difference was was speculated that the enhanced cellular activity of compound 14
observed when the benzene ring was replaced with aromatic may be partly due to the improved lipophilicity and membrane
heterocyclic ring (10-13). Among them, the pyridyl substituted permeability as well as the nature of cells.
Table 3 Cytotoxic activities of benzochalcones against MCF-7, MDA-MB-231, LCC6/P-GP and MCF-7 cell lines.
IC50 values (μM)
IC50 values (μM)
No
No
MDA-MB-
231
46.2 ± 5.3
LCC6/
P-gp
> 100
MCF-7/
1B1
32.3 ± 4.5
MDA-MB-
231
19.8 ± 2.8
LCC6/
P-gp
8.0 ± 2.3
MCF-7/
1B1
12.7 ± 1.7
MCF-7
MCF-7
25.9 ± 3.2
25.8 ± 4.1
29.1 ± 3.9
48.1 ± 3.7
20.6 ± 2.2
22.3 ± 1.2
38.5 ± 2.9
26.9 ± 3.1
43.4 ± 1.6
1.8 ± 0.3
11
12
7.6 ± 0.7
20.5 ± 1.3
39.6 ± 4.1
1.9 ± 0.4
24.7 ± 2.1
22.5 ± 2.8
20.3 ± 1.2
6.3 ± 1.4
> 100
1
2
37.8 ± 2.8
66.4 ± 4.4
79.6 ± 4.9
43.5 ± 3.6
37.6 ± 1.8
65.3 ± 4.2
43.9 ± 4.0
75.3 ± 6.8
14.3 ± 0.8
> 100
29.8 ± 2.6
67.3 ± 3.9
48.6 ± 2.9
12.2 ± 2.7
20.9 ± 2.0
45.7 ± 5.3
25.1 ± 3.8
43.9 ± 4.4
7.7 ± 0.9
65.1 ± 4.1
33.8 ± 4.1
> 100
25.7 ± 2.5
48.7 ± 3.3
62.2 ± 5.7
34.1 ± 3.0
35.2 ± 2.2
35.5 ± 3.4
15.8 ± 2.2
> 100
15.8 ± 2.4
> 100
14.8 ± 1.9
> 100
13
3
14
3.9 ± 0.4
19.4 ± 2.7
30.8 ± 1.9
80.9 ± 6.2
9.6 ± 1.3
> 100
43.8 ± 6.3
18.2 ± 1.8
45.5 ± 3.5
23.8 ± 3.1
15.1 ± 1.8
> 100
4
42.4 ± 4.2
41.3 ± 3.2
48.4 ± 4.2
26.4 ± 4.5
> 100
15
5
16
6
17
7
18
8
19
9
11.3 ± 0.8
> 100
20
46.8 ± 4.7
> 100
48.9 ± 4.1
93.8 ± 6.1
> 100
20.4 ± 3.3
> 100
10
ANF
80.7 ± 7.6
82.3 ± 8.5
5-FU
> 100
Modification of naphthalene and linker parts could also affect However, introduction of three methoxyl groups to the naphthalene
their activities of 15-20. A remarkable change was not observed for moiety (18) led to a significant increase in cytotoxic activities against
removal of the free hydroxyl group on the naphthalene part of these selected cancer cells, with IC50 values ranging from 6.3 to 15.8
compound 1 (15), conversion of this free hydroxyl group to an ether μM. Coincidentally, as discussed above, this compound also exerted
(16), or replacement of this substituent with an amino group (17). strong CYP1B1 inhibitory ability, suggesting that compound 18 could
This journal is © The Royal Society of Chemistry 2019
Med. Chem. Commun., 2019, 00, 1-11 | 5
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be considered as a potential lead compound for developing for molecular docking study based on 3PM0. As shown in Fig. 2, 18
View Article Online
multifunctional drugs with a broad range of applications for cancer tightly fitted the active site of CYP1B1, and DitOs In: a10p.h10th39a/leCn9eMDpa0r0t2w58aHs
prevention and treatment. It is interesting to note that both 14 and surrounded by hydrophobic residues such as Val126, Phe268,
18 bearing multiple methoxyl groups showed promising cytotoxicity, Leu264, Thr325 and Gly329, as well as was covered by the phenyl
which is in agreement with previous study made by Valdameri et ring of Phe231. In this case, introduction of three methoxyl groups to
al22. As seen with compound 19, reduction of the carbon- the naphthalene moiety could increase the hydrophobicity and
carbon double bond of compound 1 resulted in a significant loss of electron density, which provided a favorable hydrophobic contact
its activity. Additionally, pyrimidine ring connected derivative 20 was with these hydrophobic residues and an enhanced π-π stacking
also less active as compared to the corresponding α, β-unsaturated interaction with Phe231. The distance between the closest carbon
ketone connected derivative 15. Built upon these phenomena, we atom on the phenyl ring of ANF and heme iron is 4.2 Å, while the
can conclude that the α, β-unsaturated ketone moiety of chalcones corresponding part of 18 is much closer to the heme iron, with
may be served as an important pharmacophore for cytotoxic activity. distance of 3.5 Å, which may play a more important role in affecting
the formation of reactive heme iron-oxo intermediate during
catalysis. Alternatively, the presence of a hydroxyl group provided an
additional interaction with Gly329 via formation of a hydrogen bond,
with a distance of 2.1 Å. All of these factors could contribute to
improvement in the enzyme inhibition activity of CYP1B1.
2.4. Molecular docking
The crystal structure of CYP1B1 in complex with ANF is also
known (PDB 3PM0). To gain insight into the potential binding modes
and rationalize the observed efficiency of CYP1B1 inhibition by these
chalcones, compound 18 as a potent CYP1B1 inhibitor was selected
Fig.2 The binding model of compound 18 with CYP1B1 (hydrogen bonding is depicted by red dashed lines, whereas yellow dashed lines
represent distance).
3. Conclusions
In summary, structural modification of ANF gave a series of benzochalcone against four selected cancer cell lines was also
benzochalcone derivatives, which exhibited varied level of inhibitory determined, and compounds 10, 11 and 14 showed remarkable
potency on CYP1 enzymes. Compared with ANF, almost all growth inhibitory effects not only on wild-type MCF-7 cell line, but
compounds presented an enhanced selectivity for inhibition of also on the drug resistant LCC6/P-gp cell line. Notably, compounds
CYP1B1 over CYP1A2. It was clearly observed that among all 10 and 11 were also found to be effective for drug resistant MCF-
benzochalcone, compounds 18 and 20 were identified as the most 7/1B1 cell line, which provide an additional clue for developing drugs
potent CYP1B1 inhibitors, with IC50 values in the single-digit to overcome drug resistance. Consistent with the aim of our
nanomolar range. In addition, the cytotoxic activity of these research, compound 18 exhibited good biological activities
6 | J. Name., 2012, 00, 1-3
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in both enzymatic and cellular assay, which provides an important administration of drugs. Based on this observation, the identification
View Article Online
lead compound for developing multi-functional agents to surmount of compounds with more potent inhibitoryDaOctI:iv1i0ty.10fo39r/bCo9tMhDe0n0z2y5m8He
drug-drug interactions that frequently occur during the combined and cell is continuing and will be reported in due course.
4. Experimental
4.1 Chemistry
(101 MHz, CDCl3) δ 193.20, 164.47, 163.12, 160.58, 141.11, 137.78,
137.41, 132.13, 132.05, 130.24, 130.06, 127.39, 125.92, 124.57,
124.54, 124.50, 123.88, 123.17, 123.10, 118.28, 116.47, 116.25.
HRMS (ESI) calcd for [C19H13FO2 + H]+ 293.0978, found 293.09768.
4.2.3. (E)-3-(3-fluorophenyl)-1-(1-hydroxynaphthalen-2-yl)prop-2-
en-1-one (3)
Commercially available starting materials, reagents, and dry
solvents were used as supplied. During the synthesis reaction
progress was monitored by thin-layer chromatography TLC (silica gel
GF254) and visualized with UV light (254 or 365 nm). Column
chromatography was conducted on silica gel (300 - 400 mesh) from
Qingdao Ocean Chemical Factory. 1H-NMR and 13C-NMR spectra
were recorded on a Varian Mercury-300 (400 MHz) spectrometer
using DMSO-d6 or CDCl3 as solvents and TMS as an internal standard.
All chemical shifts are given in ppm and coupling constants are given
in Hz. Mass spectra were recorded with HRMS (ESI, Agilent 6540
QTOF).
1
Yellow solid; yield 52%; H NMR (400 MHz, CDCl3) δ 14.74 (s,
1H), 8.50 (d, J = 8.3 Hz, 1H), 7.91 (d, J = 15.4 Hz, 1H), 7.85 – 7.75 (m,
2H), 7.71 (d, J = 15.5 Hz, 1H), 7.65 (t, J = 7.3 Hz, 1H), 7.54 (t, J = 7.6
Hz, 1H), 7.49 – 7.35 (m, 3H), 7.31 (d, J = 8.7 Hz, 1H), 7.14 (t, J = 7.8 Hz,
1H); 13C NMR (101 MHz, CDCl3) δ 192.88, 164.52, 161.83, 143.47,
137.44, 130.60, 130.52, 130.33, 127.40, 126.00, 124.74, 124.52,
123.76, 121.76, 118.34, 117.70, 117.49, 114.72, 114.50. HRMS (ESI)
calcd for [C19H13FO2 + H]+ 293.0978, found 293.09770
4.2 General procedure for the preparation of compounds 1-18
To a mixture of 2.5 mmol of selected naphthyl methyl ketone
and 2.5 mmol of selected benzaldehyde in 100 ml round bottom flask
30 ml ethanol and 40 mmol of KOH was added. The mixture was
stirred at room temperature for 24 h. After completion of the
reaction as indicated by TLC, the mixture was poured in ice water and
acidified with dilute HCl. Precipitated product was filtered and then
was purified by flash chromatography on silica to give the
corresponding title compound.
4.2.4. (E)-3-(4-fluorophenyl)-1-(1-hydroxynaphthalen-2-yl)prop-2-
en-1-one (4)
1
Yellow solid; yield 55%; H NMR (400 MHz, CDCl3) δ 14.80 (s,
1H), 8.49 (d, J = 8.3 Hz, 1H), 7.93 (d, J = 16 Hz, 1H), 7.82 (d, J = 8.9 Hz,
1H), 7.77 (d, J = 8.1 Hz, 1H), 7.72 – 7.61 (m, 4H), 7.54 (t, J = 7.6 Hz,
1H), 7.30 (d, J = 8.9 Hz, 1H), 7.13 (t, J = 8.1 Hz, 2H); 13C NMR (101
MHz, CDCl3) δ 192.99, 165.47, 164.42, 163.00, 143.69, 137.37,
130.60, 130.52, 130.23, 127.37, 125.95, 124.49, 123.79, 120.18,
118.24, 116.31, 116.10, 114.96, 113.37. HRMS (ESI) calcd for
[C19H13FO2 + H]+ 293.0978, found 293.09751
4.2.1. (E)-1-(1-hydroxynaphthalen-2-yl)-3-phenylprop-2-en-1-one (1)
1
Yellow solid; yield 53%; H NMR (400 MHz, CDCl3) δ 14.85 (s,
1H), 8.50 (d, J = 8.3 Hz, 1H), 7.97 (d, J = 15.4 Hz, 1H), 7.83 (d, J = 8.8
Hz, 1H), 7.79 – 7.67 (m, 4H), 7.63 (t, J = 7.5 Hz, 1H), 7.53 (t, J = 7.6 Hz,
1H), 7.44 (s, 3H), 7.30 (d, J = 8.9 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ
193.19, 164.39, 145.03, 137.37, 134.75, 130.81, 130.19, 129.01,
128.65, 127.38, 125.91, 125.47, 124.49, 123.89, 120.44, 118.21,
113.44. HRMS (ESI) calcd for [C19H14O2 + H]+ 275.1072, found
275.10760.
4.2.5. (E)-3-(2-chlorophenyl)-1-(1-hydroxynaphthalen-2-yl)prop-2-
en-1-one (5)
1
Yellow solid; yield 51%; H NMR (400 MHz, CDCl3) δ 14.74 (s,
1H), 8.50 (d, J = 8.3 Hz, 1H), 8.36 (d, J = 15.5 Hz, 1H), 7.87 – 7.74 (m,
3H), 7.71 (d, J = 15.5 Hz, 1H), 7.64 (t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.5
Hz, 1H), 7.47 (d, J = 7.3 Hz, 1H), 7.39 – 7.27 (m, 3H); 13C NMR (101
MHz, CDCl3) δ 192.93, 164.52, 140.72, 137.42, 135.70, 133.11,
131.38, 130.39, 130.30, 127.95, 127.38, 127.09, 125.97, 125.44,
124.54, 123.83, 123.12, 118.30, 113.37. HRMS (ESI) calcd for
[C19H13ClO2 + H]+ 309.0682, found 309.06844.
4.2.2. (E)-3-(2-fluorophenyl)-1-(1-hydroxynaphthalen-2-yl)prop-2-
en-1-one (2)
1
Yellow solid; yield 55%; H NMR (400 MHz, CDCl3) δ 14.79 (s,
1H), 8.49 (d, J = 8.2 Hz, 1H), 8.00 (d, 15.8 Hz, 1H), 7.88 – 7.71 (m, 3H),
7.70 – 7.58 (m, 2H), 7.53 (t, J = 7.2 Hz, 1H), 7.44 – 7.33 (m, 1H), 7.29
(d, J = 9.0 Hz, 1H), 7.21 (t, J = 7.4 Hz, 1H), 7.18 – 7.08 (m, 1H); 13C NMR
4.2.6. (E)-3-(3-chlorophenyl)-1-(1-hydroxynaphthalen-2-yl)prop-2-
en-1-one (6)
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Med. Chem. Commun., 2019, 00, 1-11 | 7
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1
Yellow solid; yield 59%; H NMR (400 MHz, CDCl3) δ 14.73 (s, 4.2.10. (E)-1-(1-hydroxynaphthalen-2-yl)-3-(pyridin-2-yl)prop-2-en-
View Article Online
DOI: 10.1039/C9MD00258H
1H), 8.52 – 8.44 (m, 1H), 7.88 (d, J = 15.5 Hz, 1H), 7.81 (d, J = 8.9 Hz, 1-one (10)
1
1H), 7.79 – 7.71 (m, 2H), 7.70 – 7.61 (m, 3H), 7.56 – 7.51 (m, 2H), 7.41
Yellow solid; yield 48%; H NMR (400 MHz, CDCl3) δ 14.75 (s,
– 7.38 (m, 1H), 7.31 (d, J = 8.9 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 1H), 8.83 – 8.63 (m, 1H), 8.58 – 8.43 (m, 1H), 8.42 – 8.28 (m, 1H), 8.05
192.81, 164.51, 143.21, 137.45, 136.59, 135.05, 130.55, 130.33, – 7.85 (m, 2H), 7.82 – 7.69 (m, 2H), 7.62 (d, J = 6.9 Hz, 1H), 7.56 – 7.43
130.24, 128.00, 127.40, 126.99, 125.99, 124.88, 124.53, 124.43, (m, 2H), 7.30 – 7.20 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 193.46,
123.77, 121.81, 118.34. HRMS (ESI) calcd for [C19H13ClO2 + H]+ 164.55, 152.85, 150.05, 142.53, 137.54, 137.09, 130.29, 127.38,
309.0682, found 309.06790.
4.2.7. (E)-3-(4-chlorophenyl)-1-(1-hydroxynaphthalen-2-yl)prop-2- 113.65. HRMS (ESI) calcd for [C18H13NO2 + H]+ 276.1025, found
en-1-one (7) 276.10255.
125.85, 125.36, 124.77, 124.58, 124.51, 124.48, 124.30, 118.30,
1
Yellow solid; yield 55%; H NMR (400 MHz, CDCl3) δ 14.77 (s, 4.2.11. (E)-1-(1-hydroxynaphthalen-2-yl)-3-(pyridin-3-yl)prop-2-en-
1H), 8.49 (d, J = 8.2 Hz, 1H), 7.96 – 7.88 (m, 1H), 7.81 (d, J = 8.9 Hz, 1-one (11)
1H), 7.77 (d, J = 8.1 Hz, 1H), 7.72 (s, 1H), 7.66 – 7.60 (m, 3H), 7.56 –
Yellow solid; yield 49%; 1H NMR (400 MHz, DMSO-d6) δ 14.91 (s,
7.52 (m, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 9.0 Hz, 1H); 13C NMR 1H), 9.08 (s, 1H), 8.67 – 8.59 (m, 1H), 8.44 (d, J = 7.9 Hz, 1H), 8.39 –
(101 MHz, CDCl3) δ 192.91, 164.46, 143.47, 137.41, 136.73, 133.26, 8.26 (m, 3H), 8.01 – 7.90 (m, 2H), 7.73 (t, J = 7.6 Hz, 1H), 7.60 (t, J =
130.28, 129.74, 129.30, 127.38, 125.97, 124.52, 123.76, 120.97, 7.6 Hz, 1H), 7.54 – 7.48 (m, 1H), 7.46 (d, J = 9.1 Hz, 1H); 13C NMR (101
118.28, 113.37, 109.98, 77.30, 76.98, 76.66. HRMS (ESI) calcd for MHz, DMSO-d6) δ 193.78, 163.81, 151.81, 151.18, 142.28, 137.62,
[C19H13ClO2 + H]+ 309.0682, found 309.0678.
4.2.8. (E)-1-(1-hydroxynaphthalen-2-yl)-3-(2-hydroxyphenyl)prop-2- 124.14, 123.42, 118.81, 113.75. HRMS (ESI) calcd for [C18H13NO2 + H]+
en-1-one (8) 276.1025, found 276.10353.
135.95, 131.08, 130.77, 128.10, 126.72, 125.51, 124.85, 124.41,
Yellow solid; yield 52%; 1H NMR (400 MHz, DMSO-d6) δ 15.15 (s, 4.2.12. (E)-1-(1-hydroxynaphthalen-2-yl)-3-(pyridin-4-yl)prop-2-en-
1H), 10.39 (s, 1H), 8.35 (d, J = 8.0 Hz, 1H), 8.28 (d, J = 15.4 Hz, 1H), 1-one (12)
1
8.21 (d, J = 9.0 Hz, 1H), 8.09 (d, J = 15.2 Hz, 1H), 7.98 (d, J = 7.5 Hz,
Yellow solid; yield 46%; H NMR (400 MHz, CDCl3) δ 14.61 (s,
1H), 7.92 (d, J = 7.6 Hz, 1H), 7.72 (t, J = 7.3 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 8.71 (dd, J = 4.5, 1.6 Hz, 2H), 8.49 (dd, J = 8.3, 0.6 Hz, 1H), 7.84
1H), 7.45 (d, J = 9.0 Hz, 1H), 7.29 (d, J = 7.2 Hz, 1H), 6.95 (d, J = 8.2 Hz, (d, J = 1.7 Hz, 2H), 7.80 – 7.74 (m, 2H), 7.69 – 7.62 (m, 1H), 7.57 – 7.52
1H), 6.90 (t, J = 7.3 Hz, 1H); 13C NMR (101 MHz, DMSO-d6) δ 194.16, (m, 1H), 7.50 (dd, J = 4.5, 1.6 Hz, 2H), 7.31 (d, J = 8.9 Hz, 1H). 13C NMR
163.57, 158.06, 141.17, 137.44, 133.14, 130.82, 129.50, 128.07, (101 MHz, CDCl3) δ 192.47, 164.78, 150.66, 141.87, 141.68, 137.54,
126.61, 125.28, 124.98, 124.97, 124.08, 121.67, 119.93, 118.68, 130.60, 127.45, 126.15, 125.34, 124.76, 124.58, 123.61, 122.09,
116.78, 113.79. HRMS (ESI) calcd for [C19H14O3 + H]+ 291.1021, found 118.55, 113.25. HRMS (ESI) calcd for [C18H13NO2 + H]+ 276.1025,
291.10289.
4.2.9. (E)-1-(1-hydroxynaphthalen-2-yl)-3-(2-methoxyphenyl)prop-2- 4.2.13. (E)-1-(1-hydroxynaphthalen-2-yl)-3-(1H-pyrrol-2-yl)prop-2-
en-1-one (9) en-1-one (13)
Yellow solid; yield 54%; H NMR (400 MHz, CDCl3) δ 14.96 (s, Yellow solid; yield 51%; H NMR (400 MHz, CDCl3) δ 15.06 (s,
found 276.10329.
1
1
1H), 8.50 (d, J = 8.3 Hz, 1H), 8.29 (d, J = 15.5 Hz, 1H), 7.91 – 7.82 (m, 1H), 8.76 (brs, 1H), 8.48 (d, J = 8.7 Hz, 1H), 7.88 (d, J = 15.1 Hz, 1H),
2H), 7.77 (d, J = 7.9 Hz, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.63 (t, J = 7.5 Hz, 7.77 (dd, J = 13.6, 8.4 Hz, 2H), 7.62 (t, J = 7.2 Hz, 1H), 7.52 (t, J = 7.4
1H), 7.53 (t, J = 7.5 Hz, 1H), 7.41 (t, J = 7.9 Hz, 1H), 7.30 (d, J = 8.8 Hz, Hz, 1H), 7.30 (d, J = 16.7 Hz, 2H), 7.03 (s, 1H), 6.78 (s, 1H), 6.37 (s,
1H), 7.02 (t, J = 7.5 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 3.96 (s, 3H); 13
C
1H); 13C NMR (101 MHz, CDCl3) δ 192.60, 164.01, 137.16, 134.32,
NMR (101 MHz, CDCl3) δ 193.80, 164.27, 159.02, 140.70, 137.30, 129.87, 129.37, 127.29, 125.77, 125.59, 124.40, 123.74, 123.57,
132.06, 130.00, 129.57, 127.34, 125.79, 125.53, 124.46, 124.08, 117.94, 116.04, 113.80, 113.45, 111.85. HRMS (ESI) calcd for
123.83, 121.14, 120.79, 118.04, 113.61, 111.31, 55.59. HRMS (ESI) [C17H13NO2 + H]+ 264.1025, found 264.10236.
calcd for [C20H16O3+ H]+ 305.1178, found 305.11768.
4.2.14.
(E)-1-(1-hydroxynaphthalen-2-yl)-3-(1,4,5,8-
tetramethoxynaphthalen-2-yl)prop-2-en-1-one (14)
8 | J. Name., 2012, 00, 1-3
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1
Yellow solid; yield 45%; H NMR (400 MHz, CDCl3) δ 14.93 (s,
brown solid; yield 43%; 1H NMR (400 MHz, CDCl3) δ 12.83 (s, 1H),
1H), 8.49 (d, J = 14.6 Hz, 2H), 7.91 (d, J = 8.3 Hz, 1H), 7.80 (d, J = 18.1 7.86 (d, J = 15.4 Hz, 1H), 7.75 (d, J = 15.8 Hz, 1H), 7.66 (d, J = 6.7 Hz,
Hz, 2H), 7.68 – 7.60 (m, 1H), 7.59 – 7.49 (m, 1H), 7.32 (d, J = 8.4 Hz, 2H), 7.46 – 7.38 (m, 3H), 7.27 (s, 1H), 6.98 (d, J = 8.6 Hz, 1H), 6.88 (d,
1H), 7.14 (s, 1H), 6.93 (q, J = 8.2 Hz, 2H), 4.04 (s, 3H), 3.98 (s, 3H), 3.93 J = 8.3 Hz, 1H), 4.03 (s, 3H), 3.94 (s, 3H), 3.91 (s, 3H); 13C NMR (101
(s, 3H), 3.86 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 193.43, 164.33, MHz, CDCl3) δ 191.93, 152.81, 151.22, 148.74, 143.32, 135.21,
153.46, 152.09, 151.37, 151.26, 140.11, 137.32, 130.07, 127.36, 130.29, 130.13, 128.87, 128.50, 128.42, 124.33, 118.52, 117.36,
125.85, 125.53, 124.99, 124.50, 124.07, 123.00, 122.24, 121.31, 112.02, 108.22, 107.46, 58.06, 57.80, 57.01. HRMS (ESI) calcd for
118.07, 113.61, 110.87, 108.75, 104.95, 63.68, 57.91, 57.42 , 57.09. [C22H20O5 + H]+ 365.1389, found 365.13991.
HRMS (ESI) calcd for [C27H24O6 + H]+ 445.1651, found 445.16585.
4.2.19. 1-(1-hydroxynaphthalen-2-yl)-3-phenylpropan-1-one (19)
Compound 1 (0.5 mmol, 137 mg) was suspended in ethanol (50
4.2.15. (E)-1-(naphthalen-2-yl)-3-phenylprop-2-en-1-one (15)
Pale white solid; yield 72%; 1H NMR (400 MHz, CDCl3) δ 8.53 (s, ml), and a mixture of water solution (8 mL) containing 1.07 g (20
1H), 8.11 (d, J = 8.6 Hz, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.94 – 7.82 (m, mmol) ammonium chloride at room temperature was added. The
3H), 7.73 – 7.63 (m, 3H), 7.62 – 7.51 (m, 2H), 7.43 (d, J = 4.6 Hz, 3H); mixture is stirred vigorously, and 0.195 g (3 mmol) zinc powder
13C NMR (101 MHz, CDCl3) δ 190.18, 144.71, 135.54, 135.48, 134.97, added in three equal portions at intervals of 15min. The progress of
132.57, 130.54, 129.95, 129.53, 128.97, 128.57, 128.51, 128.39, the reaction was monitored by TLC. When the reaction was finished,
127.83, 126.78, 124.49, 122.09. HRMS (ESI) calcd for [C19H14O + H]+ The suspended material was removed by filtration and the filtrate
259.1123, found 259.11315.
was evaporated under reduced pressure. The obtained residue was
4.2.16. (E)-1-(1-methoxynaphthalen-2-yl)-3-phenylprop-2-en-1-one poured into water, and the precipitated crude product was filtered
(16)
and recrystallized from ethanol to give pure compound 19 as pale
1
1
Pale white solid; yield 56%; H NMR (400 MHz, CDCl3) δ 8.31 – white solid (116 mg, yield 84%) . H NMR (400 MHz, CDCl3) δ 14.04
8.23 (m, 1H), 7.87 – 7.71 (m, 3H), 7.67 – 7.50 (m, 6H), 7.40 – 7.29 (m, (s, 1H), 8.47 (d, J = 8.3 Hz, 1H), 7.75 (d, J = 8.2 Hz, 1H), 7.68 – 7.59 (m,
3H), 3.92 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 192.68, 156.98, 143.99, 2H), 7.53 (t, J = 7.5 Hz, 1H), 7.35 – 7.27 (m, 4H), 7.27 – 7.21 (m, 3H),
136.58, 134.99, 130.47, 128.96, 128.53, 128.17, 128.10, 128.06, 3.40 (t, J = 7.7 Hz, 2H), 3.13 (t, J = 7.7 Hz, 2H); 13C NMR (101 MHz,
126.67, 126.50, 126.06, 124.11, 123.17, 64.03. HRMS (ESI) calcd for CDCl3) δ 205.12, 162.57, 140.81, 137.27, 130.02, 128.58, 128.38,
[C20H16O2 + H]+ 289.1229, found 289.1232.
127.36, 126.28, 125.92, 125.34, 124.44, 124.07, 118.34, 112.76,
4.2.17.
(E)-1-(1-aminonaphthalen-2-yl)-3-phenylprop-2-en-1-one 40.34, 30.21. HRMS (ESI) calcd for [C19H16O2 + H]+ 277.1229, found
(17)
277.1220.
brown solid; yield 46%; 1H NMR (400 MHz, CDCl3) δ 7.95 (d, J = 4.2.20. 4-(naphthalen-2-yl)-6-phenylpyrimidin-2-amine (20)
8.2 Hz, 1H), 7.88 (d, 1H), 7.79 – 7.71 (m, 3H), 7.69 – 7.64 (m, 2H), 7.60 To a mixture of compound 15 (1 mmol, 258 mg) and guanidine
– 7.55 (m, 1H), 7.53 – 7.47 (m, 1H), 7.45 – 7.33 (m, 3H), 7.10 (d, J = hydrochloride (2 mmol, 191 mg) in 100 ml round bottom flask 15 ml
8.9 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 191.11, 150.12, 142.35, ethanol and 50% aqueous KOH solution (15 mL) was added. The
136.36, 135.49, 129.93, 129.01, 128.86, 128.45, 128.22, 126.47, mixture was stirred at reflux temperature for 1.5 h. Then, 30%
125.41, 123.75, 123.35, 121.75, 115.42, 112.33. HRMS (ESI) calcd for aqueous H2O2 (5 ml) was added to the above mixture in small
[C19H15NO + H]+ 274.1232, found 274.12352.
4.2.18. (E)-1-(1-hydroxy-4,5,8-trimethoxynaphthalen-2-yl)-3- indicated by TLC, he ethanol was removed under reduced pressure,
phenylprop-2-en-1-one (18) and water was added to the residue. The precipitated title compound
portions over a period of 1.5 h. After completion of the reaction as

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ARTICLE
was filtered off, and washed with water for several times. The crude 4.4. Cytotoxic activity assay
solid was recrystallized from ethanol to give pure title compound 20 According to the reported procedure, MCF-7/1B1 cells were
as pale white solid (240 mg, yield 81%). 1H NMR (400 MHz, CDCl3) δ obtained by coincubation of parental MCF-7 cells with 2,3,7,8-
8.58 (s, 1H), 8.16 (d, J = 8.5 Hz, 1H), 8.13 – 8.05 (m, 2H), 8.02 – 7.92 tetrachlorodibenzo-p-dioxin (TCDD, 10 nM) for five days. LCC6/P-gp
(m, 2H), 7.91 – 7.83 (m, 1H), 7.60 (s, 1H), 7.57 – 7.46 (m, 5H), 5.43 (s, cell line was obtained from The Hong Kong Polytechnic University.
2H); 13C NMR (101 MHz, CDCl3) δ 166.26, 166.04, 163.62, 137.73, MCF-7, MDA-MB-231 and MCF-7/1B1 cells were grown in RPMI-1640
134.94, 134.44, 133.23, 130.49, 128.93, 128.78, 128.50, 127.72, medium, while LCC6/P-gp were grown in DMEM/high glucose
127.20, 127.17, 127.14, 126.48, 124.19, 104.50. HRMS (ESI) calcd for medium. cell growth inhibition was determined by MTT cytotoxicity
[C20H15N3 + H]+ 298.1344, found 298.13599.
assay. Generally, Approximately 4000 cells, suspended medium,
were seeded into each well of a 96-well plate and incubated at 37 oC
in a humidified atmosphere with 5% CO2 for 24 h. Then, different
4.3. Enzyme assays
The recombinant human CYP1B1, CYP1A1, and CYP1A2 concentrations of synthesized compounds (the final concentrations
enzymes, each equipped with P450 reductase (Supersomes),were were 0.5, 1, 5, 10, 25, 50 and 100 μM, respectively) were added
purchased from BD Genetest. 7-Ethoxyresorufin (7-ER) was obtained (except vehicle control group). The final volume in each well was 200
from Sigma-Aldrich. Nicotinamide-adenine dinucleotide phosphate μl and the final concentration of DMSO in any assay system did not
(NADP+), D-glucose-6-phosphate (G-6-P) and glucose-6-phosphate exceed 0.1% (v/v). After 48 h incubation, the culture medium was
dehydrogenase (G-6-PD) were purchased from Biosharp. The removed and refreshed by RPMI-1640 medium (200 μL). Next, MTT
inhibitory activities of these prepared compounds against CYP1B1, solution (20 μL, 5 mg/mL) was added to each well and incubated for
CYP1A1, and CYP1A2 enzymes were determined using the EROD an additional 4 h. Medium was discarded and and the resulting
23
assay as reported earlier.19,
Generally, a mixture with a final formazan crystals were dissolved in DMSO (150 μL/well). Absorbance
volume of 200 μl containing different concentrations (the final values were determined by a microplate reader at 570 nm. Three
concentrations were 0.5, 1, 5, 10, 50, 100, 500 and 1000 nM, replicates of each concentration were used in each assay and all
respectively) of tested compounds (except positive and negative assays were repeated three times. IC50 values were calculated with
control wells), an enzyme source (20 fmol CYP1B1, 10 fmol CYP1A1 the GraphPad Prism Software (Version 5.0) using the non-linear
or 60 fmol CYP1A2), 150 nM 7-ER,1.3 mM NADP+, 3.3 mM G-6-P, 0.5 regression formula, log (inhibitor) vs. normalized response-variable
U/ml, G-6-PD and 3.3 mM MgCl2 was incubated in a black 96-well slope.
flat-bottomed microplate at 37 oC for varied time durations
(incubation time for CYP1B1, CYP1A1, and CYP1A2 was 35, 15 and 50 4.5. Molecular docking
min, respectively). Then, the reaction was stopped by the addition of
The molecule docking experiment was carried out using the
100 μl of methanol to all wells. Lastly, the fluorescence intensity with docking program in MOE 2008 based on the crystal structures of
excitation and emission filters at 544 and 590 nm, respectively. For CYP1B1 (PDB ID, 3PM0). The initial 3D conformation of compound 18
each concentration, three replicates were used and each test was were optimized in ChemBio3D Ultra using the MM2 energy
repeated three times. The IC50 value for each compound was minimization method. All molecules were removed from the crystal
calculated with the GraphPad Prism Software (Version 5.0) using the structures before the experiment. Hydrogens and partial charges
non-linear regression formula, log (inhibitor) vs. normalized were added with the protonate 3D application. The residues within
response-variable slope.
a radius of 8.0 Å around the ligands were selected as docking sites.

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ARTICLE
Docking parameters were kept at default, except for the first
scoring function, where ASE Scoring was used instead of the default
London dG. The best pose was characterized by the scoring results.
10. M. Sudhakaran, S. Sardesai and A. I. Doseff. Antioxidants (Basel).
2019,8.
11. J. Dong, Q. Zhang, Q. Meng, Z. Wang, S. Li and J. Cui, Mini. Rev.
Med. Chem., 2018, 18, 1714-1732.
Conflicts of interest
The authors declare no conflicts of interest.
12. T. Shimada, H. Yamazaki, M. Foroozesh, N. E. Hopkins, W. L.
Alworth and F. P. Guengerich, Chem. Res. Toxicol., 1998, 11, 1048-
1056.
Acknowledgements
Research reported in this publication were supported by the
13. N. Mateeva, S. V. K. Eyunni, K. K. Redda, U. Ononuju, T. D.
Hansberry, C. Aikens and A. Nag, Bioorg. Med. Chem. Lett., 2017, 27,
2350-2356.
Fundamental Research Funds for the Central University
(No. ZH2018QNB15), the Shanghai Natural Science Fund (No.
16ZR1418100), the Shanghai Science and Technology Innovation
Program (No. 15431900700) and the National Natural Science
Foundation of China (Grant No. 21602132). We want to express our
gratitude to Dr. Iris L. K. Wong and Prof. Larry M. C. Chow in the Hong
Kong Polytechnic University for the provision of the well-cultured
LCC6 drug-resistant cancer cells.
14. I. S. Williams, P. Joshi, L. Gatchie, M. Sharma, N. K. Satti, R. A.
Vishwakarma, B. Chaudhuri and S. B. Bharate, Bioorg. Med. Chem.
Lett., 2017, 27, 3683-3687.
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Ruparelia, L. Gatchie, V. R. Sonawane, I. S. Williams, H. L. Tan, P. Joshi,
S. S. Bharate, V. Kumar, S. B. Bharate and B.Chaudhuri, Eur. J. Med.
Chem., 2017, 129, 159-174.
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DOI: 10.1039/C9MD00258H
O
D
C ring-
opening
B
R
O
C
Ar
A
Structural
modification
O
benzochalcones
ANF
Good activity in inhibiting CYP1B1
enzyme and/or cytotoxicity,including
towards drug-resistant cancer cells
CYP1 inhibitors
low cytotoxicity against
cancer cells