Development of 2-arylbenzo[: H] quinolone analogs as selective CYP1B1 inhibitors

Article abstract of DOI:10.1039/c8ra00465j

The CYP1B1 enzyme is regarded as a potential target for cancer prevention and therapy. Based on the structure of α-naphthoflavone (ANF), diverse 2-arylbenzo[h]quinolone derivatives were designed, synthesized and evaluated as selective CYP1B1 inhibitors. Compared with ANF, although few of the title compounds possessed comparable or slightly higher CYP1B1 inhibitory activity, these compounds displayed a significantly increased selectivity toward CYP1B1 over CYP1A2. Among them compounds 5e, 5g and 5h potently inhibited the activity of CYP1B1 with IC₅₀ values of 3.6, 3.9 and 4.1 nM respectively, paralleled by an excellent selectivity profile. On the basis of predicted clogP values, these target compounds may exhibit improved water-solubility compared to ANF. In particular, 5h showed a great superiority in the reversal of CYP1B1-mediated docetaxel resistance in vitro. The current study may serve as a good starting point for the further development of more potent as well as specific CYP1B1 inhibitors capable of reversing CYP1B1-mediated anticancer-drug resistance.

Full text of DOI:10.1039/c8ra00465j

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PAPER
Development of 2-arylbenzo[h]quinolone analogs
as selective CYP1B1 inhibitors†
Cite this: RSC Adv., 2018, 8, 15009
Jinyun Dong,
Zengtao Wang, Qingqing Meng, Qijing Zhang, Guang Huang,
*
*
Jiahua Cui and Shaoshun Li
The CYP1B1 enzyme is regarded as a potential target for cancer prevention and therapy. Based on the
structure of a-naphthoflavone (ANF), diverse 2-arylbenzo[h]quinolone derivatives were designed,
synthesized and evaluated as selective CYP1B1 inhibitors. Compared with ANF, although few of the title
compounds possessed comparable or slightly higher CYP1B1 inhibitory activity, these compounds
displayed a significantly increased selectivity toward CYP1B1 over CYP1A2. Among them compounds 5e,
5g and 5h potently inhibited the activity of CYP1B1 with IC50 values of 3.6, 3.9 and 4.1 nM respectively,
paralleled by an excellent selectivity profile. On the basis of predicted clog P values, these target
compounds may exhibit improved water-solubility compared to ANF. In particular, 5h showed a great
superiority in the reversal of CYP1B1-mediated docetaxel resistance in vitro. The current study may serve
as a good starting point for the further development of more potent as well as specific CYP1B1 inhibitors
capable of reversing CYP1B1-mediated anticancer-drug resistance.
Received 17th January 2018
Accepted 3rd April 2018
DOI: 10.1039/c8ra00465j
rsc.li/rsc-advances
Subsequent oxidation of 4-OHE2 to E2-3,4-quinone promotes
the formation of a covalent quinone-DNA adduct, which is
1 Introduction
responsible for estrogen-related carcinogenesis. Conversely, the
other two members of the CYP1 family are prone to generate 2-
hydroxy estradiol (2-OHE2); its further-oxidized product E2-2,3-
quinone has been proven to be non-mutagenic.6,7 On the other
hand, the overexpressed CYP1B1 isoform can speed up the
metabolic inactivation of a structurally diverse range of anti-
cancer drugs, such as docetaxel, doxorubicin, paclitaxel, and
mitoxantrone.8 As a result, the cellular efficacy of cytotoxic
drugs is decreased and cancer cells will eventually develop
resistance to these chemotherapeutic agents. Many studies have
conrmed that the CYP1B1 isoform is signicantly and
consistently overexpressed in a wide variety of tumor tissues
such as mammary, prostate, ovary, uterus, pituitary, and also in
skin and lung cancers, while it is expressed at very low levels or
not at all in normal tissues.9–12 Hence, selective inhibition of
this enzyme will have a great inuence on cancer prevention
and the reversal of drug resistance.
Human cytochrome P450 enzymes (CYPs) are composed of
different families of hemeproteins that play a predominant role
in the oxidative metabolism of both exogenous and endogenous
substances. The CYP1 subfamily of enzymes (CYP1s) contains
three extensively studied members, CYP1A1, CYP1A2, and
CYP1B1, due to their critical roles in pro-carcinogen-elicited
tumorigenesis and inducing drug resistance.1 They are
expressed in a tissue-specic manner, and are induced via the
aryl hydrocarbon receptor (AhR), which is a ligand-activated
transcription factor.2 Planar aromatic molecules such as
2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD) and 7,12-dime-
thylbenz[a]anthracene (DMBA) have been identied as potent
AhR ligands that induce the overexpression of CYP1 enzymes.3–5
A large number of pro-carcinogens such as polycyclic aromatic
hydrocarbons (PAHs), polyhalogenated aromatic hydrocarbons
(PHAHs), aryl amines, heterocyclic aryl amines and estrogens
can be converted into cytotoxic, mutagenic and carcinogenic
chemicals in the presence of overexpressed CYP1 enzymes.1
Among the three isoforms mentioned above, CYP1B1 is the
most promising target. On one hand, it has been regarded as an
important enzyme in the carcinogenic action of 17b-estradiol
(E2) by catalyzing the hydroxylation of E2 at C-4 regiospecically
to generate the carcinogenic form 4-hydroxy estradiol (4-OHE2).
a-Naphthoavone (ANF),
a synthetic avonoid, bears
a phenyl ring annulated at C-7,8 of the A-ring of avone. It has
been recognized as a potent CYP1 inhibitor which exhibits
strong inhibitory effects on recombinant human CYP1B1 and
CYP1A2 with IC50 values of 5 and 6 nM respectively, but to
a much lesser extent on CYP1A1 (with an IC50 value of 60 nM).13
Evidence has accumulated during the past several years that
exposure to ANF can reduce drug resistance and enhance the
sensitivity of CYP1B1-expressing cell lines to anticancer
drugs.14,15 However, its further development is still restricted
due to its poor water solubility and very limited selectivity
School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai,
China. E-mail: cpucjh@sjtu.edu.cn; ssli@sjtu.edu.cn; Fax: +86 21 34204775; Tel: +86
21 34204775
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra00465j
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toward CYP1B1 over CYP1A2.8 From the X-ray crystallographic addition of antimony trichloride (SbCl3) in CH3CN.21 Consid-
structures of CYP1 enzymes and ANF, it can be observed that the ering its toxicity, we also made efforts in exploring safe catalysts
amino acid sequences and the orientations of ANF in the such as ZnCl2 and AlCl3, nevertheless, none of these Lewis acids
binding cavity are different among CYP1 members.16–18 Asp333 were identied as an efficient catalyst for this intramolecular
on the I helix of CYP1B1 is adjacent to the oxygen atom in the C- aza-Michael reaction. On the other hand, oxidation of 10a–d by
˚
ring of ANF, with a short distance of 4.6 A, while Asp320 on the I alkaline hydrogen peroxide afforded the stable 2-amino-
helix of CYP1A1 or CYP1A2 is close to the carbonyl oxygen atom chalcone epoxide derivatives 11a,b in very good yields,22 which
of ANF. Inspired by a hypothesis that an alkaline group can be were cyclized to 3-hydroxy-2-aryl-2,3-dihydrobenzo[h]quinolin-
introduced at a suitable location to enhance interactions with 4(1H)-ones 2a,b via an intramolecular nucleophilic reaction by
the acidic amino acid Asp333, replacement of the oxygen atom addition of a catalytic amount of AlCl3. It is of note that dilute
on the C-ring of ANF with a bioisosteric hydrophilic imino HCl and HOAc as catalysts were also used in this reaction, but
group was carried out with the aim to improve its selectivity we failed to obtain any title compound.
towards CYP1B1 and water solubility. In continuation of our
For the preparation of 3-hydroxy-2-arylbenzo[h]quinolin-
previous work in this area,8 we synthesized and biologically 4(1H)-ones 3a–d, we used the synthetic strategy described in
evaluated different classes of 2-arylbenzo[h]quinolones as novel Scheme 2. 1-Amino-2-naphthonitril (7) was hydrolyzed with
CYP1 inhibitors (Fig. 1).
20% NaOH to provide the corresponding L-amino-2-naphthoic
acid (12) in 62% yield. Treatment of 12 with 2-bromo-1-
arylethan-1-ones 13a–d in the presence of K2CO3 in DMF gave
intermediates 14a–d in excellent yields, which was followed by
a cyclization under acidic conditions using polyphosphoric acid
2 Results and discussion
2.1 Synthesis
ꢀ
(PPA) at 130 C to yield title compounds 3a–d.
The 2-aryl-2,3-dihydrobenzo[h]quinolin-4(1H)-ones 1a–d and
their 3-hydroxy derivatives 2a,b were prepared from 1-nitro-
naphthalene (6) (Scheme 1). Treatment of 6 with ethyl cyanoa-
cetate and KCN in the presence of KOH in DMF at 55 ꢀC for 36 h
formed an intermediate, which was followed by hydrolysis to
furnish 1-amino-2-naphthonitril (7) in 40% yield.19 Several
attempts to improve the yield of compound 7 including varia-
tion of time, temperature and replacement of ethyl cyanoacetate
with dicyanomethane were made by our research group–
unfortunately the yield was not obviously improved. Subse-
quently, intermediate 7 was reacted with methylmagnesium
iodide, and this was followed by a hydrolysis reaction to give 1-
amino-2-acetylnaphthalene (8).20 The 2-aminochalcone deriva-
tives 10a–d were obtained by the condensation of 8 with benz-
aldehydes 9a–d under alkaline conditions in THF. On one hand,
the cyclizations of 10a–d to the corresponding 2-aryl-2,3-dihy-
drobenzo[h]quinolin-4(1H)-ones 1a–d were accomplished by the
The 2-arylbenzo[h]quinolin-4(1H)-one series 3e and 5a–j
was prepared using the synthetic method described in
Scheme 3. Ethyl benzoylacetate derivatives 17a–j as key
intermediates were obtained by the reaction of potassium
ethyl malonate with different benzoic acids 16a–j in the
presence of 1,10-carbonyl-bis-1H-imidazole (CDI) in an
acceptable yield.23 1-Naphthylamine (18) was commercially
available and its derivative 20 was prepared effectively from
the starting material 19 using our previously reported
method.24 The condensation reaction of these naphthylamine
derivatives with 17a–j in the presence of PPA provided title
compounds in low yields. It is interesting to note that 4a, the
isomer of 3e, also could be isolated in high yield during the
process for the preparation of 3e. Lastly, methylation of 4a
was carried out using methyl iodide under alkaline conditions
to give 4b in high yield.
Fig. 1 Design of a series of 2-arylbenzo[h]quinolones as CYP1 inhibitors.
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Scheme 1 Synthetic routes for compounds 1a–d and 2a,b. Reagents and conditions: (a) KCN, NCCH2COOEt, KOH, DMF, 60 ꢀC for 24 h and then
reflux in NaOH–H2O (5%); (b) Mg, CH3I, Et2O, N2, HCl (2 N); (c) CH3ONa, THF; (d) SbCl3, CH3CN, 80 ꢀC; (e) H2O2, NaOH, MeOH; (f) AlCl3, DCM.
in inhibiting CYP1B1 and CYP1A1, with IC50 values of 20.2 and
44.6 nM respectively, but was much less effective in inhibiting
CYP1A2. However, addition of a hydroxyl group at the C3 posi-
tion (2b) dramatically reduced the inhibitory effects on CYP1B1
and CYP1A1. On the basis of the results above, we presume that
the double bond at the C2–C3 position may be an indispensable
structural feature for inhibitory action, while the hydroxyl group
at the C3 position is an unfavorable structural characteristic for
exerting an inhibitory effect on CYP1s.
Next we prepared and evaluated a series of 3-hydroxy-2-
arylbenzo[h]quinolin-4(1H)-ones 3a–d and a 2-arylbenzo[h]qui-
nolin-4(1H)-one (3e) with a C2–C3 double bond. As can be
appreciated from Table 2, the inhibitory activities of
compounds 3a–d against CYP1B1 were signicantly higher than
those against CYP1A2 (IC50 > 5000 nM). Also, they poorly
inhibited the catalytic activity of CYP1A1. It seems that
substituents on the B-ring exert no obvious effect on CYP1
inhibition, since compounds 3b–d displayed similar potencies
2.2 CYP1 enzyme inhibitory activities
The ethoxyresorun-O-deethylase (EROD) assay, a widely used
method for the evaluation of CYP1 activities, was used to
determine the inhibitory abilities of these synthesized
compounds toward recombinant human CYP1B1, CYP1A1, and
CYP1A2 enzymes. Initially, we synthesized several 2-aryl-2,3-
dihydrobenzo[h]quinolin-4(1H)-ones and their 3-hydroxy deriv-
atives that were lacking the C2–C3 double bond and/or
a hydroxyl group at the C3 position on the C-ring (1a–d and
2a,b), with the view that this would possibly improve the poor
water solubility of ANF by reducing its molecular planarity. The
results are presented in Table 1. Generally, it was observed that
all compounds displayed selective inhibition of CYP1B1 over
CYP1A1 and CYP1A2, which was consistent with our expecta-
tion. However, all of the tested compounds showed moderate to
poor inhibitory activities toward CYP1s, and their inhibitory
activities decreased dramatically compared to that of ANF.
Among them, 1b was identied as the most potent compound
Scheme 2 Synthetic routes for compounds 3a–d. Reagents and conditions: (a) NaOH solution (20%), EtOH, reflux, conc. HCl; (b) K2CO3, DMF,
90 ꢀC; (c) PPA, 120 ꢀC.
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Scheme 3 Synthetic routes for compounds 3e, 4a, 4b and 5a–j. Reagents and conditions: (a) (CH3)2SO4, NaOH, THF; (b) HNO3, HOAc; (c)
NH2NH2–H2O, Zn; (d) CDI, MgCl2, TEA, THF; (e) PPA, 130 ꢀC; (f) NaH, CH3I, DMF.
against the CYP1s to that of 3a. It was noteworthy that 3a, with of ANF. Interestingly, compared with ANF this compound had
a double bond introduced at the C2–C3 position, was 3 times an excellent selectivity prole, as it was 27.9-fold selective for
more potent than 2a in the CYP1B1 enzymatic inhibition assay. CYP1B1 over CYP1A2, suggesting that 3e is a promising lead
Compound 3e, a 3-dehydroxylated compound of 3a, exhibited compound for the further development of selective and potent
an increased ability to inhibit CYP1B1, with an IC50 value of CYP1B1 inhibitors. Together with the data presented in Tables 1
17.3 nM, and is about 5–8 times more potent than 3a and 1a. and 2, it can be concluded that the C2–C3 double bond
However, comparison of the activities of 3e and ANF against the contributes to improving the inhibitory efficiency toward CYP1
CYP1s showed that replacement of the oxygen atom in ANF with enzymes, while the C3-hydroxyl group results in a loss in
a nitrogen atom in 3e led to a loss of inhibitory potency. This potency. To our surprise, compound 4a as a chemical isomer of
result may be explained by the fact that introduction of 3e could strongly inhibit the activities of CYP1B1 and CYP1A1
a nitrogen atom resulted in a reduced planarity of the backbone with IC50 values of 7.6 and 18.5 nM respectively, and was 2 and 5
Table 1 Inhibitory potency of 2-aryl-2,3-dihydrobenzo[h]quinolin-4(1H)-ones 1a–d and their 3-hydroxy derivatives 2a,b against CYP1B1, 1A1,
and 1A2
IC50 values (nM)
1B1
IC50 ratio
1A1/1B1
Compound
R1
R
1A1
404.6
44.6
577.5
493.2
587.9
1161
1A2
1A2/1B1
1a
1b
1c
1d
2a
2b
ANF
H
H
H
H
OH
OH
—
H
131.4
20.2
139.3
98.6
259.6
302.5
5.6
952.8
>5000
1097
1045
1411
>5000
14.1
3.1
2.2
4.1
5.0
2.3
7.3
>247.5
7.9
10.6
5.4
>16.5
2.5
3,4,5-TriOCH3
o-F
m-F
H
3,4,5-TriOCH3
—
3.8
11.2
62.8
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Table
2
Inhibitory potency of 3-hydroxy-2-arylbenzo[h]quinolin- Table 3 Inhibitory potency of 6,10-dimethoxy-2-arylbenzo[h]quino-
4(1H)-ones 3a–d, 2-arylbenzo[h]quinolin-4(1H)-one 3eand its lin-4(1H)-ones 5a–j against CYP1B1, CYP1A1, and CYP1A2
chemical isomers 4a,b against CYP1B1, CYP1A1, and CYP1A2
IC50 values (nM)
IC50 ratio
IC50 values (nM)
1B1 1A1 1A2
IC50 ratio
Compound R1
R
1B1
1A1
1A2
1A1/1B1 1A2/1B1
Compound
R
1A1/1B1 1A2/1B1
3a
3b
3c
3d
3e
4a
4b
OH
OH
OH
OH
H
H
o-F
86.7
85.9
897.7 >5000 10.4
1783 >5000 20.8
>57.7
>58.2
>35.3
>53.6
27.9
19.3
—
5a
5b
5c
5d
5e
5f
5g
5h
5i
H
o-F
m-F
m-Cl
m-Br
m-OCH3
p-F
p-OCH3
p-CF3
16.6 12.6 147.6
27.9 26.4 174
7.8 39.3 330.9
27.6 29.4 84.5
3.6 19.8 587.6
7.1 35.8 190.1
3.9 10.5 2107
0.8
0.9
5.0
1.1
5.5
5.0
2.7
8.9
6.2
42.4
3.1
163.2
26.8
540.3
>1219.5
38.9
>30.5
p-OH 141.7 525.1 >5000 3.7
p-F
H
—
—
93.3
17.3
7.6
333.5 >5000 3.6
103.2 483.5 6.0
OH
OCH3
18.5
NI
146.9 2.4
NI
NIa
—
a
4.1 64.7 >5000 15.8
34.4 300.4 1337
NI ¼ no inhibition.
8.7
5j
3,4,5-TriOCH3 163.9 809
>5000 4.9
times more potent than 3e. Additionally, it showed 19.3-fold
selectivity for CYP1B1 over CYP1A2 (with an IC50 value of
146.9 nM for CYP1A2). However, when this hydroxyl group was
methylated, the obtained compound 4b was no longer a CYP1
inhibitor since its concentration-dependent activity was not
observed. It can be speculated that the hydroxyl group may form
an essential hydrogen bond with other residue(s).
The crystal structure of human CYP1B1 with ANF disclosed
that p–p stacking between the naphthalene part of ANF and
Phe231 plays an inuential role in maintaining the high
binding affinity of ANF with human CYP1B1.16 Considering that
enhancing the electron density of the naphthalene part will
intensify the p–p stacking interaction with Phe231, a series of
were introduced into the B-ring, the resulting compound 5j
suffered dramatic potency losses in CYP1 inhibitory activities.
This might be due to the fact that the substituted groups can
form steric clashes which affect 5j binding to the active site
cavities of CYP1 enzymes with a suitable conformation.
As shown in Fig. 2, on the basis of the inhibitory potency and
selectivity of the aforementioned 2-arylbenzo[h]quinolones as
CYP1B1 inhibitors, we could draw the following conclusions
related to their structure–activity relationships (SARs): (1)
introduction of methoxy groups at C-6 and C-10 on the naph-
thalene ring greatly increased the inhibitory potency towards
CYP1B1; (2) the double bond between C-2 and C-3 contributed
to the CYP1B1 inhibitory efficiency; (3) the nitrogen atom in the
C-ring caused detrimental effects to the CYP1B1 inhibitory
efficiency, but the presence of this atom had somewhat positive
effects towards water solubility; (4) introduction of the C-3
hydroxyl group led to a great loss in the potency of CYP1 inhi-
bition; (5) the nature of the substituents on the B-ring also has
a strong inuence on both CYP1B1 potency and selectivity.
Balancing all of the above factors, we have chosen 4a and 5e for
further molecular docking investigations.
6,10-dimethoxy-2-arylbenzo[h]quinolin-4(1H)-ones
were
synthesized. The results are summarized in Table 3. In general,
it was observed that the potency loss caused by replacing the
oxygen atom in ANF with a nitrogen atom was able to be
partially or completely restored by introducing two methoxy
groups on the naphthalene moiety. The resulting inhibitors
(5a–j) suppressed the CYP1 activities in
a substituent-
dependent manner. Consistent with our previous results, all
of the inhibitors except 5a, 5b and 5d showed high selectivity
toward CYP1B1 over CYP1A1 and CYP1A2. Among them,
compounds 5e and 5g exhibited the highest potency for inhi-
bition of CYP1B1 with IC50 values of 3.6 and 3.9 nM respectively,
and were more potent than ANF. Furthermore, these two
compounds also displayed very high selectivity toward CYP1B1
over CYP1A2 (more than 163 and 540 times higher than those
observed for CYP1A2, respectively), more than 65 and 216 times
higher than that of ANF. Besides, compounds 5c, 5f and 5h
displayed similar potencies and notable selectivity against
CYP1B1 to those of 5e and 5g with IC50 values of 7.8, 7.1 and
4.1 nM, respectively. Conversely, when three methoxy groups
2.3 Molecular docking
To investigate the binding modes and rationalize the observed
efficiency and selectivity of CYP1 inhibition by compound 4a
and the most potent inhibitor 5e, molecular docking studies
were carried out based on the crystal structures 3PM0 and 2HI4,
which are from CYP1B1 and CYP1A2 complexed with a-naph-
thoavone. These two crystal structures were obtained from the
Protein Data Bank (RCSB PDB). As shown in Fig. 3(A and B),
compound 4a tightly tted the active site of CYP1B1, and the
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Fig. 2 The SARs for the 2-arylbenzo[h]quinolones.
naphthalene part of this molecule was covered by the phenyl
ring of Phe231. In addition to this p–p stacking interaction, two
hydrogen bonds formed by the hydroxyl group of 4a and Asp326
2.4 Reversal of drug-resistance in MCF-7 cells
Docetaxel as an effective chemotherapeutic agent has been
extensively applied for the treatment of solid tumors in clinics.
Nevertheless, aer a positive initial response to treatment with
these chemotherapeutic drugs, a large proportion of patients
relapse. This event is mainly attributed to the intrinsic or
acquired drug resistance that greatly compromises the efficacy
of anticancer drugs. Previous studies demonstrated that
acquired resistance to docetaxel is due in particular to the
overexpression of the CYP1B1 enzyme in cancer cells.25 Taking
into account the fact that the level of CYP1B1 expression is
higher in hormone-dependent tissues such as those of the
uterus, ovary and breast,26 MCF-7 cells were chosen as a model
in which CYP1B1 expression was studied. In addition, early
research clearly indicated its cytotoxicity can be restored by co-
incubation of the CYP1B1-overexpressing cells with the CYP1B1
inhibitor ANF.14 Stimulated by this nding, we selected six
potent inhibitors 4a, 5e, 5g, 5c, 5f and 5h to evaluate their
potential to reverse docetaxel resistance in MCF-7 cells.
˚
(with lengths of 1.7 and 2.4 A, respectively) provided an
enhanced binding interaction. Also, it offered a reasonable
explanation for the phenomenon that methylation of this
hydroxyl group led to a dramatically decreased binding affinity
of compound 4b with CYP1B1. However, when 4a was bound to
CYP1A2, no hydrogen bond was formed and its binding affinity
arose mainly from the p–p stacking interaction with Phe226.
Compound 5e, a potent CYP1B1 inhibitor, exhibited excel-
lent selectivity over CYP1A2. Molecular docking provided
a plausible explanation for this observation. The results sug-
gested that the binding affinity of this compound to CYP1B1 is
much higher than that to CYP1A2 (Fig. 4(A and B)). A hydrogen
bond was formed between the carbonyl group of 5e and Asp326
˚
of CYP1B1 with a distance of 3.0 A. Additionally, the alkaline
nitrogen atom at the 1-position was adjacent to the acidic
˚
amino acid Asp333 with a short distance of 3.6 A, which may
play an essential role in improving its binding affinity to
CYP1B1. Besides, the p–p stacking interaction between 5e and
Phe231 also contributed to this binding affinity. However, in the
case of CYP1A2, the p–p stacking interaction between 5e and
Phe226 appeared to be the only direct interaction.
It is well known that CYP1B1 expression is substantially
induced by exposure of the MCF-7 cell line to TCDD.27,28
According to the reported methods and our previously pre-
sented study, CYP1B1-expressing MCF-7 cells (dened as MCF-
7/1B1) could be generated by treatment of parental MCF-7 cells
Fig. 3 The binding models: compound 4a with CYP1B1 (A) and CYP1A2 (B).
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Fig. 4 The binding models: compound 5e with CYP1B1 (A) and CYP1A2 (B) (hydrogen bonding is depicted by red dashed lines, whereas yellow
dashed lines represent distance only).
with a low concentration of TCDD for several days. As a result, plausible explanation was that 5h may possess a better water-
an enhanced expression of CYP1B1 protein is readily measur- solubility than 5e, as the predicted clog P value of the former
able in MCF-7/1B1 cells by western-blot techniques as is lower than that of the latter. Finally, the IC50 values of
compared to parental cells.8,27 In this study, MCF-7/1B1 cells docetaxel in combination with 10 mM 5h in MCF-7/1B1 cells
were obtained using the same method. Subsequently, the anti- were determined. As described in Fig. 5, compound 5h was
proliferation assay was carried out to conrm whether the cells shown to be more active than ANF in the reversal of CYP1B1-
were resistant to docetaxel. Not surprisingly, the results indi- mediated docetaxel resistance. This may be ascribed to
cated that MCF-7/1B1 cells were 5-fold more resistant to doce- a potent CYP1B1 inhibitory activity and an improved water-
taxel than parental MCF-7 cells (the IC50 values for docetaxel in solubility. Overall, the co-incubation of MCF-7/1B1 cells with
parental MCF-7 cells and MCF-7/1B1 cells were 29.5 and 158.6 10 mM 5h caused a complete reversal of docetaxel-resistance,
mM, respectively).
since the IC50 value was decreased to 33.9 ꢁ 5.1 mM, which
Initially, MCF-7/1B1 cells were treated with 20 mM docetaxel was comparable to that in the parental MCF-7 cells.
alone or in combination with 10 mM CYP1B1 inhibitors to
preliminarily evaluate the chemosensitization effects of these
selected compounds. A percentage inhibition value for each
3 Experimental
combination was obtained aer testing twice, and the results
3.1 Chemistry: general procedure
are summarized in Table 4. Generally, the percentage inhibition
Chemicals and solvents were used as purchased from
commercial suppliers. All anhydrous solvents were dried
according to standard methods. Column chromatography was
conducted on silica gel (200–300 mesh) from Qingdao Ocean
values for combinations of docetaxel with a CYP1B1 inhibitor
were higher than that for treatment with docetaxel alone. To our
surprise, among these tested compounds it was 5h–but not the
most potent CYP1B1 inhibitor 5e–that was found to be the most
promising compound, since it exhibited the highest ability to
restore the cytotoxicity of docetaxel. Next, to further conrm
whether this effect was caused by the cytotoxicity of 5e, MCF-7/
1B1 cells were exposed to varying concentrations of 5e alone,
but a signicant antiproliferative activity was not observed. A
Table 4 Percentage inhibition values of MCF-7/1B1 cell proliferation
with treatment of 10 mM CYP1B1 inhibitors and 20 mM docetaxel
%
Compound
clog Pa
inhibition
Medium
5h
5g
5c
5e
5f
4a
ANF
—
7.6
33.3
22.5
21.6
26.1
20.1
20.5
26.3
3.5
3.7
3.7
4.4
3.5
5.6
4.7
Fig. 5 Reversal of docetaxel resistance in combination with 10 mM 5h
in MCF-7/1B1 cells. IC50 values of docetaxel are shown as the mean (ꢁ
SD) of three independent measurements. Significant differences from
IC50 values are marked with an asterisk (P < 0.01).
a
clog P values were predicted by ChemBioDraw Ultra 12.0.
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1
Chemical Factory. The H and 13C NMR spectra were obtained added SbCl3 (0.5 equivalents). The reaction mixture was heated
ꢀ
on a Varian Mercury-300 (400 MHz) spectrometer using DMSO- at 80 C. Upon completion of the process (monitored by TLC),
d6, CDCl3 and CD3OD as solvents and TMS as an internal the reaction mixture was decomposed with water, extracted with
standard. All chemical shis are given in ppm and coupling EtOAc, and washed with saturated aqueous NaCl. The
constants are given in Hz. Mass spectra were recorded with combined organic phases were dried over Na2SO4 and concen-
HRMS (ESI, Agilent). Reaction progress was monitored by thin- trated under reduced pressure. The residue was puried by
layer chromatography TLC (silica gel GF254) and visualized with silica gel column chromatography to afford 1a–d as yellow
UV light (254 or 365 nm). The purity of compounds was deter- solids.
mined with an Agilent 1260 HPLC system (column: Eclipse plus
3.1.5. 2-Phenyl-2,3-dihydrobenzo[h]quinolin-4(1H)-one (1a).
Yellow solid; yield: 53%; 1H NMR (400 MHz, CDCl3) d 7.85 (d, J ¼
8.7 Hz, 1H), 7.73 (t, J ¼ 9.1 Hz, 2H), 7.54–7.46 (m, 3H), 7.43–7.32
C18, 3.5 mm, 4.6 ꢂ 100 mm (Agilent); ow rate: 0.8 ml minꢃ1
;
mobile phase: A: MeOH, B: H2O).
3.1.1. Procedure for the preparation of 1-amino-2- (m, 4H), 7.14 (d, J ¼ 8.7 Hz, 1H), 5.34 (s, 1H), 4.83 (dd, J ¼ 14.2,
naphthonitril (7). This compound was prepared according to 3.8 Hz, 1H), 3.01–2.90 (m, 1H), 2.79 (dd, J ¼ 16.2, 3.7 Hz, 1H);
a procedure described in the literature.19 1H NMR (400 MHz, HRMS (ESI) calcd for C19H16NO [M + H]+ 274.1232, found
DMSO-d6) d 8.33 (d, J ¼ 8.4 Hz, 1H), 7.80 (d, J ¼ 7.6 Hz, 1H), 7.59 274.1214. HPLC purity ¼ 99.1%.
(t, J ¼ 7.3 Hz, 1H), 7.51 (t, J ¼ 7.1 Hz, 1H), 7.31 (d, J ¼ 8.1 Hz,
1H), 7.10 (d, J ¼ 8.3 Hz, 1H), 6.86 (s, 2H).
3.1.6. 2-(3,4,5-Trimethoxyphenyl)-2,3-dihydrobenzo[h]qui-
nolin-4(1H)-one (1b). Yellow solid; yield: 46%; 1H NMR (400
3.1.2. Procedure for the preparation of 1-amino-2- MHz, CDCl3) d 7.90 (d, J ¼ 8.7 Hz, 1H), 7.80 (dd, J ¼ 17.9,
acetylnaphthalene (8). Dry magnesium turnings (0.9 g, 37.5 8.2 Hz, 2H), 7.58 (t, J ¼ 7.4 Hz, 1H), 7.47 (t, J ¼ 7.8 Hz, 1H), 7.21
mmol) were added to a 250 ml, two-necked, round-bottomed (d, J ¼ 8.7 Hz, 1H), 6.75 (s, 2H), 5.39 (s, 1H), 4.80 (dd, J ¼ 14.4,
ask equipped with one rubber septum and
a reux 3.2 Hz, 1H), 3.89 (s, 6H), 3.87 (s, 3H), 3.04–2.94 (m, 1H), 2.88 -
condenser. The reaction system was stirred under nitrogen, and 2.79 (m, 1H); HRMS (ESI) calcd for C22H22NO4 [M + H]+
then a solution of methyl iodide (3.5 g, 25 mmol) in anhydrous 364.1549, found 364.1528. HPLC purity ¼ 99.3%.
ethyl ether (40 ml) was added very slowly into the ask through
3.1.7. 2-(2-Fluorophenyl)-2,3-dihydrobenzo[h]quinolin-4(1H)-
a syringe. The reaction mixture was kept stirring at room one (1c). Yellow solid; yield: 58%; 1H NMR (400 MHz, CDCl3)
temperature for 2 h. Then, a solution of 7 (2.5 g, 15 mmol) in d 7.90 (d, J ¼ 8.5 Hz, 1H), 7.86–7.74 (m, 2H), 7.67–7.55 (m, 2H),
anhydrous ethyl ether (50 ml) was added slowly through 7.51–7.44 (m, 1H), 7.38–7.30 (m, 1H), 7.23–7.17 (m, 2H), 7.15–7.09
a syringe. Aer the addition was nished, the mixture was (m, 1H), 5.42 (s, 1H), 5.35–5.22 (m, 1H), 3.17–2.85 (m, 2H); HRMS
heated to reux for 2 h, and then aqueous HCl (2 N, 50 ml) was (ESI) calcd for C19H15FNO [M + H]+ 292.1138, found 292.1121.
ꢀ
added. The resulting mixture was stirred at 30 C for 2 h. Aer HPLC purity ¼ 95.6%.
the solution was neutralized with aqueous NaOH, the ether
3.1.8. 2-(3-Fluorophenyl)-2,3-dihydrobenzo[h]quinolin-4(1H)-
layer was separated and the water layer was extracted with one (1d). Yellow solid; yield: 51%; 1H NMR (400 MHz, CDCl3)
EtOAc. The combined organic layers were washed with satu- d 7.90 (d, J ¼ 8.7 Hz, 1H), 7.80 (dd, J ¼ 12.6, 8.5 Hz, 2H), 7.58 (t, J ¼
rated aqueous NaCl and then dried over Na2SO4. The organic 7.3 Hz, 1H), 7.52–7.45 (m, 1H), 7.44–7.36 (m, 1H), 7.32–7.25 (m,
layer was concentrated under reduced pressure, and the residue 2H), 7.22 (d, J ¼ 9.0 Hz, 1H), 7.14–7.02 (m, 1H), 5.38 (s, 1H), 4.95–
was puried by ash chromatography on silica to give 8 as 4.82 (m, 1H), 3.07–2.78 (m, 2H); HRMS (ESI) calcd for C19H15FNO
1
a yellow solid. H NMR (400 MHz, DMSO-d6) d 8.58 (brs, 2H), [M + H]+ 292.1138, found 292.1120. HPLC purity ¼ 100.0%.
8.37 (d, J ¼ 8.7 Hz, 1H), 7.75 (t, J ¼ 9.9 Hz, 2H), 7.60 (t, J ¼ 6.0 Hz,
3.1.9. General procedure for the preparation of 11a,b.
1H), 7.48 (t, J ¼ 6.0 Hz, 1H), 7.00 (d, J ¼ 8.8 Hz, 1H), 2.59 (s, 3H). Sodium hydroxide (0.5 ml, 10%) and hydrogen peroxide (1.2 ml,
3.1.3. General procedure for the preparation of 10a–d. To 30%) were added to a suspension of 10a–d (2 mmol) in CH3OH
a solution of 8 (1 equivalent) in THF was added sodium meth- (10 ml). The reaction mixture was stirred at room temperature
oxide (1.1 equivalents) and benzaldehyde 9a–d (1 equivalent). for 48 h, and then the yellow crystals were ltered off to give
Aer being stirred overnight at room temperature, THF was 11a,b in excellent yields. Representative spectral data of 11a, (1-
evaporated under reduced pressure. The mixture was poured amino-2-naphthyl)(3-phenyloxiran-2-yl)methanone: 1H NMR
into saturated NH4Cl, extracted with EtOAc, and washed with (400 MHz, DMSO-d6) d 8.74 (s, 2H), 8.42 (d, J ¼ 8.2 Hz, 1H), 7.76
saturated NaHCO3 and aqueous NaCl. The combined organic (dd, J ¼ 8.5, 4.6 Hz, 2H), 7.63 (t, J ¼ 7.4 Hz, 1H), 7.55–7.33 (m,
phases were dried over Na2SO4 and concentrated under reduced 6H), 6.99 (d, J ¼ 9.1 Hz, 1H), 4.77 (d, J ¼ 2.0 Hz, 1H), 4.12 (d, J ¼
pressure. The residue was puried by silica gel column chro- 2.0 Hz, 1H).
matography to give 10a–d as yellow solids. Representative
3.1.10. General procedure for the preparation of 2a,b. To
spectral data of 10d, (E)-1-(1-aminonaphthalen-2-yl)-3-(3- a stirred solution of 11a,b (1 equivalent) in dry DCM was added
uorophenyl)prop-2-en-1-one: 1H NMR (400 MHz, CDCl3) AlCl3 (0.5 equivalents). The reaction mixture was stirred at room
d 7.96 (d, J ¼ 8.7 Hz, 1H), 7.86 (d, J ¼ 8.9 Hz, 1H), 7.81 (s, 1H), temperature overnight, and then the reaction mixture was
7.76 (d, J ¼ 8.3 Hz, 1H), 7.74 (s, 2H), 7.60 (t, J ¼ 7.3 Hz, 1H), 7.50 decomposed with water, extracted with EtOAc and washed with
(t, J ¼ 7.3 Hz, 1H), 7.45–7.39 (m, 1H), 7.36 (d, J ¼ 10.6 Hz, 1H), saturated aqueous NaCl. The combined organic layers were
7.18–7.03 (m, 2H).
dried over Na2SO4 and concentrated under reduced pressure.
3.1.4. General procedure for the preparation of 1a–d. To The residue was puried by silica gel column chromatography
a stirred solution of 10a–d (1 equivalent) in dry acetonitrile was to afford 2a,b as yellow solids.
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3.1.11. (2S,3R)
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C19H14NO2 [M + H]+ 288.1025, found 288.1016. HPLC purity ¼
99.0%.
3-Hydroxy-2-phenyl-2,3-dihydrobenzo[h]
quinolin-4(1H)-one (2a). Yellow solid; yield: 53%; 1H NMR (400
MHz, DMSO-d6) d 8.32 (d, J ¼ 8.2 Hz, 1H), 7.80 (s, 2H), 7.64 (d,
J ¼ 8.3 Hz, 1H), 7.49 (t, J ¼ 7.5 Hz, 1H), 7.42–7.32 (m, 3H), 7.31–
7.22 (m, 3H), 6.46 (s, 1H), 5.91 (d, J ¼ 6.9 Hz, 1H), 4.31 (t, J ¼
6.5 Hz, 1H), 4.11 (d, J ¼ 6.3 Hz, 1H); HRMS (ESI) calcd for
3.1.17. 2-(2-Fluorophenyl)-3-hydroxybenzo[h]quinolin-4(1H)-
one (3b). Purple solid; yield: 32%; 1H NMR (400 MHz, DMSO-d6)
d 11.73 (s, 1H), 8.99–8.89 (m, 1H), 8.15 (d, J ¼ 8.8 Hz, 1H), 8.02 (m,
1H), 7.91 (s, 1H), 7.81–7.75 (m, 1H), 7.68–7.64 (m, 2H), 7.62–7.56
(m, 2H), 7.37 (d, J ¼ 7.0 Hz, 1H), 7.27–7.23 (m, 1H); HRMS (ESI)
calcd for C19H13FNO2 [M + H]+ 306.0930, found 306.0910. HPLC
purity ¼ 100.0%.
C
19H16NO2 [M + H]+ 290.1181, found 290.1160. HPLC purity ¼
99.4%.
3.1.12. (2S,3R) 3-Hydroxy-2-(3,4,5-trimethoxyphenyl)-2,3-
3.1.18. 3-Hydroxy-2-(4-hydroxyphenyl)benzo[h]quinolin-
4(1H)-one (3c). Purple solid; yield: 39%; 1H NMR (400 MHz,
DMSO-d6) d 11.32 (s, 1H), 9.85 (s, 1H), 8.99 (d, J ¼ 7.7 Hz, 1H),
8.16 (d, J ¼ 8.9 Hz, 1H), 8.01 (d, J ¼ 7.2 Hz, 1H), 7.80–7.55 (m,
6H), 6.96 (d, J ¼ 8.2 Hz, 2H); HRMS (ESI) calcd for C19H14NO3
[M + H]+ 304.0974, found 304.0955. HPLC purity ¼ 91.5%.
3.1.19. 2-(4-Fluorophenyl)-3-hydroxybenzo[h]quinolin-4(1H)-
one (3d). Purple solid; yield: 41%; 1H NMR (400 MHz, DMSO-d6)
d 11.47 (s, 1H), 8.99 (s, 1H), 8.21–8.11 (m, 1H), 8.08–8.00 (m, 1H),
7.88 (s, 2H), 7.76–7.55 (m, 4H), 7.47–7.36 (m, 2H); HRMS (ESI)
calcd for C19H13FNO2 [M + H]+ 306.0930, found 306.0915. HPLC
purity ¼ 95.3%.
dihydrobenzo[h]quinolin-4(1H)-one (2b). Yellow solid; yield:
48%; 1H NMR (400 MHz, CDCl3) d 7.88 (d, J ¼ 8.8 Hz, 1H), 7.84–
7.78 (m, 2H), 7.63 (t, J ¼ 7.6 Hz, 1H), 7.54–7.48 (m, 1H), 7.24 (s,
1H), 6.87 (s, 2H), 5.40 (s, 1H), 4.58 (d, J ¼ 7.3 Hz, 1H), 4.01 (s,
1H), 3.98 (s, 1H), 3.93 (s, 6H), 3.90 (s, 3H); HRMS (ESI) calcd for
C
22H22NO5 [M + H]+ 380.1498, found 380.1480. HPLC purity ¼
99.2%.
3.1.13. Procedure for the preparation of 1-amino-2-
naphthoic acid (12). To a stirred solution of 7 (1.68 g, 10
mmol) in EtOH (50 ml) was added NaOH solution (20%, 16 ml).
The mixture was heated to reux for 10 h. Aer cooling, the
EtOH was removed under reduced pressure and water (100 ml)
was added. The mixture was extracted with EtOAc twice, and the
organic layers were discarded. Then the water layer was brought
to pH 6.5 with conc. aqueous HCl to furnish a white precipitate.
The solid was collected and dried under vacuum to give 12 in
62% yield, which was used in the next step without further
3.1.20. General procedure for the preparation of ethyl
benzoylacetate derivatives 17a–j. To a mixture of benzoic acids
16a–j (10 mmol) and carbonyldiimidazole (11 mmol), dry THF
was added. The reaction mixture was stirred at room tempera-
ture for 6 h, and then a mixture of ethyl potassium malonate
(15, 10 mmol) [which had been prepared from ethyl hydrogen
malonate (10 g) and KOH (4 g) in absolute ethyl alcohol 40 ml],
MgCl2 (20 mmol) and triethylamine (3.4 ml) were added. The
reaction mixture was stirred at room temperature for 24 h.
Evaporation of the solvent provided a residue that was diluted
in HCl aqueous solution (2 M). The mixture was extracted with
EtOAc. The combined organic layers were washed with satu-
rated aqueous NaHCO3, saturated aqueous NaCl, and then
dried over Na2SO4. Evaporation of the solvent provided a crude
product that was puried by silica gel column chromatography
to give 17a–j as an oil in good yields. They could be used in the
next step without further purication.
3.1.21. General procedure for the preparation of 3e, 4a and
5a–j. A mixture of naphthylamine derivative (1 equivalent), ethyl
benzoylacetate derivatives 17a–j (2 equivalents), and PPA was
heated at 130 ꢀC with stirring. Aer the reaction was completed,
the mixture was cooled to room temperature and neutralized
with aqueous NaOH. The resulting mixture was extracted with
EtOAc. The combined organic extracts were washed with satu-
rated NaCl aqueous, dried over Na2SO4 and the solvent removed
under vacuum. Residues were puried by silica gel column
chromatography to give the title compounds.
1
purication. H NMR (400 MHz, CD3OD) d 8.15 (d, J ¼ 8.3 Hz,
1H), 7.82 (d, J ¼ 8.9 Hz, 1H), 7.71 (d, J ¼ 7.9 Hz, 1H), 7.53 (t, J ¼
7.5 Hz, 1H), 7.44 (t, J ¼ 7.6 Hz, 1H), 6.99 (d, J ¼ 8.7 Hz, 1H).
3.1.14. General procedure for the preparation of 14a–d. To
a solution of 12 (374 mg, 2 mmol) in DMF (5 ml), K2CO3
(414 mg, 3 mmol) was added. The mixture was heated to 90 ꢀC
and stirred for 1.5 h. Aer cooling to room temperature, 2-
bromo-1-arylethan-1-one 13a–d (2 mmol) was added. Then, the
ꢀ
reaction mixture was heated to 60 C and stirred for 2 h. Aer
that, the mixture was poured into ice water (20 ml). The ltered
precipitate, aer washing with water and drying, afforded 14a–
d in excellent yields as white solids. Representative spectral data
of 14a, (2-oxo-2-phenylethyl-1-amino-2-naphthoate): 1H NMR
(400 MHz, DMSO-d6) d 8.36 (d, J ¼ 8.6 Hz, 1H), 8.04 (d, J ¼
7.5 Hz, 2H), 7.86 (d, J ¼ 8.7 Hz, 1H), 7.81 (s, 1H), 7.79 (s, 2H),
7.72 (t, J ¼ 7.5 Hz, 1H), 7.67–7.56 (m, J ¼ 7.3 Hz, 3H), 7.51 (t, J ¼
8.8 Hz, 1H), 7.06 (d, J ¼ 8.8 Hz, 1H), 5.72 (s, 2H).
3.1.15. General procedure for the preparation of 3a–d.
Compound 14a–d was added to polyphosphoric acid (PPA) and
stirred at 120 ꢀC for 3 h. Aer that, the mixture was neutralized
by aqueous NaOH. The resulting mixture was extracted with
EtOAc several times. The combined organic extracts were
washed with saturated aqueous NaCl, dried over Na2SO4, and
the solvent removed under vacuum. The residue was puried by
silica gel column chromatography to afford 3a–d as purple
solids.
3.1.16. 3-Hydroxy-2-phenylbenzo[h]quinolin-4(1H)-one (3a).
Purple solid; yield: 40%; 1H NMR (400 MHz, DMSO-d6) d 11.47 (s,
1H), 8.97 (s, 1H), 8.17–8.09 (m, 1H), 8.03–7.93 (m, 1H), 7.78 (s,
2H), 7.72–7.59 (m, 3H), 7.54 (m, 4H); HRMS (ESI) calcd for
3.1.22. 2-Phenylbenzo[h]quinolin-4(1H)-one (3e). Pale
white solid; yield: 38%;1H NMR (400 MHz, DMSO-d6) d 12.24 (s,
1H), 8.96 (d, J ¼ 6.1 Hz, 1H), 7.97 (d, J ¼ 7.4 Hz, 1H), 7.74–7.65
(m, 2H), 7.65–7.54 (m, 4H), 7.54–7.49 (m, 2H), 7.46–7.28 (m,
1H), 6.53 (s, 1H); HRMS (ESI) calcd for C19H14NO [M + H]+
272.1075, found 272.1052. HPLC purity ¼ 98.9%.
3.1.23. 2-Phenylbenzo[h]quinolin-4-ol (4a). Pale white
solid; yield: 60%; H NMR (400 MHz, DMSO-d6) d 9.28 (s, 1H),
1
8.23 (s, 2H), 8.10 (d, J ¼ 8.5 Hz, 1H), 8.01 (s, 1H), 7.84 (d, J ¼
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8.5 Hz, 1H), 7.74 (s, 2H), 7.63–7.55 (m, 2H), 7.54–7.29 (m, 2H);
HRMS (ESI) calcd for C19H14NO [M + H]+ 272.1075, found
272.1052. HPLC purity ¼ 98.2%.
3.1.32. 6,10-Dimethoxy-2-(4-(triuoromethyl)phenyl)benzo
[h]quinolin-4(1H)-one (5i). Brown solid; yield: 21%; 1H NMR
(400 MHz, DMSO-d6) d 11.75 (s, 1H), 7.94 (d, J ¼ 7.4 Hz, 2H), 7.84
(d, J ¼ 8.0 Hz, 1H), 7.80 (d, J ¼ 7.4 Hz, 2H), 7.66 (t, J ¼ 7.9 Hz,
1H), 7.35 (d, J ¼ 8.0 Hz, 1H), 6.66 (s, 1H), 6.58 (s, 1H), 4.18 (s,
3H), 3.76 (s, 3H); HRMS (ESI) calcd for C22H17F3NO3 [M + H]+
400.1161, found 400.1153. HPLC purity ¼ 100.0%.
3.1.24. 6,10-Dimethoxy-2-phenylbenzo[h]quinolin-4(1H)-
one (5a). Brown solid; yield: 22%; 1H NMR (400 MHz, DMSO-
d6) d 11.65 (s, 1H), 7.78 (d, J ¼ 8.3 Hz, 1H), 7.65–7.50 (m, 6H),
7.29 (d, J ¼ 7.9 Hz, 1H), 6.70 (s, 1H), 6.49 (s, 1H), 4.16 (s, 3H),
3.71 (s, 3H); HRMS (ESI) calcd for C21H18NO3 [M + H]+
332.1287, found 332.1271. HPLC purity ¼ 99.1%.
3.1.33. 6,10-Dimethoxy-2-(3,4,5-trimethoxyphenyl)benzo[h]
1
quinolin-4(1H)-one (5j). Brown solid; yield: 17%; H NMR (400
3.1.25. 2-(2-Fluorophenyl)-6,10-dimethoxybenzo[h]quinolin-
4(1H)-one (5b). Brown solid; yield: 17%; 1H NMR (400 MHz,
DMSO-d6) d 11.81 (s, 1H), 7.92–7.83 (m, 1H), 7.73–7.67 (m, 1H),
7.65–7.59 (m, 1H), 7.59–7.51 (m, 1H), 7.49–7.42 (m, 2H), 7.41–
7.35 (m, 1H), 6.61 (s, 1H), 6.53 (s, 1H), 4.21 (s, 3H), 3.74 (s, 3H);
HRMS (ESI) calcd for C21H17FNO3 [M + H]+ 350.1192, found
350.1174. HPLC purity ¼ 98.0%.
MHz, DMSO-d6) d 11.78 (s, 1H), 7.86 (d, J ¼ 8.4 Hz, 1H), 7.66 (t,
J ¼ 8.0 Hz, 1H), 7.35 (d, J ¼ 7.9 Hz, 1H), 6.97 (s, 1H), 6.82 (s, 2H),
6.58 (s, 1H), 4.17 (s, 3H), 3.80 (s, 6H), 3.79 (s, 3H), 3.72 (s, 3H);
HRMS (ESI) calcd for C24H24NO6 [M + H]+ 422.1604, found
422.1579. HPLC purity ¼ 99.2%.
3.1.34. 4-Methoxy-2-phenylbenzo[h]quinoline (4b). Sodium
hydride (120 mg, 5 mmol) was added to a solution of 4a (678 mg,
2.5 mmol) in 5 ml of anhydrous DMF. The reaction mixture was
stirred at room temperature for 15 min and methyl iodide
(426 mg, 3 mmol) was added slowly. Aer being stirred for 2 h,
the mixture was poured into ice water. Filtering the precipitate
aer washing, drying and recrystallization from CH3OH affor-
3.1.26. 2-(3-Fluorophenyl)-6,10-dimethoxybenzo[h]quinolin-
4(1H)-one (5c). Brown solid; yield: 19%; 1H NMR (400 MHz,
DMSO-d6) d 11.76 (s, 1H), 7.86 (d, J ¼ 8.5 Hz, 1H), 7.71–7.59 (m,
2H), 7.48–7.32 (m, 4H), 6.73 (s, 1H), 6.56 (s, 1H), 4.19 (s, 3H), 3.77
(s, 3H); HRMS (ESI) calcd for C21H17FNO3 [M + H]+ 350.1192,
found 350.1180. HPLC purity ¼ 100.0%.
1
ded 4b as a pale white solid in 89% yield. H NMR (400 MHz,
3.1.27. 2-(3-Chlorophenyl)-6,10-dimethoxybenzo[h]quinolin-
4(1H)-one (5d). Brown solid; yield: 15%; 1H NMR (400 MHz,
DMSO-d6) d 11.65 (s, 1H), 7.78 (d, J ¼ 8.2 Hz, 1H), 7.69–7.57 (m,
4H), 7.52 (s, 1H), 7.32 (d, J ¼ 7.9 Hz, 1H), 6.61 (s, 1H), 6.52 (s, 1H),
4.15 (s, 3H), 3.72 (s, 3H); HRMS (ESI) calcd for C21H17ClNO3 [M +
H]+ 366.0897, found 366.0876. HPLC purity ¼ 99.2%.
DMSO-d6) d 9.34 (d, J ¼ 6.5 Hz, 1H), 8.46 (d, J ¼ 7.2 Hz, 2H),
8.14–7.97 (m, 2H), 7.90 (d, J ¼ 8.8 Hz, 1H), 7.83–7.69 (m, 3H),
7.64–7.57 (m, 2H), 7.54 (d, J ¼ 6.9 Hz, 1H), 4.21 (s, 3H); HRMS
(ESI) calcd for C20H15NO [M + H]+ 286.1232, found 286.1245.
HPLC purity ¼ 98.4%.
3.1.28. 2-(3-Bromophenyl)-6,10-dimethoxybenzo[h]quinolin-
4(1H)-one (5e). Brown solid; yield: 18%; 1H NMR (400 MHz,
3.2 Enzyme assays
DMSO-d6) d 11.75 (s, 1H), 7.93–7.87 (m, 1H), 7.86–7.79 (m, 1H), The recombinant human CYP1B1, CYP1A1, and CYP1A2
7.76 (s, 1H), 7.69–7.61 (m, 1H), 7.58–7.53 (m, 1H), 7.49 (s, 1H), enzymes, each equipped with P450 reductase (Supersomes),
7.40–7.31 (m, 1H), 6.69 (s, 1H), 6.55 (s, 1H), 4.18 (s, 3H), 3.76 (s, were purchased from BD Genetest. 7-Ethoxyresorun (7-ER) was
3H); HRMS (ESI) calcd for C21H17BrNO3 [M + H]+ 410.0392, found obtained from Sigma-Aldrich. NADP+, D-glucose-6-phosphate
410.0369. HPLC purity ¼ 100.0%.
(G-6-P) and glucose-6-phosphate dehydrogenase (G-6-PD) were
3.1.29. 6,10-Dimethoxy-2-(3-methoxyphenyl)benzo[h]quino- purchased from Biosharp. Other solvents and reagents used in
1
lin-4(1H)-one (5f). Brown solid; yield: 16%; H NMR (400 MHz, the biological evaluation were of the highest quality and
DMSO-d6) d 11.61 (s, 1H), 7.76 (d, J ¼ 7.9 Hz, 1H), 7.58 (t, J ¼ commercial availability. In the enzyme assay, FlexStation 3
7.2 Hz, 1H), 7.54–7.46 (m, 1H), 7.26 (d, J ¼ 7.4 Hz, 1H), 7.15–7.01 apparatus was used for recording the uorescence intensity
(m, 3H), 6.73 (s, 1H), 6.50 (s, 1H), 4.14 (s, 3H), 3.86 (s, 3H), 3.71 with excitation and emission lters at 544 and 590 nm,
(s, 3H); HRMS (ESI) calcd for C22H20NO4 [M + H]+ 362.1392, respectively.
found 362.1399. HPLC purity ¼ 94.6%.
The inhibitory activities of these prepared compounds
3.1.30. 2-(4-Fluorophenyl)-6,10-dimethoxybenzo[h]quinolin- against CYP1B1, CYP1A1, and CYP1A2 enzymes were deter-
4(1H)-one (5g). Brown solid; yield: 22%; 1H NMR (400 MHz, mined using the EROD assay as reported earlier.29 All tested
DMSO-d6) d 11.73 (s, 1H), 7.85 (d, J ¼ 8.3 Hz, 1H), 7.69–7.58 (m, compounds were dissolved as stock solutions (10 mM) in DMSO
3H), 7.42 (t, J ¼ 8.1 Hz, 2H), 7.35 (d, J ¼ 7.8 Hz, 1H), 6.73 (s, 1H), and diluted to working solutions with a Tris–HCl buffer at pH
6.53 (s, 1H), 4.18 (s, 3H), 3.77 (s, 3H); HRMS (ESI) calcd for 7.4, and the nal concentration of organic solvent was <1% (v/v)
C21H17FNO3 [M + H]+ 350.1192, found 350.1175. HPLC purity ¼ in all cases. Next, a mixture with a nal volume of 200 ml con-
95.1%.
taining different concentrations of tested compounds (except
3.1.31. 6,10-Dimethoxy-2-(4-methoxyphenyl)benzo[h]quino- positive and negative control wells), an enzyme source (20 fmol
lin-4(1H)-one (5h). Brown solid; yield: 19%; 1H NMR (400 MHz, CYP1B1, 10 fmol CYP1A1 and 60 fmol CYP1A2), 150 nM 7-ER,
DMSO-d6) d 11.70 (s, 1H), 7.85 (d, J ¼ 8.4 Hz, 1H), 7.66 (t, J ¼ 1.3 mM NADP+, 3.3 mM G-6-P, 0.5 U mlꢃ1 G-6-PD and 3.3 mM
8.1 Hz, 1H), 7.50 (d, J ¼ 7.5 Hz, 2H), 7.34 (d, J ¼ 7.4 Hz, 1H), 7.13 MgCl2 was ꢀincubated in a black 96-well at-bottomed micro-
(d, J ¼ 8.5 Hz, 2H), 6.86 (s, 1H), 6.48 (s, 1H), 4.18 (s, 3H), 3.86 (s, plate at 37 C for different times (incubation time for CYP1B1,
3H), 3.78 (s, 3H); HRMS (ESI) calcd for C22H20NO4 [M + H]+ CYP1A1, and CYP1A2 was 35, 15 and 50 min, respectively).
362.1392, found 362.1376. HPLC purity ¼ 95.6%.
Finally, the reaction was stopped by the addition of 100 ml of
methanol to all wells. The IC50 value for each compound was
15018 | RSC Adv., 2018, 8, 15009–15020
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calculated with the GraphPad Prism Soware (Version 5.0) electron-density of the naphthalene part to intensify the p–p
using the non-linear regression formula: log (inhibitor) vs. stacking interaction. It is noteworthy that 2-arylbenzo[h]quino-
normalized response-variable slope.
lones have shown a signicantly increased selectivity toward
CYP1B1 over CYP1A2. SAR analysis indicated that the double
bond at the C2–C3 position is an indispensable structural
feature for inhibitory action, while the hydroxyl group at the C3
position is an unfavorable structural characteristic. A molecular
docking study disclosed that the most potent compound 5e
tightly tted the binding cavity of CYP1B1; hydrogen bonds,
hydrophobic interactions and p–p stacking interaction
contribute to this high binding affinity, while these interactions
were markedly reduced when it bound to CYP1A2. On the basis
of the predicted clog P values, these target compounds may
exhibit an improved water-solubility compared to ANF. Among
them, 5h was found to be a potential compound to reverse
CYP1B1-mediated drug resistance, since the cytotoxicity of
docetaxel could be restored by pretreating MCF-7/1B1 cells with
5h at 10 mM. All of these results indicated that further devel-
opment of these 2-arylbenzo[h]quinolones as more potent and
specic CYP1B1 inhibitors may result in the discovery of
promising candidates to reverse CYP1B1-mediated anticancer-
drug resistance.
3.3 Cancer cell growth inhibition assays
RPMI-1640 culture medium, fetal bovine serum (FBS), and PBS
(Hyclone) were purchased from Thermo Fisher Scientic. TCDD
was obtained from AccuStandard. The MCF-7 cell line was
cultured in RPMI 1640 medium with 10% FBS at 37 ꢀC in
a humidied atmosphere with 5% CO2. MCF-7/1B1 cells were
obtained by coincubation of parental MCF-7 cells with TCDD
(10 nM) for ve days. The in vitro cytotoxicity was determined by
combination of docetaxel with CYP1B1 inhibitors using the
standard MTT assay.30,31 Approximately 3000 cells, suspended in
RPMI 1640 medium, were seeded into each well of a 96-well
ꢀ
plate and incubated at 37 C in a humidied atmosphere with
5% CO2 for 24 h. Then, six different concentrations of docetaxel
with or without 10 mM CYP1B1 inhibitors were added. Absor-
bance values were determined by a microplate reader at 490 nm,
and the IC50 values were calculated with the GraphPad Prism
Soware (Version 5.0) using the non-linear regression formula:
log (inhibitor) vs. normalized response-variable slope. Student’s
t tests were performed to compare their IC50 values using the
PASW Statistics 18 soware.
Conflicts of interest
3.4 Molecular docking
There are no conicts to declare.
In order to understand the binding mode and selectivity of the
title compounds with both CYP1B1 and CYP1A2, we performed
a molecule docking experiment using the docking program in
MOE 2008 based on the crystal structures of CYP1B1 (PDB ID:
3PM0) and CYP1A2 (PDB ID: 2HI4). The initial 3D conformation
of compounds 4a and 5e were optimized in ChemBio3D Ultra
using the MM2 energy minimization method. All molecules
were removed from the crystal structures before the experiment.
Hydrogens and partial charges were added with the protonate
Acknowledgements
This study was supported by the National Natural Science
Foundation of China (Grant No. 21602132), the Shanghai
Science and Technology Innovation Program (No. 15431900700)
and the Shanghai Natural Science Fund (No. 16ZR1418100).
Jinyun Dong also gratefully acknowledges the help and
encouragement of Haixia Zhang.
˚
3D application. The residues within a radius of 8.0 A around the
ligands were selected as docking sites. Docking parameters
were kept at default, except for the rst scoring function, where
ASE Scoring was used instead of the default London dG. The
best pose was characterized by the scoring results. To test
whether the docking program is suitable for the ligands binding
to human CYP1B1 and CYP1A2, the CYP1B1-ANF and CYP1A2-
ANF complexes (PDB ID: 3PM0 and PDB ID: 2HI4, respectively)
were initially chosen as models. The results revealed that the
docking compounds with ANF were comparable to their corre-
sponding crystallographic structures. It was implied that the
MOE docking program used is suitable for the current study.
Notes and references
1 J. Dong, Q. Zhang, Q. Cui, G. Huang, X. Pan and S. Li,
ChemMedChem, 2016, 11, 2102–2118.
2 H. Doostdar, M. D. Burke and R. T. Mayer, Toxicology, 2000,
144, 31–38.
3 J. Mimura and Y. Fujii-Kuriyama, Biochim. Biophys. Acta,
2003, 1619, 263–268.
4 T. Muto, T. Watanabe, M. Moto, M. Okamura, Y. Kashida,
Y. Kanai, K. Mitsumori and H. Endou, J. Toxicol. Pathol.,
2003, 16, 287–290.
5 B. N. Zordoky and A. O. El-Kadi, Toxicol. In Vitro, 2010, 24,
863–871.
4 Conclusions
The structural modication of a-naphthoavone gave new 2-
arylbenzo[h]quinolone derivatives, which exhibited varying
potency and selectivity for CYP1B1 inhibition. Compared with
ANF, replacement of the oxygen atom on the C-ring by
a nitrogen atom caused a mild potency loss. However, this
decreased inhibitory activity could be restored by enhancing the
6 A. J. Lee, M. X. Cai, P. E. Thomas, A. H. Conney and B. T. Zhu,
Endocrinology, 2003, 144, 3382–3398.
7 J. G. Liehr and M. J. Ricci, Proc. Natl. Acad. Sci. U. S. A., 1996,
93, 3294–3296.
8 J. Cui, Q. Meng, X. Zhang, Q. Cui, W. Zhou and S. Li, J. Med.
Chem., 2015, 58, 3534–3547.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 15009–15020 | 15019

View Article Online
RSC Advances
Paper
9 G. I. Murray, M. C. Taylor, M. C. McFadyen, J. A. McKay, 20 W. Zhang, K. F. Koehler, B. Harris, P. Skolnick and
W. F. Greenlee, M. D. Burke and W. T. Melvin, Cancer Res.,
1997, 57, 3026–3031.
10 R. D. Bruno and V. C. Njar, Bioorg. Med. Chem., 2007, 15,
5047–5060.
J. M. Cook, J. Med. Chem., 1994, 37, 745–757.
21 R. Bhattacharya, P. Kundu and G. Maiti, Synth. Commun.,
2010, 40, 476–481.
22 J. A. Donnelly and D. F. Farrell, J. Org. Chem., 1990, 55, 1757–
1761.
11 M. C. E. McFadyen, M. E. Cruickshank, I. D. Miller,
H. McLeod, W. T. Melvin, N. E. Haites, D. Parkin and 23 K. Machida, Y. Hirose, S. Fuse, T. Sugawara and
G. I. Murray, Br. J. Cancer, 2001, 85, 242–246. T. Takahashi, Chem. Pharm. Bull., 2010, 58, 87–93.
12 L. H. Patterson and G. I. Murray, Curr. Pharm. Des., 2002, 8, 24 J. Dong, Q. Zhang, G. Huang, Q. Meng and S. Li, Russ. J. Gen.
1335–1347. Chem., 2017, 87, 837–841.
13 T. Shimada, H. Yamazaki, M. Foroozesh, N. E. Hopkins, 25 I. Chang, Y. Mitsui, S. Fukuhara, A. Gill, D. K. Wong,
W. L. Alworth and F. P. Guengerich, Chem. Res. Toxicol.,
1998, 11, 1048–1056.
S. Yamamura, V. Shahryari, Z. L. Tabatabai, R. Dahiya and
D. M. Shin, Oncotarget, 2015, 6, 7774–7787.
14 M. C. McFadyen, H. L. McLeod, F. C. Jackson, W. T. Melvin, 26 D. C. Spink, C. L. Hayes, N. R. Young, M. Christou,
J. Doehmer and G. I. Murray, Biochem. Pharmacol., 2001, 62,
207–212.
T. R. Sutter, C. R. Jefcoate and J. F. Gierthy, J. Steroid
Biochem. Mol. Biol., 1994, 51, 251–258.
15 A. Datta, N. Bhasin, H. Kim, M. Ranjan, B. Rider, 27 D. C. Spink, B. C. Spink, J. Q. Cao, J. A. DePasquale,
Z. Y. A. Elmageed, D. Mondal, K. C. Agrawal and
B. T. Pentecost, M. J. Fasco, Y. Li and T. R. Sutter,
A. B. Abdel-Mageed, Cancer Lett., 2015, 362, 25–35.
Carcinogenesis, 1998, 19, 291–298.
16 A. Wang, U. Savas, C. D. Stout and E. F. Johnson, J. Biol. 28 V. Martinez, R. O’connor, Y. Liang and M. Clynes, Br. J.
Chem., 2011, 286, 5736–5743. Cancer, 2008, 98, 564–570.
17 S. Sansen, J. K. Yano, R. L. Reynald, G. A. Schoch, 29 S. Yamaori, M. Kushihara, I. Yamamoto and K. Watanabe,
K. J. Griffin, C. D. Stout and E. F. Johnson, J. Biol. Chem.,
2007, 282, 14348–14355.
18 A. A. Walsh, G. D. Szklarz and E. E. Scott, J. Biol. Chem., 2013,
288, 12932–12943.
Biochem. Pharmacol., 2010, 79, 1691–1698.
30 W. Zhou, Y. Peng and S. Li, Eur. J. Med. Chem., 2010, 45,
6005–6011.
´
31 A. Regielfutyra, M. Kusliskiewicz, V. Sebastian, S. Irusta,
19 Y. Tomioka, K. Ohkubo and M. Yamazaki, Chem. Pharm.
Bull., 1985, 33, 1360–1366.
M. Arruebo, A. Kyzioł and G. Stochel, RSC Adv., 2017, 7,
52398–52413.
15020 | RSC Adv., 2018, 8, 15009–15020
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