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

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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 IC₅₀values 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-OHE₂to 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.^6,7On 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.⁸As 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.^9–12Hence, 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.¹They are

expressed in a tissue-specic manner, and are induced via the

aryl hydrocarbon receptor (AhR), which is a ligand-activated

transcription factor.²Planar 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 E₂at 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 IC₅₀values of 5 and 6 nM respectively, but to

a much lesser extent on CYP1A1 (with an IC₅₀value 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.^14,15However, 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 CYP1A2.⁸From the X-ray crystallographic addition of antimony trichloride (SbCl₃) in CH₃CN.²¹Consid-

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 ZnCl₂and AlCl₃, nevertheless, none of these Lewis acids

binding cavity are diﬀerent among CYP1 members.^16–18Asp333 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,²²which

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,⁸we 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 K₂CO₃in 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.¹⁹Several

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).²⁰The 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.²³1-Naphthylamine (18) was commercially

available and its derivative 20 was prepared eﬀectively from

the starting material 19 using our previously reported

method.²⁴The 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 IC₅₀values 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 IC₅₀value 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 IC₅₀values 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

IC₅₀values (nM)

1B1

IC₅₀ratio

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

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

IC₅₀values (nM)

IC₅₀ratio

IC₅₀values (nM)

1B1 1A1 1A2

IC₅₀ratio

Compound R1

R

1B1

1A1

1A2

1A1/1B1 1A2/1B1

Compound

R

1A1/1B1 1A2/1B1

3a

3b

3c

3d

3e

4a

4b

OH

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

NI^a

—

a

4.1 64.7 >5000 15.8

34.4 300.4 1337

NI ¼ no inhibition.

8.7

5j

3,4,5-TriOCH₃163.9 809

>5000 4.9

times more potent than 3e. Additionally, it showed 19.3-fold

selectivity for CYP1B1 over CYP1A2 (with an IC₅₀value 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 CYP1B1.¹⁶Considering 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 IC₅₀values 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 IC₅₀values 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.²⁵Taking

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,²⁶MCF-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.¹⁴Stimulated 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 IC₅₀values of

compared to parental cells.^8,27In 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 IC₅₀values 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 IC₅₀value 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 P^a

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

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. IC₅₀values of docetaxel are shown as the mean (ꢁ

SD) of three independent measurements. Signiﬁcant diﬀerences from

IC₅₀values 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, CDCl₃and 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 Na₂SO₄and 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 min^ꢃ1

;

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.¹⁹¹H 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 NaHCO₃and 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 Na₂SO₄and 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 Na₂SO₄and 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 Na₂SO₄and 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.²⁹All 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 ml^ꢃ1G-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 MgCl₂was _ꢀ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 IC₅₀value 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.^30,31Approximately 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% CO₂for 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 IC₅₀values 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 IC₅₀values 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.

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Article Doi

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

DOI: 10.1039/c8ra00465j

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Article abstract of DOI:10.1039/c8ra00465j

Full text of DOI:10.1039/c8ra00465j

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