Synthesis and structure-activity relationship studies of α-naphthoflavone derivatives as CYP1B1 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.111938

Cytochrome P450 1B1(CYP1B1) has been recognized as an important target for cancer prevention and drug resistance reversal. In order to obtain potent and selective CYP1B1 inhibitors, a series of forty-one α-naphthoflavone (ANF) derivatives were synthesized, characterized, and evaluated for CYP1B1, CYP1A1 and CYP1A2 inhibitory activities. A closure look into the structure-activity relationship for the inhibitory effects on CYP1B1 indicated that modification of the C ring of ANF would decrease the CYP1B1 inhibitory potency, while incorporation of substituent(s) into the different positions of the B ring yielded analogues with varying CYP1B1 inhibitory capacity. Among these derivatives, compounds 9e and 9j were identified as the most potent two selective CYP1B1 inhibitors with IC₅₀ values of 0.49 and 0.52 nM, respectively, which were 10-fold more potent than the lead compound ANF. In addition, molecular docking and a reasonable 3D-QSAR (three-dimensional quantitative structure-activity relationship) study were performed to provide a better understanding of the key structural features influencing the CYP1B1 inhibitory activity. The results achieved in this study would lay a foundation for future development of selective, potent, low-toxic and water-soluble CYP1B1 inhibitors.

Full text of DOI:10.1016/j.ejmech.2019.111938

Journal Pre-proof
Synthesis and structure-activity relationship studies of α-naphthoflavone derivatives
as CYP1B1 inhibitors
Jinyun Dong, Zengtao Wang, Jiahua Cui, Qingqing Meng, Shaoshun Li
PII:
S0223-5234(19)31090-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.111938
EJMECH 111938
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 August 2019
Revised Date: 28 November 2019
Accepted Date: 30 November 2019
Please cite this article as: J. Dong, Z. Wang, J. Cui, Q. Meng, S. Li, Synthesis and structure-activity
relationship studies of α-naphthoflavone derivatives as CYP1B1 inhibitors, European Journal of
Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2019.111938.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Masson SAS.

Graphical abstract:

Synthesis and structure-activity relationship studies of α-naphthoflavone
derivatives as CYP1B1 inhibitors
Jinyun Dong, Zengtao Wang, Jiahua Cui, Qingqing Meng*, Shaoshun Li*
*School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai,
China. E-mail: ssli@sjtu.edu.cn ; Fax: +86 021-34204775; Tel: +86 021-34204775
Abstract
Cytochrome P450 1B1(CYP1B1) has been recognized as an important target for cancer
prevention and reversing drug-resistance. In order to obtain potent and selective CYP1B1
inhibitors, a series of forty-one α-naphthoflavone (ANF) derivatives were synthesized,
characterized, and evaluated for CYP1B1, CYP1A1 and CYP1A2 inhibitory activities. A
closure look into the structure-activity relationship for the inhibitory effects against
CYP1B1 indicated that modification of the C ring of ANF would decrease the CYP1B1
inhibitory potency, while incorporation of substituent(s) into the different positions of the
B ring yielded analogs with varying CYP1B1 inhibitory capacity. Among these
derivatives, compounds 9e and 9j were identified as the most potent two selective
CYP1B1 inhibitors with IC50 values of 0.49 and 0.52 nM, which were 10-fold more
potent than the lead compound ANF. In addition, Molecular docking and a reasonable
3D-QSAR (three-dimensional quantitative structure-activity relationship) study were
performed to provide a better understanding the key structural features influencing the
CYP1B1 inhibitory activity. The results achieved in this study would lay a foundation for
future development of selective, potent, low-toxic and water-soluble CYP1B1 inhibitors.
Key words: α-naphthoflavones, CYP1B1 inhibitors, SARs, 3D-QSAR
1. Introduction
Cytochrome P450 enzymes (CYPs) have been recognized as a large ubiquitous
super-family of hemoprotein monooxygenases that are involved in a variety of reduction
and oxidation reactions on both endogenic and xenobiotic compounds. Among them,
CYP1 subfamily contains three members known as CYP1A1, CYP1A2, and CYP1B1.
They are expressed in a tissue-specific manner: CYP1A1 and CYP1B1 are extrahepatic
enzymes, while CYP1A2 is an intrahepatic enzyme. These three isoenzymes,
particularly CYP1B1, are constitutively expressed in a wide variety of malignant tumors,
such as the majority of mammary tumors, lung, and colon[1]. In fact, various planar
aromatic molecules including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and
7,12-dimethylbenz[a]anthracene (DMBA) induce the overexpression of CYP1 enzymes
via binding to the aryl hydrocarbon receptor (AhR), a ligand-activated transcription
factor[2-5].
CYP1B1 enzyme has been extensively studied because of its predominant role in

metabolizing xenobiotics and endogenous compounds into carcinogenic forms and
inducing drug resistance. A large number of pro-carcinogens such as exogenous
polycyclic aromatic hydrocarbons (PAHs), polyhalogenated aromatic hydrocarbons
(PHAHs), aryl and heterocyclic aryl amines as well as endogenous estrogens can be
converted into cytotoxic, mutagenic and carcinogenic chemicals in presence of the
overexpressed CYP1enzymes[6]. Additionally, CYP1B1 can catalyze the
4-hydroxylation of 17β-estradiol (E2) subsequently leading to the formation of mutagenic
E2-3,4-quinone that binds covalently to DNA. In contrast, CYP1A1 and CYP1A2
mediate hydroxylation of 17β-estradiol mainly to 2-hydroxyestradiol (2-OHE2), while its
further-oxidized product E2-2,3-quinone is not considered carcinogenic[6]. Moreover, it
should be noted that CYP1B1 can also metabolically inactivate a structurally diverse
array of anti-cancer drugs such as docetaxel, tamoxifen, and paclitaxel, leading to drug
resistance[7, 8]. All these findings suggest the clinical importance of CYP1B1 inhibitors
in cancer prevention and treatment.
Recently, much emphasis has been on developing new and potent CYP1 inhibitors,
and distinct families of chemicals containing flavonoids, trans-stilbenes, coumarins,
alkaloids, and anthraquinone derivatives, have been characterized as CYP1 inhibitors[9,
10]. Among them, α-naphthoflavone (ANF), also known as 7,8-benzoflavone, is one of
the most potent CYP1 inhibitors. It exhibits strong inhibitory effects on recombinant
human CYP1B1, CYP1A2, and CYP1A1 with IC50 values of 5, 6 and 60 nM,
respectively[11]. Evidence has accumulated during the past several years that ANF not
only acts as a chemopreventive agent, but also enhances the sensitivity of
CYP1B1-expressing cells to some anticancer drugs. Apart from its excellent biological
activities, ANF also possesses a broad safety profile and health benefits[12]. All these
promising properties indicate that ANF is an attractive anticancer drug for further study.
Nevertheless, its further development is still restricted owing to its poor water solubility
and very limited selectivity toward CYP1B1 over CYP1A2.
To address these problems, researchers including our research group have made
great efforts to develop novel ANF derivatives, to the best of our knowledge, however,
no such an ideal compound has ever been reported[13-16]. According to our previous
results, we found that (1) introduction of hydrophobic and electron donor groups to the
naphthalene moiety is beneficial for CYP1 inhibitory potency; (2) improvement of water
solubility by reducing the C2-C3 double bond of ANF or introducing C3-hydroxyl group,
or cleavage of the C ring of ANF results in a somewhat loss activity detrimental effect.
Bearing in mind the above considerations, we consider that the B and C rings of ANF
can be served as two additional sites for further modification and exploring the
structure-activity relationships (SARs). As part of our continuous search for more potent
and selective CYP1B1 inhibitors with improved water solubility, we synthesized various
kinds of ANF derivatives.
2. Results and discussion
2.1 Chemistry
The synthetic routes adopted for the synthesis of ANF derivatives are illustrated in

Scheme 1A. The starting ethyl benzoylacetate derivatives 1 were synthesized from
benzoic acid and ethyl potassium malonate according to reported method[15].
Commercially available naphthalene-1-thiol (2) was treated with ethyl benzoylacetate
derivative 1 in the presence of polyphosphoric acid (PPA) at 120 °C to afford
2-substituted phenyl-4H-benzo[h]thiochromen-4-one derivatives 3a-e, which were
treated with 30% aqueous hydrogen peroxide solution in the presence of acetic acid at
60 °C to provide the corresponding 2-substituted phenyl-4H-benzo[h]thiochromen-4-one
1,1-dioxide derivatives 4a-b[17]; ANF derivatives 9a-w, 10a-h and 11a-c were
synthesized as illustrated in Scheme 1B. Using Fries rearrangement reaction, acetylation
of available naphthalen-1-ol (5) was carried out with acetic acid and ZnCl2 at 120 °C to
afford 1-(1-hydroxynaphthalen-2-yl)ethan-1-one (6) in good yields[18]. Intermediate 6
was further allowed to react with benzoic acid derivative in the presence of
N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide
4-(dimethylamino)pyridine (DMAP) to give ester derivative 7, which was treated with
NaH to undergo Baker-Venkataraman rearrangement to yield
hydrochloride
(EDCI)
and
1-(1-hydroxynaphthalen-2-yl)-3-phenylpropane-1,3-dione derivative 8. Intermediate 8
was further cyclized in the presence of H2SO4-EtOH solution (10%) at 90 °C to generate
the 2-substituted phenyl-4H-benzo[h]chromen-4-one derivatives 9a-w. The 2-substituted
phenyl-4H-benzo[h]chromen-4-thione derivatives 10a-h were next obtained by refluxing
the corresponding flavone derivatives 9a-w with Lawesson’s reagent in dry toluene.
Oximation of the corresponding 4-thioflavones 10a-h by hydroxylamine hydrochloride
in pyridine gave compounds 11a-c.

Scheme 1. Reagents and conditions: i) PPA, 120 °C; ii) H2O2, HOAc; iii) ZnCl2, HOAc; iv) benzoic
acid derivative, EDCI, DMAP; v) NaH; vi) H2SO4-EtOH, 90 °C; vii) Lawesson’s reagent, toluene,
reflux; viii) hydroxylamine hydrochloride, Py, reflux. See Tables 1-3 for the R group.
2.1 CYP1 enzyme inhibition activities
The synthesized compounds in this study were examined for their inhibitory effects
on recombinant human CYP1 enzymes using the EROD (7-ethoxyresorufin O-deethylase)
assay. Our previous study disclosed that replacement of the “O” atom at position-1 on the
C-ring of ANF with a bioisosteric “NH” group caused a mild potency loss, indicating
that a hydrophilic group would not be suitable for this substitution. Considering that
sulfur atom is more lipophilic than oxygen atom, we converted this “O” atom at
position-1 into a bioisosteric sulfur atom, intending to get novel compounds with
modified biological behaviors. The results are presented in Table 1. Compounds 3a-e
showed moderate inhibitory activity toward CYP1B1, with decreased activity compared
with that of ANF. Surprisingly, all of them displayed selective inhibition of CYP1A1
over CYP1B1 and CYP1A2. To verify whether the decreased inhibitory activities were
caused by the relatively less electronegativity of sulfur atom, further oxidation of two
potent CYP1B1 inhibitors 3a and 3d was carried out. However,
a further decrease in CYP1B1 inhibitory activity was observed when a sulfone group was
introduced at the 1-position (compounds 4a and 4b) in comparison to the corresponding
sulfur-substituted forms. Based on these data, we can conclude that the oxygen atom at
this site in ANF is very important for achieving high affinity for ligand binding, although
it does not appear to be making any interactions with any amino acid residue in the
crystal structure of ANF bound to CYP1B1.
Table 1 Inhibitory activities of sulfur substituted α-naphthoflavones on CYP1 enzymes
IC50 values (nM)
IC50 ratio
Compd.
n
R
1B1
57.0
1A1
32.6
84.6
45.8
13.6
9.0
1A2
1A1/1B1 1A2/1B1
0
0
0
0
0
2
2
H
>1000
582.2
>1000
295.8
211.9
169.6
698.7
0.6
0.8
0.5
0.3
0.2
0.6
>4.0
>17.5
5.6
3a
3b
3c
3d
3e
4a
4b
o-F
103.6
89.7
o-Cl
m-Cl
p-Cl
H
>11.1
7.3
40.5
51.9
4.0
222.9
250.4
129.2
>1000
0.8
m-Cl
2.8

IC50 values (nM)
IC50 ratio
Compd.
n
-
R
-
1B1
5.9
1A1
80.3
1A2
18.0
1A1/1B1 1A2/1B1
13.6
3.1
ANF
Similarly, we turned our attention to the C4-position of ANF to confirm whether
modification at this position could provide more potent CYP1B1 inhibitors. As a general
trend, replacement of the oxygen atom at C4-position of ANF with a sulfur atom led to
compounds 10a-h, which exhibited more potent CYP1B1 inhibitory activity in
comparison with that of substitution at 1- oxygen atom as shown in Table 2. Among
them, compounds 10a, 10c, 10d, 10e, and 10g possessed single digit nanomolar IC50
values toward CYP1B1 (IC50 values of 7.8, 5.0, 9.4, 9.1 and 1.6 nM, respectively). Of
particular note, although most of them were identified as potent CYP1B1 inhibitors, only
compound 10g showed a 3-fold improvement in the CYP1B1 biochemical assay when
compared with ANF. Therefore, we speculate that substitution with a sulfur atom at this
site may has a very limited potential for improving CYP1B1 inhibitory efficiency.
Next, compounds 11a-c were designed by oximation of the corresponding
representative compounds 10a, 10d, and 10g for the following reasons: (1) as a hydrogen
bond acceptor and donor, oxime group may form hydrogen bond interactions with some
residues; (2) oxime group can be metabolized by CYP1B1, indicating that
oxime-containing derivatives may have high affinity for ligand binding; (3) hydrophilic
oxime group not only contributes to improving water solubility, but also provides
reaction site for further modification. We evaluated the newly designed oxime-containing
derivatives 11a-c and found that they still showed similar CYP1B1 inhibitory activities
with IC50 values ranging from 3.2 to 10.4 nM. In this respect, compound 11a was slightly
more active than ANF in CYP1B1 inhibition, while introduction of a substituent to the
B-ring (compounds 11b and 11c) diminished its CYP1 inhibitory activity. Together with
these findings, it is implied that modification at C4-position generally had a slight effect
in improving inhibitory efficiency toward CYP1B1, whereas had a most pronounced
effect in their selectivity. Compared with ANF, all of these compounds displayed an
increased selectivity between CYP1B1and CYP1A2, but a decreased selectivity between
CYP1B1and CYP1A1.
Table 2 Inhibitory activities of C4 sulfur/oxime substituted α-naphthoflavones on CYP1 enzymes
IC50 values (nM)
IC50 ratio
Compd.
R1
S
R
H
1B1
7.8
1A1
65.4
1A2
179.0
1A1/1B1 1A2/1B1
8.4
22.9
10a

IC50 values (nM)
IC50 ratio
Compd.
R1
R
1B1
10.6
5.0
1A1
31.9
25.1
31.7
72.8
12.3
1A2
1A1/1B1 1A2/1B1
S
S
S
S
S
o-F
m-F
51.8
134.1
304.2
304.7
42.8
3.0
5.0
3.4
8.0
0.9
4.9
26.8
32.4
33.5
3.2
10b
10c
10d
10e
10f
m-Cl
m-OCH3
p-F
9.4
9.1
13.3
S
S
p-Cl
1.6
18.8
3.2
9.6
53.2
>1000
74.6
6.0
1.5
33.3
>53.2
23.3
77.3
9.4
10g
10h
11a
3,4,5-triOCH3
28.2
32.1
94.9
44.4
80.3
H
m-Cl
p-Cl
-
10.0
9.1
=N-OH
=N-OH
10.4
8.0
803.6
75.2
11b
11c
=N-OH
-
5.6
5.9
18.0
13.6
3.1
ANF
From the above observations, it can be reasonably concluded that oxygen atoms at
positions 1 and 4 of ANF are important for CYP1B1 inhibitory activity, so it seems to be
very hard to obtain a compound with significantly increased CYP1B1 inhibitory activity
by replacement of these two oxygen atoms. Analysis of the crystal structure of CYP1B1
in complex with ANF showed that the phenyl ring (B ring) of ANF comes into close
contact with heme[19]. As a result, introduction of substituents to the phenyl ring may
affect the formation of the reactive heme iron-oxo intermediate during catalysis. Based
on this idea, we therefore turned our attention to the phenyl ring of ANF. As shown in
Table 3, at the start of this study, synthesis and evaluation of halogen- and methoxyl-
substituted ANF analogs 9a-h was intended to identify the optimum location on the
phenyl moiety. There was an increase in potency against CYP1B1 and CYP1A1,while a
decrease in potency against CYP1A2 for most of the substituted derivatives when
compared to the unsubstituted parent compound ANF. In particular, a significant increase
in CYP1B1 inhibitory activity was observed when a chlorine atom was introduced at the
meta-position (compound 9e). This compound showed an IC50 value of 0.49 nM, which
was a 12-fold increase over lead compound ANF (IC50 value of 5.9 nM). Furthermore, 9e
displayed a significantly increased selectivity toward CYP1B1 over CYP1A2 when
compared to ANF (150.2 vs 3.1). However, to our surprise, moving this chlorine atom
from the meta- to ortho-position led to a significant loss in CYP1 inhibitory potency.
Analysis of the biological data of compounds 9a-c (F derivatives), 9d-f (Cl derivatives)
and 9g-h (OCH3 derivatives), we can preliminarily conclude that meta-position was
optimal for substitution on the phenyl ring.
Next, meta-substituted compounds 9i-k were synthesized in order to explore the
effect of hydrophilic/lipophilic groups on the CYP1 inhibitory activity. Among them,
compound 9j bearing a lipophilic “CF3” group was identified as the most potent

CYP1B1 inhibitors with IC50 value of 0.52 nM, which was equipotent to the
meta-chlorine substituted derivative 9e. Dimethylamino substituted derivative 9k showed
a similar level of CYP1B1 inhibitory activity to ANF, while a hydrophilic group of
hydroxyl 9i resulted in a nearly 3-fold decrease in activity (IC50 = 14.8 nM), suggesting
that lipophilic group(s) may be beneficial for CYP1B1 inhibitory activity. Compared
with meta-fluorine substituted compound 9b, meta-trifluoromethyl substituted derivative
9j was found to be 4-fold more potent in blocking CYP1B1 activity. Based on this
observation, we hypothesized that introduction of more lipophilic groups to the phenyl
ring may increase their inhibitory activity toward CYP1B1. Thus, compounds 9l-o
bearing lipophilic groups both at meta-position and other position(s) were synthesized.
However, it was found that, compared to 9e or 9j, all of these multi-substituted
derivatives exhibited a slight decrease in CYP1B1 inhibitory potency with IC50 values
ranging from 3.2 to 5.6 nM. The positive correlation between lipophilicity and
biochemical activity was not observed, indicating that steric hindrance may be an
important factor in blocking CYP1 enzymes. To test our hypothesis, a larger naphthalene
ring was introduced to give compound 9p. As expected, compared with ANF, a
significantly decreased potency in blockage of CYP1B1 and CYP1A2 was observed for
compound 9p with IC50 values of 48.4 and more than 1000 nM, respectively, supporting
our hypothesis.
As we all know, ANF has quite low water solubility owing to its planar and
hydrophobic structure, which limits its application rang. Although our above results
pointed to the importance of lipophilicity on CYP1B1 inhibitory activity, we still want to
introduce some hydrophilic functional groups to improve its water solubility. Thus,
nitrogen-containing compounds 9q-t were prepared since hydrophilic piperazine and
morpholine groups have been widely recognized as ‘privileged scaffolds’ in medicinal
chemistry. Derivative 9r incorporated with a 4-methyl-piperazin-1-yl group at the
meta-position of phenyl ring showed more potent than para-substituted analogue 9q on
inhibition of CYP1B1 (IC50 values of 24.6 and 63.8 nM, respectively), which is in
agreement with our previous results. Addition of a chlorine atom to the meta-position of
9r affording analogue 9s resulted in a 2-fold decrease in IC50 value from 24.6 to 47.7 nM
for CYP1B1 inhibition, however, replacement of the 3-(4-methyl-piperazin-1-yl) group
of 9r with a 4-morpholinyl group giving analogue 9t, led to a 12-fold increase in IC50
value from 24.6 to 2.0 nM against CYP1B1. Therefore, we speculated that the enhanced
CYP1B1 inhibitory activity of compound 9t may be partly due to the reduced steric
hindrance. Of particular note, CYP1A2 enzyme appears to be even more sensitive to
steric hindrance than CYP1B1 according to the relationship between structural features
of compounds 9n-t and biological activities of CYP1A2 inhibition (IC50 values ranging
from submicromolar to micromolar levels). It may be ascribed to the smallest active site
cavity volume of CYP1A2 among CYP1 family members[19-21]. For the purpose of
improving its water solubility, physiologically acceptable salts of piperazine substituted
ANF derivative 9q were prepared by adding different kinds of acids. To our surprise,
the sulfate form of compound 9q exhibited a significantly increased water solubility
(more than 20 mg/ml). However, in the subsequent experiment of reversing CYP1B1

induced drug-resistance, we found this compound also displayed a remarkable
cytotoxicity towards MCF-7 breast cancer cells when compared with ANF. We speculate
that its enhanced cytotoxicity may be associated with the piperazinyl group on the B
ring.
Given that an ideal ANF derivative with improved CYP1B1 inhibitory activity and
water solubility cannot be obtained by introduction of nitrogen-containing aliphatic
heterocycle to the phenyl ring of ANF, we attempted to replace this phenyl ring with
alkaline pyridine ring (analogues 9u-w), which can be used for further modification to
improve water solubility. To our surprise, compounds 9u-w showed that pyridine
substitution was tolerated with slight increase or only minimal loss of CYP1B1
inhibitory activity as compared with ANF. With regard to this series, 2-pyridine
derivative 9u was found to be the most potent CYP1B1 inhibitor with IC50 value of 3.5
nM, while 2- and 3-pyridine derivatives 9v and 9w were slightly less active at 6.2 and
9.3 nM, respectively, suggesting the importance of the position of nitrogen atom in CYP1
inhibitory potency. It should be noted that compared with ANF, these three compounds
also exhibited an enhanced inhibitory activity toward CYP1A1, but a decreased
inhibitory activity toward CYP1A2, which contributes to improving the selectivity
between CYP1B1 and CYP1A2. Based on this finding, it could be envisaged that some
other aromatic heterocycles may also be tolerated for the B ring of ANF, and
significantly affect their inhibitory activity toward CYP1 isoenzymes. Moreover, their
cytotoxicity was not significantly augmented by replacing the phenyl ring with
nitrogen-containing aromatic heterocycle as compared to ANF, suggesting the great
potential of B ring in structural modification.
Table 3 Inhibitory activities of B ring modified α-naphthoflavones against CYP1s
IC50 values (nM)
IC50 ratio
Compd.
Ar
1B1
5.9
1A1
80.3
12.2
16.7
6.7
1A2
1A1/1B1 1A2/1B1
-
18.0
23.7
62.2
12.1
113.2
73.6
10.6
177.8
13.6
3.7
3.1
7.2
ANF
9a
2-fluorophenyl
3-fluorophenyl
4-fluorophenyl
2-chlorophenyl
3-chlorophenyl
4-chlorophenyl
3-methoxyphenyl
3.3
2.2
7.6
28.3
4.5
9b
9c
2.7
2.5
21.6
0.49
4.2
227.9
5.2
10.6
10.6
1.0
5.2
9d
9e
150.2
2.5
4.3
9f
3.8
35.8
9.4
46.8
9g

IC50 values (nM)
IC50 ratio
Compd.
Ar
1B1
1A1
1A2
1A1/1B1 1A2/1B1
4-methoxyphenyl
3-hydroxyphenyl
7.3
30.9
53.1
16.7
98.2
91.5
94.2
4.2
3.6
13.5
6.2
9h
9i
14.8
0.52
3-(trifluoromethyl)
phenyl
32.1
181.2
9j
3-(dimethylamino)
phenyl
4.1
9.6
183.3
2.3
44.7
9k
3,5-dichlorophenyl
4.2
5.6
5.1
25.5
11.8
3.0
156.9
28.6
6.1
2.1
0.6
37.4
5.1
9l
9m
9n
3,4-difluorophenyl
3,4,5-trimethoxypheny
>1000
>196.1
l
3-chloro-5-methoxyph
enyl
3.2
163.9
>1000
51.2
312.5
9o
2-naphthyl
48.4
63.8
46.4
>1000
>1000
1.0
2.4
>20.7
15.7
9p
9q
4-(4-methyl-piperazin-
1-yl) phenyl
153.4
3-
24.6
47.7
2.0
34.6
37.9
30.9
761.6
>1000
318.5
1.4
0.8
31.0
21.0
9r
9s
9t
(4-methyl-piperazin-1-
3-Cl-5-(4-methyl-pipe
razin-1-yl) phenyl
3-(4-morpholinyl)
phenyl
15.5
159.3
2-pyridyl
3-pyridyl
4-pyridyl
3.5
6.2
9.3
37.9
25.3
18.7
25.1
23.2
36.4
10.8
4.1
7.2
3.7
3.9
9u
9v
2.0
9w
2.2 Molecular docking
To gain insight into the potential binding modes and rationalize the observed
efficiency and selectivity of CYP1 inhibition by the most potent CYP1B1 inhibitor 9e,
we performed a molecular docking study based on the crystal structures of CYP1B1 or
CYP1A2 in complex with ANF (PDB IDs of 3PM0 and 2HI4, respectively). As can be
seen from part A of Figure 1, similar to ANF, compound 9e tightly fitted well into the
active sites of both CYP1B1 and CYP1A2, while it adopted a different binding pose to
ANF within the corresponding ligand binding pocket. ANF forms only one hydrogen
bond to Asp326 of CYP1B1 with a distance of 2.8 Å. However, the carbonyl group of
compound 9e could form three hydrogen bonds to the amino and hydroxyl groups of
Ser131 as well as the carbonyl group of Asp326 with distances of 3.5, 2.1 and 2.8 Å,
respectively, which may play an essential role in improving its binding affinity to
CYP1B1(Figure 1B). In addition to the common π-π stacking interaction formed by
naphthalene part and phenyl ring of Phe231, the T-shaped π-π stacking interaction
between the phenyl moiety provided an additional binding affinity. Also, compound 9e is

buried in hydrophobic contacts with the residues such as Leu264, Phe268, Phe120,
Ala330 and Ala121. The presence of a chlorine atom not only affected the binding pose
of 9e in CYP1B1, but also provide an extra hydrophobic contact with Ile399. All of these
factors could contribute to the improvement in the enzyme inhibition activity of CYP1B1.
However, in the case of compound 9e bound to CYP1A2 (Figure 1D), the most
significant difference between 9e and ANF is that no hydrogen bond or water-mediated
hydrogen bond was formed between compound 9e and CYP1A2 (ANF could form a
water-mediated hydrogen bond with Gly316[20]). As a result, the binding affinity of 9e for
CYP1A2 arises mainly from a π-π stacking interaction with Phe226
and hydrophobic contacts with many hydrophobic residues such as Val227, Phe226,
Phe260, Thr498, Leu382, Ile386, Thr321 and Ala317. This observation may provide a
reasonable explanation for a lower activity of compound 9e against CYP1A2.
Figure 1 The binding models of compound 9e with CYP1B1(A) and CYP1A2 (C). (Hydrogen bonding
is depicted by yellow dashed lines; π-π stacking interaction is depicted by green dashed lines; T-shaped
π-π stacking interaction is depicted by red dashed lines; and, hydrophobic contacts is depicted by grey
dashed lines. In parts A and C, ANF was shown in cyan, while 9e was shown in yellow )
2.3 3D-QSAR study
In the current work, 3D-QSAR (three-dimensional quantitative structure-activity
relationship) models CoMFA (comparative molecular field analysis) and CoMSIA
(comparative molecular similarity indices analysis) studies were undertaken to figure out
the effects of steric, electrostatic, hydrophobic, and hydrogen bonding properties of these
compounds on CYP1B1 inhibitory activity, and to obtain reliable models to predict the
inhibitory activity of structurally similar compounds on CYP1B1. Based on the variety
of activity and structure diversity, all the forty-two compounds were divided into a
training set comprising 33 compounds and an external test set comprising nine

compounds: the test set consists of compounds 10d, 3b, 4b, 9a, 9e, 9k, 9m, 9w and ANF;
the remaining compounds were used as training set. Molecular alignment of compounds
is a crucial step in the development of 3D-QSAR model. Compound 9j, one of the most
active molecule, was chosen as the template molecule and all the other molecules were
aligned to it using the database alignment method in SYBYL-X 2.0. The aligned
molecules are shown in Figure 2.
Figure 2 Database alignment superposition of training set used for 3D-QSAR analysis based on
compound 9j.
Based on the molecular alignment, CoMFA and CoMSIA were applied to derive a
correlation relationship between the CYP1B1 inhibitor biological activity and the
structural properties. A partial least squares (PLS) approach was used to derive the
3D-QSAR model. Initial CoMFA PLS analysis with all 33 compounds in the training set
afforded model with very low cross-validated q2 value. After omitting four possible
outliers, compounds 10g, 11b, 9i and 9r, a better q2 value of 0.526 was obtained from a
training set of 29 compounds. Meanwhile, the corresponding conventional correlation
coefficient r2, standard error of the estimate (SEE), and the variance ratio F(n1=6, n2=22)
were also calculated as 0.981, 0.094 and 191.581, respectively. The IC50 values of these
ANF derivatives were predicted to validate the stability and predictive ability of CoMFA
model. As shown in Figure 3 and Table 4, the predicted values were in good agreement
with the experimental data for the training set indicated the reliability of CoMFA model.
Also, this figure shows a good agreement between experimental and predicted values for
the test set within a statistically tolerable error range.
Table 4 Experimental and predictive activities of CoMFA for the 38 α-naphthoflavone analogues.
pIC50
Predicted
pIC50
Predicted
Residu
al
Compd.
Compd.
Residual
Actual
8.108
7.975
8.301
8.027
Actual
7.666
9.31
10a
10b
10c
9d
9e#
9f
8.105
7.902
8.375
8.316
0.003
0.073
-0.074
-0.289
7.923
8.872
8.32
-0.257
0.438
0.057
-0.078
8.377
8.42
10d#
9g
8.498

Compd.
pIC50
Residu Compd.
pIC50
Residual
-0.042
0.053
-0.48
10e
9h
8.041
7.876
7.726
8.495
8.097
7.244
6.985
7.047
7.393
7.285
6.652
6.601
8.481
8.658
8.569
8.091
7.912
7.771
8.377
8.165
7.273
7.209
7.109
7.339
7.282
6.654
6.966
8.142
8.65
-0.05
-0.036
-0.045
0.118
-0.068
-0.029
-0.224
-0.062
0.054
0.003
-0.002
-0.365
0.339
0.008
0.101
8.137
9.284
8.387
8.377
8.252
8.292
8.495
7.315
7.195
7.321
8.699
8.456
8.208
8.032
8.229
8.179
9.231
8.867
8.313
8.597
8.229
8.54
10f
10h
11a
11c
3a
9j
9k#
9l
0.064
-0.345
0.063
-0.045
0.105
0.069
0.027
-0.006
0.127
-0.13
9m#
9n
3b#
9o
3c
9p
7.21
3d
9q
7.126
7.294
8.705
8.329
8.338
8.273
8.24
3e
9s
4a
9t
4b#
9a#
9b
9u
9v
9w#
ANF#
-0.241
-0.011
9c
8.468
# represents the test set compounds
Figure 3 Correlation plot of the predicted pIC50 with the experimental pIC50 values generated through
CoMFA .
CoMFA and CoMSIA contour maps can be applied to rationalize the regions in 3D
space around the molecules where changes in the particular physicochemical properties
are predicted to increase or decrease the activity. In general, the molecular force field of
the CoMFA model includes the steric and electrostatic fields. However, in our CoMFA
study, no electrostatic contour map was found, and only the steric contour map was
generated with 9j as a reference structure (Figure 4), indicating that the interaction of
steric field is extremely important for potency (the contribution of steric is 100%). The
steric interaction is denoted by green and yellow contours. A large green contour near the

meta-position of the benzene ring indicates that a bulky substituent at this site would
increase the CYP1B1 inhibitory potency. This is consistent with the fact that most
compounds bearing substituents at meta position of the benzene ring generally showed
an enhanced CYP1B1 inhibitory activity. Inversely, a small yellow contour near the
ortho-position of the benzene ring means that steric bulk is unfavored at this site.
Consistent to our experimental results, ortho-substituted derivatives generally showed a
decreased CYP1B1 inhibitory activity when compared with meta- or para-substituted
analogs. Similarly, the presence of a yellow contour around the 1-position oxygen atom
of C ring indicates this site is a disfavored region for steric bulk and replacement of this
oxygen atom with other groups (such as sulfur and sulfone groups) should be avoided in
future design.
Figure 4 The steric contour map of CoMFA for α-naphthoflavone analogues.
Considering that CoMSIA model, including five molecular force fields (steric,
electrostatic, hydrophobic, hydrogen bond donor, and hydrogen bond acceptor fields),
could provide a more comprehensive understanding of the key structural requirements
responsible for the biological activity. Using the similar method mentioned above, we
also established a reasonable CoMSIA model with satisfactory statistical values
including q2, r2, and SEE values (0.598, 0.937, and 0.179, respectively). It should be
noted that the training set for CoMSIA model consists of 34 compounds, and the
remaining eight compounds are test set (10g, 11a, 9e, 9i, 9u, 9v, 9w and ANF). The
relative field contributions of the hydrophobic, hydrogen bond acceptor, steric and
electrostatic fields were 27.4, 38.7, 8.6 and 25.2%, respectively, suggesting that
hydrophobic, hydrogen bond acceptor and electrostatic interactions play relatively more
important roles in the CYP1B1 inhibitory activity. The contour maps for CoMSIA are
depicted in Figure 5. A prominent magenta colored contour near the carbonyl oxygen
atom indicates that a hydrogen bond acceptor is favored at this position (Figure 5a). The
red contours present near 1-position oxygen atom shows unfavorable activity for the
presence of a hydrogen bond acceptor group. In Figure 5b, the hug yellow contour
surrounding the phenyl ring reflects the increase in activity found by introducing
hydrophobic group in this region. Seen from Figure 5c, the phenyl moiety is surrounded
by a large red contour, which means that electron-withdrawing increase the activity

substituent in this region is beneficial to the activity. A green contour centralizes on the
meta-position of the phenyl ring reveals around this region bulky substituent is favored
(Figure 5d), which is also in consistent with the above mentioned CoMFA steric field. In
addition, a yellow contour was also found around this phenyl ring suggesting the
existence of multiple substituents at the phenyl ring is unfavorable for CYP1B1
inhibitory activity.
Figure 5 Contour maps of CoMSIA model: hydrogen-bond acceptor field (A), hydrophobic field (B),
electrostatic field (C), and steric field (D).
3 Conclusion
In this study, structural modification of ANF gave a series of forty-one derivatives,
which exhibited varied levels of inhibitory potencies on CYP1B1, CYP1A1 and
CYP1A2 enzymes. Among them, compounds 9e and 9jwere found to be the most potent
two CYP1B1 inhibitors with IC50 values of 0.49 and 0.52 nM, which were 10-fold more
potent than the lead compound ANF. Meanwhile, these two compounds also presented an
enhanced selectivity for inhibition of CYP1B1 over CYP1A2. To rationalize the
biological results and gain more information on the binding mode of these compounds, a
molecular docking study was performed and the result revealed that the hydrogen bond
and hydrophobic interactions are two important factors for increasing the binding affinity
at CYP1B1 active site. To get enough information for better understanding the
structure-activity relationship and the key structural features influencing the activity, a
3D-QSAR study on CYP1B1 inhibition was also conducted. The reasonable CoMFA and
CoMSIA models showed satisfactory correlations and predictive abilities. Their contour
maps emphasized the importance of oxygen atom at 1 position, carbonyl oxygen atom at
4 position, as well as hydrophobic and electron-withdrawing substituent at meta-position
of the phenyl ring for CYP1B1 inhibitory activity. Based on the observation that

variation of the phenyl ring of ANF has a significant impact on the CYP1 inhibitory
activity and aromatic heterocycles are also well tolerated for this part, we have
successfully developed many derivatives with more potent inhibition of CYP1B1, high
selectivity and improved aqueous solubility by expanding the phenyl and naphthyl, and
they will be reported in due course.
4. Experimental
4.1. Chemistry
All starting materials and reagents were either obtained from commercial suppliers
or prepared according to literature reported procedures. All purchased chemicals and
solvents were used without further purification unless otherwise indicated. All anhydrous
solvents were dried according to standard methods. Column chromatography was
1
conducted on silica gel (200-300 mesh) from Qingdao Ocean Chemical Factory. The H
13
and C NMR spectra were obtained on a Varian Mercury-300 (400 MHz) spectrometer
using DMSO-d6 and CDCl3 as solvents and TMS as an internal standard. All chemical
shifts (δ) are given in ppm and coupling constants are given in Hz. Reactions progress
was monitored by thin-layer chromatography TLC (silica gel GF254) and visualized with
UV light (254 or 365 nm). Mass spectra were recorded with HRMS (ESI, Agilent 6540
QTOF).
4.1.1
General
procedure
for
the
preparation
of
2-substituted
phenyl-4H-benzo[h]thiochromen-4-one derivatives 3a-e
Ethyl benzoylacetate derivative 1 was prepared according to reported methods[15].
A mixture of commercially available naphthalene-1-thiol (2, 1 equivalent), ethyl
benzoylacetate derivative 1 (2 equivalents), and polyphosphoric acid (PPA ) was heated
at 120 °C with stirring. After the reaction was completed, the mixture was cooled to
room temperature and neutralized with NaOH aqueous. Then, the resulting mixture was
extracted with EtOAc. The combined organic extracts were washed with saturated NaCl
aqueous, dried over Na2SO4 and the solvent removed under vacuum. The crude residue
was subjected to silica gel column chromatography to give compounds 3a-e.
4.1.1.1 2-phenyl-4H-benzo[h]thiochromen-4-one (3a). Pale white solid, yield: 58%;
1H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J = 7.2 Hz, 1H), 8.38 (d, J = 8.7 Hz, 1H), 8.24
– 8.08 (m, 2H), 8.02 – 7.90 (m, 2H), 7.88 – 7.77 (m, 2H), 7.71 – 7.57 (m, 3H), 7.43 (s,
1H). 13C NMR (101 MHz, CDCl3) δ 181.01, 151.13, 137.26, 136.67, 133.99, 130.77,
129.56, 129.46, 129.30, 128.99, 128.94, 127.94, 127.30, 127.12, 124.08, 123.78, 123.56.
HRMS (ESI) calcd for [C19H12SO + H]+ 289.0687, found 289.06861.
4.1.1.2 2-(2-fluorophenyl)-4H-benzo[h]thiochromen-4-one (3b). Pale white solid,
yield: 55%; 1H NMR (400 MHz, CDCl3) δ 8.47 (d, J = 8.5 Hz, 1H), 8.29 (d, J = 7.9 Hz,
1H), 7.86 (t, J = 9.5 Hz, 2H), 7.69 – 7.51 (m, 3H), 7.43 (s, 1H), 7.29 – 7.18 (m, 3H). 13C
NMR (101 MHz, CDCl3) δ 180.65, 149.18, 145.08 (d, J = 2.0 Hz), 137.63, 133.96,
132.16, 132.08, 130.28 (d, J = 2.0 Hz), 129.48, 129.32, 129.00 (d, J = 2.0 Hz), 128.02,
127.48 (d, J = 4.0 Hz), 127.32, 124.78 (d, J = 4.0 Hz), 123.76, 123.52, 116.88, 116.66.
HRMS (ESI) calcd for [C19H11FSO + H]+ 307.0593, found 307.05954.

4.1.1.3 2-(2-chlorophenyl)-4H-benzo[h]thiochromen-4-one (3c). Pale white solid,
yield: 48%; 1H NMR (400 MHz, CDCl3) δ 8.47 (d, J = 8.6 Hz, 1H), 8.23 (d, J = 8.2 Hz,
1H), 7.84 (t, J = 8.4 Hz, 2H), 7.65 – 7.52 (m, 2H), 7.49 – 7.40 (m, 2H), 7.39 – 7.28 (m,
2H), 7.09 (s, 1H). 13C NMR (101 MHz, CDCl3) δ 180.53, 148.61, 137.85, 135.22, 133.95,
132.72, 131.17, 130.86, 130.47, 129.52, 129.28, 129.05, 128.96, 128.03, 127.74, 127.33,
127.13, 123.76, 123.50. HRMS (ESI) calcd for [C19H11ClSO + H]+ 323.0297, found
323.02967.
4.1.1.4 2-(3-chlorophenyl)-4H-benzo[h]thiochromen-4-one (3d). Pale white solid,
yield: 52%; 1H NMR (400 MHz, CDCl3) δ 8.44 (d, J = 8.8 Hz, 1H), 8.33 (d, J = 7.9 Hz,
1H), 7.91 – 7.79 (m, 2H), 7.68 (s, 1H), 7.66 – 7.53 (m, 3H), 7.49 – 7.35 (m, 2H), 7.25 (s,
13
1H). C NMR (101 MHz, CDCl3) δ 180.81, 149.36, 138.34, 136.94, 135.35, 134.01,
130.73, 130.56, 129.52, 129.37, 129.07, 129.02, 128.16, 127.42, 127.23, 125.27, 124.52,
123.71, 123.52. HRMS (ESI) calcd for [C19H11ClSO + H]+ 323.0297, found 323.02927.
4.1.1.5 2-(4-chlorophenyl)-4H-benzo[h]thiochromen-4-one (3e). Pale white solid,
yield: 45%; 1H NMR (400 MHz, CDCl3) δ 8.43 (d, J = 8.8 Hz, 1H), 8.31 (d, J = 6.9 Hz,
1H), 7.93 – 7.78 (m, 2H), 7.65 – 7.56 (m, 4H), 7.43 (d, J = 8.1 Hz, 2H), 7.24 (s, 1H). 13C
NMR (101 MHz, CDCl3) δ 180.84, 149.70, 137.09, 136.93, 135.03, 134.00, 129.57,
129.46, 129.35, 129.04, 129.02, 128.32, 128.11, 127.39, 124.12, 123.70, 123.50. HRMS
(ESI) calcd for [C19H11ClSO + H]+ 323.0297, found 323.02919.
4.1.2
General
procedure
for
the
preparation
of
2-substituted
phenyl-4H-benzo[h]thiochromen-4-one 1,1-dioxide derivatives 4a-b
To a solution of thio naphthoflavone (3, 2 mmol) in acetic acid (4 ml) was added 1g
of 30% aqueous hydrogen peroxide solution. The mixture was heated for 10 hours at
60 °C. After cooling, the reaction mixture was poured into an ice-water solution and then
resulting solid was filtered and washed with water. Recrystallization from ethanol gave
compounds 4a-b.
4.1.2.1 2-phenyl-4H-benzo[h]thiochromen-4-one 1,1-dioxide (4a). yellow-green
1
solid, yield: 78%; H NMR (400 MHz, CDCl3) δ 8.87 (d, J = 7.9 Hz, 1H), 8.15 (d, J =
8.7 Hz, 1H), 8.05 (d, J = 8.6 Hz, 1H), 7.96 – 7.81 (m, 3H), 7.76 – 7.61 (m, 2H), 7.58 –
13
7.41 (m, 3H), 6.82 (s, 1H). C NMR (101 MHz, CDCl3) δ 178.59, 153.92, 138.06,
136.28, 133.52, 131.77, 129.86, 129.20, 129.11, 129.08, 128.72, 127.96, 127.13, 126.46,
126.11, 122.14. HRMS (ESI) calcd for [C19H11ClSO+ H]+ 323.0297, found 323.02919.
HRMS (ESI) calcd for [C19H12SO3+ Na]+ 343.0405, found 343.04019.
4.1.2.2 2-(3-chlorophenyl)-4H-benzo[h]thiochromen-4-one 1,1-dioxide (4b).
yellow-green solid, yield: 75%; 1H NMR (400 MHz, CDCl3) δ 8.85 (d, J = 7.6 Hz, 1H),
8.14 (d, J = 8.3 Hz, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 6.7 Hz, 1H), 7.84 (s, 1H),
7.79 – 7.63 (m, 3H), 7.49 (d, J = 7.9 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 6.80 (s, 1H). 13C
NMR (101 MHz, CDCl3) δ 178.34, 152.52, 137.83, 136.31, 135.24, 133.67, 131.82,
130.45, 130.28, 130.00, 129.30, 129.12, 129.10, 128.63, 127.33, 127.03, 126.39, 126.05,
122.13. HRMS (ESI) calcd for [C19H11ClSO3+ Na]+ 377.0015, found 377.00095.
4.1.3
phenyl-4H-benzo[h]chromen-4-one derivatives 9a-w
The 1-(1-hydroxynaphthalen-2-yl)ethan-1-one (6) as a key intermediate was
General
procedure
for
the
preparation
of
2-substituted

obtained from commercially available naphthalen-1-ol (5) according to the literature
procedure[18].
To a stirred solution of 1-(1-hydroxynaphthalen-2-yl)ethan-1-one 6 (2 mmol) in dry
DMF (10 ml), benzoic acid derivative (2.2 mmol, commercially available or prepared
according to the reported literature procedure), EDCI (3 mmol) and DMAP (3 mmol)
were added and the resultant solution stirred for overnight at room temperature. After
completion of the reaction as indicated by TLC, the reaction mixture was poured into an
ice-water solution. the resulting mixture was extracted with EtOAc. The combined
organic extracts were washed with saturated NaCl aqueous, dried over Na2SO4. the
solvent was removed under vacuum and the crude residue was subjected to silica gel
column chromatography to give intermediate 7.
To a stirred solution of intermediate 7 (1mmol) in dry DMF (5 ml), NaH (10 mmol)
was added and the resultant solution stirred for 5 hours at room temperature. After
completion of the reaction as indicated by TLC, the reaction mixture was quenched with
saturated ammonium chloride ice water solution and the precipitate obtained was filtered.
After drying, this crude product 8 can be used directly without further purification.
Intermediate 8 (0.5 mmol) was dissolved in H2SO4-EtOH solution (10%, 25 ml).
The resultant solution was kept for stirring at 90 °C till completion of the reaction (TLC
monitoring). Solvents were removed under vacuum and the residue was purified by
column chromatography using silica gel to give compounds 9a-w.
4.1.3.1 2-(2-fluorophenyl)-4H-benzo[h]chromen-4-one (9a). Pale white solid, yield:
1
63%; H NMR (400 MHz, CDCl3) δ 8.43 (d, J = 7.1 Hz, 1H), 8.06 (d, J = 8.6 Hz, 1H),
7.92 (t, J = 7.3 Hz, 1H), 7.82 (d, J = 7.1 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.63 – 7.52 (m,
2H), 7.46 (d, J = 5.7 Hz, 1H), 7.29 (t, J = 7.4 Hz, 1H), 7.17 (d, J = 9.9 Hz, 1H), 6.97 (s,
1H). 13C NMR (101 MHz, CDCl3) δ 178.10, 160.46 (d, J = 254.0 Hz), 158.25 (d, J = 4.0
Hz), 153.66, 135.95, 132.90 (d, J = 9.0 Hz), 129.32, 128.94 (d, J = 2.0 Hz), 128.17,
127.19, 125.39, 124.78 (d, J = 3.0 Hz), 123.98, 122.32, 120.62, 120.33 (d, J = 10.0 Hz),
120.05, 117.12 (d, J = 22.0 Hz), 113.29 (d, J = 10.0 Hz). HRMS (ESI) calcd for
[C19H11FO2 + H]+ 291.0821, found 291.08226; calcd for [C19H11FO2 + Na]+ 313.0641,
found 313.06454.
4.1.3.2 2-(3-fluorophenyl)-4H-benzo[h]chromen-4-one (9b). Pale white solid, yield:
1
67%; H NMR (400 MHz, CDCl3) δ 8.49 (d, J = 3.2 Hz, 1H), 8.08 (d, J = 8.7 Hz, 1H),
7.86 (d, J = 5.2 Hz, 1H), 7.71 (d, J = 8.3 Hz, 2H), 7.64 (d, J = 7.0 Hz, 3H), 7.48 (dd, J =
14.2, 7.1 Hz, 1H), 7.19 (d, J = 5.9 Hz, 1H), 6.87 (s, 1H). 13C NMR (101 MHz, CDCl3) δ
178.03, 163.07 (d, J = 246.0 Hz), 161.15 (d, J = 3.0 Hz), 153.45, 136.04, 134.08 (d, J =
8.0 Hz), 130.91 (d, J = 8.0 Hz), 129.40, 128.28, 127.30, 125.58, 123.97, 122.25, 121.93
(d, J = 3.0 Hz), 120.64, 120.25, 118.48 (d, J = 21.0 Hz), 113.37 (d, J = 24.0 Hz), 109.34.
HRMS (ESI) calcd for [C19H11FO2 + H]+ 291.0821, found 291.08264; calcd for
[C19H11FO2 + Na]+ 313.0641, found 313.06429.
4.1.3.3 2-(4-fluorophenyl)-4H-benzo[h]chromen-4-one (9c). Pale white solid, yield:
1
68%; H NMR (400 MHz, CDCl3) δ 8.45 (d, J = 6.9 Hz, 1H), 8.05 (d, J = 8.6 Hz, 1H),
8.00 – 7.87 (m, 2H), 7.83 (d, J = 5.4 Hz, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.61 (d, J = 6.8
Hz, 2H), 7.18 (t, J = 8.1 Hz, 2H), 6.81 (s, 1H). 13C NMR (101 MHz, CDCl3) δ 178.02,

164.68 (d, J = 252.0 Hz), 161.64, 153.39, 135.98, 129.29, 128.41, 128.32, 128.25,
128.07(d, J = 3.0 Hz), 127.20, 125.43, 123.95, 122.18, 120.65, 120.08, 116.54, 116.32,
108.49. HRMS (ESI) calcd for [C19H11FO2 + H]+ 291.0821, found 291.08240; calcd for
[C19H11FO2 + Na]+ 313.0641, found 313.06420.
4.1.3.4 2-(2-chlorophenyl)-4H-benzo[h]chromen-4-one (9d). Pale white solid, yield:
1
63%; H NMR (400 MHz, CDCl3) δ 8.46 (d, J = 8.0 Hz, 1H), 8.11 (d, J = 8.7 Hz, 1H),
7.85 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 8.6 Hz, 1H), 7.68 – 7.54 (m, 3H), 7.51 (d, J = 7.7
Hz, 1H), 7.45 – 7.33 (m, 2H), 6.75 (s, 1H). 13C NMR (101 MHz, CDCl3) δ 177.99,
162.02, 154.04, 135.97, 132.93, 131.88, 131.82, 130.99, 130.90, 129.39, 128.10, 127.27,
127.22, 125.47, 124.09, 122.51, 120.64, 120.09, 114.06. HRMS (ESI) calcd for
[C19H11ClO2 + H]+ 307.0526, found 307.05328; calcd for [C19H11ClO2 + Na]+ 329.0345,
found 329.03510.
4.1.3.5 2-(3-chlorophenyl)-4H-benzo[h]chromen-4-one (9e). Pale white solid, yield:
58%; 1H NMR (400 MHz, DMSO-d6) δ 8.71 – 8.58 (m, 1H), 8.29 – 8.17 (m, 2H), 8.14 –
8.05 (m, 1H), 7.98 (d, J = 8.6 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.82 (dd, J = 6.0, 3.1 Hz,
2H), 7.74 – 7.61 (m, 2H), 7.28 (s, 1H). 13C NMR (101 MHz, DMSO-d6) δ 177.23, 160.78,
153.20, 135.90, 134.55, 133.78, 131.89, 131.53, 130.09, 128.68, 128.21, 126.42, 125.99,
125.52, 123.83, 122.77, 120.33, 120.07, 109.47. HRMS (ESI) calcd for [C19H11ClO2 +
H]+ 307.0526, found 307.05352; calcd for [C19H11ClO2 + Na]+ 329.0345, found
329.03540.
4.1.3.6 2-(4-chlorophenyl)-4H-benzo[h]chromen-4-one (9f). Pale white solid, yield:
1
59%; H NMR (400 MHz, CDCl3) δ 8.46 (s, 1H), 8.06 (d, J = 7.0 Hz, 1H), 7.96 – 7.77
(m, 3H), 7.77 – 7.56 (m, 3H), 7.46 (d, J = 6.8 Hz, 2H), 6.85 (s, 1H). 13C NMR (101 MHz,
CDCl3) δ 177.97, 161.52, 153.44, 137.85, 136.03, 130.34, 129.50, 129.35, 128.28,
127.43, 127.24, 125.52, 123.97, 122.19, 120.65, 120.17, 108.83. HRMS (ESI) calcd for
[C19H11ClO2 + H]+ 307.0526, found 307.05344; calcd for [C19H11ClO2 + Na]+ 329.0345,
found 329.03518.
4.1.3.7 2-(3-methoxyphenyl)-4H-benzo[h]chromen-4-one (9g). Pale white solid,
yield: 75%; 1H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 8.03 (d, J = 7.2 Hz, 1H), 7.79 (s,
1H), 7.64 (d, J = 8.0 Hz, 1H), 7.58 (s, 2H), 7.46 (d, J = 6.5 Hz, 1H), 7.37 (d, J = 7.4 Hz,
2H), 6.98 (d, J = 6.8 Hz, 1H), 6.82 (s, 1H), 3.81 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
178.15, 162.30, 160.00, 153.40, 135.92, 133.14, 130.23, 129.20, 128.15, 127.13, 125.29,
124.01, 122.25, 120.64, 120.18, 118.55, 116.76, 111.85, 108.89, 55.44. HRMS (ESI)
calcd for [C20H14O3 + H]+ 303.1021, found 303.10284; calcd for [C20H14O3 + Na]+
325.0841, found 325.08477.
4.1.3.8 2-(4-methoxyphenyl)-4H-benzo[h]chromen-4-one (9h). Pale white solid,
yield: 72%; 1H NMR (400 MHz, CDCl3) δ 8.44 (s, 1H), 8.12 – 7.97 (m, 1H), 7.93 – 7.75
13
(m, 3H), 7.71 – 7.46 (m, 3H), 6.95 (s, 2H), 6.77 (d, J = 5.1 Hz, 1H), 3.80 (s, 3H). C
NMR (101 MHz, CDCl3) δ 178.12, 162.30, 153.35, 135.90, 129.09, 128.17, 127.84,
126.94, 125.12, 124.07, 122.24, 120.71, 120.06, 114.55, 107.25, 55.45. HRMS (ESI)
calcd for [C20H14O3 + H]+ 303.1021, found 303.10316; calcd for [C20H14O3 + Na]+
325.0841, found 325.08477.
4.1.3.9 2-(3-hydroxyphenyl)-4H-benzo[h]chromen-4-one (9i). Pale white solid, yield:

1
54%; H NMR (400 MHz, DMSO-d6) δ 9.91 (s, 1H), 8.59 (d, J = 4.9 Hz, 1H), 8.14 –
8.04 (m, 1H), 7.97 (d, J = 8.6 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 4.8 Hz, 2H),
7.67 – 7.56 (m, 8.8 Hz, 2H), 7.40 (t, J = 7.7 Hz, 1H), 7.05 (s, 1H), 7.01 (d, J = 7.8 Hz,
1H). 13C NMR (101 MHz, DMSO-d6) δ 177.26, 162.41, 158.44, 153.14, 135.91, 132.89,
130.85, 130.01, 128.77, 128.16, 125.81, 123.94, 122.48, 120.46, 120.09, 119.23, 117.60,
113.19, 108.50. HRMS (ESI) calcd for [C19H12O3 + H]+ 289.0865, found 289.08708.
4.1.3.10 2-(3-(trifluoromethyl)phenyl)-4H-benzo[h]chromen-4-one (9j). Pale white
1
solid, yield: 52%; H NMR (400 MHz, CDCl3) δ 8.44 (d, J = 3.4 Hz, 1H), 8.16 (s, 1H),
8.12 – 8.01 (m, 2H), 7.91 – 7.81 (m, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.69 (d, J = 8.9 Hz,
13
1H), 7.67 – 7.59 (m, 3H), 6.89 (s, 1H). C NMR (101 MHz, CDCl3) δ 177.81, 160.88,
153.47, 136.09, 132.89, 132.02, 131.69, 129.86, 129.43, 129.31, 128.30, 127.98 (d, J =
4.0 Hz), 127.38, 125.67, 123.94, 122.98 (q, J = 4.0 Hz), 122.13, 120.62, 120.28, 109.60.
HRMS (ESI) calcd for [C20H11F3O2 + H]+ 341.0789, found 341.07987.
4.1.3.11 2-(3-(dimethylamino)phenyl)-4H-benzo[h]chromen-4-one (9k). Pale white
1
solid, yield: 54%; H NMR (400 MHz, CDCl3) δ 8.57 (s, 1H), 8.16 (d, J = 8.6 Hz, 1H),
7.91 (s, 1H), 7.76 (d, J = 8.5 Hz, 1H), 7.69 (s, 2H), 7.45 – 7.31 (m, 2H), 7.25 – 7.19 (m,
13
1H), 7.01 – 6.85 (m, 2H), 3.06 (s, 6H). C NMR (101 MHz, CDCl3) δ 178.41, 163.65,
153.56, 150.61, 135.96, 132.69, 129.83, 129.15, 128.21, 127.12, 125.22, 124.19, 122.33,
120.75, 120.25, 115.55, 114.56, 109.70, 108.78, 40.60.
4.1.3.12 2-(3,5-dichlorophenyl)-4H-benzo[h]chromen-4-one (9l). Pale white solid,
yield: 60%; 1H NMR (400 MHz, CDCl3) δ 8.47 (d, J = 5.2 Hz, 1H), 8.08 (d, J = 8.6 Hz,
1H), 7.89 (d, J = 6.7 Hz, 1H), 7.79 (s, 2H), 7.73 (d, J = 8.5 Hz, 1H), 7.70 – 7.65 (m, 2H),
7.49 (s, 1H), 6.84 (s, 1H). 13C NMR (101 MHz, CDCl3) δ 177.66, 159.72, 153.47, 136.14,
136.11, 134.95, 131.20, 129.52, 128.33, 127.44, 125.80, 124.60, 123.90, 122.20, 120.61,
120.35, 110.02. HRMS (ESI) calcd for [C19H10Cl2O2 + H]+ 341.0136, found 341.01366.
4.1.3.13 2-(3,4-difluorophenyl)-4H-benzo[h]chromen-4-one (9m). Pale white solid,
1
yield: 47%; H NMR (400 MHz, DMSO-d6) δ 8.74 (d, J = 8.3 Hz, 1H), 8.42 – 8.31 (m,
1H), 8.21 – 8.09 (m, 2H), 7.98 (q, J = 8.6 Hz, 2H), 7.86 – 7.80 (m, 2H), 7.70 (dd, J =
13
18.3, 8.8 Hz, 1H), 7.29 (s, 1H). C NMR (101 MHz, CDCl3) δ 177.9, 160.4, 153.4,
152.34 (dd, J = 254.0 Hz, J = 13.0 Hz), 150.75 (dd, J = 250.0 Hz, J = 13.0 Hz), 136.09,
129.46, 129.03, 128.34, 127.36, 125.69, 123.90, 122.82 (dd, J = 7.0 Hz, J = 4.0 Hz),
122.16, 120.63, 120.16, 118.37 (d, J = 18.0 Hz), 115.56 (d, J = 20.0 Hz), 109.12. HRMS
(ESI) calcd for [C19H10F2O2 + H]+ 309.0727, found 309.07285.
4.1.3.14 2-(3,4,5-trimethoxyphenyl)-4H-benzo[h]chromen-4-one (9n). Pale white
1
solid, yield: 58%; H NMR (400 MHz, CDCl3) δ 8.38 (d, J = 6.9 Hz, 1H), 8.03 (d, J =
8.7 Hz, 1H), 7.82 (d, J = 5.5 Hz, 1H), 7.65 (d, J = 9.1 Hz, 1H), 7.62 – 7.54 (m, 2H), 7.09
13
(s, 2H), 6.78 (s, 1H), 3.91 (s, 6H), 3.88 (s, 3H). C NMR (101 MHz, CDCl3) δ 177.99,
162.41, 153.60, 153.32, 141.10, 135.95, 129.17, 128.28, 127.18, 127.02, 125.31, 123.96,
121.94, 120.64, 120.10, 108.43, 103.60, 61.04, 56.32. HRMS (ESI) calcd for [C22H18O5 +
H]+ 363.1232, found 363.12361.
4.1.3.15 2-(3-chloro-5-methoxyphenyl)-4H-benzo[h]chromen-4-one (9o). Pale white
solid, yield: 61%; 1H NMR (400 MHz, CDCl3) δ 8.57 – 8.44 (m, 1H), 8.09 (d, J = 8.6 Hz,
1H), 7.87 (s, 1H), 7.72 (d, J = 9.2 Hz, 1H), 7.68 – 7.59 (m, 2H), 7.51 (s, 1H), 7.33 (s,

1H), 7.01 (s, 1H), 6.84 (s, 1H), 3.85 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 177.02,
160.14, 159.73, 152.53, 135.09, 135.03, 133.41, 128.42, 127.29, 126.34, 124.61, 123.01,
121.30, 119.66, 119.30, 117.65, 115.81, 109.84, 108.57, 54.82. HRMS (ESI) calcd for
[C20H13ClO3 + H]+ 337.0631, found 337.06357.
4.1.3.16 2-(naphthalen-2-yl)-4H-benzo[h]chromen-4-one (9p). Pale white solid,
yield: 61%; 1H NMR (400 MHz, CDCl3) δ 8.45 (d, J = 6.8 Hz, 1H), 8.31 (s, 1H), 8.02 (d,
J = 8.6 Hz, 1H), 7.91 – 7.68 (m, 5H), 7.67 – 7.52 (m, 3H), 7.46 (d, J = 4.1 Hz, 2H), 6.90
(s, 1H). 13C NMR (101 MHz, CDCl3) δ 178.07, 162.36, 153.40, 135.92, 134.50, 132.80,
129.21, 129.01, 128.99, 128.85, 128.19, 127.97, 127.78, 127.13, 127.07, 126.61, 125.28,
124.01, 122.31, 122.28, 120.66, 120.17, 108.88. HRMS (ESI) calcd for [C23H14O2 + H]+
323.1072, found 323.10747.
4.1.3.17 2-(4-(4-methylpiperazin-1-yl)phenyl)-4H-benzo[h]chromen-4-one (9q).
1
Pale yellow solid, yield: 69%; H NMR (400 MHz, DMSO-d6) δ 8.69 – 8.59 (m, 1H),
8.16 – 8.05 (m, 3H), 7.97 (d, J = 8.6 Hz, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.83 – 7.76 (m,
2H), 7.17 (d, J = 8.7 Hz, 2H), 7.06 (s, 1H), 4.06 (brs, 2H), 3.46 (brs, 2H), 3.30 – 3.20
(brs, 2H), 3.15 (brs, 2H), 2.80 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 177.06, 162.72,
153.00, 152.13, 135.82, 129.88, 128.74, 128.20, 128.12, 125.61, 123.94, 122.63, 121.57,
120.53, 120.04, 115.49, 106.36, 52.16, 44.64, 42.36. HRMS (ESI) calcd for [C24H22N2O2
+ H]+ 371.1760, found 371.17762.
4.1.3.18 2-(3-(4-methylpiperazin-1-yl)phenyl)-4H-benzo[h]chromen-4-one (9r).
Pale white solid, yield: 66%; 1H NMR (400 MHz, CDCl3) δ 8.48 – 8.38 (m, 1H), 8.05 (d,
J = 8.6 Hz, 1H), 7.87 – 7.73 (m, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.62 – 7.55 (m, 2H), 7.42
– 7.30 (m, 3H), 7.02 (d, J = 7.8 Hz, 1H), 6.82 (s, 1H), 3.32 – 3.18 (m, 4H), 2.59 – 2.50
13
(m, 4H), 2.31 (s, 3H). C NMR (101 MHz, CDCl3) δ 178.24, 163.20, 153.47, 151.75,
135.93, 132.78, 129.88, 129.17, 128.19, 127.12, 125.25, 124.09, 122.25, 120.70, 120.22,
119.15, 117.44, 113.20, 108.84, 54.94, 48.84, 46.12. HRMS (ESI) calcd for [C24H22N2O2
+ H]+ 371.1760, found 371.17770.
4.1.3.19 2-(3-chloro-5-(4-methylpiperazin-1-yl)phenyl)-4H-benzo[h]chromen-4-one
1
(9s). Pale white solid, yield: 64%; H NMR (400 MHz, CDCl3) δ 8.55 – 8.44 (m, 1H),
8.09 (d, J = 8.7 Hz, 1H), 7.91 – 7.84 (m, 1H), 7.73 (d, J = 8.7 Hz, 1H), 7.69 – 7.62 (m,
2H), 7.38 (s, 1H), 7.25 (s, 1H), 6.99 (s, 1H), 6.84 (s, 1H), 3.33 – 3.20 (m, 4H), 2.64 –
13
2.52 (m, 4H), 2.33 (s, 3H). C NMR (101 MHz, CDCl3) δ 178.09, 162.00, 153.54,
152.51, 136.05, 136.00, 134.16, 129.35, 128.29, 127.31, 125.52, 124.05, 122.28, 120.68,
120.30, 118.32, 116.82, 111.34, 109.45, 54.72, 48.37, 46.04. HRMS (ESI) calcd for
[C24H21ClN2O2 + H]+ 405.1370, found 405.13835.
4.1.3.20 2-(3-morpholinophenyl)-4H-benzo[h]chromen-4-one (9t). Pale white solid,
yield: 69%; 1H NMR (400 MHz, CDCl3) δ 8.52 – 8.40 (m, 1H), 8.07 (d, J = 8.5 Hz, 1H),
7.88 – 7.79 (m, 1H), 7.68 (d, J = 8.6 Hz, 1H), 7.64 – 7.57 (m, 2H), 7.45 (d, J = 7.4 Hz,
1H), 7.42 – 7.32 (m, 2H), 7.04 (d, J = 7.7 Hz, 1H), 6.84 (s, 1H), 3.92 – 3.79 (m, 4H),
13
3.27 – 3.12 (m, 4H). C NMR (101 MHz, CDCl3) δ 178.21, 163.04, 153.50, 151.63,
135.97, 132.93, 130.01, 129.22, 128.24, 127.18, 125.32, 124.10, 122.26, 120.72, 120.25,
118.86, 117.99, 113.02, 108.97, 66.73, 49.16. HRMS (ESI) calcd for [C23H19NO3 + H]+
358.1443, found 358.14512.

4.1.3.21 2-(pyridin-2-yl)-4H-benzo[h]chromen-4-one (9u). Pale white solid, yield:
43%; 1H NMR (400 MHz, CDCl3) δ 8.70 (d, J = 4.2 Hz, 1H), 8.57 – 8.47 (m, 1H), 8.15
(d, J = 7.8 Hz, 1H), 8.10 (d, J = 8.7 Hz, 1H), 7.92 – 7.81 (m, 2H), 7.70 (d, J = 8.7 Hz,
1H), 7.63 (dd, J = 6.2, 3.2 Hz, 2H), 7.49 (s, 1H), 7.39 (dd, J = 7.1, 4.8 Hz, 1H). 13C NMR
(101 MHz, CDCl3) δ 178.31, 160.79, 153.33, 150.16, 149.56, 148.07, 137.21, 136.03,
129.29, 128.27, 127.18, 125.57, 125.47, 124.05, 122.27, 120.88, 120.85, 110.09. HRMS
(ESI) calcd for [C18H11NO2 + H]+ 274.0868, found 274.08778.
4.1.3.22 2-(pyridin-3-yl)-4H-benzo[h]chromen-4-one (9v). Pale white solid, yield:
55%; 1H NMR (400 MHz, CDCl3) δ 9.19 (s, 1H), 8.72 (s, 1H), 8.42 (s, 1H), 8.16 (s, 1H),
8.03 (d, J = 7.9 Hz, 1H), 7.82 (s, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.61 (s, 2H), 7.45 (s, 1H),
13
6.86 (s, 1H). C NMR (101 MHz, CDCl3) δ 177.60, 160.15, 153.44, 152.09, 147.37,
136.01, 133.39, 129.43, 128.25, 127.96, 127.32, 125.64, 123.86, 123.81, 122.12, 120.57,
120.27, 109.59. HRMS (ESI) calcd for [C18H11NO2 + H]+ 274.0868, found 274.08748.
4.1.3.23 2-(pyridin-4-yl)-4H-benzo[h]chromen-4-one (9w). Pale white solid, yield:
59%; 1H NMR (400 MHz, CDCl3) δ 8.81 (d, J = 4.7 Hz, 2H), 8.59 – 8.46 (m, 1H), 8.10
(d, J = 8.7 Hz, 1H), 7.97 – 7.88 (m, 1H), 7.82 (d, J = 4.8 Hz, 2H), 7.75 (d, J = 8.8 Hz,
13
1H), 7.71 – 7.64 (m, 2H), 6.99 (s, 1H). C NMR (101 MHz, CDCl3) δ 177.69, 159.79,
153.47, 150.94, 139.38, 136.19, 129.60, 128.38, 127.44, 125.89, 123.97, 122.19, 120.61,
120.54, 119.74, 110.60. HRMS (ESI) calcd for [C18H11NO2 + H]+ 274.0868, found
274.08763.
4.1.4
General
procedure
for
the
preparation
of
2-substituted
phenyl-4H-benzo[h]chromen-4-thione derivatives 10a-h
To a solution of compound 9 (1 equivalent) in dry toluene (20 ml), Lawesson's
reagent (2 equivalents) was added and the resultant solution was kept for stirring at
110 °C till completion of the reaction (TLC monitoring). Solvents were removed under
vacuum and the residue was purified by column chromatography using silica gel to give
compounds 10a-h.
4.1.4.1 2-phenyl-4H-benzo[h]chromene-4-thione (10a). Pale yellow solid, yield:
82%; 1H NMR (400 MHz, DMSO-d6) δ 8.78 (s, 1H), 8.46 (d, J = 7.9 Hz, 1H), 8.35 (d, J
= 6.7 Hz, 2H), 8.11 (d, J = 10.3 Hz, 2H), 8.00 (d, J = 8.4 Hz, 1H), 7.87 (s, 2H), 7.67 (d, J
13
= 6.3 Hz, 3H). C NMR (101 MHz, DMSO-d6) δ 200.16, 153.99, 148.47, 135.97,
132.53, 130.86, 130.52, 129.86, 128.67, 128.46, 127.23, 126.87, 126.53, 123.90, 123.55,
123.43, 120.78. HRMS (ESI) calcd for [C19H12OS + H]+ 289.0687, found 289.06936.
4.1.4.2 2-(2-fluorophenyl)-4H-benzo[h]chromene-4-thione (10b). Pale yellow solid,
yield: 74%; 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 8.7 Hz, 2H), 8.02 – 7.94 (m, 1H),
7.89 (s, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H), 7.62 (t, J = 7.2 Hz, 2H),
7.52 – 7.42 (m, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.20 – 7.15 (m, 1H). 13C NMR (101 MHz,
CDCl3) δ 201.18, 160.54 (d, J = 256.0 Hz), 149.67 (d, J = 5.0 Hz), 148.65, 136.07,
133.09 (d, J = 9.0 Hz), 129.69, 129.08, 128.18, 127.46, 126.97, 126.33, 125.17 (d, J =
11.0 Hz), 124.95 (d, J = 4.0 Hz), 123.96 (d, J = 18.0 Hz), 123.07, 119.86 (d, J = 10.0 Hz),
117.25 (d, J = 22.0 Hz), 109.99. HRMS (ESI) calcd for [C19H11FOS + H]+ 307.0593,
found 307.05965.
4.1.4.3 2-(3-fluorophenyl)-4H-benzo[h]chromene-4-thione (10c). Pale yellow solid,

yield: 73%; 1H NMR (400 MHz, CDCl3) δ 8.57 – 8.45 (m, 2H), 7.84 (d, J = 5.7 Hz, 1H),
7.76 (d, J = 12.0 Hz, 2H), 7.72 – 7.60 (m, 4H), 7.53 – 7.44 (m, 1H), 7.23 (d, J = 8.2 Hz,
1H). 13C NMR (101 MHz, CDCl3) δ 200.98, 163.17 (d, J = 246.0 Hz), 152.01 (d, J = 2.0
Hz), 148.42, 136.15, 133.55 (d, J = 8.0 Hz), 131.04 (d, J = 8.0 Hz), 129.77, 128.28,
127.56, 127.07, 126.47, 124.01, 123.86, 122.95, 122.08 (d, J = 3.0 Hz), 121.64, 118.64
(d, J = 21.0 Hz), 113.38 (d, J = 24.0 Hz). HRMS (ESI) calcd for [C19H11FOS + H]+
307.0593, found 307.05952.
4.1.4.4 2-(3-chlorophenyl)-4H-benzo[h]chromene-4-thione (10d). Pale yellow solid,
1
yield: 78%; H NMR (400 MHz, DMSO-d6) δ 8.78 – 8.70 (m, 1H), 8.43 (d, J = 9.0 Hz,
1H), 8.35 (s, 1H), 8.31 (d, J = 7.8 Hz, 1H), 8.17 – 8.08 (m, 2H), 7.99 (d, J = 8.9 Hz, 1H),
13
7.92 – 7.81 (m, 2H), 7.74 (d, J = 8.1 Hz, 1H), 7.68 (t, J = 7.9 Hz, 1H). C NMR (101
MHz, DMSO-d6) δ 200.53, 152.60, 148.58, 136.07, 134.75, 133.22, 132.21, 131.71,
130.69, 128.74, 128.61, 127.12, 126.81, 126.78, 126.02, 123.92, 123.73, 123.38, 121.48.
HRMS (ESI) calcd for [C19H11ClOS + H]+ 323.0297, found 323.03025.
4.1.4.5 2-(3-methoxyphenyl)-4H-benzo[h]chromene-4-thione (10e). Pale yellow
1
solid, yield: 80%; H NMR (400 MHz, CDCl3) δ 8.59 – 8.23 (m, 2H), 7.84 – 7.65 (m,
13
2H), 7.65 – 7.43 (m, 4H), 7.41 – 7.22 (m, 2H), 7.07 – 6.93 (m, 1H), 3.80 (s, 3H). C
NMR (101 MHz, CDCl3) δ 200.63, 160.09, 153.21, 148.35, 135.98, 132.40, 130.35,
129.58, 128.13, 127.39, 126.84, 126.18, 123.98, 123.85, 122.91, 121.32, 118.79, 117.25,
111.69, 55.51. HRMS (ESI) calcd for [C20H14O2S + H]+ 319.0793, found 319.08003.
4.1.4.6 2-(4-fluorophenyl)-4H-benzo[h]chromene-4-thione (10f). Pale yellow solid,
yield: 75%; 1H NMR (400 MHz, CDCl3) δ 8.46 (d, J = 8.7 Hz, 2H), 8.02 – 7.91 (m, 2H),
13
7.81 (d, J = 6.1 Hz, 1H), 7.73 (s, 1H), 7.70 – 7.55 (m, 3H), 7.23 – 7.17 (m, 2H). C
NMR (101 MHz, CDCl3) δ 200.73, 163.17 (d, J = 253.0 Hz), 152.57, 148.36, 136.07,
129.65, 128.63, 128.54, 128.25, 127.56, 127.45, 126.77, 126.29, 123.98, 123.91, 122.86,
121.02, 116.76, 116.54. HRMS (ESI) calcd for [C19H11FOS + H]+ 307.0593, found
307.05930.
4.1.4.7 2-(4-chlorophenyl)-4H-benzo[h]chromene-4-thione (10g). Pale yellow solid,
yield: 64%; 1H NMR (400 MHz, CDCl3) δ 8.54 (t, J = 7.9 Hz, 2H), 7.96 (d, J = 8.1 Hz,
2H), 7.88 (d, J = 7.0 Hz, 1H), 7.82 (s, 1H), 7.73 (d, J = 9.0 Hz, 1H), 7.71 – 7.64 (m, 2H),
7.50 (d, J = 8.4 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 201.39, 152.82, 148.44, 138.07,
136.15, 129.79, 129.74, 129.70, 128.32, 127.62, 127.53, 126.97, 126.44, 124.04, 123.92,
122.93, 121.31. HRMS (ESI) calcd for [C19H11ClOS + H]+ 323.0297, found 323.02968.
4.1.4.8 2-(3,4,5-trimethoxyphenyl)-4H-benzo[h]chromene-4-thione (10h). Pale
yellow solid, yield: 77%; 1H NMR (400 MHz, CDCl3) δ 8.48 (dd, J = 16.2, 8.1 Hz, 2H),
7.85 (d, J = 6.7 Hz, 1H), 7.76 (s, 1H), 7.71 – 7.61 (m, 3H), 7.18 (s, 2H), 3.94 (s, 6H),
13
3.90 (s, 3H). C NMR (101 MHz, CDCl3) δ 200.39, 153.78, 153.56, 148.43, 141.49,
136.12, 129.59, 128.34, 127.49, 126.75, 126.29, 126.25, 124.01, 122.66, 121.11, 103.87,
61.07, 56.44. HRMS (ESI) calcd for [C22H18O4S + H]+ 379.1004, found 379.10178.
4.1.5
General
procedure
for
the
preparation
of
2-substituted
phenyl-4H-benzo[h]chromen-4-thione derivatives 11a-c
To a solution of compound 10 (1 equivalent) in dry pyridine (20 ml), hydroxylamine
hydrochloride (2 equivalents) was added and the resultant solution was kept for stirring

at 120 °C till completion of the reaction (TLC monitoring). Solvents were removed
under vacuum and the residue was purified by column chromatography using silica gel to
give compounds 11a-c.
4.1.5.1 (E)-2-phenyl-4H-benzo[h]chromen-4-one oxime (11a). Pale white solid,
1
yield: 78%; H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 8.48 (d, J = 7.7 Hz, 1H),
8.11 (d, J = 6.8 Hz, 2H), 8.03 (d, J = 7.7 Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.82 (d, J =
8.7 Hz, 1H), 7.77 – 7.65 (m, 2H), 7.64 – 7.53 (m, 3H), 7.27 (s, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 153.75, 146.77, 142.88, 134.28, 132.62, 130.94, 129.53, 128.45, 128.12,
127.72, 125.85, 125.23, 124.02, 121.71, 119.61, 114.66, 94.72. HRMS (ESI) calcd for
[C19H13NO2 + H]+ 288.1025, found 288.10357.
4.1.5.2 (E)-2-(3-chlorophenyl)-4H-benzo[h]chromen-4-one oxime (11b). Pale white
1
solid, yield: 81%; H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.54 – 8.35 (m, 1H),
8.00 (s, 1H), 7.93 – 7.87 (m, 3H), 7.61 – 7.58 (m, 1H), 7.57 (d, J = 6.2 Hz, 4H), 7.55 –
7.53 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 168.29, 161.77, 151.56, 135.34, 134.34,
131.54, 131.40, 130.44, 128.42, 128.11, 126.72, 126.35, 125.78, 125.73, 123.94, 123.30,
120.77, 110.07, 101.64. HRMS (ESI) calcd for [C19H12ClNO2 + H]+ 322.0635, found
322.06382.
4.1.5.3 (E)-2-(4-chlorophenyl)-4H-benzo[h]chromen-4-one oxime (11c). Pale white
solid, yield: 74%; 1H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H), 8.46 (s, 1H), 8.01 (d,
13
J = 7.2 Hz, 2H), 7.93 (d, J = 9.0 Hz, 2H), 7.75 – 7.57 (m, 5H), 7.53 (s, 1H). C NMR
(101 MHz, DMSO-d6) δ 168.14, 161.90, 151.50, 135.31, 135.23, 129.67, 128.87, 128.42,
128.20, 128.08, 126.35, 125.70, 123.91, 123.26, 120.77, 110.08, 101.50. HRMS (ESI)
calcd for [C19H12ClNO2 + H]+ 322.0635, found 322.06414.
4.2 CYP1 enzyme inhibition assay
EROD (7-ethoxyresorufin O-deethylase) has been used for determining the
inhibitory activity of these synthesized compounds against recombinant human CYP1
enzymes, according to our previously reported procedure[14, 16]. The recombinant
human CYP1 enzymes (CYP1B1, CYP1A1, and CYP1A2, each equipped with P450
reductase (Supersomes)), were purchased from BD Genetest (Corning).
7-Ethoxyresorufin (7-ER) was available from Sigma-Aldrich. Nicotinamide-adenine
dinucleotide
phosphate
(NADP+),
D-glucose-6-phosphate
(G-6-P)
and
glucose-6-phosphate dehydrogenase (G-6-PD) were obtained from the company of
Biosharp. Generally, a mixture with a final volume of 200 µl containing different
concentrations (the final concentrations were 0.5, 1, 5, 10, 50, 100, 500 and 1000 nM,
respectively) of tested compounds (except positive and negative control wells), an
enzyme source (20 fmol CYP1B1, 10 fmol CYP1A1 or 60 fmol CYP1A2), substrate
(7-ER, 150 nM), a NADPH regeneration system containing NADP+ (1.3 mM), G-6-P
(3.3 mM), G-6-PD (0.5 U/ml) and MgCl2 (3.3 mM) was incubated in a black 96-well
flat-bottomed microplate at 37 oC for varied time durations (incubation time for CYP1B1,
CYP1A1, and CYP1A2 was 35, 15 and 50 min, respectively). Then, the reaction was
quenched by addition of 100 µl of pre-cooled methanol to all wells. Lastly, the
fluorescence intensity was measured by FlexStation 3 apparatus with excitation and

emission filters at 544 and 590 nm, respectively. Three replicates were used for each
concentration. The IC50 value for each compound was calculated with the GraphPad
Prism Software (Version 5.0) using the non-linear regression formula, log (inhibitor) vs.
normalized response-variable slope.
4.3 Molecular docking procedure
The X-ray structure coordinates of ANF bound with CYP1B1 (PDB ID 3PM0) or
CYP1A2 (PDB ID 2HI4) were downloaded from RCSB protein data bank
(http://www.rcsb.org/). The modelling experiment described in this study was performed
by using the docking program of MOE 2008 as our previously described[14]. All water
molecules in the crystal structure of CYP1B1 were removed prior to docking. However,
in light of the importance of water molecules in formation of water-mediated hydrogen
bond, water molecules in the crystal structure of CYP1A2 were retained for docking
study.
4.4 CoMFA and CoMSIA analysis
In this work, predictive 3D-QSAR models CoMFA and CoMSIA were performed
using SYBYL-X 2.0 software from Tripos, Inc. The IC50 values were transformed into
pIC50 (-log 10 (IC50*10-9)) values and used as the dependent variable in CoMFA and
CoMSIA analysis. The 3D structures of these CYP1B1 inhibitors were constructed with
SYBYL/Sketch module and energy minimization were implemented using the Tripos
force field with convergence criterion of 0.005 kcal/(Å· mol) and a maximum of 1000
iterations and Gasteiger-Hückel charges. Based on the variety of activity and structure
diversity, all the forty-two ANF derivatives were divided into training set and test set. As
one of the most active molecule, compound 9j was chosen as a main template and the
other molecules were aligned to it. In CoMFA study, all aligned molecules were located
at the center of grid box with a 3D regularly spaced grid of 2.0 Å using an sp3 hybridized
carbon atom as a probe with +1.0 charge to calculate steric and electrostatic fields. The
default cut-off energy value was set at 30 kcal/mol for both steric and electrostatic fields.
Partial least squares (PLS) analysis was used to linearly correlate CoMFA fields with the
activity values. Cross-validation was performed using the leave-one-out(LOO) method.
CoMSIA similarity index descriptors were calculated using a similar lattice box as in
CoMFA. For CoMSIA descriptors, five similarity index fields (steric, electrostatic,
hydrophobic, hydrogen bond donor, and hydrogen bond acceptor) were calculated using
an sp3 carbon with a charge, hydrophobic interaction, and hydrogen-bond donor and
acceptor properties of +1.0 was used as a probe at every grid point to measure the five
above-mentioned fields. The default value of 0.3 was used as attenuation factor. All of
the contours represented the default 80% and 20% level contributions for favored and
disfavored areas, respectively.
Conflicts of interest
The authors declare no conflicts of interest.

Acknowledgements
The research reported in this publication was supported by the Interdisciplinary
Program of Shanghai Jiao Tong University (project number: ZH2018QNB15), the
Fundamental Research Funds for the Central University (No. ZH2018QNB15), the
Shanghai Natural Science Fund (No. 16ZR1418100), as well as the Shanghai Science
and Technology Innovation Program (No. 15431900700). We also want to express our
gratitude to associate professor Xiaoyan Pan and professor Jie Zhang in the Xi’an
Jiaotong University for providing SYBYL-X 2.0 software.
References
[1] G. I. Murray, M. C. Taylor, M. C. McFadyen, J. A. McKay, W. F. Greenlee, M. D.
Burke, W. T. Melvin, Tumor-specific expression of cytochrome P450 CYP1B1, Cancer
Res., 57 (1997) 3026-3031.
[2] M. Nakajima, M. Iwanari, T. Yokoi, Effects of histone deacetylation and DNA
methylation on the constitutive and TCDD-inducible expressions of the human CYP1
family in MCF-7 and HeLa cells, Toxicol. Lett., 144 (2003) 247-256.
[3]
M.
W.
Haque,
S.
P.
Pattanayak,
Taxifolin
Inhibits
7,12-Dimethylbenz(a)anthracene-induced Breast Carcinogenesis by Regulating
AhR/CYP1A1 Signaling Pathway, Pharmacogn. Mag., 13 (2018) S749-S755.
[4] J. C. Rowlands, L. He, R. Hakkak, M. J. Ronis, T. M. Badger, Soy and whey proteins
downregulate DMBA-induced liver and mammary gland CYP1 expression in female rats,
J. Nutr., 131 (2001) 3281-3287.
[5] E. H. Han, Y. P. Hwang, T. C. Jeong, S. S. Lee, J. G. Shin, H. G. Jeong, Eugenol
inhibit 7,12-dimethylbenz[a]anthracene-induced genotoxicity in MCF-7 cells:
Bifunctional effects on CYP1 and NAD(P)H: quinone oxidoreductase, FEBS Lett., 581
(2007) 749-756.
[6] J. Dong, Q. Zhang, Q. Cui, G. Huang, X. Pan, S. Li, Flavonoids and
Naphthoflavonoids: Wider Roles in the Modulation of Cytochrome P450 Family 1
Enzymes, ChemMedChem, 11 (2016) 2102-2118.
[7] M. C. McFadyen, H. L. McLeod, F. C. Jackson, W. T. Melvin, J. Doehmer, G. I.
Murray, Cytochrome P450 CYP1B1 protein expression: a novel mechanism of
anticancer drug resistance, Biochem Pharmacol., 62 (2001) 207-212.
[8] B. Rochat, J. M. Morsman, G. I. Murray, W. D. Figg, H. L. McLeod, Human
CYP1B1 and anticancer agent metabolism: mechanism for tumor-specific drug
inactivation?, J. Pharmacol. Exp. Ther., 296 (2001) 537-541.
[9] R. Dutour, F. Cortes-Benitez, J. Roy, D. Poirier, Structure-Based Design and
Synthesis of New Estrane-Pyridine Derivatives as Cytochrome P450 (CYP) 1B1
Inhibitors, ACS Med. Chem. Lett., 8 (2017) 1159-1164.
[10] R. Dutour, D. Poirier, Inhibitors of cytochrome P450 (CYP) 1B1, Eur. J. Med.
Chem., 135 (2017) 296-306.
[11] T. Shimada, H. Yamazaki, M. Foroozesh, N. E. Hopkins, W. L. Alworth, F. P.
Guengerich, Selectivity of polycyclic inhibitors for human cytochrome P450s 1A1, 1A2,

and 1B1, Chem. Res. Toxicol., 11 (1998) 1048-1056.
[12] A. Datta, N. Bhasin, H. Kim, M. Ranjan, B. Rider, Z.Y. Abd Elmageed, D. Mondal,
K.C. Agrawal, A. B. Abdel-Mageed, Selective targeting of FAK-Pyk2 axis by
alpha-naphthoflavone abrogates doxorubicin resistance in breast cancer cells, Cancer
Lett., 362 (2015) 25-35.
[13] M. Kubo, K. Yamamoto, T. Itoh, Design and synthesis of selective CYP1B1
inhibitor via dearomatization of α-naphthoflavone, Bioorg. Med. Chem., 27 (2019)
285-304.
[14] J. Cui, Q. Meng, X. Zhang, Q. Cui, W. Zhou, S. Li, Design and synthesis of new
α-naphthoflavones as cytochrome P450 (CYP) 1B1 inhibitors to overcome
docetaxel-resistance associated with CYP1B1 overexpression, J. Med. Chem., 58 (2015)
3534-3547.
[15] J. Y. Dong, Z. T. Wang, Q. Q. Meng, Q. J. Zhang, G. Huang, J. H. Cui, S. S. Li,
Development of 2-arylbenzo[h]quinolone analogs as selective CYP1B1 inhibitors, RSC
Adv., 8 (2018) 15009-15020.
[16] J. Dong, J. Cui, Y. Wu, S. Li, Q. Meng, Q. Zhang, Z. Wang, G. Huang, Development
of benzochalcone derivatives as selective CYP1B1 inhibitors and anticancer agents,
MedChemComm, 10 (2019) 1606-1614.
[17] H. Nakazumi, T. Ueyama, Kitao, Teijiro, Synthesis and antibacterial activity of
2-phenyl-4H-benzo [b] thiopyran-4-ones (Thioflavones) and related compounds, J.
Heterocycl. Chem., 21 (1984) 193-196.
[18] D. Vyawahare, M. Ghodke, A. P. Nikalje, Green synthesis and pharmacological
screening of novel 1, 5-benzothiazepines as CNS agents, Int. J. Pharm. Pharm. Sci., 2
(2010) 27-29.
[19] A. Wang, U. Savas, C. D. Stout, E. F. Johnson, Structural characterization of the
complex between alpha-naphthoflavone and human cytochrome P450 1B1, J. Biol.
Chem., 286 (2011) 5736-5743.
[20] S. Sansen, J. K. Yano, R. L. Reynald, G. A. Schoch, K. J. Griffin, C. D. Stout, E. F.
Johnson, Adaptations for the oxidation of polycyclic aromatic hydrocarbons exhibited by
the structure of human P450 1A2, J. Biol. Chem., 282 (2007) 14348-14355.
[21] A. A. Walsh, G. D. Szklarz, E. E. Scott, Human cytochrome P450 1A1 structure and
utility in understanding drug and xenobiotic metabolism, J. Biol. Chem., 288 (2013)
12932-12943.

Highlights
‹ Discovery of two potent CYP1B1 inhibitors with high selectivity;
‹ Provided comprehensive structure-activity relationships of α
derivatives as CYP1 inhibitors;
-
naphthoflavone
‹ Expanding SAR with a range of phenyl ring replacements would lay a foundation for
development of ANF-based water-soluble CYP1B1 inhibitors.

Conflicts of interest
The authors declare no conflicts of interest.