Discovery of heterocycle-containing α-naphthoflavone derivatives as water-soluble, highly potent and selective CYP1B1 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112895

Cytochrome P450 1B1 (CYP1B1) has been well validated as an attractive target for cancer prevention and drug resistance reversal. In continuation of our interest in this area, herein, a set of forty-six 6,7,10-trimethoxy-α-naphthoflavone derivatives varying in B ring was synthesized and screened against CYP1 enzymes, leading to the identification of fluorine-containing compound 15i as the most potent and selective CYP1B1 inhibitor (IC₅₀ value of 0.07 nM), being 84-fold more potent than that of the template molecule ANF. Alternatively, the amino-substituted derivative 13h not only possessed a potent inhibitory effect on CYP1B1 (IC₅₀ value of 0.98 nM), but also had a substantially increased water solubility as compared with the lead ANF (311 μg/mL for 13h and <5 μg/mL for ANF). The current study expanded the structural diversity of CYP1B1 inhibitors, and compound 13h could be considered as a promising starting point with great potential for further studies.

Full text of DOI:10.1016/j.ejmech.2020.112895

Journal Pre-proof
Discovery of heterocycle-containing α-naphthoflavone derivatives as water-soluble,
highly potent and selective CYP1B1 inhibitors
Jinyun Dong, Guang Huang, Qing Cui, Qingqing Meng, Shaoshun Li, Jiahua Cui
PII:
S0223-5234(20)30867-9
DOI:
https://doi.org/10.1016/j.ejmech.2020.112895
EJMECH 112895
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 June 2020
Revised Date: 2 September 2020
Accepted Date: 24 September 2020
Please cite this article as: J. Dong, G. Huang, Q. Cui, Q. Meng, S. Li, J. Cui,, Discovery of heterocycle-
containing α-naphthoflavone derivatives as water-soluble, highly potent and selective CYP1B1 inhibitors,
European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112895.
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Graphical abstract:

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Discovery of heterocycle-containing α-naphthoflavone derivatives as
water-soluble, highly potent and selective CYP1B1 inhibitors
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Jinyun Dong1,2, Guang Huang1, Qing Cui1, Qingqing Meng1, Shaoshun Li1* and Jiahua Cui1, 3**
1 School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, China
2 College of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, China
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School of Environmental Science and Engineering, Shanghai Jiao Tong University, 800
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Dongchuan Road, Shanghai, China
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*Corresponding Author; **Corresponding Author.
E-mail addresses: cpucjh@sjtu.edu.cn (J. Cui), ssli@sjtu.edu.cn (S. Li).
Abstract
Cytochrome P450 1B1 (CYP1B1) has been well validated as an attractive target for cancer
prevention and drug resistance reversal. In continuation of our interest in this area, herein, a set of
forty-six 6,7,10-trimethoxy-α-naphthoflavone derivatives varying in B ring was synthesized and
screened against CYP1 enzymes, leading to the identification of fluorine-containing compound
15i as the most potent and selective CYP1B1 inhibitor (IC50 value of 0.07 nM), which was
84-fold more potent than that of the template molecule ANF. In addition, the amino-substituted
derivative 13h not only possessed a potent inhibitory effect on CYP1B1 (IC50 value of 0.98 nM),
but also had a substantially increased water solubility as compared with the lead ANF (311
μg/mL for 13h and < 5 μg/mL for ANF). The current study expanded the structural diversity of
CYP1B1 inhibitors, and compound 13h could be considered as a promising starting point with
great potential for further studies.
Keywords: CYP1 enzymes, CYP1B1 inhibitors, α-naphthoflavone derivatives, SARs.
1. Introduction
Cytochrome P450 enzymes (CYPs) belong to a superfamily of heme-dependent
monooxygenases that are involved in the metabolic biotransformation of a wide variety of
structurally diverse endogenic and xenobiotic chemicals, including many approved drugs [1].
Among them, the cytochrome P450 subfamily 1 enzymes (CYP1s) including CYP1A1, CYP1A2,
and CYP1B1 have long been of interest for their dominant roles in the bioactivation of numerous
procarcinogens compounds (such as polycyclic aromatic hydrocarbons) to mutagenic and
carcinogenic derivatives [2]. They are expressed in a tissue-specific manner: CYP1A2 is located
primarily in the liver, while CYP1A1 and CYP1B1 are mainly expressed in extrahepatic tissues
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such as breast, prostate and endometrium [3, 4]. Various planar aromatic molecules including
7,12- dimethylbenz[a]anthracene (DMBA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
could stimulate the expression levels of CYP1s by binding to the aryl hydrocarbon receptor
(AhR), a ligand-activated transcription factor [5-7].
CYP1B1 isoform is the most attractive therapeutic target among the three CYP1 members
mentioned above for the following reasons: (1) CYP1B1 is constitutively overexpressed to a
notable extent in tumors (breast, testis, colon, lung, etc. ), although the expression levels of these
enzymes differ between tumor and corresponding normal tissues [8], (2) besides its role in the
bioactivation of many exogenous procarcinogens, CYP1B1 catalyzes the 4-hydroxylation of
endogenic 17β-estradiol (E2) leading to the formation of E2-3,4-quinone, a mutagenic compound
able to bind DNA covalently, thereby contributing to estrogen carcinogenesis, and (3) it confers
drug resistance by metabolically inactivating structurally diverse anticancer drugs such as
docetaxel, mitoxantrone, doxorubicin and paclitaxel [9]. Therefore, strategy that contributes to
the selective inhibition of CYP1B1 could be therapeutically beneficial for preventing mammary
tumor formation and countering CYP1B1-associated drug resistance [9].
Over the past years, various kinds of compounds including flavonoids, trans-stilbenes,
coumarins, phytoestrogens and alkaloids have been characterized as CYP1 inhibitors [4, 10, 11].
Among them, flavonoids have been extensively studied since they have diverse medicinal
applications and are less likely to induce toxicity [12, 13]. α-Naphthoflavone (ANF), a synthetic
flavonoid, has long been determined as one of the most potent CYP1 inhibitors. Shimada et al.
found ANF could considerably inhibit the recombinant human CYP1B1, CYP1A2 and CYP1A1
with IC50 values of 5, 6 and 60 nM, respectively [14]. As a consequence, ANF not only functions
as a chemopreventive agent, but also as a chemosensitizing agent to reverse the CYP1B1-
mediated drug resistance [15]. Similar to the most flavonoids, ANF possesses a broad safety
profile [16]. All these promising properties of ANF make it a potential candidate for further study.
Nevertheless, poor water solubility and low selectivity toward CYP1B1 over the other CYP1
isoforms (especially CYP1A2 isoform) remain the limitations to its applications as both chemical
tools and therapeutics [17].
To this end, our research group recently has made great efforts toward the development of
potent and selective CYP1B1 inhibitors with improved water solubility by structural modification
of ANF (shown in Fig.1), however, such a compound has not yet been discovered [11, 17-19]. We
introduced three methoxy groups to the naphthalene moiety of ANF and found that their CYP1
inhibition ability obviously enhanced compared with ANF [17]. Notably, compound 4c (structure
shown in Fig.1) exhibited strong CYP1B1 inhibitory activity with an IC50 value as low as 0.043
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nM. Regrettably, its water solubility is much lower for successful use in cell-based study.
Structural modification of the C ring of ANF including introduction of functional groups to the
C3 position, reduction of the C2-C3 double bond, or isosteric replacement of oxygen atom at
1-position with “NH” or “S” led to varying degrees of loss of CYP1 inhibition, indicating C ring
is indispensable for maintaining the blocking activity. [18, 19]. However, incorporation of
substituents into the B ring pronouncedly affects their inhibitory capacity and selectivity toward
CYP1B1 [17, 18]. In addition, aromatic heterocycles are tolerated for potency in CYP1 inhibition
[18]. Based on these findings, herein we describe our efforts to identify and characterize novel
aromatic heterocycle substituted α-naphthoflavone derivatives as water-soluble, potent and
selective CYP1B1 inhibitors.
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Fig 1. Summary of our previous and current work.
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2. Results and discussion
2.1. Chemistry
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All the compounds studied were synthesized according to the procedures described in
Scheme 1 (the synthetic routes for preparation of some aromatic acid intermediates were not
shown here). Methylation of commercially available 1,5-dihydroxynaphthalene (1) followed by
bromination and methoxylation provided 1,4,5,8-tetramethoxynaphthalene (4). Vilsmeier reaction
of
4
with
N,N-dimethylformamide
and
phosphorus
oxychloride
gave
2-formyl-1,4,5,8-tetramethoxynaphthalene (5), which was reacted with Grignard reagent
(CH3MgI) to give the alcohol 6. Oxidation of 6 with active manganese dioxide afforded the
corresponding ketone derivative 7, which was subjected to selective demethylation in the
presence of aluminum chloride to yield the key intermediate 8. The ester derivatives 9, obtained
by the esterification reaction of 8 with various aromatic acids in the presence of EDCI (or HATU)
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and DMAP at room temperature, were treated with NaH to undergo Baker-Venkataraman
rearrangement to furnish the corresponding 1,3-dione derivatives 10. Acid-catalyzed cyclization
reaction of 10 provided the final compounds in good yields.
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Scheme 1. Reagents and conditions: (a) KOH, (CH3)2SO4, rt.; (b) CH3CN, NBS, -10 oC; (c) CH3ONa, CuI,
reflux; (d) POCl3, DMF, reflux; (e) CH3MgI, Et2O, NH4Cl, rt.; (f) MnO2, DCM, reflux; (g) AlCl3, CH3CN, 60
oC; (h) EDCI/DMAP or HATU/DMAP; (i) NaH, DMF; (j) 10% H2SO4-EtOH, reflux.
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2.2. CYP1 enzyme inhibitory activities
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The inhibitory effects of these studied compounds on recombinant human CYP1 enzymes
were measured by the EROD (7-ethoxyresorufin O-deethylation) assay as described previously
[17]. In our previous study, we found that introduction of a piperazine moiety to the B ring of
ANF resulted in a slight decrease of CYP1B1 inhibitory activity, while morpholine ring was
shown to be tolerated (two- and single-digit nanomolar IC50 values, respectively). In light of the
importance of nitrogen-containing heterocycles in improving water solubility, hence, at the start
of this study, we attempted to introduce three methoxy groups into the naphthalene moiety of
ANF, aiming to increase their CYP1B1 inhibitory potency. The results are presented in Table 1.
Out of our expectation, all of these piperazine-substituted derivatives (compounds 11a-b and 11d)
suffered a great loss of inhibitory effects on CYP1 enzymes (IC50 values of > 500 nM).
Consistently, morpholine-substituted derivative (11c) was the most potent one among them,
however, compared with ANF or the previously reported compound that does not contain three
methoxy groups, analog 11c still showed a somewhat loss of activity in blocking CYP1B1
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enzyme (IC50 values of 5.9, 2 and 14.9 nM, respectively) [18]. From these results we speculate
that the introduction of multi-methoxy groups would lead to unfavorable conformations of these
aliphatic heterocycles within the active sites.
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Table 1. Inhibitory activities of 6,7,10-trimethoxy-α-naphthoflavone derivatives against CYP1s
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IC50 values (nM)
1A1 1A2
IC50 ratio
Compd.
R
1B1
1A1/1B1 1A2/1B1
3-
11a
(4-methyl-piperazi
n-1-yl)
>1000 >1000
>1000
-
-
3-Cl-5-(4-methyl-p
iperazin-1-yl)
3-(4-morpholinyl)
11b
519.4
14.9
>1000
132.5
>1000
>1000
>1.9
8.9
>1.9
11c
11d
>67.1
4-(4-methyl-pipera
>1000
5.9
>1000
80.3
>1000
18.0
-
-
zin-1-yl)
-
13.6
3.1
ANF
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Considering that aliphatic heterocycles may not be suitable for ANF-based skeleton, we
turned our attention to replacing the phenyl ring with various aromatic heterocycles. Herein, we
synthesized a variety of aromatic heterocycle ring-substituted α-naphthoflavone derivatives to
determine an optimal one. In this part, an initial set of heteroaromatic fused-ring derivatives
including 12a-h was designed and synthesized to evaluate whether a bigger substituent could
enhance the CYP1B1 inhibitory activity. As shown in Table 2, all of these analogs, except 12f,
exhibited potent inhibition of CYP1B1 with IC50 values ranging from 0.87 to 15.6 nM, while
showing weak or no inhibitory effect toward CYP1A2 with IC50 values more than 1000 nM.
Compound 12a incorporating an indole ring was found to potently inhibit CYP1B1 with an IC50
value of 0.95 nM. Based on this compound, we observed that: (1) reversal of the indole ring
resulted in 4-fold decrease in CYP1B1 inhibitory potency (compound 12g, IC50 = 4.1 nM),
whereas bioisosteric replacement of the nitrogen atom of 12g with an oxygen atom afforded
compound 12h with a comparable activity with 12a (IC50 = 0.87 nM); (2) addition of a nitrogen
atom to the indole ring yielding compounds 12b-c and 12f caused a differential decrease in
inhibitory activity toward CYP1B1, especially compound 12f, which showed more than100-fold
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loss in potency against CYP1B1, however, out of our expectation, it was able to selectively
inhibit CYP1A1(IC50 values of 189.4 and 23.8 nM for CYP1B1 and CYP1A2, respectively); (3)
addition of a carbon atom to the pyrrole ring of indole providing the corresponding quinoline
derivative 12d that showed only a minimal loss of CYP1B1 inhibitory activity with an IC50 value
of 1.8 nM and, interestingly, the activity was increased when its quinoline functionality was
reversed (compound 12e, IC50 = 1.0 nM). Taking these observations together, we could postulate
that CYP1B1 and CYP1A1 inhibitory efficacies are affected by the position and number of
nitrogen atom on the B ring, but, of note, the nitrogen atom is not an essential structural element
for these compounds.
Some single-ring substituted derivatives including 12i-l and 13a-15a were also synthesized
and evaluated to explore the structure-activity relationship (SAR) in this series. As presented in
Table 2, all of these five- and six-membered ring substituted derivatives had a potent inhibitory
effect on CYP1B1 with IC50 values ranging from sub-nanomole to nanomole levels. However,
compared with the fused-ring derivatives above, all of these single-ring derivatives, except 12l,
showed a significant increase in activity against CYP1A2, suggesting that CYP1A2 may be more
sensitive to steric hindrance than that of the other two isozymes. Removal of the phenyl moiety of
compound 12h led to furan derivative 12i, which showed a comparable potency against CYP1B1
(IC50 = 1.2 nM). However, replacement of the oxygen atom of compound 12i with a sulfur atom
was unfavorable for CYP1B1 inhibition (12j, IC50 = 4.8 nM), but it was able to be partially
restored by introducing a nitrogen atom (12k, IC50 = 2.6 nM), suggesting the importance of
physicochemical properties of aromatic heterocycles such as electron cloud density and
lipophilicity. To improve water solubility, we further explored four nitrogen-containing
six-membered ring derivatives 12l and 13a-15a. Three pyridine substituted analogs 13a-15a
showed similar CYP1B1 inhibitory activities with IC50 values around 1 nM, which was more
potent than pyridazine derivative 12l (IC50 = 2.0 nM). In light of their CYP1B1 inhibitory
potency, selectivity and water-soluble potential, our attention was drawn to the development of
pyridine substituted derivatives as CYP1B1 inhibitors.
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Table 2. Inhibitory activities of heterocyclic ring-substituted 6,7,10-trimethoxy-α-naphthoflavone
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derivatives toward CYP1 enzymes
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IC50 values (nM)
IC50 ratio
Compd.
Ar
1B1
0.95
1A1
1A2
1A1/1B1 1A2/1B1
141.9
>1000
>1000
>1000
>1000
149.4
2.7
>1052.6
>64.1
12a
12b
12c
15.6
2.0
42.4
1.6
0.8
>500
1.8
20.9
11.6
>555.6
12d
12e
1.0
117.6
23.8
>1000
>1000
117.6
0.1
>1000
>5.3
189.4
12f
4.1
0.87
1.2
16.5
10.1
13.8
4.4
>1000
>1000
67.2
4.0
11.6
11.5
0.9
>243.9
>1149.4
56.0
12g
12h
12i
4.8
40.4
8.4
12j
12k
2.6
3.1
29.2
1.2
11.2
2.0
1.0
125.7
7.6
>1000
117.3
62.9
7.6
>500
117.3
12l
13a
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IC50 values (nM)
IC50 ratio
Compd.
Ar
1B1
1.2
1A1
1A2
1A1/1B1 1A2/1B1
40.4
199.8
33.7
166.5
14a
0.6
5.9
5.4
107.6
18.0
9.0
179.3
3.1
15a
-
80.3
13.6
ANF
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A series of pyridin-3-yl substituted α-naphthoflavone derivatives was first synthesized and
their biological data were shown in Table 3. In our previous studies, the meta-position of B ring
in α-naphthoflavone scaffold was found to be very critical for CYP1B1 inhibitory potency.
Therefore, we initially focused our efforts on introducing several substituents into the
meta-position (C-5’) of pyridin-3-yl ring. Compounds 13b-d bearing halogen or methyl group
showed a noticeable potency in blocking CYP1B1 with a very high selectivity profile over
CYP1A2, being five-fold more potent than ANF (their IC50 values around 1 nM). By contrast,
analogs 13e-g attaching hydrophilic functionalities such as amino, carboxyl and hydroxy showed
a drop in CYP1B1 inhibitory activity, with IC50 values at tens of nanomolar concentrations. To
improve their activity against CYP1B1, we moved these hydrophilic substituents to the C-4’ or
C-2’ positions. To our delight, compound 13h, whose structure was characterized by an amino
substituent at C-4’ position, considerably increased the inhibitory effect on CYP1B1 with an IC50
value of 0.98 nM. Furthermore, it maintained high selectivity over the other two isozymes.
Surprisingly, a dramatic drop in enzymatic activity was observed when the amino was replaced
by a hydroxy (13i, IC50 = 658.3 nM), or was moved to C-2’ position (13k, IC50 = 175.2 nM). Of
note, substitutions on the C-2’ position (13j-l) were less active than the corresponding C-5’ or
C-4’ substituted derivatives in blocking CYP1B1, suggesting that substitution at the 2’-position
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was detrimental to the activity.
Table 3. Inhibitory activities of pyridin-3-yl substituted 6,7,10-trimethoxy-α-naphthoflavone derivatives
toward CYP1s
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IC50 values (nM)
IC50 ratio
Compd.
R
1B1
1.0
1A1
7.6
1A2
1A1/1B1 1A2/1B1
H
5’-F
117.3
140.6
742.0
160.7
628.5
>1000
>1000
108.0
>1000
>1000
>1000
965.5
18.0
7.6
18.1
7.3
117.3
143.5
674.5
123.6
27.4
>33.2
>97.1
110.2
>1.5
-
13a
13b
13c
13d
13e
13f
0.98
1.1
17.7
8.0
5’-Br
5’-CH3
5’-NH2
5’-COOH
5’-OH
4’-NH2
4’-OH
2’-OH
2’-NH2
2’-Br
1.3
6.9
5.3
22.9
30.1
10.3
0.98
658.3
>1000
175.2
63.9
5.9
18.8
85.8
80.9
30.8
>1000
151.9
137.1
60.7
80.3
0.8
2.9
7.9
13g
13h
13i
31.4
>1.5
0.2
13j
0.8
>5.7
15.1
3.1
13k
13l
0.9
-
13.6
ANF
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Next, a set of pyridin-4-yl substituted α-naphthoflavone derivatives was carried out to
confirm the effect of the type of pyridine ring on CYP1 enzymes. Similarly, we also focused our
optimization efforts on the meta-position (3’-position) of B ring in α-naphthoflavone scaffold and
the results are summarized in Table 4. Compared with ANF, halogen group substituted analogs
14b-d were more potent and selective in blocking CYP1B1 enzyme (IC50 values ranging from
0.46 to 1.1 nM), while hydroxy- and amino-substituted derivatives 14e and 14f were significantly
less potent (IC50 values of 61.5 and 20.6, respectively). Analog 14g, obtained by removing the
amino to the 2’-position, showed a slight loss of CYP1B1 inhibitory activity compared to 14f,
which confirmed our hypothesis of the importance of structural modification at 3’-position.
Among them, compound 14d attaching a bromine atom at 3’-position was characterized as the
most potent and selective CYP1B1 inhibitor, with an IC50 value of 0.46 nM. Consistent with the
SAR observed in Table 3, hydrophobic-substituted derivatives were generally more potent than
the corresponding hydrophilic ones. It is interesting to note that a significant reduction in
CYP1A2 inhibitory efficacy was observed as compared with 14a and the other compounds
presented in Table 4, indicating that larger substituents in the B ring would not be accommodated
by the active site of CYP1A2.
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Table 4. Inhibitory activities of pyridin-4-yl substituted 6,7,10-trimethoxy-α-naphthoflavone derivatives
toward CYP1s
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IC50 values (nM)
IC50 ratio
Compd.
R
1B1
1.2
1A1
40.4
38.9
91.6
26.9
232.0
15.3
31.6
80.3
1A2
1A1/1B1 1A2/1B1
H
199.8
518.0
>1000
>1000
>1000
>1000
>1000
18.0
33.7
41.8
83.3
58.5
3.8
166.5
557.0
>909.1
>2173.9
>16.3
>48.5
>40.3
3.1
14a
14b
14c
14d
14e
14f
3’-F
0.93
1.1
3’-Cl
3’-Br
3’-OH
3’-NH2
2’-NH2
-
0.46
61.5
20.6
24.8
5.9
0.7
1.3
14g
ANF
13.6
216
217
218
With the goal of gaining more potent CYP1B1 inhibitors and exploring SAR, we finally
turned our attention to expanding a series of pyridin-2-yl substituted α-naphthoflavone
derivatives as CYP1B1 inhibitors. Several meta-position (3’-position) substituted derivatives
including 15b-f were synthesized and enzyme inhibition data are given in Table 5. Consistent
with the above results, hydrophobic functionality-containing analogs 15d-f were more active than
the hydrophilic-substituted derivatives 15b and 15c in suppressing CYP1 enzymes. Particularly,
halogen-substituted compounds 15e and 15f were found to have outstanding ability to inhibit
CYP1B1 with IC50 values of 0.38 and 0.3 nM, respectively. To determine whether CYP1B1
inhibitory ability could be improved by modification at the other positions of pyridin-2-yl ring,
we synthesized another two compounds 15g and 15h with chlorine atom incorporated at the 4’-
and 5’-position, respectively. To our surprise, compound 15g was found to be more potent than
15h and 15f in inhibiting CYP1B1 enzyme with an IC50 value of 0.14 nM, indicating the
importance of 4’-position in CYP1 enzymes. Keeping this in mind, an attempt has been made to
find out more potent CYP1B1 inhibitors by introducing fluorine and bromine atoms at 4’-position
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of the pyridin-2-yl ring. Gratifyingly, fluorine-substituted derivative 15i showed two-fold more
potent CYP1B1 inhibitory activity than 15g with an IC50 value as low as 0.07 nM, being the most
potent CYP1B1 inhibitor. Analysis of the data presented in Tables 3-5, we can easily conclude
that amino-substituted derivatives generally displayed higher CYP1B1 inhibitory activity than the
corresponding hydroxyl-substituted compounds. In light of the critical roles of amino group in
improving water solubility and further chemical modification, herein, we continued to introduce
an amino group to the 4’-position. However, the resulting compound 15k still remained a
comparable level of CYP1B1 inhibitory activity to that of ANF.
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Table 5. Inhibitory activities of pyridin-2-yl substituted 6,7,10-trimethoxy-α-naphthoflavone derivatives
toward CYP1s
243
IC50 values (nM)
IC50 ratio
Compd.
R
1B1
5.9
1A1
80.3
5.4
1A2
1A1/1B1 1A2/1B1
-
18.0
13.6
9.0
3.1
179.3
ANF
15a
15b
15c
15d
15e
15f
H
0.6
107.6
>1000
>1000
148.2
668.4
348.6
208.5
>1000
>1000
622.8
>1000
3’-NH2
3’-OH
3’-CH3
3’-F
6.5
56.8
322.6
19.9
2.3
8.7
>153.8
>129.9
123.5
7.7
41.9
16.6
6.1
1.2
0.38
0.30
0.14
0.84
0.07
1.3
1758.9
1162
3’-Cl
4’-Cl
5’-Cl
4’-F
5.7
19.0
11.4
11.8
240
29.9
11.1
1.6
1489.3
>1190.5
>14285.7
479.1
15g
15h
15i
9.9
16.8
38.9
66.6
4’-Br
4’-NH2
15j
6.0
>166.7
15k
244
245
246
3. Molecular docking studies
In an attempt to gain insight into the probable binding modes and rationalize the observed
11

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CYP1B1 inhibitory efficacy and selectivity of the most potent compound 15i, molecular docking
studies have been carried out using GOLD 5.3.0 software (Cambridge, UK). The crystal
structures of ANF in complex with CYP1B1 (PDB ID: 3PM0) and CYP1A2 (PDB ID: 2HI4)
were used for this study. As shown in parts A to C in Fig. 2, compound 15i snugly fits well into
the active site of CYP1B1 and it is involved in hydrophobic contacts with residues F231, L264,
F268, G329, A330, A133, I399, F134, V126 as well as L509. An enhanced π-π stacking
interaction between the naphthalene part and phenyl ring of F231 caused by increased electron
density of the naphthalene part provides a strong binding affinity. Also, the methoxy group forms
a hydrogen bond with D333 with a distance of 2.6 Å. The docked complex was structurally
superimposed on the ligand ANF (Fig 2B), and it showed that the pyridine moiety of compound
15i is closer to heme than that of ANF (distances of 3.0 and 4.3 Å, respectively), which is
unfavorable for formation of the reactive heme iron-oxo intermediate during catalysis. The
surface map of docked complex indicated that the pyridine moiety of compound 15i is
surrounded by a hydrophobic region (colored by brown), which is favorable for hydrophobic
contact with fluorine substituent. Further, it also provides an explanation for the fact that
compounds incorporating a hydrophobic substituent on the pyridine ring generally showed better
CYP1B1 inhibitory effects than those with hydrophilic substituents. As can be seen from parts D
and E in Fig. 2, compound 15i docked in the active site of CYP1A2 with a similar pose to that in
CYP1B1. It binds to a hydrophobic pocket formed by amino acid residues F226, L497, I386,
T124, A317, G316 and F260, with the pyridine moiety being closer to heme than that of ANF.
However, it does not form a hydrogen bond (or water-mediated hydrogen bond[20]) with any
residues as compared with ANF (ANF forms a water-mediated hydrogen bond with Gly316),
which may provide a partial explanation for a lower activity of compound 15i against CYP1A2.
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273
274
Fig 2. Molecular docking study of compound 15i in CYP1B1(A-C) and CYP1A2 (D-E) (compound 15i: yellow
stick; ANF: green stick; yellow dash line: hydrogen bond; green dash line: π-π stacking interaction; cyan dash
line: distance)
Considering that the total score (expressed as -log(Kd), including crash score and polar score)
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277
278
279
280
281
282
283
284
285
286
287
288
289
generated by Surflex-Dock may directly predict the binding affinity of the ligand-protein
complex, therefore we calculated their parameters using another molecular docking software
SYBYL-X 2.0 (Tripos Inc., St. Louis, USA) to confirm the observed biological results again.
Compound 15i and the template molecule ANF were docked into the active sites of CYP1B1 and
CYP1A2 and the highest scores are presented in Table 6. Surprisingly, a high correlation was
found between the total scores and the activities (-logIC50). The total score of compound 15i in
CYP1B1 was much higher than that in CYP1A2 with values of 8.5969 and 4.1880, respectively,
while ANF possessed similar total scores in CYP1B1 and CYP1A2 with values of 6.9669 and
7.1981, respectively. Furthermore, compound 15i showed a higher total score than ANF in
CYP1B1 with values of 8.5969 and 6.9669, respectively, whereas it displayed a lower total score
than ANF in CYP1B1 with values of 4.1880 and 7.1981, respectively. These results are
consistent with experimentally observed trends in CYP1 inhibitory potency. It should be noted
that compound 15i reached a very low crash score of -7.4502, indicating a high degree of
inappropriate penetration by this compound into the binding site of CYP1A2. Based on the
previous finding that the volume of the CYP1A2 cavity is smaller than that of CYP1B1 (size
13

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292
values of 375 and 398 Å3, respectively) [21], therefore, it might be speculated that steric
hindrance is another critical factor for contributing to the lower CYP1A2 inhibitory efficacy of
compound 15i.
293
294
Table 6. Surflex-Dock scores of compound 15i and ANF
IC50 values (nM)
Docking score in CYP1B1
Docking score in CYP1A2
Compd.
Total
Score
Crash
Score
Polar
Score
Total
Score
Crash
Score
Polar
Score
CYP1B1 CYP1A2
0.07
5.9
>1000
18.0
8.5969 -2.5712 0.0001
6.9699 -0.3755 0.0000
4.1880
7.1981
-7.4502 1.1228
-0.3857 0.1418
15i
ANF
295
296
297
298
(Total score, expressed as -log(Kd), represent binding affinities; Crash score, degree of inappropriate
penetration by the ligand into the protein and interpenetration between ligand atoms that are separated by
rotatable bonds of compounds. c) Polar score, the contribution of polar non-hydrogen bonding interactions to
the total score)
299
300
301
4. Determination of water solubility
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
A suitable water solubility of a compound (drug) is one of the most important properties for
obtaining reliable in vitro and in vivo assay results in early drug discovery and for ensuring
sufficient concentration in circulation to get a desired therapeutic exposure [22]. ANF is a potent
CYP1 inhibitor, but it has poor water solubility that may limit its efficacy and further
development [17]. In this study, some of these synthesized α-naphthoflavone derivatives have
been selected to determine whether their water solubility was improved. Compound 15i as the
most potent and selective CYP1B1 inhibitor among them was first selected to evaluate its water
solubility by a standard HPLC method. Unfortunately, similar to ANF, the saturated solution
concentration of compound 15i in water was undetectable, indicating poor water solubility
(presented in Table 7). As a result, we transformed the compound 15i to its hydrochloride and
sulfate forms. Regrettably, an enhanced water solubility still cannot be achieved, for its salt forms
were unstable in water and they could restore to the prototype form immediately.
In light of the fact that the amino-substituted α-naphthoflavone derivatives are more potent
than the corresponding hydroxyl-substituted derivatives in blocking CYP1B1, and
simultaneously they are more alkaline and less hydrophobic (lower ClogP, calculated by
Chembiodraw Ultra 14.0 ) than compound 15i, in spite of a slight loss of CYP1B1 inhibitory, we
turned our attention to the compound 13h, the most potent CYP1B1 inhibitor among the
amino-substituted series. To our surprise, compound 13h showed more than 60-fold of
improvement in water solubility as compared with ANF and compound 15i (311 μg/mL for 13h,
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325
326
< 5 μg/mL for ANF and 15i). It is interesting to note that a remarkably increased water solubility
was also observed for its salt form, for example, the water solubility of its sulfate form was up to
4910 μg/mL. it suggested that compound 13h may be a promising starting point with great
potential for further studies.
Table 7. Water solubility of the selected compounds.
Water solubility (μg/mL)
Compd.
ClogP
Free base
< 5
Salt form
4.65
3.75
2.99
/
ANF
15i
< 5
< 5
311
4910
13h
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
4. Conclusion
In this study, structural modification of ANF gave
a
series of forty-six
6,7,10-trimethoxy-α-naphthoflavone derivatives, which exhibited varying levels of inhibitory
potency on CYP1 enzymes. SAR analysis indicated that the CYP1 inhibitory efficacy was greatly
determined by the type and position of the substituents on the B ring. Among them, the
fluorine-containing derivative 15i showed excellent selectivity for CYP1B1 over the other two
CYP1 isoforms and was characterized as the most potent inhibitor for CYP1B1 with an IC50
value of 0.07 nM, with 84-fold more potent inhibition than ANF as the lead compound.
Molecular modeling study revealed that appropriate binding site conformation, hydrophobic
interactions and an enhanced π-π stacking interactions were the predominant factors contributing
to its high CYP1B1 inhibitory activity. Additionally, the amino-substituted derivative 13h not
only possessed a potent inhibitory effect on CYP1B1, but also had a substantially increased water
solubility as compared with ANF as determined by solubility assay, suggesting that it may have a
great potential for further study. The follow-up study of reversal of drug resistance by compound
13h in vitro and in vivo is currently underway and will be reported upon completion.
5. Experimental section
5.1. Chemistry
All starting materials and reagents were either obtained from commercial suppliers or
prepared according to literature reported procedures. All solvents were used as supplied without
further purification unless otherwise indicated. Anhydrous solvents were dried according to
standard methods. Proton nuclear magnetic resonance (1H NMR) and 13C NMR spectra were
15

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359
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361
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365
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367
368
369
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373
374
375
376
377
378
379
380
381
382
383
recorded with Varian Mercury-300 (400 MHz) spectrometer. Chemical shifts (δ scale) are
reported in parts per million (ppm) downfield from tetramethylsilane (TMS) and coupling
constants (J) are expressed in hertz (Hz). Multiplicities are shown as the following abbreviations:
s (singlet), brs (broad singlet), d (doublet), t (triplet), m (multiplet). Reactions were monitored by
thin-layer chromatography (TLC, silica gel GF254) and visualized with UV light (254 or 365 nm).
Column chromatography was conducted on silica gel (200-300 mesh). High resolution mass
spectrometry (HRMS) were obtained on Agilent 6540 QTOF (ESI).
5.2. General procedure for preparation of all the α-naphthoflavone derivatives
The intermediates 2-8 were easily prepared from 1,5-dihydroxynaphthalene (1) according to
our
previously
reported
procedures[23].
To
a
stirred
solution
of
1-(1-hydroxy-4,5,8-trimethoxynaphthalen-2-yl)ethan-1-one (8, 2 mmol) in dry DMF (10 mL),
commercially available or laboratory prepared aromatic benzoic acid derivative (2.2 mmol),
condensing agent EDCI/DMAP (3 mmol/3mmol) or HATU/DMAP were added. The resultant
solution was stirred at ambient temperature for overnight when TLC showed that the reaction was
complete. The reaction mixture was poured into an ice-water solution and extracted with EtOAc.
The combined organic layers were washed with saturated NaCl aqueous, and dried over Na2SO4.
The solvent was removed under reduced pressure and the crude residue was subjected to silica gel
column chromatography to give intermediate 9.
NaH was added to a mixture of intermediate 9 (1 mmol) in dry DMF (5 mL) under N2 and
The mixture was stirred at ambient temperature for 5 h. After being quenched with acetic acid
ice-water solution and the precipitate obtained was filtered. After drying, the red crude product 10
can be used directly without further purification.
Intermediate 10 (0.5 mmol) was suspended to H2SO4-EtOH solution (10%, 25 mL) and the
resultant mixture was kept for stirring at temperature of 90 °C till the completion of the reaction
as indicated by TLC. After removing solvents under vacuum, the residue was dissolved in water,
neutralized by NaHCO3 and extracted with EtOAc. The organic layers were washed with
saturated brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The
residue was purified by column chromatography using silica gel to give all the studied
compounds in this work.
5.2.2.
6,7,10-trimethoxy-2-(3-(4-methylpiperazin-1-yl)phenyl)-4H-benzo[h]benzopyran-4-one
(11a)
Yellow solid; yield 69%; 1H NMR (400 MHz, CDCl3) δ 7.63 – 7.55 (m, 2H), 7.46 (d, J = 6.5 Hz,
1H), 7.36 (t, J = 8.0 Hz, 1H), 7.07 (d, J = 8.8 Hz, 1H), 7.02 (d, J = 5.7 Hz, 2H), 6.91 (s,1H), 4.01
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386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
(s, 6H), 3.88 (s, 3H), 3.29 – 3.24 (m, 4H), 2.61 – 2.56 (m, 4H), 2.33 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 177.73, 163.59, 154.39, 151.88, 151.75, 151.39, 149.17, 133.25, 129.62, 122.35,
121.51, 118.68, 117.78, 114.11, 113.13, 109.70, 109.56, 107.28, 98.95, 58.20, 57.03, 56.61, 54.98,
49.03, 46.03. HRMS (ESI) calcd for [C27H28N2O5 + H]+ 461.2076, found 461.20977.
5.2.2. 2-(3-chloro-5-(4-methylpiperazin-1-yl)phenyl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran
-4-one (11b).
1
Yellow solid; yield 62%; H NMR (400 MHz, CDCl3) δ 7.58 (s, 1H), 7.31 (s, 1H), 7.18 (s, 1H),
6.95 (d, J = 8.8 Hz, 1H), 6.91 – 6.84 (m, 2H), 6.79 (s, 1H), 3.96 (s, 3H), 3.93 (s, 3H), 3.81 (s, 3H),
13
3.24 – 3.16 (m, 4H), 2.54 – 2.50 (m, 4H), 2.30 (s, 3H). C NMR (101 MHz, CDCl3) δ 177.39,
161.62, 154.38, 152.30, 151.46, 151.02, 148.85, 135.61, 134.04, 122.09, 121.31, 118.03, 117.89,
117.38, 112.87, 111.05, 108.72, 106.97, 98.53, 57.91, 56.43, 56.28, 54.76, 48.50, 46.04. HRMS
(ESI) calcd for [C27H27ClN2O5 + H]+ 495.1687, found 495.17089.
5.2.3. 6,7,10-trimethoxy-2-(3-morpholinophenyl)-4H-benzo[h]benzopyran-4-one (11c).
1
Yellow solid; yield yield73%; H NMR (400 MHz, CDCl3) δ 7.72 – 7.61 (m, 2H), 7.50 (s, 1H),
7.44 (t, J = 7.8 Hz, 1H), 7.17 – 7.02 (m, 3H), 6.96 (s, 1H), 4.06 (s, 3H), 4.04 (s, 3H), 3.93 (s, 3H),
3.93 – 3.86 (m, 4H), 3.31 – 3.22 (brs, 4H). 13C NMR (101 MHz, CDCl3) δ 177.66, 163.33,
154.43, 151.76, 151.54, 151.39, 149.12, 133.34, 129.73, 122.28, 121.48, 118.63, 118.59, 118.40,
113.89, 112.99, 109.75, 107.28, 98.84, 66.76, 58.15, 57.01, 56.58, 49.54. HRMS (ESI) calcd for
[C26H25NO6 + H]+ 448.1760, found 448.17656.
5.2.4.
6,7,10-trimethoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-4H-benzo[h]benzopyran-4-one
(11d).
1
Yellow solid; yield 65%; H NMR (400 MHz, CDCl3) δ 7.84 (d, J = 8.6 Hz, 2H), 7.39 (s, 1H),
6.93 (d, J = 8.8 Hz, 1H), 6.85 (d, J = 9.0 Hz, 3H), 6.72 (s, 1H), 3.95 (s, 3H), 3.91 (s, 3H), 3.81 (s,
3H), 3.35 – 3.22 (m, 4H), 2.54 – 2.45 (m, 4H), 2.28 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
177.43, 163.30, 154.03, 152.88, 151.51, 151.06, 148.79, 127.56, 121.99, 121.76, 121.24, 118.31,
114.40, 112.49, 108.70, 104.70, 99.00, 57.93, 56.49, 56.44, 54.71, 47.53, 46.07. HRMS (ESI)
calcd for [C27H28N2O5 + H]+ 461.2076, found 461.20804.
5.2.5 2-(1H-Indol-6-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (12a)
Brown solid; yield 74%; 1H NMR (400 MHz, DMSO-d6) δ 11.63 (s, 1H), 8.36 (s, 1H), 7.82 (dd, J
= 8.5, 1.4 Hz, 1H), 7.73 – 7.65 (m, 1H), 7.62 – 7.51 (m, 1H), 7.36 – 7.29 (m, 2H), 7.28 – 7.23 (m,
17

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420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
1H), 7.12 (s, 1H), 6.53 (s, 1H), 4.17 (s, 3H), 3.92 (s, 3H), 3.83 (s, 3H). 13C NMR (101 MHz,
DMSO-d6) δ 176.30 , 164.42 , 154.45 , 151.30 , 151.22 , 148.54, 136.17 , 130.76 , 129.27 ,
124.48 , 121.56 , 121.22 , 120.99 , 118.04 , 117.50 , 113.46 , 110.48 , 110.40 , 105.74 , 102.16 ,
98.63 , 57.89 , 57.41 , 56.60 . HRMS (ESI) calcd for [C24H19NO5 + H]+ 402.1341, found
402.13443.
5.2.6. 2-(1H-benzo[d]imidazol-5-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (12b)
Brown solid; yield 68%; 1H NMR (400 MHz, DMSO-d6) δ 13.14 – 12.69 (m, 1H), 8.67 (brs, 1H),
8.42 (brs, 1H), 8.11 (brs, 1H), 7.79 (brs, 1H), 7.38 – 7.27 (m, 3H), 7.24 (s, 1H), 4.19 (s, 3H), 3.96
(s, 3H), 3.86 (s, 3H). HRMS (ESI) calcd for [C23H18N2O5 + H]+ 403.1294, found 403.12986.
5.2.7. 2-(1H-indazol-5-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (12c)
1
Brown solid; yield 45%; H NMR (400 MHz, DMSO-d6) δ 13.34 (s, 1H), 8.66 (s, 1H), 8.26 (s,
1H), 8.10 (d, J = 8.7 Hz, 1H), 7.68 (d, J = 8.7 Hz, 1H), 7.30 – 7.15 (m, 3H), 7.09 (s, 1H), 4.08 (s,
3H), 3.88 (s, 3H), 3.79 (s, 3H). HRMS (ESI) calcd for [C23H18N2O5 + H]+ 403.1294, found
403.12917.
5.2.8. 6,7,10-Trimethoxy-2-(quinolin-6-yl)-4H-benzo[h]benzopyran-4-one (12d)
1
Brown solid; yield 72%; H NMR (400 MHz, CDCl3) δ 8.95 (d, J = 4.0 Hz, 1H), 8.50 (s, 1H),
8.24 – 8.10 (m, 3H), 7.44 (dd, J = 8.2, 4.2 Hz, 1H), 7.40 (s, 1H), 7.10 – 7.04 (m, 1H), 7.03 – 6.95
(m, 2H), 4.08 (s, 3H), 4.00 (s, 3H), 3.90 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 177.37, 161.69,
154.48, 151.81, 151.43, 151.26, 149.12, 149.00, 136.75, 130.09, 130.07, 127.83, 126.51, 126.17,
122.25, 121.98, 121.44, 118.19, 112.99, 109.02, 107.52, 98.69, 58.00, 56.53, 56.46. HRMS (ESI)
calcd for [C25H19NO5 + H]+ 414.1341, found 414.13359.
5.2.9. 6,7,10-Trimethoxy-2-(quinolin-2-yl)-4H-benzo[h]benzopyran-4-one (12e)
Brown solid; yield 69%; 1H NMR (400 MHz, DMSO-d6) δ 8.74 (s, 1H), 8.51 (s, 1H), 8.11 (d, J =
24.7 Hz, 2H), 7.89 (s, 1H), 7.73 (s, 1H), 7.52 (s, 1H), 7.42-7.26 (m, 3H), 4.17 (s, 3H), 3.96 (s,
3H), 3.86 (s, 3H). 13C NMR (101 MHz, pyridine) δ 176.87, 161.37, 155.11, 151.63, 151.59,
149.88, 149.61, 149.35, 148.87, 148.00, 137.35, 130.42, 130.07, 128.92, 127.99, 127.92, 122.59,
118.42, 113.40, 109.62, 108.53, 99.12, 57.64, 56.48, 56.07. HRMS (ESI) calcd for [C25H19NO5 +
H]+ 414.1341, found 414.13420.
5.2.10. 6,7,10-Trimethoxy-2-(1H-pyrrolo[2,3-b] pyridin-3-yl)-4H-benzo[h]benzopyran-4-one
18

452
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454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
(12f)
1
Brown solid; yield 52%; H NMR (400 MHz, DMSO-d6) δ 12.55 (s, 1H), 8.57 (d, 1H), 8.41 –
8.37 (m, 2H), 7.37 (s, 1H), 7.34 – 7.30 (m, 1H), 7.30 – 7.25 (m, 2H), 6.92 (s, 1H), 4.05 (s, 3H),
3.95 (s, 3H), 3.86 (s, 3H). HRMS (ESI) calcd for [C23H18N2O5 + H]+ 403.1294, found 403.12922.
5.2.11. 2-(1H-Indol-3-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (12g)
Brown solid; yield 68%; 1H NMR (400 MHz, DMSO-d6) δ 12.00 (s, 1H), 8.22 (s, 1H), 8.04 (d, J
= 7.3 Hz, 1H), 7.55 (d, J = 7.3 Hz, 1H), 7.30 (s, 1H), 7.26 – 7.20 (m, 2H), 7.16 (s, 2H), 6.79 (s,
13
1H), 3.99 (s, 3H), 3.89 (s, 3H), 3.79 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 175.80, 161.41,
154.07, 151.21, 151.10, 148.22, 137.53, 129.61, 123.87, 122.99, 121.70, 121.40, 120.91, 120.32,
117.84, 113.13, 112.92, 109.94, 108.75, 104.98, 98.80, 57.73, 56.98, 56.49. HRMS (ESI) calcd
for [C24H19NO5 + H]+ 402.1341, found 402.13442.
5.2.12. 2-(benzofuran-2-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (12h)
Yellow solid; yield 59%; 1H NMR (400 MHz, DMSO-d6) δ 7.92 (d, J = 7.5 Hz, 1H), 7.80 – 7.66
(m, 2H), 7.51 (t, J = 7.5 Hz, 1H), 7.39 (t, J = 7.2 Hz, 1H), 7.30 (s, 3H), 6.91 (s, 1H), 4.16 (s, 3H),
3.94 (s, 3H), 3.85 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 176.74, 155.68, 154.56, 154.55, 151.79,
151.27, 148.83, 148.46, 127.81, 126.69, 123.71, 122.26, 122.06, 121.57, 118.06, 113.21, 111.78,
109.37, 108.62, 106.68, 98.74, 58.05, 56.87, 56.52. HRMS (ESI) calcd for [C24H18O6 + H]+
403.1182, found 403.11872.
5.2.13. 2-(furan-2-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (12i)
Yellow solid; yield 61%; 1H NMR (400 MHz, DMSO-d6) δ 8.00 (s, 1H), 7.18 (d, J = 3.3 Hz, 1H),
7.16 (s, 1H), 7.14 – 7.09 (m, 2H), 6.78 (dd, J = 3.2, 1.6 Hz, 1H), 6.59 (s, 1H), 3.96 (s, 3H), 3.86
(s, 3H), 3.78 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 175.53, 154.48, 154.31, 151.21, 150.99,
147.91, 147.17, 146.44, 121.39, 121.17, 117.53, 113.62, 113.40, 113.28, 110.13, 104.41, 98.26,
57.68, 56.96, 56.43. HRMS (ESI) calcd for [C20H16O6 + H]+ 353.1025, found 353.10283.
5.2.14. 6,7,10-Trimethoxy-2-(thien-2-yl)-4H-benzo[h]benzopyran-4-one (12j)
Yellow solid; yield 63%; 1H NMR (400 MHz, DMSO-d6) δ 8.00 (d, J = 3.7 Hz, 1H), 7.93 (dd, J =
4.9, 0.9 Hz, 1H), 7.27 (dd, J = 4.9, 3.8 Hz, 1H), 7.24 (s, 1H), 7.21 – 7.16 (m, 2H), 7.00 (s, 1H),
4.04 (s, 3H), 3.89 (s, 3H), 3.79 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 175.79, 158.58, 154.56,
151.30, 150.95, 148.10, 135.29, 132.09, 129.38, 129.17, 121.46, 121.20, 117.55, 113.46, 109.88,
105.31, 98.43, 57.79, 56.80, 56.50. HRMS (ESI) calcd for [C20H16O5S + H]+ 369.0797, found
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369.08019.
5.2.15. 6,7,10-Trimethoxy-2-(thiazol-4-yl)-4H-benzo[h]benzopyran-4-one (12k)
Brown solid; yield 49%; 1H NMR (400 MHz, DMSO-d6) δ 9.33 (s, 1H), 8.33 (s, 1H), 7.31 – 7.19
(m, 3H), 7.00 (s, 1H), 4.10 (s, 3H), 3.92 (s, 3H), 3.83 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ
176.17, 158.07, 157.45, 154.65, 151.35, 151.11, 148.50, 148.32, 122.44, 121.53, 121.39, 117.67,
113.59, 110.33, 107.17, 98.36, 57.81, 57.28, 56.57; HRMS (ESI) calcd for [C19H15NO5S + H]+
370.0749, found 370.07509.
5.2.16. 6,7,10-Trimethoxy-2-(pyridazin-4-yl)-4H-benzo[h]benzopyran-4-one (12l)
Brown solid; yield 41%; 1H NMR (400 MHz, DMSO-d6) δ 9.94 (s, 1H), 9.55 (d, J = 6.1 Hz, 1H),
8.32 (s, 1H), 7.57 (s, 1H), 7.35 – 7.19 (m, 3H), 4.09 (s, 3H), 3.91 (s, 3H), 3.81 (s, 3H). HRMS
(ESI) calcd for [C20H16N2O5 + H]+ 365.1137, found 365.11316.
5.2.17. 6,7,10-Trimethoxy-2-(pyridin-3-yl)-4H-benzo[h]benzopyran-4-one (13a)
1
Brown solid; yield 59%; H NMR (400 MHz, DMSO-d6) δ 9.45 (s, 1H), 8.79 (s, 1H), 8.61 (d,
1H), 7.74 – 7.66 (m, 1H), 7.41 – 7.26 (m, 4H), 4.10 (s, 3H), 3.96 (s, 3H), 3.86 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 177.01, 160.14, 154.59, 151.69, 151.46, 151.16, 148.94, 147.66, 133.18,
128.12, 123.45, 122.23, 121.45, 118.03, 113.09, 108.84, 107.47, 98.58, 57.96, 56.45, 56.42.
HRMS (ESI) calcd for [C21H17NO5 + H]+ 364.1185, found 364.11871.
5.2.18. 6,7,10-Trimethoxy-2-(pyridin-4-yl)-4H-benzo[h]benzopyran-4-one (14a)
Brown solid; yield 52%; 1H NMR (400 MHz, DMSO-d6) δ 8.87 (d, J = 5.7 Hz, 2H), 8.16 (d, J =
5.6 Hz, 1H), 7.43 (s, 1H), 7.35 – 7.23 (m, 1H), 4.12 (d, J = 2.7 Hz, 1H), 3.94 (s, 1H), 3.85 (s, 1H).
13C NMR (101 MHz, CDCl3) δ 177.22, 159.81, 154.83, 151.57, 151.32, 150.62, 149.09, 139.65,
122.40, 121.76, 119.71, 118.17, 113.35, 109.24, 108.57, 98.54, 58.08, 56.60, 56.55. HRMS (ESI)
calcd for [C21H17NO5 + H]+ 364.1185, found 364.11881.
5.2.19. 6,7,10-Trimethoxy-2-(pyridin-2-yl)-4H-benzo[h]benzopyran-4-one (15a)
Brown solid; yield 62%; 1H NMR (400 MHz, DMSO-d6) δ 8.80 (d, J = 3.6 Hz, 1H), 8.40 (d, J =
7.7 Hz, 1H), 8.22 (t, J = 7.6 Hz, 1H), 7.69 – 7.61 (m, 1H), 7.36 – 7.14 (m, 4H), 4.14 (s, 3H), 3.95
(s, 3H), 3.86 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 177.60, 161.10, 154.37, 151.34, 151.12,
149.94, 149.68, 148.63, 136.66, 125.07, 122.09, 121.88, 121.01, 118.04, 112.83, 108.86, 107.97,
98.78, 57.91, 56.44, 56.42. HRMS (ESI) calcd for [C21H17NO5 + H]+ 364.1185, found 364.11897.
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5.2.20. 2-(5-fluoropyridin-3-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (13b)
Yellow solid; yield 67%; 1H NMR (400 MHz, DMSO-d6) δ 9.26 (s, 1H), 8.77 (d, J = 2.7 Hz, 1H),
8.41 (d, J = 9.7 Hz, 1H), 7.38 (s, 1H), 7.29 – 7.21 (m, 3H), 4.02 (s, 3H), 3.90 (s, 3H), 3.81 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 176.95, 160.77, 158.57, 158.20, 154.84, 151.45, 151.29, 148.99,
143.10, 140.16, 139.92, 129.90, 122.36, 121.62, 120.37, 120.16, 113.28, 108.98, 108.16, 98.57,
58.05, 56.56, 56.42. HRMS (ESI) calcd for [C21H16FNO5 + H]+ 382.1091, found 382.10865.
5.2.21. 2-(5-Bromopyridin-3-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (13c)
1
Yellow solid; yield 64%; H NMR (400 MHz, DMSO-d6) δ 9.36 (s, 1H), 8.89 (s, 1H), 8.80 (s,
13
1H), 7.42 (s, 1H), 7.32 – 7.23 (m, 3H), 4.08 (s, 3H), 3.91 (s, 3H), 3.82 (s, 3H). C NMR (101
MHz, CDCl3) δ 176.96, 158.45, 154.89, 152.48, 151.58, 151.33, 149.10, 145.29, 136.23, 129.90,
122.45, 121.68, 121.11, 118.04, 113.53, 108.95, 108.07, 98.71, 58.20, 56.62, 56.42. HRMS (ESI)
calcd for [C21H16BrNO5 + H]+ 442.0290, found 442.02815.
5.2.22. 6,7,10-Trimethoxy-2-(5-methylpyridin-3-yl)-4H-benzo[h]benzopyran-4-one (13d)
1
Yellow solid; yield 63%; H NMR (400 MHz, DMSO-d6) δ 9.15 (s, 1H), 8.58 (s, 1H), 8.31 (s,
13
1H), 7.29 –7.11 (m, 4H), 4.02 (s, 3H), 3.91 (s, 3H), 3.83 (s, 3H), 2.43 (s, 3H). C NMR (101
MHz, CDCl3) δ 177.20, 160.52, 154.63, 152.21, 151.61, 151.24, 149.13, 144.87, 133.84, 133.23,
127.83, 122.32, 121.57, 118.20, 113.27, 108.94, 107.55, 98.74, 58.13, 56.57, 56.46, 18.55.
HRMS (ESI) calcd for [C22H19NO5 + H]+ 378.1341, found 378.13487.
5.2.23. 2-(5-aminopyridin-3-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (13e)
1
Brown solid; yield 54%; H NMR (400 MHz, DMSO-d6) δ 8.49 (s, 1H), 8.17 – 8.02 (m, 1H),
7.54 (s, 1H), 7.26 – 7.08 (m, 3H), 7.01 – 6.87 (m, 1H), 5.61 (s, 2H), 3.98 (s, 3H), 3.87 (s, 3H),
3.78 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 176.14, 161.37, 154.55, 151.12, 151.06, 148.54,
145.21, 139.31, 135.38, 128.03, 121.47, 121.20, 117.73, 116.68, 113.42, 110.17, 107.25, 98.20,
57.72, 57.08, 56.45. HRMS (ESI) calcd for [C21H18N2O5 + H]+ 379.1294, found 379.12887.
5.2.24. 5-(6,7,10-trimethoxy-4-oxo-4H-benzo[h]chroman-2-yl) nicotinic acid (13f)
Dark brown solid; yield 39%; 1H NMR (400 MHz, DMSO-d6) δ 9.57 (s, 1H), 9.21 (d, J = 2.2 Hz,
1H), 9.06 (d, J = 2.7 Hz, 1H), 7.47 (s, 1H), 7.41 – 7.12 (m, 3H), 4.15 (s, 3H), 3.92 (s, 3H), 3.82 (s,
3H). HRMS (ESI) calcd for [C22H17NO7 + H]+ 408.1083, found 408.14514.
5.2.25. 2-(5-hydroxypyridin-3-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (13g)
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Yellow solid; yield 58%; H NMR (400 MHz, DMSO-d6) δ 8.82 (s, 1H), 8.31 (s, 1H), 7.91 (s,
13
1H), 7.27 – 7.20 (m, 3H), 7.18 (s, 1H), 4.02 (s, 3H), 3.89 (s, 3H), 3.80 (s, 3H). C NMR (101
MHz, DMSO-d6) δ 176.24, 160.51, 154.68, 154.35, 151.15, 151.11, 148.56, 140.93, 138.51,
128.78, 121.52, 121.27, 119.72, 117.75, 113.56, 110.35, 107.77, 98.21, 57.78, 57.02, 56.53.
HRMS (ESI) calcd for [C21H17NO6 + H]+ 380.1134, found 380.11340.
5.2.26. 2-(6-aminopyridin-3-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (13h)
Brown solid; yield 48%; 1H NMR (400 MHz, DMSO-d6) δ 8.84 (d, J = 2.3 Hz, 1H), 8.11 (dd, J =
8.8, 2.4 Hz, 1H), 7.29 (s, 1H), 7.24 (d, J = 2.0 Hz, 2H), 6.93 (s, 1H), 6.78 (s, 2H), 6.57 (d, J = 8.8
13
Hz, 1H), 4.02 (s, 3H), 3.90 (s, 3H), 3.81 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 175.97 ,
162.49 , 162.04 , 154.32 , 151.17 , 151.10 , 148.18 , 135.00 , 121.41, 121.10 , 117.87 , 115.58 ,
113.18 , 110.16 , 109.99, 108.16 , 103.68 , 98.64 , 57.79 , 57.00 , 56.55. HRMS (ESI) calcd for
[C21H18N2O5 + H]+ 379.1294, found 379.12959.
5.2.27. 2-(6-hydroxypyridin-3-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (13i)
Yellow solid; yield 59%; 1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 7.75 (d, J = 8.8 Hz, 1H),
7.30 (s, 1H), 7.20 (s, 2H), 6.58 (s, 1H), 5.94 (d, J = 9.6 Hz, 1H), 4.01 (s, 3H), 3.89 (s, 3H), 3.80 (s,
3H). HRMS (ESI) calcd for [C21H17NO6 + H]+ 380.1134, found 380.11406.
5.2.28. 2-(2-hydroxypyridin-3-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (13j)
1
Yellow solid; yield 53%; H NMR (400 MHz, DMSO-d6) δ 12.37 (s, 1H), 8.69 (dd, J = 7.5, 2.1
Hz, 1H), 7.90 (s, 1H), 7.73 (d, J = 6.0 Hz, 1H), 7.37 – 7.17 (m, 3H), 6.64 (t, J = 6.8 Hz, 1H), 4.05
(s, 3H), 3.90 (s, 3H), 3.81 (s, 3H). HRMS (ESI) calcd for [C21H17NO6 + H]+ 380.1134, found
380.11364.
5.2.29. 2-(2-aminopyridin-3-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (13k)
Brown solid; yield 51%; 1H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 10.22 (s, 1H), 8.73 (dd,
J = 4.4, 1.8 Hz, 1H), 8.45 (dd, J = 7.9, 1.8 Hz, 1H), 7.38 (dd, J = 7.9, 4.5 Hz, 1H), 7.05 – 6.97 (m,
2H), 6.95 – 6.90 (m, 1H), 6.40 – 6.32 (m, 1H), 3.98 (s, 3H), 3.80 (s, 3H), 3.78 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 177.42, 153.45, 151.52, 151.26, 150.67, 150.41, 149.34, 146.75, 134.76,
120.49, 120.10, 119.53, 117.23, 115.41, 110.92, 109.99, 109.39, 107.39, 57.69, 57.56, 57.36.
HRMS (ESI) calcd for [C21H18N2O5 + H]+ 379.1294, found 379.13068.
5.2.30. 2-(2-bromopyridin-3-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (13l)
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Yellow solid; yield 56%; 1H NMR (400 MHz, DMSO-d6) δ 8.71 (d, J = 7.2 Hz, 1H), 7.93 (s, 1H),
7.76 (d, J = 4.6 Hz, 1H), 7.34 – 7.25 (m, 3H), 6.67 (t, J = 6.7 Hz, 1H), 4.08 (s, 3H), 3.93 (s, 3H),
3.85 (s, 3H).
5.2.31. 2-(2-fluoropyridin-4-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (14b)
Yellow solid; yield 62%; 1H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J = 5.2 Hz, 1H), 7.74 (d, J =
5.2 Hz, 1H), 7.64 (s, 1H), 7.40 (s, 1H), 7.33 – 7.27 (m, 3H), 4.10 (s, 3H), 3.95 (s, 3H), 3.85 (s,
3H). 13C NMR (101 MHz, CDCl3) δ 177.34, 164.85, 159.93, 154.70, 151.65, 151.19, 149.04,
147.62, 142.14, 122.42, 121.68, 118.13, 113.37, 112.43, 108.94, 108.42, 108.31, 98.58, 58.10,
56.54, 56.36.
5.2.32. 2-(2-chloropyridin-4-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (14c)
Yellow solid; yield 61%; 1H NMR (400 MHz, DMSO-d6) δ 8.71 (d, J = 5.1 Hz, 1H), 8.35 (s, 1H),
8.22 (d, J = 5.1 Hz, 1H), 7.55 (s, 1H), 7.36 – 7.34 (m, 2H), 7.32 (s, 1H), 4.15 (s, 3H), 3.96 (s, 3H),
3.86 (s, 3H). HRMS (ESI) calcd for [C21H16ClNO5 + H]+ 398.0795, found 398.0793.
5.2.33. 2-(2-Bromo-pyridin-4-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (14d)
1
Yellow solid; yield 65%; H NMR (400 MHz, CDCl3) δ 8.55 (d, J = 5.3 Hz, 1H), 8.41 (s, 1H),
7.76 (d, J = 5.4 Hz, 1H), 7.49 (d, J = 10.2 Hz, 1H), 7.17 (d, J = 8.6 Hz, 1H), 7.11 (d, J = 8.5 Hz,
1H), 7.05 (s, 1H), 4.18 (s, 3H), 4.07 (s, 3H), 3.95 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 177.06,
158.10, 155.06, 151.61, 151.32, 150.76, 149.05, 143.45, 142.21, 129.69, 124.82, 122.53, 121.86,
118.38, 117.92, 113.64, 109.06, 98.49, 58.17, 56.62, 56.47.
5.2.34. 2-(2-hydroxypyridin-4-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (14e)
1
Yellow solid; yield 66%; H NMR (400 MHz, DMSO-d6) δ 11.91 (s, 1H), 7.59 (d, J = 6.8 Hz,
1H), 7.38 – 7.26 (m, 5H), 6.92 (d, J = 6.8 Hz, 1H), 4.09 (s, 3H), 3.95 (s, 1H), 3.86 (s, 1H). HRMS
(ESI) calcd for [C21H17NO6 + H]+ 380.1134, found 380.11428.
5.2.35. 2-(2-aminopyridin-4-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (14f)
1
Brown solid; yield 54%; H NMR (400 MHz, DMSO-d6) δ 8.15 (s, 1H), 7.32 (s, 3H), 7.24 (s,
13
1H), 7.18 (s, 1H), 7.11 (s, 1H), 6.24 (s, 2H), 4.09 (s, 3H), 3.95 (s, 3H), 3.85 (s, 3H). C NMR
(101 MHz, CDCl3) δ 177.43, 160.49, 159.01, 154.77, 151.65, 151.41, 149.11, 148.46, 141.74,
122.45, 121.76, 118.36, 113.39, 110.28, 109.55, 108.53, 105.29, 98.66, 58.15, 56.80, 56.60.
HRMS (ESI) calcd for [C21H18N2O5 + H]+ 379.1294, found 379.12943.
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5.2.36. 2-(3-aminopyridin-4-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (14g)
Brown solid; yield 59%; 1H NMR (400 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.41 (d, J = 5.0 Hz, 1H),
7.89 (d, J = 4.9 Hz, 1H), 7.11 – 6.98 (m, 2H), 6.98 – 6.91 (m, 1H), 6.48 (s, 1H), 3.98 (s, 3H), 3.82
(d, J = 13.4 Hz, 3H), 3.79 (s, 3H). HRMS (ESI) calcd for [C21H18N2O5 + H]+ 379.1294, found
379.12930. HRMS (ESI) calcd for [C21H18N2O5 + H]+ 379.1294, found 379.12930.
5.2.37. 2-(6-aminopyridin-2-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (15b)
1
Brown solid; yield 55%; H NMR (400 MHz, DMSO-d6) δ 7.76 – 7.64 (m, 1H), 7.57 (d, J = 7.5
Hz, 1H), 7.27 (s, 1H), 7.25 (s, 2H), 7.16 (s, 1H), 6.65 (d, J = 7.9 Hz, 1H), 6.29 (s, 2H), 4.05 (s,
13
3H), 3.90 (s, 3H), 3.81 (s, 3H). C NMR (101 MHz, CDCl3) δ 177.95, 161.84, 158.04, 154.34,
151.78, 151.30, 148.40, 138.27, 122.37, 122.03, 118.51, 117.98, 113.83, 113.07, 111.80, 110.99,
109.18, 99.15, 58.20, 56.79, 56.60. HRMS (ESI) calcd for [C21H18N2O5 + H]+ 379.1294, found
379.12958.
5.2.38. 2-(6-hydroxypyridin-2-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (15c)
1
Yellow solid; yield 61%; H NMR (400 MHz, DMSO-d6) δ 11.00 (s, 1H), 7.80 (s, 1H), 7.53 (s,
1H), 7.34 – 6.96 (m, 4H), 6.72 (d, J = 7.9 Hz, 1H), 4.00 (s, 3H), 3.85 (s, 3H), 3.77 (s, 3H). HRMS
(ESI) calcd for [C21H17NO6 + H]+ 380.1134, found 380.11321.
5.2.39. 6,7,10-Trimethoxy-2-(6-methylpyridin-2-yl)-4H-benzo[h]benzopyran-4-one (15d)
Yellow solid; yield 64%; 1H NMR (400 MHz, DMSO-d6) δ 8.19 (d, J = 7.7 Hz, 1H), 8.07 (t, J =
7.8 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.36 – 7.26 (m, 4H), 4.12 (s, 3H), 3.94 (s, 3H), 3.85 (s, 3H),
2.60 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 177.76, 161.57, 158.68, 154.32, 151.49, 151.14,
149.17, 148.75, 136.74, 124.85, 122.16, 121.91, 118.19, 118.06, 112.89, 108.89, 107.88, 98.90,
58.00, 56.52, 56.46, 24.53. HRMS (ESI) calcd for [C22H19NO5 + H]+ 378.1341, found 378.13490.
5.2.40. 2-(6-fluoropyridin-2-yl)-6,7,10 trimethoxy-4H-benzo[h]benzopyran-4-one (15e)
Yellow solid; yield 60%; 1H NMR (400 MHz, DMSO-d6) δ 8.43 (d, J = 7.7 Hz, 1H), 8.32 – 8.25
(m, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.36 – 7.24 (m, 3H), 7.17 (s, 1H), 4.14 (s, 3H), 3.95 (s, 3H),
3.86 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 177.47, 164.07, 161.67, 159.47, 154.57, 151.39,
151.24, 148.64, 141.82, 122.20, 121.96, 118.32, 118.03, 113.03, 111.78, 111.41, 109.06, 98.75,
57.96, 56.56, 56.46. HRMS (ESI) calcd for [C21H16FNO5 + H]+ 382.1091, found 382.10968.
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5.2.41. 2-(6-chloropyridin-2-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (15f)
Yellow solid; yield 56%; 1H NMR (400 MHz, DMSO-d6) δ 8.26 (d, J = 7.0 Hz, 2H), 7.74 (d, J =
13
7.0 Hz, 1H), 7.27 (s, 3H), 7.16 (s, 1H), 4.09 (s, 3H), 3.91 (s, 1H), 3.82 (s, 1H). C NMR (101
MHz, CDCl3) δ 177.45, 159.53, 154.61, 151.53, 151.44, 151.29, 150.49, 148.69, 139.28, 125.87,
122.04, 119.34, 118.11, 113.11, 109.96, 109.13, 108.73, 98.79, 58.05, 56.64, 56.50. HRMS (ESI)
calcd for [C21H16ClNO5 + H]+ 398.0795, found 398.08009.
5.2.42. 2-(5-chloropyridin-2-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (15g)
1
Yellow solid; yield 57%; H NMR (400 MHz, DMSO-d6) δ 8.86 (s, 1H), 8.42 – 8.30 (m, 2H),
7.34 – 7.20 (m, 4H), 4.11 (s, 3H), 3.92 (s, 1H), 3.82 (s, 1H).
5.2.43. 2-(4-chloropyridin-2-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (15h)
1
Yellow solid; yield 49%; H NMR (400 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.41 (s, 1H), 7.81 (s,
1H), 7.30 (m, 4H), 4.13 (s, 3H), 3.95 (s, 3H), 3.86 (s, 3H). HRMS (ESI) calcd for [C21H16ClNO5
+ H]+ 398.0795, found 398.07919.
5.2.44. 2-(5-fluoropyridin-2-yl)-6,7,10 trimethoxy-4H-benzo[h]benzopyran-4-one (15i)
Yellow solid; yield 56%; 1H NMR (400 MHz, CDCl3) δ 8.53 (s, 1H), 8.43 – 8.35 (m, 1H), 7.54 (s,
2H), 7.47 (s, 1H), 7.10 (d, J = 9.0 Hz, 1H), 7.04 (d, J = 8.9 Hz, 1H), 4.05 (s, 3H), 4.01 (s, 3H),
3.89 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 177.62, 161.53, 160.41, 158.93, 154.62, 151.58,
151.49, 148.70, 146.38, 138.73, 138.48, 123.37, 123.19, 122.40, 122.03, 118.37, 113.21, 109.37,
108.14, 99.10, 58.20, 56.84, 56.60. HRMS (ESI) calcd for [C21H16FNO5 + H]+ 382.1091, found
382.10886.
5.2.45. 2-(5-Bromopyridin-2-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (15j)
1
Yellow solid; yield 53%; H NMR (400 MHz, DMSO-d6) δ 8.83 (s, 1H), 8.42 (s, 1H), 8.14 (s,
1H), 7.28 – 7.17 (m, 4H), 4.03 (s, 3H), 3.88 (s, 3H), 3.80 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
177.47, 160.19, 154.53, 151.27, 150.87, 149.77, 148.54, 148.38, 139.28, 136.71, 122.59, 122.00,
121.03, 117.97, 112.87, 108.98, 108.10, 98.69, 57.93, 56.52, 56.47. HRMS (ESI) calcd for
[C21H16BrNO5 + H]+ 442.0290, found 442.02948.
5.2.46. 2-(5-aminopyridin-2-yl)-6,7,10-trimethoxy-4H-benzo[h]benzopyran-4-one (15k)
1
Brown solid; yield 50%; H NMR (400 MHz, DMSO-d6) δ 8.14 – 8.00 (m, 2H), 7.30 (s, 1H),
7.28 – 7.22 (m, 2H), 7.16 – 7.10 (m, 1H), 7.02 (s, 1H), 6.19 (brs, 2H), 4.07 (s, 3H), 3.91 (s, 3H),
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3.82 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 176.02, 162.89, 154.43, 151.19, 148.06, 147.77,
137.22, 135.97, 122.65, 121.52, 121.49, 119.31, 117.90, 113.21, 110.27, 104.03, 98.73, 57.85,
57.20, 56.59. HRMS (ESI) calcd for [C21H18N2O5 + H]+ 379.1294, found 379.12981.
5.3. CYP1 enzyme inhibition assay
The inhibitory activity of these synthesized compounds against recombinant human CYP1
enzymes was measured by EROD (7-ethoxyresorufin O-deethylase) assay according to our
previously reported procedure[17, 18]. The recombinant human CYP1 enzymes including
CYP1B1, CYP1A1, and CYP1A2, each equipped with P450 reductase (Supersomes), were
obtained from BD Genetest (Corning). Substrate 7-Ethoxyresorufin (7-ER) and MgCl2 were
purchased from Sigma-Aldrich. D-glucose-6-phosphate (G-6-P), glucose-6-phosphate
dehydrogenase (G-6-PD) and nicotinamide-adenine dinucleotide phosphate (NADP+) were
purchased from Biosharp Co., Ltd. In brief, a mixture with a final volume of 200 μl containing
various concentrations 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) and a NADPH regeneration system (1.3 mM NADP+, 3.3 mM G-6-P, 0.5 U/mL G-6-PD and
o
3.3 mM MgCl2) was incubated in a black 96-well flat-bottomed microplate at 37 C for varied
time durations (CYP1B1 35 min; CYP1A1 15 min; CYP1A2 50 min). Then, the reaction was
quenched by addition of 100 μl of pre-cooled methanol to all wells, and the fluorescence intensity
was determined by FlexStation 3 apparatus with excitation and emission filters at 544 and 590
nm, respectively. Three replicates were used for each concentration. The IC50 values were
calculated from the non-linear regression formula, log (inhibitor) vs. normalized
response-variable slope, by the GraphPad Prism Software (Version 5.0).
5.4 Molecular docking procedure
The X-ray structure coordinates of ANF bound with CYP1B1 (PDB entry 3PM0) or
CYP1A2 (PDB entry 2HI4) were achieved from RCSB protein data bank (http://www.rcsb.org/).
The modelling experiments described in this study were performed by the docking programs of
GOLD 5.3.0 and SYBYL-X 2.0. All water molecules in the crystal structure of CYP1B1 were
removed prior to docking, but water molecules in the crystal structure of CYP1A2 were retained
for docking study owing to its possible role in formation of water-mediated hydrogen bond. For
docking with GOLD 5.3.0, the ligands were drawn in ChemBio 3D Ultra software (2014) and
subjected to energy minimization using the MM2 force field. The docking protocol was validated
by reproducing the pose of ANF at the binding site in CYP1B1 or CYP1A2. For docking with
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SYBYL-X 2.0, ligands were constructed with Sybyl/Sketch module and optimized applying
Powell’s method with Tripos force field with convergence criterion set at 0.05 kcal/(Å mol), and
assigned with Gasteiger-Hückel method. Hydrogen and missing atoms of the protein were added
and the co-crystallized ligand (ANF) served as a template to generate protomol with a threshold
of 0.50 as default setting. Docking calculations were carried out with Surflex-Dock with other
docking parameters as default.
5.5 Determination of water-solubility
A high-performance liquid chromatographic (HPLC) method (Eclipse plus C18, 3.5 μm,
4.6×100 mm (Agilent)) was used to determine their water solubilities. Briefly, Standard stock
solutions were prepared by dissolving 5 mg of each compound in 1 mL of the mixture solvent of
DCM and MeOH. Then, the stock solution was diluted by MeOH to yield concentrations of 2000,
1500, 1000, 500, 350, 250 and 125 μg/mL or 5000, 4000, 3000, 2000,1500, 1000 and 500 μg/mL
(for the salt form of compound 13h) were injected into HPLC (by dilution if necessary) and
detected at wavelength of 360 nm. Next, a volume of 10 μL supersaturated solution of the
compounds (by dilution if necessary) in water were injected into HPLC for analysis (detection
wavelength at 360 nm). The water solubility was calculated according to the linear regression
equation.
Declaration of competing interest
The authors declare no conflicts of interest.
Acknowledgements
This program was supported by National Natural Science Foundation of China (Grant No.
21602132 and No. 81673281), the Interdisciplinary Program of Shanghai Jiao Tong University
(project number: ZH2018QNB15) and Zhejiang Chinese Medical University Startup Funding
(2020ZR12). We also want to express our gratitude to associate professor Xiaoyan Pan and
professor Jie Zhang in the Xi’an Jiaotong University for providing molecular docking software.
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