Synthesis and bioevaluation of novel benzodipyranone derivatives as P-glycoprotein inhibitors for multidrug resistance reversal agents

Article abstract of DOI:10.1016/j.ejmech.2016.03.070

Multidrug resistance (MDR) is a phenomenon in which cells become resistant to structurally and mechanistically unrelated drugs, and it is one of the emerging problems in cancer therapy today. The relation between overexpression of the ABC transporter subf

Full text of DOI:10.1016/j.ejmech.2016.03.070

Accepted Manuscript
Synthesis and Bioevaluation of Novel Benzodipyranone Derivatives as P-Glycoprotein
Inhibitors for Multidrug Resistance Reversal Agents
Chien-Yu Chen, Nai-Yu Liu, Hui-Chang Lin, Chih-Yu Lee, Chin-Chuan Hung, Chih-
Shiang Chang
PII:
S0223-5234(16)30252-5
10.1016/j.ejmech.2016.03.070
EJMECH 8495
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 December 2015
Revised Date: 9 March 2016
Accepted Date: 25 March 2016
Please cite this article as: C.-Y. Chen, N.-Y. Liu, H.-C. Lin, C.-Y. Lee, C.-C. Hung, C.-S. Chang,
Synthesis and Bioevaluation of Novel Benzodipyranone Derivatives as P-Glycoprotein Inhibitors for
Multidrug Resistance Reversal Agents, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.03.070.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and Bioevaluation of Novel Benzodipyranone Derivatives as
P-Glycoprotein Inhibitors for Multidrug Resistance Reversal Agents
Chien-Yu Chena, Nai-Yu Liua, Hui-Chang Lina, Chih-Yu Leea, Chin-Chuan Hunga, *,
Chih-Shiang Changa, *
aSchool of Pharmacy, College of Pharmacy, China Medical University, 91,
Hsueh-Shih Road, Taichung 404, Taiwan, Republic of China.
To whom correspondence should be addressed.
Corresponding Authors
*For C.S.Chang.: phone, +886-4-22053366#5615; fax, +886-4-22078083;
E-mail: chihshiang@mail.cmu.edu.tw.
*For C.C.Hung.: phone, +886-4-22053366#5155; fax, +886-4-22078083;
E-mail: cc0206hung@gmail.com.

ACCEPTED MANUSCRIPT
ABSTRACT
Multidrug resistance (MDR) is a phenomenon in which cells become resistant to
structurally and mechanistically unrelated drugs, and it is one of the emerging
problems in cancer therapy today. The relation between overexpression of the ABC
transporter subfamily B member 1 (ABCB1/P-glycoprotein) and resistant cancers has
been well characterized. In the present study, we successfully synthesized 52 novel
benzodipyranone analogs and evaluated for their P-gp inhibitory activity in a P-gp
transfected cell line, ABCB1/Flp-InTM-293. Among these derivatives, 5a bearing on
the 3-methylphenyl substituent, displayed the most potent P-gp inhibitory activity,
which can enable the increase of the intracellular accumulation of P-gp substrate
Calcein-AM. 5a exhibited more potency on promoted anticancer drugs cytotoxicity
by reversing P-gp-mediated drug resistance in both ABCB1/Flp-InTM-293 and KBvin
cell lines. In particular, the compound 5a sensitized ABCB1/Flp-InTM-293 cells toward
paclitaxel, vincristine, and doxorubicin by 16.1, 21.0, and 1.6-fold at 10 ꢀM,
respectively. Further, 5a dramatically sensitized the resistant cell line KBvin toward
paclitaxel and vincristine by 23.1 and 29.7-fold at 10 ꢀM, respectively. It’s possible
that its mechanism of MDR inhibition can restore the intracellular accumulation of
drugs and eventually chemosensitize cancer cells to anticancer drugs and reduce
ABCB1 mRNA expression level.
Keywords: multidrug resistance, P-glycoprotein, benzodipyranone, reversal agent.

ACCEPTED MANUSCRIPT
1. Introduction
One of the critical obstacles related to cancer chemotherapy is resistance against
anticancer drugs. Therefore, drug resistance is one of the major causes of death in
cancer patients. Multidrug resistance (MDR) is a phenomenon in which cancer cells
become resistant to structurally and mechanistically unrelated drugs [1-3]. The most
widely studied mechanisms with known clinical significance are (a) pumping drugs
out of cells via the activation of efflux pumps, such as ATP-dependent transporters [4];
(b) resistance being mediated by reduced drug uptake, such as through cellular
endocytosis [5]; (c) activation of detoxifying proteins, such as phase I, II, and III
metabolic enzymes [6]; (d) cells activating mechanisms that repair drug-induced DNA,
RNA, or protein damage; and (e) disruptions in apoptotic signalling pathways
(especially p53 and Bcl-2) allowing cells to become resistant to drug-induced cell
apoptosis [7]. Among these mechanisms that have been reported to result in MDR, the
most widely studied mechanism is related to the overexpression of P-glycoprotein
(P-gp, also known as MDR1 or ABCB1), belonging to the ATP-binding cassette
(ABC) family of transport proteins which use the hydrolysis of ATP as the energy
source for extruding chemotherapeutic drugs out of cells [8], resulting in lowered
intracellular anticancer drug concentrations and leading to drug resistance [9].
Identifying the many agents that can modulate the P-gp transporter that interfere with
P-gp by the competitive or noncompetitive inhibition of drug efflux has recently been
studied. On the basis of their affinity, specificity, and toxicity, P-gp inhibitors are
classified into three generations. First-generation agents such as verapamil [10],
cyclosporin A [11], reserpine, quinidine [12], tamoxifen, and trifluoperazine (Figure
1), attempt to block P-gp and have been highly successful in doing just that. But these
agents often produce disheartening results in vivo because their low binding affinities
necessitate the use of high doses, resulting in unacceptable toxicity.
Second-generation P-gp inhibitors include cyclosporine A analogue valspodar [13],
pipecolinate derivative biricodar and verapamil analogue dexverapamil [14], and
these agents have a higher specificity than the first generation inhibitors and are also
less toxic. However, they are confounded by unforeseeable pharmacokinetic
interactions and interactions with other ABC transporter proteins [15]. Finally,
third-generation agents such as anthranilamide tariquidar [16], acridonecarboxamide
elacridar [17, 18], and cyclopropyldibenzosuberane zosuquidar that specifically and
potently inhibit P-gp function have been developed by using quantitative
structure-activity relationships (QSAR) and combinatorial chemistry to conquer the

ACCEPTED MANUSCRIPT
restrictions of the second generation P-gp inhibitors (Figure 2). One of the most
hopeful third-generation P-gp inhibitors is tariquidar, but its use was discontinued due
to unfavorable toxicity reports in phase III clinical trials [19]. Unfortunately, among
these inhibitors, none of them have been approved for clinical use [20].
Among the heterocyclic compounds, benzodipyranone scaffold have rarely been
used for research with current information. This class of compounds is available from
both chemical synthesis or from natural products [21]. However, only a few studies
have explored their bioactivity, such as their antiallergic activity [22]. It is with that in
mind that, we developed an interest in benzodipyranone derivatives against
P-gp-mediated MDR. In this study, we synthesized a series of new benzodipyranone
derivatives as P-gp inhibitors. These novel benzodipyranone analogues were
evaluated for their ability to influence the expression of ABCB1 mRNA, P-gp function
inhibitory potency and MDR-reversing activity in P-gp overexpressing cell lines.
Figure 1 herein
Figure 2 herein
2. Results and discussion
2.1. Chemistry
The preparation of benzodipyranone derivatives was illustrated in Scheme 1.
The synthesis of 3 started from the commercial of phloroglucinol (1) and appropriate
acetonitrile (2) in borontrifluoride etherate (BF3ꢀEt2O) was purged of HCl gas and
stirred at room temperature for 12-24 h to obtain imine intermediate, which was then
hydrolyzed to the corresponding 3. Subsequently, the preparation of 4a-31a and
4b-31b started from the synthesis of a benzodipyranone nucleus by a
Vilsmeier–Haack reaction that involved 3, methanesulfonyl chloride (MsCl), and
N,N-dimethylformamide (DMF) in borontrifluoride etherate stirred at 120 oC for 2-4 h
to eventually afford asymmetrical benzodipyrano-4,10-dione 4a-31a, and symmetrical
benzodipyrano-4,6-dione 4b-31b of benzodipyranone derivatives.
Scheme 1 herein
2.2. Biological activity
In order to establish the ability of the benzodipyranone derivatives to inhibit the
activity of P-gp, all the synthesized analogues were evaluated in vitro against MDR

ACCEPTED MANUSCRIPT
cell lines using the following assays: (a) Calcein-AM assay; (b) MDR reversal activity;
and (c) ABCB1 mRNA expression assay.
2.2.1. Enhancement of Calcein-AM uptake into ABCB1/Flp-InTM-293 cells
The effect of the analogues on the activities of P-gp was determined by
evaluating Calcein accumulation in Flp-InTM-293 cells that were transfected
overexpressing the P-gp transporters. The Calcein-AM assay utilized for the
evaluation involved measuring the fluorescent intensity imparted by Calcein, which
accumulated in the cells due to the inhibition of P-gp efflux by the benzodipyranone
derivatives. In Table 1 and Table 2, the P-gp inhibition fold is reported for each of the
tested compounds, with verapamil being used as a reference compound for the P-gp
inhibition test. In asymmetrical benzodipyrano-4,10-dione 4a-31a, the activity results
show that the introduction of an electron donating group, compound 4a, 5a, 6a, 7a, 9a,
and 11a inhibited P-gp function from 1.53 to 2.05 fold at 5 ꢀM (Table 1). However,
no improved P-gp inhibitory activity was observed among the 3-methoxyphenyl (8a),
3-hydroxyphenyl (10a), 3,4,5-trimethoxyphenyl (12a), and 3,4-methylenedioxyphenyl
(13a) substitution. Furthermore, compound 14a-27a were introduced as a series of
electron withdrawing substituents, respectively, all resulting in a decreased inhibition
of P-gp activity. Further, compound 28a-31a substituted with thiopen-2-yl (28a),
1-naphthyl (29a), phenyl (30a), and cyclopropyl (31a), respectively, were lost against
P-gp activity. These results suggest that the substitution of electron donating groups
plays an important factor in determining P-gp inhibitory activity.
Table 1 herein
In addition, the symmetrical benzodipyrano-4,6-dione 4b-31b were investigated
for their P-gp inhibitory activity (Table 2). However, all symmetrical
benzodipyranone derivatives were inactive toward P-gp modulating effect. These
results illustrate that the benzodipyrano-4,10-dione 4a-31a possibly have a higher
degree of reaching a conformation that is more suitable for matching the P-gp
substrate binding sites than the corresponding benzodipyrano-4,6-dione 4b-31b.
Table 2 herein
2.2.2. Chemo-sensitizing effect of target compounds
The assessment of the ability of a given compound tested in our study to enhance
the growth inhibitory effects of the anticancer drugs paclitaxel, vincristine, and
doxorubicin in resistant cell lines displaying MDR due to P-gp overexpression proved

ACCEPTED MANUSCRIPT
useful in the quantification and characterization of MDR reversal by inhibition of the
MDR phenotype. The reversal effects of selected compounds 5a, 9a, and 11a on the
MDR phenotype were investigated on the P-gp-transfected cell line
ABCB1/Flp-InTM-293 and its parent Flp-InTM-293. The MDR reversal effect of
benzodipyranone derivatives was compared by measuring the reversal fold (RF),
defined as the ratio of IC50 without a modulator to IC50 with a modulator. Alone, 5a,
9a, and 11a weakly inhibited ABCB1/Flp-InTM-293 cell growth (ꢁ10%) at 8 and 10
ꢀM. Preliminarily, we found that ABCB1/Flp-InTM-293 cells were about 1196.9, 137.9
and 9.9-fold more resistant toward paclitaxel, vincristine, and doxorubicin than their
parental Flp-InTM-293 cells, respectively. Then the MDR-reversal activity of the
studied compounds was tested using the same method in the presence of paclitaxel,
vincristine, or doxorubicin (Table 3). For paclitaxel cytotoxicity, 5a (RF = 16.1)
exhibited a higher modulating activity than 9a (RF = 8.2) and 11a (RF= 9.5) at 10 ꢀM.
For vincristine cytotoxicity, 5a (RF = 21.0) exhibited a greater modulating activity
than 9a (RF = 1.6) and 11a (RF= 9.7) at 10 ꢀM. For doxorubicin cytotoxicity, 5a (RF
= 1.6), 9a (RF = 1.7) and 11a (RF= 1.5) at 10 ꢀM were roughly equal in their ability
to reverse doxorubicin resistance in ABCB1/Flp-InTM-293 cells.
Table 3 herein
Furthermore, the MDR cancer cell line KBvin and the parental cancer cell line
HeLaS3 were employed in this study (Table 4). For paclitaxel cytotoxicity, 5a
displayed a potent P-gp modulating activity with a RF of 23.1 at 10 ꢀM. For
vincristine cytotoxicity, 5a displayed a powerful reversal activity with RF of 29.7 at
10 ꢀM. Additionally, 5a at 10 ꢀM with vincristine co-administered showed a
moderate modulating activity with a RF of 8.8 in the parental cancer cell line HeLaS3.
Table 4 herein
2.2.3. ABCB1 mRNA detected by RT-qPCR
In the ABCB1 mRNA, expression was detected by real-time quantitative reverse
transcription PCR (Figure 3). 5a and 11a significantly decreased ABCB1 mRNA
expression in the P-gp-transfected cell line ABCB1/Flp-InTM-293. 9a showed no effect
on ABCB1 mRNA expression level in both the P-gp-transfected cell line
ABCB1/Flp-InTM-293 and its parental cell line Flp-InTM-293, while little ABCB1 gene
expression was detected in the parental cell line Flp-InTM-293.
Figure 3 herein

ACCEPTED MANUSCRIPT
2.2.4. Docking interaction of compound 5a and 5b with P-gp.
The compound 5a and 5b was docked into the binding site of drug binding
domain (DBD) to provide the interaction between the ligand and the receptor (Figure
4a, 4b). Docking was done using Discovery Studio 3.1 software. Remarkably, 5a is
stabilized through specific interactions such as hydrogen bonding with residues in the
drug binding domain of P-gp and nonspecific interactions such as hydrophobic ꢂ-ꢂ
stacking aromatic interactions. The hydrogen bond acceptor oxygen atom at the 1, 5,
and 10-position of the benzodipyrano-4,10-dione showed hydrogen bonding
interaction with the side chain of TYR306 and SER725, respectively. The hydrogen
bond donor at the 5-position of the hydroxy group showed hydrogen bonding
interaction with the side chain of the SER725. The phenyl rings of the 3-m-tolyl group
interacted with PHE71 and PHE728 through π−π stacking aromatic interactions.
However, compound 5b only generated a hydrogen bond interaction with TYR306.
These results was verified our SAR deduction that benzodipyrano-4,10-dione 4a-31a
have a higher degree of reaching a conformation that is high affinity for matching the
drug binding sites of P-gp than the corresponding benzodipyrano-4,6-dione 4b-31b.
Figure 4 herein
3. Conclusion
In this study, we synthesized a novel series of benzodipyranone derivatives and
evaluated their inhibitory activity of P-gp by the ABCB1/Flp-InTM-293 cell line
fluorescence efflux of a specific P-gp substrate Calcein-AM. The structure activity
relationship (SAR) study suggests that asymmetrical benzodipyrano-4,10-dione
4a-31a bearing on the R substituents with electron donating group exhibited a better
inhibitory activity of P-gp efflux function (between 1.53 to 2.05 fold) than electron
withdrawing substituents at 5 ꢀM. These results illustrates that the substituent of an
electron donating group play an important factor in determining P-gp inhibitory
activity. However, the symmetrical benzodipyrano-4,6-dione 4b-31b were inactive
toward P-gp inhibitory effect. According to the SAR and molecule docking results,
the benzodipyrano-4,10-dione derivatives (4a-31a) have a higher degree of reaching a
conformation that is more suitable for matching with the drug-binding pocket of P-gp
than the corresponding benzodipyrano-4,6-dione derivatives (4b-31b). At the higher
dose tested (10 ꢀM), 5a, 9a, and 11a were able to significantly (p<0.05) reverse
paclitaxel, vincristine, and doxorubicin resistance. 5a was the most potent compound

ACCEPTED MANUSCRIPT
in that it significantly sensitized ABCB1/Flp-InTM-293 cells to various anticancer
drugs including paclitaxel, vincristine, and doxorubicin with nanomolar IC50 values
(IC50 ranged from 39.11 to 586.26 nM). In addition, 5a at 10 ꢀM was able to sensitize
MDR cell line KBvin toward paclitaxel and vincristine by 23.1-fold and 29.7-fold,
respectively.
The present study demonstrates that these synthetic derivatives of
benzodipyranone derivatives can be employed as effective inhibitors of P-gp mediated
drug resistance in cancer cells, and their mechanism of MDR inhibition is associated
with an increasing intracellular accumulation of a given anticancer drug induced by
directly blocking P-gp-mediated drug efflux or reducing ABCB1 mRNA expression
level. In conclusion, we have defined the structure-activity relationships of these new
series of P-gp inhibitors and identified 5a as having a good efficacy, which might
prove useful down the line as a lead for the development of new inhibitors of
P-gp-mediated MDR.
4. Experimental section
4.1. Chemistry
4.1.1. Materials and methods
Reagents and solvents were obtained commercially and used without further
purification. Reactions were monitored by TLC, using Merck plates with fluorescent
indicator (TLC Silica Gel 60 F254). Flash column chromatography was performed on
silica gel (Merck Silica Gel 60, 400-630 mesh). Melting points were determined on a
1
Yanaco MP-500D melting point apparatus and were uncorrected. H and 13C NMR
spectra were obtained on a Bruker Ultrashield 500 plus FT-NMR Spectrometer (500
MHz) in DMSO-d6. The following abbreviations are used: s, singlet; d, doublet; t,
triplet; m, multiplet. EI-HRMS and EIMS were measured with a Finnigan/Thermo
Quest MAT 95XL instrument, ESIMS was measured with a Finnigan/Thermo LCQ
ion-trap mass spectrometer. The purity of active compounds was assessed using a
HPLC. The column used was a C18 reverse phase column (NUCLEODUR C18 HTec,
250 mm × 4.6 mm, 5 ꢀm) attached to a Jasco 851-AS autosample and Jasco PU-980
pump coupled to a Jasco MD-910 photodiode array (PDA) detector was used. Each
sample was injected at a volume of 20 ꢀL and eluted with an isocratic mobile phase
(methanol/water), and the flow rate was 1 mL/min. All tested compounds were
confirmed to be ≥ 97% pure based on the area of the major peak when compared to
the total combined area. Note: compound 19a, 21a, 23a, 24a, 25a, 26a, 6b, 9b, 15b,
16b, 18b, 19b, 24b, 25b, and 27b difficult to dissolve in DMSO, DMF and pyridine,

ACCEPTED MANUSCRIPT
so it is unable to obtain 13C NMR spectrum.
4.1.2. General procedure for synthesis of benzodipyranone derivatives 4a,b-31a,b
To a solution of phloroglucinol (1, 1 mmol) and appropriate acetonitrile (2, 2.4
mmol) dissolved in borontrifluoride etherate (15 mL) was purged HCl gas. The
mixture was stirred at room temperature for 10-14 h followed by the addition of 1 M
HCl (15 mL). The resulting mixture was stirred at reflux for 4 h, and after completion
as monitored by TLC, the reaction residue was diluted with ethyl acetate. The ethyl
acetate layer was then washed with saturated NaHCO3 aqueous and brine. The organic
fractions were dried over anhydrous MgSO4 and evaporated under vacuum. The
residue was purified by flash chromatography on silica gel using ethyl
acetate−dichloromethane as eluent to provide corresponding products 3.
A mixture of the appropriate 3 (1 mmol), methanesulfonyl chloride (6 mmol) and
o
DMF (6 mmol) in borontrifluoride etherate (1 mL) was stirred at 120 C for 2-4 h.
After reaction completion as monitored by TLC, the resulting mixture was cooled at
o
60 C with vigorous stir, and the reaction mixture was diluted with ethyl acetate, the
suspension was filtered and the solid washed with ethyl acetate to afford symmetrical
benzodipyrano-4,6-dione 4b-31b as a solid. Subsequently, the filtrate was then
washed with 1 M HCl and brine. The organic fractions were dried over anhydrous
MgSO4 and evaporated under vacuum. The residue was subjected to flash
chromatography on silica gel used a mixture of ethyl acetate−n-hexane as eluent to
provide asymmetrical benzodipyrano-4,10-dione 4a-31a.
4.1.3. 5-hydroxy-3,9-di-o-tolylpyrano[2,3-f]chromene-4,10-dione (4a)
o
1
Orange solid. Yield 8%. mp 111.1-111.8 C; H NMR (500 MHz, DMSO-d6) δ
13.78 (s, 1 H, OH), 8.66 (s, 1 H, 2-H), 8.28 (s, 1 H, 8-H), 7.04-7.37 (m, 8 H, ArH),
5.74 (s, 1 H, 6-H), 2.21 (s, 3 H, CH3), 2.18 (s, 3 H, CH3); 13C NMR (125 MHz,
DMSO-d6) δ 180.3, 172.5, 163.9, 161.5, 156.7, 156.2, 152.7, 137.6, 131.5, 130.7,
130.6, 129.8, 129.8, 129.7, 128.7, 128.3, 126.7, 125.7, 125.6, 125.4, 108.8, 106.6,
99.5, 99.5,19.6, 19.5. EIMS m/z 410.2 (M+). HPLC purity 99.8% (λmax 280 nm).
4.1.4. 5-hydroxy-3,9-di-m-tolylpyrano[2,3-f]chromene-4,10-dione (5a)
o
Yellow solid. Yield 33%. mp 146.5-147-2 C; 1H NMR (500 MHz, DMSO-d6) δ
13.84 (s, 1 H, OH), 8.78 (s, 1 H, 2-H), 8.40 (s, 1 H, 8-H), 7.40-7.44 (m, 2 H, 6;, 6”-H),
7.30-7.37 (m, 4 H, 2’, 4’, 2”, 4”-H), 7.20-7.25 (m, 2 H, 5’, 5”-H), 6.97 (s, 1 H, 6-H),
2.36 (s, 3 H, CH3), 2.35 (s, 3 H, CH3); 13C NMR (125MHz, DMSO-d6) δ 180.4, 172.6,
163.9, 161.2, 156.0, 155.9, 152.2, 137.4, 137.2, 131.2, 129.7, 129.6, 129.5, 129.1,
128.6, 128.2, 128.0, 126.1, 126.1, 125.4, 124.4, 108.9, 106.5, 99.4, 21.0, 21.0. EIMS

ACCEPTED MANUSCRIPT
m/z 410.2 (M+). HPLC purity 99.6% (λmax 280 nm).
4.1.5. 5-hydroxy-3,9-di-p-tolylpyrano[2,3-f]chromene-4,10-dione (6a)
o
1
White solid. Yield 19%. mp 202.5-203.1 C; H NMR (500 MHz, DMSO-d6) δ
13.85 (s, 1 H, OH), 8.78 (s, 1 H, 2-H), 8.37 (s, 1 H, 8-H), 7.52 (d, J = 8.1 Hz, 2 H, 2’,
6’-H), 7.43 (d, J = 8.1 Hz, 2 H, 2’’, 6’’-H), 7.27 (d, J = 8.0 Hz, 2 H, 3’, 5’-H), 7.23 (d,
J = 8.0 Hz, 2 H, 3’’, 5’’-H), 6.95 (s, 1 H, 6-H), 2.35 (d, J = 5.3 Hz, 6 H, 4’, 2×CH3);
13C NMR (125MHz, DMSO-d6) δ 180.5, 172.7, 163.9, 161.2, 156.0, 155.9, 152.0,
138.0, 137.3, 128.9, 128.9, 128.8, 128.8, 128.7, 128.7, 128.3, 126.9, 125.2, 124.2,
108.9, 106.5, 99.3, 99.3, 20.8, 20.8, 20.8. EIMS m/z 410.1 (M+). HPLC purity 99.3%
(λmax 252 nm).
4.1.6. 5-hydroxy-3,9-bis(2-methoxyphenyl)pyrano[2,3-f]chromene-4,10-dione (7a)
Brown powder. Yield 16%. mp 216.8-217.6 oC; 1H NMR (500 MHz, DMSO-d6)
δ 13.81 (s, 1 H, OH), 8.65 (s, 1 H, 2-H), 8.25 (s, 1 H, 8-H), 7.38-7.45 (m, 2 H, ArH),
7.32 (dd, J = 1.6, 7.5 Hz, 1 H, ArH), 7.24 (dd, J = 1.6, 7.4 Hz, 1 H, ArH), 7.13 (d, J =
8.2 Hz, 1 H, ArH), 7.09 (d, J = 8.1 Hz, 1 H, ArH), 7.00-7.05 (m, 2 H, ArH), 6.98 (s, 1
H, 6-H), 3.76 (s, 3 H, OCH3), 3.73 (s, 3 H, OCH3); 13C NMR (125 MHz, DMSO-d6) δ
180.2, 172.3, 163.8, 161.4, 157.5, 157.4, 156.8, 156.0, 152.5, 131.6, 131.5, 130.4,
129.9, 124.0, 122.5, 120.5, 120.3, 120.2, 118.7, 111.5, 111.3, 108.7, 106.5, 99.5, 55.7,
55.6. EIMS m/z 442.1 (M+). HPLC purity 99.3% (λmax 272 nm).
4.1.7. 5-hydroxy-3,9-bis(3-methoxyphenyl)pyrano[2,3-f]chromene-4,10-dione (8a)
White powder. Yield 23%. mp 185.9-186.8 oC; 1H NMR (500 MHz, DMSO-d6) δ
13.84 (s, 1 H, OH), 8.84 (s, 1 H, 2-H), 8.43 (s, 1 H, 8-H), 7.37-7.40 (m, 1 H, 5’-H),
7.33-7.36 (m, 1 H, 5”-H), 7.21-7.22 (m, 2 H, 2’, 4’-H), 7.11-7.14 (m, 2 H, 2”, 4”-H),
7.01 (dd, J = 2.1, 8.3 Hz, 1 H, 6’-H), 6.98 (s, 1 H, 6-H), 6.96 (dd, J = 2.1, 8.3 Hz, 1 H,
13
6”-H), 3.80 (s, 3 H, OCH3), 3.79 (s, 3 H, OCH3); C NMR (125MHz, DMSO-d6) δ
180.4, 172.5, 164.0, 161.1, 159.1, 159.0, 156.3, 155.9, 152.5, 132.6, 131.1, 129.4,
129.1, 125.1, 124.1, 121.2, 121.2, 114.6, 114.6, 114.0, 113.7, 108.9, 106.6, 99.4, 55.1,
55.1. EIMS m/z 442.1 (M+).
4.1.8. 5-hydroxy-3,9-bis(4-methoxyphenyl)pyrano[2,3-f]chromene-4,10-dione (9a)
Brown powder. Yield 39%. mp 163.2-164.0 oC; 1H NMR (500 MHz, DMSO-d6)
δ 13.89 (s, 1 H, OH), 8.78 (s, 1 H, 2-H), 8.37 (s, 1 H, 8-H), 7.59 (d, J = 8.5 Hz, 2 H,
2’, 6’-H), 7.50 (d, J = 8.5 Hz, 2 H, 2’’, 6’’-H), 7.03 (d, J = 8.5 Hz, 2 H, 3’, 5’-H), 7.00
(d, J =8.5 Hz, 2 H, 3’’, 5’’-H), 6.96 (s, 1 H, 6-H), 3.80 (s, 3 H, OCH3), 3.80 (s, 3 H,
13
OCH3); C NMR (125MHz, DMSO-d6) δ 180.6, 172.9, 163.9, 161.2, 159.5, 159.2,

ACCEPTED MANUSCRIPT
156.0, 155.5, 151.7, 130.3, 130.3, 130.2, 130.2, 125.0, 123.9, 123.4, 121.9, 113.8,
113.8, 113.6, 113.6, 108.8, 106.6, 99.3, 55.2, 55.1. EIMS m/z 442.1 (M+). HPLC
purity 97.6% (λmax 248 nm).
4.1.9. 5-hydroxy-3,9-bis(3-hydroxyphenyl)pyrano[2,3-f]chromene-4,10-dione (10a)
o
1
Pale-yellow crystal. Yield 22%. mp 305.2-306.0 C; H NMR (500 MHz,
DMSO-d6) δ 13.88 (s, 1 H, 5-OH), 9.56, (s, 1 H, 3’-OH), 9.47 (s, 1 H, 3’’-OH), 8.77
(s, 1 H, 2-H), 8.38 (s, 1 H, 8-H), 7.25 (t, J = 7.9 Hz, 1 H, 5’-H), 7.22 (t, J = 7.9 Hz, 1
H, 5”-H), 7.20-7.27 (m, 2 H, ArH), 6.93-7.06 (m, 5 H, ArH), 6.78-6.84 (m, 2 H, ArH);
13C NMR (125 MHz, DMSO-d6) δ 180.4, 172.6, 164.0, 161.1, 157.1, 157.0, 156.1,
156.0, 152.2, 132.5, 131.0, 129.3, 129.1, 125.4, 124.3, 119.6, 119.6, , 116.1, 115.5,
115.0, 108.9, 106.6, 99.3, 99.3. EIMS m/z 414.1 (M+).
4.1.10.
(11a)
3,9-bis(3,4-dimethoxyphenyl)-5-hydroxypyrano[2,3-f]chromene-4,10-dione
o
1
Brown solid. Yield 6%. mp 149.1-149.9 C; H NMR (500 MHz, DMSO-d6) δ
13.91 (s, 1 H, OH), 8.84 (s, 1 H, 2-H), 8.40 (s, 1 H, 8-H), 7.26 (d, J = 1.5 Hz, 1 H,
2’-H), 7.23 (dd, J = 1.5, 8.3 Hz, 1 H, 5’-H), 7.19 (d, J = 1.5 Hz, 1 H, 2”-H), 7.11 (dd,
J = 1.5, 8.3 Hz, 1 H, 5”-H), 7.05 (d, J = 8.35 Hz, 1 H, 6’-H), 7.01 (d, J = 8.3 Hz, 1 H,
13
6”-H), 6.96 (s, 1 H, 6-H), 3.80 (s, 6 H, OCH3), 3.79 (s, 6 H, OCH3); C NMR (125
MHz, DMSO-d6) δ 180.6, 172.8, 163.9, 161.1, 155.9, 155.8, 151.9, 149.2, 148.8,
148.4, 148.3, 125.0, 124.0, 123.7, 122.2, 121.5, 121.3, 112.9, 112.7, 111.7, 111.5,
108.8, 106.5, 99.3, 55.6, 55.6, 55.6, 55.6. EIMS m/z 502.2 (M+). HPLC purity 99.3%
(λmax 212 nm).
4.1.11. 5-hydroxy-3,9-bis(3,4,5-trimethoxyphenyl)pyrano[2,3-f]chromene-4,10-dione
(12a)
o
1
White solid. Yield 20%. mp 268.0-268.7 C; H NMR (500 MHz, DMSO-d6) δ
13.87 (s, 1 H, OH), 8.89 (s, 1 H, 2-H), 8.46 (s, 1 H, 8-H), 6.98 (s, 3 H, 6, 2’, 6’-H),
6.89 (s, 2 H, 2’’, 6’’-H), 3.81 (s, 6 H, 2×OCH3), 3.80 (s, 6 H, 2×OCH3), 3.70 (s, 3 H,
13
OCH3), 3.69 (s, 3 H, OCH3); C NMR (125 MHz, DMSO-d6) δ 180.3, 172.6, 164.0,
161.1, 156.3, 155.9, 152.7, 152.5, 152.5, 152.5, 152.5, 137.8, 137.5, 126.7, 125.3,
125.1, 124.0, 108.9, 106.6, 106.6, 106.6, 106.6, 106.5, 99.4, 60.0, 60.0, 56.0, 56.0,
55.9, 55.9. EIMS m/z 562.1 (M+).
4.1.12.
3,9-bis(benzo[d][1,3]dioxol-5-yl)-5-hydroxypyrano[2,3-f]chromene-4,10-dione (13a)
o
1
Yellow solid. Yield 64%. mp 350.5-351.4 C; H NMR (500 MHz, DMSO-d6) δ

ACCEPTED MANUSCRIPT
13.63 (s, 1 H, OH), 8.70 (s, 1 H, 2-H), 8.30 (s, 1 H, 8-H), 7.21 (d, J = 1.6 Hz, 1 H,
2’-H), 7.16 (dd, J = 1.6, 8.1 Hz, 1 H, 6’-H), 7.13 (d, J = 1.6 Hz, 1 H, 2’’-H), 7.06 (dd,
J = 1.6, 8.0 Hz, 1 H, 6’’-H), 6.99 (d, J = 8.1 Hz,1 H, 5’-H), 6.95 (d, J = 8.0 Hz, 1 H,
5’’-H), 6.90 (s, 1 H, 6-H), 6.05 (s, 2 H, CH2), 6.04 (s, 2 H, CH2); 13C NMR (125 MHz,
DMSO-d6) δ 180.1, 172.3, 163.6, 160.9, 155.6, 155.2, 151.4, 147.2, 146.9, 146.8,
146.7, 124.8, 124.6, 123.8, 123.1, 122.4, 122.2, 109.1, 109.0, 108.6, 107.8, 107.6,
106.3, 100.8, 100.7, 98.9. EIMS m/z 470.0 (M+).
4.1.13. 3,9-bis(2-fluorophenyl)-5-hydroxypyrano[2,3-f]chromene-4,10-dione (14a)
o
1
White solid. Yield 13%. mp 234.3-235.2 C; H NMR (500 MHz, DMSO-d6) δ
13.6 (s, 1 H, OH), 8.83 (s, 1 H, 2-H), 8.45 (s, 1 H, 8-H), 7.43-7.55 (m, 4 H, 3’, 5’, 3”,
13
5”-H), 7.27-7.36 (m, 4 H, 4’, 6’, 4”, 6”-H), 7.06 (s, 1 H, 6-H); C NMR (125MHz,
DMSO-d6) δ 179.6, 171.7, 163.9, 161.4, 160.1 (d, J = 245.4 Hz, 1 C), 160.0 (d, J =
245.1 Hz, 1 C), 157.3, 156.0, 153.4, 132.2, 132.1 (d, J = 2.75 Hz, 1 C), 131.1 (d, J =
8.1 Hz, 1 C), 130.6 (d, J = 8.0 Hz, 1 C), 124.4 (d, J = 19.6 Hz, 1 C), 124.4 (d, J = 19.8
Hz, 1 C), 121.6, 120.3, 119.3 (d, J = 15.5 Hz, 1 C), 117.7 (d, J = 15.6 Hz, 1 C), 115.6
(d, J = 19.5 Hz, 1 C), 115.4 (d, J = 21.3 Hz, 1 C), 108.0, 106.4, 99.8. EIMS m/z 418.1
(M+).
4.1.14. 3,9-bis(3-fluorophenyl)-5-hydroxypyrano[2,3-f]chromene-4,10-dione (15a)
o
1
Brown solid. Yield 27%. mp 240.9-241.8 C; H NMR (500 MHz, DMSO-d6) δ
13.72 (s, 1 H, OH), 8.89 (s, 1 H, 2-H), 8.51 (s, 1 H, 8-H), 7.45-7.54 (m, 4 H, 2’, 2”, 4’,
4”-H), 7.40-7.43 (m, 2 H, 6’, 6”-H), 7.21-7.30 (m, 2 H, 5’, 5”-H), 7.00 (s, 1 H, 6-H);
13C NMR (125 MHz, DMSO-d6) δ 180.1, 172.3, 164.0, 161.8 (d, J = 241.6 Hz, 1 C),
161.8 (d, J = 241.1 Hz, 1 C), 161.1, 156.7, 155.8, 153.1, 133.6 (d, J = 8.6 Hz, 1 C),
132.1 (d, J = 8.5 Hz, 1 C), 130.3 (d, J = 8.4 Hz, 1 C), 130.0 (d, J = 8.4 Hz, 1 C) ,
125.0 (d, J = 2.6 Hz, 1 C), 125.0 (d, J = 2.1 Hz, 1 C), 124.0, 123.0, 115.8 (d, J = 22.4
Hz, 2 C), 115.3 (d, J = 20.8, 1 C), 114.8 (d, J =20.5 Hz, 1 C), 108.9, 106.5, 99.6, 99.6.
EIMS m/z 418.1 (M+).
4.1.15. 3,9-bis(4-fluorophenyl)-5-hydroxypyrano[2,3-f]chromene-4,10-dione (16a)
o
1
Pale-brown solid. Yield 23%. mp 273.8-274.5 C; H NMR (500 MHz,
DMSO-d6) δ 13.77 (s, 1 H, OH), 8.83 (s, 1 H, 2-H), 8.44 (s, 1 H, 8-H), 7.67-7.70 (m,
2 H, 3’, 5’-H), 7.58-7.61 (m, 2 H, 3’’, 5’’-H), 7.31 (t, J = 8.9 Hz, 2 H, 2’, 6’-H), 7.27
(t, J = 8.9 Hz, 2 H, 2’’, 6’’-H), 6.99 (s, 1 H, 6-H); 13C NMR (125 MHz, DMSO-d6) δ
180.3, 172.6, 164.0, 162.2 (d, J = 244.8 Hz, 1 C), 162.0 (d, J = 243.4 Hz, 1 C), 156.2,
156.0, 152.5, 131.2 (d, 7.8 Hz, 2 C), 131.2 (d, J = 7.9 Hz, 2 C), 127.6 (d, J = 2.8 Hz, 1
C), 126.2 (d, J= 2.63 Hz, 1 C), 124.4, 123.4, 115.3 (d, J = 21.4 Hz, 2 C), 115.0 (d, J =

ACCEPTED MANUSCRIPT
21.1 Hz, 2 C) 109.0, 106.5, 99.5, 99.5. EIMS m/z 418.0 (M+).
4.1.16. 3,9-bis(2-chlorophenyl)-5-hydroxypyrano[2,3-f]chromene-4,10-dione (17a)
White powder. Yield 22%. mp 197.9-198.8 oC; 1H NMR (500 MHz, DMSO-d6) δ
13.58 (s, 1 H, OH), 8.78 (s, 1 H, 2-H), 8.40 (s, 1 H, 8-H), 7.61 (d, J = 7.6 Hz, 1 H,
3’-H), 7.61 (d, J = 7.7 Hz, 1 H, , 3”-H), 7.42-7.52 (m, 6 H, 4’, 5’, 6’, 4”, 5”, 6”-H),
7.08 (s, 1 H, 6-H); 13C NMR (125MHz, DMSO-d6) δ 179.6, 171.7, 163.9, 161.5,
157.3, 156.0, 153.3, 133.9, 133.8, 132.6, 132.5, 130.8, 130.7, 130.2, 129.3, 129.3,
129.2, 127.2, 127.1, 125.0, 123.7, 108.8, 106.5, 99.9. EIMS m/z 415 (M-36).
4.1.17. 3,9-bis(3-chlorophenyl)-5-hydroxypyrano[2,3-f]chromene-4,10-dione (18a)
o
1
White solid. Yield 46%. mp 201.5-202.0 C; H NMR (500 MHz, DMSO-d6) δ
13.68 (s, 1 H, OH),8.88 (s, 1 H, 2-H), 8.51 (s, 1 H, 8-H), 7.72 (d, J = 0.7 Hz, 1 H,
2’-H), 7.63 (d, J = 0.7 Hz, 1 H, 2’’-H), 7.59 (m, 1 H, ArH), 7.48-7.52 (m, 3 H, ArH),
13
7.45-7.46 (m, 2 H, 6’, 6”-H), 6.99 (s, 1 H, 6-H); C NMR (125 MHz, DMSO-d6) δ
180.0, 172.3, 164.0, 161.1, 156.7, 155.9, 153.1, 133.4, 133.0, 132.7, 132.0, 130.2,
130.0, 128.7, 128.7, 128.4, 127.9, 127.6, 127.6, 123.9, 123.0, 108.9, 106.4, 99.6.
EIMS m/z 450.0 (M+).
4.1.18. 3,9-bis(4-chlorophenyl)-5-hydroxypyrano[2,3-f]chromene-4,10-dione (19a)
o
1
Pale-brown solid. Yield 18%. mp 306.8-307.6 C; H NMR (500 MHz,
DMSO-d6) δ 13.74 (s, 1 H, OH), 8.87 (s, 1 H, 2-H), 8.48 (s, 1 H, 8-H), 7.68 (d, J = 8.5
Hz, 2 H, 3’, 5’-H), 7.59 (d, J = 8.5 Hz, 2 H, 3’’, 5’’-H), 7.55 (d, J = 8.5 Hz, 2 H, 2’,
6’-H), 7.51(d, J =8.5 Hz, 2 H, 2”, 6”-H), 7.02 (s, 1 H, 6-H). EI-HRMS m/z 450.0070
(M+), calcd for C24H12Cl2O5: 450.0062.
4.1.19. 5-hydroxy-3,9-bis(2-nitrophenyl)pyrano[2,3-f]chromene-4,10-dione (20a)
o
1
White solid. Yield 35%. mp 307.9-308.7 C; H NMR (500 MHz, DMSO-d6) δ
13.25 (s, 1 H, OH), 8.96 (s, 1 H, 2-H), 8.64 (s, 1 H, 8-H), 8.18 (d, J = 8.1 Hz, 1 H,
3’-H), 8.13 (d, J = 8.1 Hz, 1 H, 3”-H), 7.84-7.91 (m, 2 H, 5’, 5”-H), 7.71-7.77 (m, 2 H,
4’, 4”-H), 7.67 (d, J = 7.3 Hz, 1 H, 6’-H), 7.62 (d, J = 7.3 Hz, 1 H, 6”-H), 7.12 (s, 1 H,
6-H); 13C NMR (125 MHz, DMSO-d6) δ 179.1, 171.5, 163.9, 161.6, 156.1, 155.9,
152.2, 149.1, 148.8, 134.2, 133.9, 132.8, 132.5, 130.5, 130.0, 125.7, 124.8, 124.5,
124.3, 124.3, 123.7, 108.2, 105.8, 100.0. EIMS m/z 426.1 (M-46).
4.1.20. 5-hydroxy-3,9-bis(4-nitrophenyl)pyrano[2,3-f]chromene-4,10-dione (21a)
o
1
Brown solid. Yield 77%. mp 343.7-344.5 C; H NMR (500 MHz, DMSO-d6) δ
13.65 (s, 1 H, OH), 9.03 (s, 1 H, 2-H), 8.66 (s, 1 H, 8-H), 8.34 (d, J = 8.7 Hz, 2 H, 3’,

ACCEPTED MANUSCRIPT
5’-H), 8.31 (d, J = 8.7 Hz, 2 H, 3’’, 5’’-H), 7.96 (d, J = 8.7 Hz, 2 H, 2’, 6’-H), 7.90 (d,
J = 8.7 Hz, 2 H, 2’’, 6’’-H), 7.11 (s, 1 H, 6-H). EI-HRMS m/z 472.0546 (M+), calcd
for C24H12N2O9: 472.0543.
4.1.21. 3,9-bis(2-chloro-6-fluorophenyl)-5-hydroxypyrano[2,3-f]chromene-4,10-dione
(22a)
o
1
Yellow solid. Yield 22%. mp 196.3-197.2 C; H NMR (500 MHz, DMSO-d6) δ
13.65 (s, 1 H, OH), 8.93 (s, 1 H, 2-H), 8.55 (s, 1 H, 8-H), 7.47-7.60 (m, 4 H, 3’, 5’, 3”,
5”-H), 7.35-7.41 (m, 2 H, 4’, 4”-H), 7.12 (s, 1 H, 6-H);13C NMR (125 MHz,
DMSO-d6) δ 179.0, 171.2, 163.9, 161.5, 160.7 (d, J = 246.0 Hz, 1 C), 160.6 (d, J =
246.4 Hz, 1 C), 158.6, 156.0, 154.7, 135.0 (d, J = 13.5 Hz, 1 C), 135.0 (d, J = 13.5 Hz,
1 C), 131.9 (d, J = 9,5 Hz, 1 C), 131.4 (d, J = 9.4 Hz, 1 C), 125.4 (d, J = 1.5 Hz, 1 C),
125.2 (d, J = 1.6 Hz,1 C), 118.9 (d, J = 19.6 Hz, 1 C), 118.8, 117.6, 117.4 (d, J = 19.4
Hz,1 C), 114.5 (d, J = 21.8 Hz,1 C), 114.4 (d, J = 22.0 Hz,1 C), 108.8, 106.4, 100.3.
EIMS m/z 451 (M-36).
4.1.22.
(23a)
3,9-bis(3,4-dichlorophenyl)-5-hydroxypyrano[2,3-f]chromene-4,10-dione
o
1
White solid. Yield 8%. mp 326.2-327.1 C; H NMR (500 MHz, DMSO-d6) δ
13.63 (s, 1 H, OH), 8.93 (s, 1 H, 2-H), 8.57 (s, 1 H, 8-H), 7.94 (d, J = 2.0 Hz, 1 H,
2’-H), 7.86 (d, J = 2.0 Hz, 1 H, 2”-H), 7.75 (d, J = 8.4 Hz, 1 H, 5’-H), 7.71 (d, J = 8.4
Hz, 1 H, 5”-H), 7.65 (dd, J = 2.0, 8.4 Hz, 1 H, 6’-H),7.58 (dd, J = 2.0, 8.4 Hz, 1 H,
6”-H), 7.04 (s, 1 H, 6-H). EI-HRMS m/z 517.9286 (M+), calcd for C24H10Cl4O5:
517.9282.
4.1.23.
5-hydroxy-3,9-bis(2-(trifluoromethyl)phenyl)pyrano[2,3-f]chromene-4,10-dione (24a)
o
1
White solid. Yield 26%. mp 118.7-119.5 C; H NMR (500 MHz, DMSO-d6) δ
13.47 (s, 1 H, OH), 8.74 (s, 1 H, 2-H), 8.37 (s, 1 H, 8-H), 7.88 (d, J = 7.8 Hz, 1 H,
6’-H), 7.85 (d, J = 7.8 Hz, 1 H, 6”-H), 7.67-7.78 (m, 4 H, 3’, 5’, 3”, 5”-H), 7.56 (d, J
= 7.6 Hz, 1 H, 4’-H), 7.48 (d, J = 7.6 Hz, 1 H, 4”-H), 7.08 (s, 1 H, 6-H). EI-HRMS
m/z 518.0585 (M+), calcd for C26H12F6O5: 518.0589.
4.1.24.
5-hydroxy-3,9-bis(3-(trifluoromethyl)phenyl)pyrano[2,3-f]chromene-4,10-dione (25a)
White crystal. Yield 48%. mp 212.6-213.3 oC; 1H NMR (500 MHz, DMSO-d6) δ
13.65 (s, 1 H, 5-OH), 8.95 (s, 1 H, 2-H), 8.58 (s, 1 H, 8-H), 8.03 (s, 1 H, 2’-H), 7.95 (s,

ACCEPTED MANUSCRIPT
1 H, 2”-H), 7.93 (d, J =7.8 Hz, 1 H, 4’-H), 7.85 (d, J =7.8 Hz, 1 H, 6’-H), 7.80 (d, J =
7.9 Hz, 1 H, 6”-H), 7.76 (d, J = 7.8 Hz, 1 H, 4”-H), 7.66-7.73 (m, 2 H, 5’, 5”-H), 7.04
(s, 1 H, 6-H). EI-HRMS m/z 518.0583 (M+), calcd for C26H12F6O5: 518.0589.
4.1.25.
5-hydroxy-3,9-bis(4-(trifluoromethyl)phenyl)pyrano[2,3-f]chromene-4,10-dione (26a)
o
1
White solid. Yield 39%. mp 202.6-203.5 C; H NMR (500 MHz, DMSO-d6) δ
13.67 (s, 1 H, OH), 8.93 (s, 1 H, 2-H), 8.55 (s, 1 H, 8-H), 7.84 (dd, J = 8.5, 9.3 Hz, 4
H, 3’, 5’, 3”, 5”-H), 7.78 (s, 4 H, 2’, 6’, 2”, 6”-H), 7.02 (s, 1 H, 6-H). EI-HRMS m/z
518.0582 (M+), calcd for C26H12F6O5: 518.0589.
4.1.26.
5-hydroxy-3,9-bis(4-(trifluoromethoxy)phenyl)pyrano[2,3-f]chromene-4,10-dione
(27a)
o
1
Yellow solid. Yield 33%. mp 175.7-176.6 C; H NMR (500 MHz, DMSO-d6) δ
13.70 (s, 1 H, OH), 8.87 (s, 1 H, 2-H), 8.49 (s, 1 H, 8-H), 7.75 (d, J = 8.8 Hz, 2 H, 3’,
5’-H), 7.67 (d, J = 8.7 Hz, 2 H, 3’’, 5’’-H), 7.46 (d, J = 8.1 Hz, 2 H, 2’, 6’-H), 7.42 (d,
13
J = 8.1 Hz, 2 H, 2’’, 6’’-H), 6.99 (s, 1 H, 6-H); C NMR (125 MHz, DMSO-d6) δ
180.1, 172.3, 164.0, 161.2, 156.5, 155.9, 152.9, 148.4, 148.1, 131.0, 131.0, 130.6,
129.1, 124.0, 123.9, 123.9, 123.1, 121.3, 121.3, 119.8 (d, J = 255.3 Hz, 1 C), 119.7 (d,
J = 232.1 Hz, 1 C), 116.2, 116.2, 108.9, 106.4, 99.6. EIMS m/z 550.0 (M+).
4.1.27. 5-hydroxy-3,9-di(thiophen-2-yl)pyrano[2,3-f]chromene-4,10-dione (28a)
o
1
Yellow solid. Yield 40%. mp 222.2-222.9 C; H NMR (500 MHz, DMSO-d6) δ
13.49 (s, 1 H, OH), 9.33 (s, 1 H, 2-H), 8.93 (s, 1 H, 8-H), 7.74 (dd, J = 1.0, 3.8 Hz, 1
H, 3’-H), 7.70 (dd, J = 0.9, 4.9 Hz, 1 H, 3’’-H), 7.62-7.63 (m, 2 H, 5’, 5’’-H), 7.19 (dd,
J = 3.8, 5.1 Hz, 1 H, 4’-H), 7.13 (dd, J = 4.1, 4.9 Hz, 1 H, 4’’-H), 7.03 (s, 1 H, 6-H);
13C NMR (125 MHz, DMSO-d6) δ 178.7, 171.5, 163.7, 160.7, 155.6, 154.7, 151.0,
131.0, 129.7, 128.3, 127.9, 126.7, 126.2, 125.3, 124.2, 119.5, 118.9, 108.6, 105.9,
99.6. EIMS m/z 394.0 (M+).
4.1.28. 5-hydroxy-3,9-di(naphthalen-1-yl)pyrano[2,3-f]chromene-4,10-dione (29a)
Orange solid. Yield 10%. mp 170.8-171.4 oC; 1H NMR (500 MHz, DMSO-d6) δ
13.74 (s, 1 H, OH), 8.78 (s, 1 H, 2-H), 8.46 (s, 1 H, 8-H), 7.98-8.05 (m, 4 H, 5’, 8’, 5”,
8”-H), 7.80 (d, J = 8.5 Hz, 1 H, 4’-H), 7.72 (d, J = 8.8 Hz, 1 H, 4”-H), 7.47-7.62 (m, 8
H, 2’, 3’, 6’, 7’, 2”, 3”, 6”, 7”-H), 7.11 (s, 1 H, 6-H); 13C NMR (125 MHz, DMSO-d6)
δ 180.8, 173.0, 164.0, 161.7, 157.1, 156.4, 153.2, 133.0, 133.0, 132.0, 131.9, 129.7,
129.1, 128.7, 128.5, 128.2, 128.0, 128.0, 128.0, 126.3, 126.1, 126.1, 125.9, 125.8,

ACCEPTED MANUSCRIPT
125.8, 125.4, 125.4, 125.4, 124.5, 109.0, 106.7, 99.6. EIMS m/z 482.1 (M+).
4.1.29. 5-hydroxy-3,9-diphenylpyrano[2,3-f]chromene-4,10-dione (30a)
o
1
White solid. Yield 51%. mp 210.9-211.5 C; H NMR (500 MHz, DMSO-d6) δ
13.84 (s, 1 H, OH), 8.83 (s, 1 H, 2-H), 8.43 (s, 1 H, 8-H), 7.64 (d, J = 7.0 Hz, 2 H, 2’,
6’-H), 7.71 (d, J = 7.0 Hz, 2 H, 2’’, 6”-H), 7.38-7.49 (m, 6 H, 3’, 4’, 5’, 3”, 4”, 5”-H),
6.99 (s, 1 H, 6-H); 13C NMR (125 MHz, DMSO-d6) δ 180.4, 172.6, 164.0, 161.2,
156.2, 156.2, 156.0, 152.4, 131.3, 129.9, 129.1,129.1, 129.1, 128.5, 128.3, 128.3,
128.1, 128.1, 125.4, 124.3, 108.9, 106.6, 99.4, 99.4. EIMS m/z 382.0 (M+).
4.1.30. 3,9-dicyclopropyl-5-hydroxypyrano[2,3-f]chromene-4,10-dione (31a)
o
1
Brown solid. Yield 7%. mp 216.0-216.9 C; H NMR (500 MHz, DMSO-d6) δ
13.78 (s, 1 H, OH), 8.37 (s, 1 H, 2-H), 7.93 (s, 1 H, 8-H), 6.81 (s, 1 H, 6-H), 1.72-1.83
(m, 2 H, 1’, 1’’-H), 0.84-0.86 (m, 2 H, 2’-H), 0.74-0.80 (m, 4 H, 3’, 2’’-H), 0.59-0.62
(m, 2 H, 3’’-H); 13C NMR (125 MHz, DMSO-d6) δ 182.0, 174.3, 163.3, 161.2, 155.7,
154.1, 150.0, 126.6, 126.0, 107.7, 105.6, 98.9, 6.5, 5.9, 5.6, 5.6, 5.5, 5.5. EIMS m/z
310.1 (M+).
4.1.31. 5-hydroxy-3,7-di-o-tolylpyrano[3,2-g]chromene-4,6-dione (4b)
o
1
Pale-brown powder. Yield 46%. mp 278.3-279.0 C; H NMR (500 MHz,
DMSO-d6) δ 14.65 (s, 1 H, OH), 8.36 (s, 2 H, 2, 8-H), 7.29-7.35 (m, 5 H, ArH),
13
7.22-7.27 (m, 4 H, ArH), 2.18 (s, 6 H, 2×CH3); C NMR (125 MHz, DMSO-d6) δ
177.5, 177.5, 164.2, 159.4, 159.4, 154.7, 154.7, 137.6, 137.6, 130.7, 130.7, 130.7,
130.7, 129.8, 129.8, 128.5, 128.5, 125.6, 125.6, 125.0, 125.0, 108.1, 108.1, 95.4, 19.6,
19.6. EIMS m/z 410.2 (M+).
4.1.32. 5-hydroxy-3,7-di-m-tolylpyrano[3,2-g]chromene-4,6-dione (5b)
o
1
Yellow solid. Yield 34%. mp 262.9-263.7 C; H NMR (500 MHz, DMSO-d6) δ
14.75 (s, 1 H, OH), 8.46 (s, 2 H, 2, 8-H), 7.31-7.36 (m, 6 H, ArH), 7.21-7.24 (m, 3 H,
13
ArH), 2.35 (s, 6 H, 2×CH3); C NMR (125 MHz, DMSO-d6) δ 177.7, 177.7, 164.5,
159.1, 159.1, 154.2, 154.2, 137.3, 137.3, 130.6, 130.6, 129.6, 129.6, 128.8, 128.8,
128.1, 128.1, 126.1, 126.1, 124.0, 124.0, 108.2, 108.2, 95.2, 21.0, 21.0. EIMS m/z
410.2 (M+).
4.1.33. 5-hydroxy-3,7-di-p-tolylpyrano[3,2-g]chromene-4,6-dione (6b)
o
1
Yellow solid. Yield 48%. mp 330.5-331.4 C; H NMR (500 MHz, DMSO-d6) δ
14.79 (s, 1 H, OH), 8.46 (s, 2 H, 2, 8-H), 7.45 (d, J = 7.4 Hz, 4 H, ArH), 7.26 (d, J =
7.4 Hz, 5 H, ArH), 2.34 (s, 6 H, 2×CH3). EI-HRMS m/z 410.1162 (M+), calcd for

ACCEPTED MANUSCRIPT
C26H18O5: 410.1154.
4.1.34. 5-hydroxy-3,7-bis(2-methoxyphenyl)pyrano[3,2-g]chromene-4,6-dione (7b)
Brown powder. Yield 8%. mp >390 oC; 1H NMR (500 MHz, DMSO-d6) δ 7.79 (s,
2 H, 2, 8-H), 7.08-7.12 (m, 2 H, ArH), 6.67 (d, J = 6.2 Hz, 2 H, ArH), 6.58-6.60 (m, 4
H, ArH), 6.24 (s, 1 H, 10-H), 3.26 (s, 6 H, 2×OCH3); 13C NMR (125 MHz, DMSO-d6)
δ 177.8, 177.8, 173.1, 160.9, 160.9, 156.5, 156.5, 152.5, 152.5, 130.5, 130.5, 128.9,
128.9, 121.3, 121.3, 120.3, 120.3, 119.4, 119.4, 112.2, 112.2, 110.4, 110.4, 89.8, 54.8,
54.8. EIMS m/z 442.1 (M+).
4.1.35. 5-hydroxy-3,7-bis(3-methoxyphenyl)pyrano[3,2-g]chromene-4,6-dione (8b)
o
1
Yellow solid. Yield 21%. mp 206.1-206.7 C; H NMR (500 MHz, DMSO-d6) δ
14.76 (s, 1 H, OH), 8.51 (s, 2 H, 2, 8-H), 7.36 (t, J = 8.2 Hz, 2 H, 5’, 5’’-H), 7.26 (s, 1
H, 10-H), 7.13-7.14 (m, 4 H, ArH), 6.97-6.99 (m, 2 H, ArH), 3.79 (s, 6 H, 2×OCH3);
13C NMR (125 MHz, DMSO-d6) δ 177.5, 177.5, 164.5, 159.0, 159.0, 159.0, 159.0,
154.4, 154.4, 131.9, 131.9, 129.2, 129.2, 123.7, 123.7, 121.3, 121.3, 114.7, 114.7,
113.7, 113.7,108.2, 108.2, 95.2, 55.1, 55.1. EIMS m/z 442.1 (M+).
4.1.36. 5-hydroxy-3,7-bis(4-methoxyphenyl)pyrano[3,2-g]chromene-4,6-dione (9b)
o
1
Brown solid. Yield 27%. mp 309.2-310.1 C; H NMR (500 MHz, DMSO-d6) δ
14.81 (s, 1 H, OH), 8.44 (s, 2 H, 2, 8-H), 7.50 (d, J = 8.6 Hz, 4 H, ArH), 7.24 (s, 1 H,
10-H), 7.01 (d, J = 8.6 Hz, 4 H, ArH), 3.79 (s, 6 H, 2×OCH3). EI-HRMS m/z
442.1045 (M+), calcd for C26H18O7: 442.1053.
4.1.37. 5-hydroxy-3,7-bis(3-hydroxyphenyl)pyrano[3,2-g]chromene-4,6-dione (10b)
o
1
Dark-green solid. Yield 18%. mp 303.8-304.7 C; H NMR (500 MHz,
DMSO-d6) δ 14.80 (s, 1 H, OH), 9.50 (s, 2 H, 2×OH), 8.45 (s, 2 H, 2, 8-H), 7.21-7.25
(m, 3 H, ArH), 6.99-6.99 (m, 2 H, ArH), 6.95 (d, J = 7.7 Hz, 2 H, ArH), 6.79-6.81 (m,
2 H, ArH); 13C NMR (125 MHz, DMSO-d6) δ 177.6, 177.6, 164.5, 159.1, 159.1,
157.1, 157.1, 154.2, 154.2, 131.8, 131.8, 129.2, 129.2, 123.9, 123.9, 119.6, 119.6,
116.1, 116.1, 115.2, 115.2, 108.2, 108.2, 95.2. EIMS m/z 414.1 (M+).
4.1.38.
(11b)
3,7-bis(3,4-dimethoxyphenyl)-5-hydroxypyrano[3,2-g]chromene-4,6-dione
o
1
Yellow needle crystal. Yield 53%. mp 282.4-283.1 C; H NMR (500 MHz,
DMSO-d6) δ 14.83 (s, 1 H, OH), 8.47 (s, 2 H, 2, 8-H), 7.24 (s, 1 H, 10-H), 7.17 (d, J =
1.9 Hz, 2 H, 2’, 2’’-H), 7.12 (dd, J = 1.9, 8.4 Hz, 2 H, 6’, 6’’-H), 7.02 (d, J = 8.4 Hz, 2
H, 5’, 5’’-H), 3.79 (s, 12 H, 4×OCH3); 13C NMR (125 MHz, DMSO-d6) δ 177.8,

ACCEPTED MANUSCRIPT
177.8, 164.5, 159.0, 159.0, 153.8, 153.8, 148.9, 148.9, 148.3, 148.3, 123.6, 123.6,
123.0, 123.0, 121.5, 121.5, 112.9, 112.9, 111.6, 111.6, 108.1, 108.1, 95.1, 55.6, 55.6,
55.5, 55.5. EIMS m/z 502.2 (M+).
4.1.39. 5-hydroxy-3,7-bis(3,4,5-trimethoxyphenyl)pyrano[3,2-g]chromene-4,6-dione
(12b)
Brown powder. Yield 8%. mp 160.3-161.2 oC; 1H NMR (500 MHz, DMSO-d6) δ
14.79 (s, 1 H, OH), 8.52 (s, 2 H, 2, 8-H), 7.28 (s, 1 H, 10-H), 6.89 (s, 4 H, 2’, 6’, 2’’,
6’’-H), 3.81 (s, 12 H, 4×OCH3), 3.70 (s, 6 H, 2×OCH3); 13C NMR (125 MHz,
DMSO-d6) δ 177.7, 177.7, 164.6, 159.0, 159.0, 154.4, 154.4, 152.6, 152.6, 152.6,
152.6, 137.6, 137.6, 126.1, 126.1, 123.8, 123.8, 108.1, 108.1, 106.7, 106.7, 106.7,
106.7, 95.3, 60.1, 60.1, 60.1, 60.1, 56.0, 56.0. EIMS m/z 562.2 (M+).
4.1.40. 3,7-bis(2-fluorophenyl)-5-hydroxypyrano[3,2-g]chromene-4,6-dione (14b)
o
1
Yellow solid. Yield 42%. mp 280.5-281.4 C; H NMR (500 MHz, DMSO-d6) δ
14.50 (s, 1 H, OH), 8.52 (s, 2 H, 2, 8-H), 7.45-7.52 (m, 4 H, ArH), 7.28-7.35 (m, 5 H,
ArH); 13C NMR (125 MHz, DMSO-d6) δ 176.7, 176.7, 164.0, 160.0 (d, J = 246.0 Hz,
2 C), 159.3, 159.3, 155.4, 155.4, 132.2 (d, J = 2.3 Hz, 2 C), 130.8 (d, J = 8.1 Hz, 2 C),
124.3 (d, J = 21.5 Hz, 2 C), 119.9, 119.9, 118,5 (d, J = 15.5 Hz, 2 C), 115.5 (d, J =
21.5 Hz, 2 C), 108.1, 108.1, 95.7. EIMS m/z 418.1 (M+).
4.1.41. 3,7-bis(3-fluorophenyl)-5-hydroxypyrano[3,2-g]chromene-4,6-dione (15b)
o
1
Green-yellow solid. Yield 48%. 287.7 C sublimation; H NMR (500 MHz,
DMSO-d6) δ 14.69 (s, 1 H, OH), 8.60 (s, 2 H, 2, 8-H), 7.48-7.53 (m, 2 H, ArH),
7.43-7.46 (m, 4 H, ArH), 7.35 (s, 1 H, 10-H), 7.24-7.28 (m, 2 H, ArH). EI-HRMS m/z
418.0659 (M+), calcd for C24H12F2O5: 418.0653.
4.1.42. 3,7-bis(4-fluorophenyl)-5-hydroxypyrano[3,2-g]chromene-4,6-dione (16b)
o
1
Green-yellow solid. Yield 68%. 283.0 C sublimation; H NMR (500 MHz,
DMSO-d6) δ 14.71 (s, 1 H, OH), 8.52 (s, 2 H, 2, 8-H), 7.60-7.63 (m, 4 H, ArH),
7.27-7.31 (m, 5 H, ArH). EI-HRMS m/z 418.0653 (M+), calcd for C24H12F2O5:
418.0653.
4.1.43. 3,7-bis(2-chlorophenyl)-5-hydroxypyrano[3,2-g]chromene-4,6-dione (17b)
o
1
Red powder. Yield 16%. mp 284.1-284.8 C; H NMR (500 MHz, DMSO-d6) δ
14.45 (s, 1 H, OH), 8.48 (s, 2 H, 2, 8-H), 7.58 (d, J = 7.7 Hz, 2 H, ArH), 7.42-7.49 (m,
6 H, ArH), 7.36 (s, 1 H, 10-H); 13C NMR (125 MHz, DMSO-d6) δ 176.7, 176.7, 164.0,
159.4, 159.4, 155.3, 155.3, 133.9, 133.9, 132.6, 132.6, 130.4, 130.4, 130.0, 130.0,

ACCEPTED MANUSCRIPT
129.3, 129.3, 127.2, 127.2, 123.4, 123.4, 108.1, 108.1, 95.8. EIMS m/z 415.0 (M-36).
4.1.44. 3,7-bis(3-chlorophenyl)-5-hydroxypyrano[3,2-g]chromene-4,6-dione (18b)
o
1
Green-yellow solid. Yield 28%. mp 322.8-323.5 C; H NMR (500 MHz,
DMSO-d6) δ 14.55 (s, 1 H, OH), 8.52 (s, 2 H, 2, 8-H), 7.66-7.67 (m, 2 H, 2’, 2’’-H),
7.54-7.56 (m, 2 H, ArH), 7.45-7.50 (m, 4 H, ArH), 7.23 (s, 1 H, 10-H). EI-HRMS m/z
450.0067 (M+), calcd for C24H12Cl2O5: 450.0062.
4.1.45. 3,7-bis(4-chlorophenyl)-5-hydroxypyrano[3,2-g]chromene-4,6-dione (19b)
o
1
Green-yellow solid. Yield 27%. mp 328.8-329.7 C; H NMR (500 MHz,
DMSO-d6) δ 14.68 (s, 1 H, OH), 8.55 (s, 2 H, 2, 8-H), 7.61 (d, J = 8.2 Hz, 4 H, ArH),
7.53 (d, J = 8.2 Hz, 4 H, ArH), 7.31 (s, 1 H, 10-H). EI-HRMS m/z 450.0068 (M+),
calcd for C24H12Cl2O5: 450.0062.
4.1.46. 5-hydroxy-3,7-bis(2-nitrophenyl)pyrano[3,2-g]chromene-4,6-dione (20b)
o
1
Brown solid. Yield 16%. mp 270.7-271.5 C; H NMR (500 MHz, DMSO-d6) δ
14.14 (s, 1 H, OH), 8.69 (s, 2 H, 2, 8-H), 8.14-8.16 (m, 2 H, ArH), 7.85-7.88 (m, 2 H,
13
ArH), 7.72-7.75 (m, 2 H, ArH), 7.62-7.63 (m, 2 H, ArH), 7.44 (s, 1 H, 10-H); C
NMR (125 MHz, DMSO-d6) δ 176.4, 176.4, 164.0, 159.5, 159.5, 154.0, 154.0, 148.9,
148.9, 134.0, 134.0, 132.6, 132.6, 130.2, 130.2, 125.1, 125.1, 124.4, 124.4, 123.3,
123.3, 107.5, 107.5, 95.0. ESIMS m/z 473.2 (M+1).
4.1.47. 3,7-bis(2-chloro-6-fluorophenyl)-5-hydroxypyrano[3,2-g]chromene-4,6-dione
(22b)
o
1
Brown solid. Yield 22%. mp 206.5-207.3 C; H NMR (500 MHz, DMSO-d6) δ
14.24 (s, 1 H, OH), 8.64 (s, 2 H, 2, 8-H), 7.53-7.58 (m, 2 H, 5’, 5’’-H), 7.49 (d, J =
13
8.1 Hz, 2 H, 3’, 3’’-H), 7.43 (s, 1 H, 10-H), 7.36-7.39 (m, 2 H, 4’, 4’’-H); C NMR
(125 MHz, DMSO-d6) δ 176.2, 176.2, 163.8, 160.7 (d, J = 246.4 Hz, 2 C), 159.5,
159.5, 156.7, 156.7, 135.1 (d, J = 3.0 Hz, 2 C), 131.7 (d, J = 9.4 Hz, 2 C), 125.4 (d, J
= 2.6 Hz, 2 C), 118.2 (d, J = 19.6 Hz, 2 C), 117.2, 117.2, 114.5 (d, J = 22.4 Hz, 2 C),
108.1, 108.1, 96.3. EIMS m/z 451.0 (M+).
4.1.48.
5-hydroxy-3,7-bis(2-(trifluoromethyl)phenyl)pyrano[3,2-g]chromene-4,6-dione (24b)
o
1
Light-yellow solid. Yield 16%. mp 192.9-193.5 C; H NMR (500 MHz,
DMSO-d6) δ 14.34 (s, 1 H, OH), 8.45 (s, 2 H, 2, 8-H), 7.86 (d, J = 7.8 Hz, 2 H, ArH),
7.75-7.78 (m, 2 H, ArH), 7.67-7.70 (m, 2 H, ArH), 7.52 (d, J = 7.5 Hz, 2 H, ArH),
7.37 (s, 1 H, 10-H). EI-HRMS m/z 518.0580 (M+), calcd for C26H12F6O5: 518.0589.

ACCEPTED MANUSCRIPT
4.1.49.
5-hydroxy-3,7-bis(3-(trifluoromethyl)phenyl)pyrano[3,2-g]chromene-4,6-dione (25b)
o
1
Brown solid. Yield 6%. mp 220.0-221.3 C; H NMR (500 MHz, DMSO-d6) δ
14.60 (s, 1 H, OH), 8.65 (s, 2 H, 2, 8-H), 7.95 (s, 2 H, 2’, 2’’-H), 7.87 (d, J = 7.5 Hz, 2
H, ArH), 7.78 (d, J = 7.7 Hz, 2 H, ArH), 7.68-7.71 (m, 2 H, 5’, 5’’-H), 7.35 (s, 1 H,
10-H). EI-HRMS m/z 518.0580 (M+), calcd for C26H12F6O5: 518.0589.
4.1.50.
5-hydroxy-3,7-bis(4-(trifluoromethoxy)phenyl)pyrano[3,2-g]chromene-4,6-dione
(27b)
o
1
Green-yellow solid. Yield 23%. mp 282.9-283.7 C; H NMR (500 MHz,
DMSO-d6) δ 14.66 (s, 1 H, OH), 8.57 (s, 2 H, 2, 8-H), 7.70 (d, J = 8.5 Hz, 4 H, ArH),
7.46 (d, J = 8.5 Hz, 4 H, ArH), 7.33 (s, 1 H, 10-H). EI-HRMS m/z 550.0482 (M+),
calcd for C26H12F6O7: 550.0487.
4.1.51. 5-hydroxy-3,7-di(thiophen-2-yl)pyrano[3,2-g]chromene-4,6-dione (28b)
o
1
Brown solid. Yield 3%. mp 344.1-344.7 C; H NMR (500 MHz, DMSO-d6) δ
14.51 (s, 1 H, OH), 8.98 (s, 2 H, 2, 8-H), 7.63-7.66 (m, 4 H, 3’, 5’, 3’’, 5’’-H), 7.35 (s,
1 H, 10-H), 7.15 (dd, J = 3.8, 4.9 Hz, 2 H, 4’, 4’’-H); 13C NMR (125 MHz, DMSO-d6)
δ 176.3, 176.3, 164.2, 158.6, 158.6, 152.9, 152.9, 130.5, 130.5, 127.8, 127.8, 126.4,
126.4, 124.6, 124.6, 118.1, 118.1, 107.6, 107.6, 95.6. EIMS m/z 393.9 (M+).
4.1.52. 5-hydroxy-3,7-di(naphthalen-1-yl)pyrano[3,2-g]chromene-4,6-dione (29b)
Orange solid. Yield 31%. mp 231.7-232.4 oC; 1H NMR (500 MHz, DMSO-d6) δ
14.60 (s, 1 H, OH), 8.53 (s, 2 H, 2, 8-H), 7.99-8.03 (m, 4 H, ArH), 7.76 (d, J = 8.4 Hz,
2 H, ArH), 7.54-7.61 (m, 4 H, ArH), 7.48-7.52 (m, 4 H, ArH), 7.43 (s, 1 H, 10-H); 13C
NMR (125 MHz, DMSO-d6) δ 178.0, 178.0, 164.2, 159.6, 159.6, 155.1, 155.1, 133.0,
133.0, 132.0, 132.0, 128.9, 128.9, 128.9, 128.9, 128.4, 128.4, 128.1, 128.1, 126.2,
126.2, 126.0, 126.0, 125.8, 125.8, 125.4, 125.4, 124.1, 124.1, 108.2, 108.2, 95.5.
EIMS m/z 482.1 (M+).
4.1.53. 5-hydroxy-3,7-diphenylpyrano[3,2-g]chromene-4,6-dione (30b)
o
1
Yellow solid. Yield 33%. mp 298.2-299.1 C; H NMR (500 MHz, DMSO-d6) δ
14.77 (s, 1 H, OH), 8.51 (s, 2 H, 2, 8-H), 7.52 (d, J = 7.1 Hz, 4 H, 2’, 6’, 2’’, 6’’-H),
7.44-7.47 (m, 4 H, 3’, 5’, 3’’, 5’’-H), 7.39-7.42 (m, 2 H, 4’, 4’’-H), 7.29 (s, 1 H, 10-H);
13C NMR (125 MHz, DMSO-d6) δ 177.6, 177.6, 164.5, 159.1, 159.1, 154.3, 154.3,
130.7, 130.7, 129.0, 1290, 129.0, 129.0, 129.0, 128.2, 128.2, 128.2, 128.2, 128.2,

ACCEPTED MANUSCRIPT
123.9, 123.9, 108.2, 108.2, 95.2. EIMS m/z 382.1 (M+).
4.1.54. 3,7-dicyclopropyl-5-hydroxypyrano[3,2-g]chromene-4,6-dione (31b)
o
1
Brown solid. Yield 29%. mp 230.8-231.7 C; H NMR (500 MHz, DMSO-d6) δ
14.69 (s, 1 H, OH), 8.02 (s, 2 H, 2, 8-H), 7.04 (s, 1 H, 10-H), 1.71-1.76 (m, 2 H, ArH),
0.78-0.82 (m, 4 H, ArH), 0.61-0.64 (m, 4 H, ArH); 13C NMR (125 MHz, DMSO-d6) δ
179.3, 179.3, 163.7, 159.0, 159.0, 152.0, 152.0, 125.0, 125.0, 107.1, 107.1, 94.8, 6.1,
6.1, 5.3, 5.3, 5.3, 5.3. EIMS m/z 310.1 (M+).
4.2. Biological evaluation
4.2.1. Cell line establishment
Flp-InTM-293 cells were cultured in DMEM medium supplemented with 10% fetal
o
bovine serum and selected with 100 ꢀg/mL zeocin at 37 C, 95% humidity and 5%
CO2. The ABCB1/pcDNA5 containing full length ABCB1 cDNA was constructed in
our previous studies [23, 24]. The constructed ABCB1/pcDNA5 plasmid and pOG44
(the Flprecombinase expression plasmid) were co-transfected into the Flp-InTM-293
cells and were selected on the basis of hygromycin B resistance. The protein and
mRNA expressions of P-gp were confirmed by Western blot analysis and real-time
quantitative RT-PCR, respectively, in our previous studies [23, 24]. Human cervical
carcinoma cell line HeLaS3 was purchased from Bioresource Collection and Research
Center (Hsinchu, Taiwan). The multidrug resistant human cervical cancer cell line
KBvin was kindly provided by Dr. Kuo-Hsiung Lee (University of North Carolina,
Chapel Hill, U.S.A) and maintained with vincristine in a fixed period.
4.2.2. Calcein-AM uptake assay
For Calcein-AM uptake study, 1×105 cells were placed on 96-well black plates
and cultured overnight. After pre-incubation with warm Hanks’ balanced salt solution
(HBSS) for 30 min, test compounds were added for 30 min in triplicate. Then,
Calcein AM (1 ꢀM) was added and incubated for another 30 min. After washed by
ice-cold HBSS, Calcein fluorescence generated within the cells was analyzed by the
SpectraMax Gemini XS microplate spectrometer (Molecular Devices Co., Sunnyvale,
CA, USA) with the excitation set at 485 nm and the emission set at 535 nm. Each
experiment was performed at least three times, each in triplicate on different days.
4.2.3. Cell viability assay
The effect of test compounds on the cell viability of Flp-InTM-293,
ABCB1/Flp-InTM-293, HeLaS3, and KBvin cells was determined by SRB assay as
previously described [25]. The influences of combinations of test compounds and

ACCEPTED MANUSCRIPT
chemotherapeutic agents on cell proliferation were further evaluated by SRB assay as
well.
4.3. Molecular modeling
4.3.1. Ligand preparation
The structures of the benzodipyrano derivatives 5a and 5b were built and energy
minimized by Discovery studio 3.1. Full minimization tool was used to generate low
energy 3D conformers of the minimized structures, default settings was used. The
resultant ligand structures were eventually docked at drug binding domain of P-gp.
4.3.2. Docking protocol
To determine the probable binding site for compound 5a and 5b, the ligand
structure was docked at drug binding domain of P-gp using the “LibDock” mode of
Discovery Studio 3.1 with the default parameters. The protein crystal structure of
mouse P-gp obtained from protein data bank (PDB ID: 4Q9I) [26].
Acknowledgments
This work was supported by grants from the Ministry of Science and Technology
of
Taiwan
(MOST104-2815-C-039-022-B;
MOST103-2815-C-039-023-B;
NSC101-2320-B-039-010-MY3) and China Medical University of Taiwan
(CMU104-SR-22; CMU103-SR-23). The multidrug resistant human cervical cancer
cell line KBvin was kindly provided by professor Kuo-Hsiung Lee (University of
North Carolina, Chapel Hill, U.S.A.).
Appendix A. Supplementary data

ACCEPTED MANUSCRIPT
References
[1] C. Ramalhete, J. Molnar, S. Mulhovo, V.E. Rosario, M.J. Ferreira, New potent
P-glycoprotein modulators with the cucurbitane scaffold and their synergistic
interaction with doxorubicin on resistant cancer cells, Bioorg. Med. Chem., 17
(2009) 6942—6951.
[2] L. Jiao, Q. Qiu, B. Liu, T. Zhao, W. Huang, H. Qian, Design, synthesis and
evaluation of novel triazole core based P-glycoprotein-mediated multidrug
resistance reversal agents, Bioorg. Med. Chem., 22 (2014) 6857—6866.
[3] G.A. Sawada, T.J. Raub, J. William Higgins, N.K. Brennan, T.M. Moore, G.
Tombline, M.R. Detty, Chalcogenopyrylium dyes as inhibitors/modulators of
P-glycoprotein in multidrug-resistant cells, Bioorg. Med. Chem., 16 (2008)
9745—9756.
[4] S.V. Ambudkar, S. Dey, C.A. Hrycyna, M. Ramachandra, I. Pastan, M.M.
Gottesman, Biochemical, cellular, and pharmacological aspects of the multidrug
transporter, Annu. Rev. Pharmacol. Toxicol., 39 (1999) 361—398.
[5] D.W. Shen, S. Goldenberg, I. Pastan, M.M. Gottesman, Decreased accumulation
of [14C]carboplatin in human cisplatin-resistant cells results from reduced
energy-dependent uptake, J. Cell Physiol., 183 (2000) 108—116.
[6] C. Xu, C.Y. Li, A.N. Kong, Induction of phase I, II and III drug
metabolism/transport by xenobiotics, Arch. Pharm. Res., 28 (2005) 249—268.
[7] S.W. Lowe, H.E. Ruley, T. Jacks, D.E. Housman, p53-dependent apoptosis
modulates the cytotoxicity of anticancer agents, Cell, 74 (1993) 957—967.
[8] E. Capparelli, L. Zinzi, M. Cantore, M. Contino, M.G. Perrone, G. Luurtsema, F.
Berardi, R. Perrone, N.A. Colabufo, SAR studies on tetrahydroisoquinoline
derivatives: the role of flexibility and bioisosterism to raise potency and
selectivity toward P-glycoprotein, J. Med. Chem., 57 (2014) 9983—9994.
[9] Z. Wang, I.L. Wong, F.X. Li, C. Yang, Z. Liu, T. Jiang, T.F. Jiang, L.M. Chow, S.B.
Wan, Optimization of permethyl ningalin B analogs as P-glycoprotein inhibitors,
Bioorg. Med. Chem., 23 (2015) 5566—5573.
[10] T. Tsuruo, H. Iida, S. Tsukagoshi, Y. Sakurai, Overcoming of vincristine
resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of
vincristine and vinblastine by verapamil, Cancer Res., 41 (1981) 1967—1972.
[11] W. Van de Vrie, E.E. Gheuens, N.M. Durante, E.A. De Bruijn, R.L. Marquet, A.T.
Van Oosterom, A.M. Eggermont, In vitro and in vivo chemosensitizing effect of
cyclosporin A on an intrinsic multidrug-resistant rat colon tumour, J. Cancer Res.

ACCEPTED MANUSCRIPT
Clin. Oncol., 119 (1993) 609—614.
[12] T. Tsuruo, H. Iida, Y. Kitatani, K. Yokota, S. Tsukagoshi, Y. Sakurai, Effects of
quinidine and related compounds on cytotoxicity and cellular accumulation of
vincristine and adriamycin in drug-resistant tumor cells, Cancer Res., 44 (1984)
4303—4307.
[13] V. Fischer, A. Rodriguez-Gascon, F. Heitz, R. Tynes, C. Hauck, D. Cohen, A.E.
Vickers, The multidrug resistance modulator valspodar (PSC 833) is metabolized
by human cytochrome P450 3A. Implications for drug-drug interactions and
pharmacological activity of the main metabolite, Drug Metab. Dispos., 26 (1998)
802—811.
[14] E. Warner, D. Hedley, I. Andrulis, R. Myers, M. Trudeau, D. Warr, K.I. Pritchard,
M. Blackstein, P.E. Goss, E. Franssen, K. Roche, S. Knight, S. Webster, R.A.
Fraser, S. Oldfield, W. Hill, R. Kates, Phase II study of dexverapamil plus
anthracycline in patients with metastatic breast cancer who have progressed on
the same anthracycline regimen, Clin. Cancer Res., 4 (1998) 1451—1457.
[15] H. Thomas, H.M. Coley, Overcoming multidrug resistance in cancer: an update
on the clinical strategy of inhibiting p-glycoprotein, Cancer control : journal of
the Moffitt Cancer Center, 10 (2003) 159—165.
[16] L. Pusztai, P. Wagner, N. Ibrahim, E. Rivera, R. Theriault, D. Booser, F.W.
Symmans, F. Wong, G. Blumenschein, D.R. Fleming, R. Rouzier, G. Boniface,
G.N. Hortobagyi, Phase II study of tariquidar, a selective P-glycoprotein inhibitor,
in patients with chemotherapy-resistant, advanced breast carcinoma, Cancer, 104
(2005) 682—691.
[17] J.S. Lagas, R.A. van Waterschoot, V.A. van Tilburg, M.J. Hillebrand, N.
Lankheet, H. Rosing, J.H. Beijnen, A.H. Schinkel, Brain accumulation of
dasatinib is restricted by P-glycoprotein (ABCB1) and breast cancer resistance
protein (ABCG2) and can be enhanced by elacridar treatment, Clin. Cancer Res.,
15 (2009) 2344—2351.
[18] I.E. Kuppens, E.O. Witteveen, R.C. Jewell, S.A. Radema, E.M. Paul, S.G.
Mangum, J.H. Beijnen, E.E. Voest, J.H. Schellens, A phase I, randomized,
open-label, parallel-cohort, dose-finding study of elacridar (GF120918) and oral
topotecan in cancer patients, Clin. Cancer Res., 13 (2007) 3276—3285.
[19] C.A. McDevitt, R. Callaghan, How can we best use structural information on
P-glycoprotein to design inhibitors?, Pharmacol. Ther., 113 (2007) 429—441.
[20] M.L. Amin, P-glycoprotein Inhibition for Optimal Drug Delivery, Drug target
insights, 7 (2013) 27—34.
[21] N.U.-D. Khan, N. Parveen, M.P. Singh, R. Singh, B. Achari, P.P.G. Dastidar, P.K.
Dutta, Two isomeric benzodipyranone derivatives from Calophyllum inophyllum,

ACCEPTED MANUSCRIPT
Phytochemistry, 42 (1996) 1181—1183.
[22] J.R. Bantick, H. Cairns, A. Chambers, R. Hazard, J. King, T.B. Lee,
Benzodipyran derivatives with antiallergic activity, J. Med. Chem., 19 (1976)
817—821.
[23] C.C. Hung, M.H. Chiou, Y.N. Teng, Y.W. Hsieh, C.L. Huang, H.Y. Lane,
Functional impact of ABCB1 variants on interactions between P-glycoprotein
and methadone, PloS one, 8 (2013) e59419.
[24] C.C. Hung, H.H. Liou, YC-1, a novel potential anticancer agent, inhibit
multidrug-resistant protein via cGMP-dependent pathway, Invest. New Drugs, 29
(2011) 1337—1346.
[25] K. Nakagawa-Goto, A. Oda, E. Hamel, E. Ohkoshi, K.H. Lee, M. Goto,
Development of a novel class of tubulin inhibitor from desmosdumotin B with a
hydroxylated bicyclic B-ring, J. Med. Chem., 58 (2015) 2378—2389.
[26] P. Szewczyk, H. Tao, A.P. McGrath, M. Villaluz, S.D. Rees, S.C. Lee, R. Doshi,
I.L. Urbatsch, Q. Zhang, G. Chang, Snapshots of ligand entry, malleable binding
and induced helical movement in P-glycoprotein, Acta Cryst. D, 71 (2015)
732-741.

ACCEPTED MANUSCRIPT
Table 1. Calcein-AM uptake assay of benzodipyranone derivatives.
P-gp inhibition fold
CPD
R
2.5 ꢀM
5 ꢀM
verapamil
4a
2.39±0.18
1.53±0.07
2.05±0.06
2.04±0.06
1.56±0.09
1.28±0.09
1.84±0.01
0.99±0.02
1.64±0.03
0.75±0.05
0.70±0.07
0.67±0.05
0.82±0.02
0.84±0.08
1.09±0.05
0.82±0.03
0.82±0.02
1.03±0.01
0.75±0.02
0.64±0.03
0.81±0.01
0.81±0.01
1.15±0.04
0.90±0.01
1.04±0.08
0.83±0.05
0.85±0.02
1.29±0.04
0.63±0.05
2-methylphenyl
3-methylphenyl
1.15±0.04
1.58±0.06
1.58±0.07
1.40±0.09
1.19±0.05
1.54±0.04
1.01±0.02
1.45±0.03
0.76±0.08
0.72±0.08
0.76±0.06
0.96±0.03
0.92±0.07
0.98±0.05
0.83±0.04
0.76±0.03
0.98±0.02
0.88±0.02
0.66±0.01
0.80±0.01
0.76±0.01
1.10±0.05
0.87±0.01
0.97±0.06
0.88±0.04
0.87±0.02
1.04±0.11
0.80±0.06
5a
4-methylphenyl
6a
2-methoxyphenyl
3-methoxyphenyl
4-methoxylphenyl
3-hydroxyphenyl
3,4-dimethoxyphenyl
3,4,5-trimethoxyphenyl
3,4-methylenedioxyphenyl
2-fluorophenyl
7a
8a
9a
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
20a
21a
22a
23a
24a
25a
26a
27a
28a
29a
30a
31a
3-fluorophenyl
4-fluorophenyl
2-chlorophenyl
3-chlorophenyl
4-chlorophenyl
2-nitrophenyl
4-nitrophenyl
2-chloro-6-fluorophenyl
3,4-dichlorophenyl
2-trifluoromethylphenyl
3-trifluoromethylphenyl
4-trifluoromethylphenyl
4-trifluoromethoxyphenyl
thiophen-2-yl
1-naphthyl
phenyl
cyclopropyl

ACCEPTED MANUSCRIPT
Table 2. Calcein-AM uptake assay of symmetrical benzodipyranone derivatives.
P-gp inhibition fold
CPD
R
2.5 ꢀM
5 ꢀM
verapamil
4b
2.39±0.18
0.81±0.02
0.93±0.05
1.81±0.12
0.82±0.05
0.85±0.04
1.30±0.02
1.19±0.09
0.86±0.02
0.72±0.01
0.89±0.05
0.75±0.02
1.30±0.01
0.85±0.01
0.87±0.09
1.08±0.04
0.83±0.02
1.06±0.06
1.36±0.07
1.18±0.10
0.63±0.12
0.67±0.02
0.64±0.02
0.90±0.05
0.86±0.03
0.87±0.05
0.86±0.03
1.62±0.10
0.76±0.02
1.00±0.06
0.87±0.06
1.33±0.06
0.81±0.01
0.80±0.03
0.92±0.06
0.60±0.03
0.85±0.08
0.71±0.01
0.50±0.02
1.06±0.05
0.85±0.05
0.95±0.02
1.32±0.07
1.27±0.08
0.62±0.02
0.61±0.04
0.72±0.03
1.04±0.08
0.86±0.02
2-methylphenyl
3-methylphenyl
4-methylphenyl
2-methoxyphenyl
3-methoxyphenyl
4-methoxylphenyl
3-hydroxyphenyl
3,4-dimethoxyphenyl
3,4,5-trimethoxyphenyl
2-fluorophenyl
5b
6b
7b
8b
9b
10b
11b
12b
14b
15b
16b
17b
18b
19b
20b
22b
24b
25b
27b
28b
29b
30b
31b
3-fluorophenyl
4-fluorophenyl
2-chlorophenyl
3-chlorophenyl
4-chlorophenyl
2-nitrophenyl
2-chloro-6-fluorophenyl
2-trifluoromethylphenyl
3-trifluoromethylphenyl
4-trifluoromethoxyphenyl
thiophen-2-yl
1-naphthyl
phenyl
cyclopropyl

ACCEPTED MANUSCRIPT
Table 3. Reversal effect of benzodipyranone derivatives on ABCB1 substrates in
Flp-InTM-293 and ABCB1/Flp-InTM-293 cell.a
Flp-InTM-293
ABCB1/Flp-InTM-293
IC50 (nM)
RF
1.0
1.5
1.8
2.7
4.2
1.0
1.3
1.0
7.8
9.1
6.6
7.1
7.8
8.2
1.0
1.2
1.6
0.9
2.1
1.2
1.3
IC50 (nM)
RF
1.0
10.8
16.1
2.1
8.2
8.8
9.5
1.0
15.6
21.0
1.4
1.6
4.1
9.7
1.0
1.4
1.6
1.6
1.7
1.5
1.5
0.5387 ± 0.0610
0.3664 ± 0.0045*
0.3065 ± 0.0149*
0.1992 ± 0.0656*
0.1278 ± 0.0173*
0.5352 ± 0.0066
0.4163 ± 0.0074
5.9705 ± 0.1791
0.7608 ± 0.0231*
0.6595 ± 0.0117*
0.9091 ± 0.0064*
0.8448 ± 0.0075*
0.7623 ± 0.0012*
0.7324 ± 0.0062*
92.1164 ± 13.6856
76.2722 ± 3.1190
58.0384 ± 1.8049*
97.5533 ± 3.0147
43.4454 ± 1.9503*
78.7184 ± 2.4492
71.0428 ± 1.7621
644.78 ± 2.60
59.6 ± 0.72*
Paclitaxel
+5a (8 ꢀM)
+5a (10 ꢀM)
+9a (8 ꢀM)
+9a (10 ꢀM)
+11a (8 ꢀM)
+11a (10 ꢀM)
Vincristine
+5a (8 ꢀM)
+5a (10 ꢀM)
+9a (8 ꢀM)
+9a (10 ꢀM)
+11a (8 ꢀM)
+11a (10 ꢀM)
Doxorubicin
+5a (8 ꢀM)
+5a (10 ꢀM)
+9a (8 ꢀM)
+9a (10 ꢀM)
+11a (8 ꢀM)
+11a (10 ꢀM)
40.12 ± 1.37*
302.78 ± 44.35*
78.82 ± 5.04*
73.14 ± 2.83*
67.67 ± 3.12*
823.13 ± 15.79
52.60 ± 0.93*
39.11 ± 1.07*
577.20 ± 11.88*
499.76 ± 19.66*
203.01 ± 29.35*
84.95 ± 8.72*
914.83 ± 15.85
664.36 ± 35.25*
586.26 ± 11.67*
575.77 ± 14.99*
544.21 ± 9.30*
597.21 ± 5.14*
594.42 ± 5.91*
aRFꢃReversal fold of benzodipyranone derivatives. RF = IC50 of anticancer drugs to the cell / IC50 of
combination with benzodipyranone derivatives and anticancer drugs. *pꢁ0.05 compared with
substrate drug transport with tested compounds.