New Seco-DSP derivatives as potent chemosensitizers

Article abstract of DOI:10.1016/j.ejmech.2020.112555

Thirty-four seco-3′R,4′R-disubstituted-2′,2′-dimethyldihydropyrano[2,3-f]chromone (seco-DSP) derivatives were designed, synthesized and evaluated for chemo-reversal activity when combined with paclitaxel or vincristine in P-gp overexpressing A2780/T and KB-VIN drug-resistant cancer cell lines. Most of the compounds displayed moderate to significant MDR reversal activities. Compound 7e showed the most potent chemo-sensitization activity with more than 1471 reversal ratio at a concentration of 10 μM, which was higher than verapamil (VRP) (212-fold). Unexpectedly the newly synthesized compounds did not show chemosensitization activities in a non-P-gp overexpressing cisplatin resistant human ovarian cancer cell line (A2780/CDDP), implying that the MDR reversal effects might be associated with P-gp overexpression. Moreover, the compounds did not exhibit significant anti-proliferative activities against non-tumorigenic cell lines (HUVEC, HOSEC and T29) compared to VRP at the tested concentration and might be safer than VRP. In preliminary pharmacological mechanism studies, the compounds increased accumulation of DOX and promoted P-gp ATPase activity in A2780/T cell lines. Western blot analysis indicated they did not affect the expression level of P-gp in the tested MDR cell lines. Thus, further studies on these seco-DSP derivatives are merited with the goal of developing a desirable chemosensitizer drug candidate.

Full text of DOI:10.1016/j.ejmech.2020.112555

Journal Pre-proof
New Seco-DSP derivatives as potent chemosensitizers
Qi Wan, Xin Jin, Yalan Guo, Zhihui Yu, Shiqi Guo, Susan Morris-Natschke, Kuo-
Hsiung Lee, Hongrui Liu, Ying Chen
PII:
S0223-5234(20)30527-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.112555
EJMECH 112555
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 January 2020
Revised Date: 28 May 2020
Accepted Date: 7 June 2020
Please cite this article as: Q. Wan, X. Jin, Y. Guo, Z. Yu, S. Guo, S. Morris-Natschke, K.-H. Lee, H.
Liu, Y. Chen, New Seco-DSP Derivatives as Potent Chemosensitizers, European Journal of Medicinal
Chemistry, https://doi.org/10.1016/j.ejmech.2020.112555.
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New Seco-DSP Derivatives as Potent Chemosensitizers
a, §
b, §
a
a
a
Qi Wan , Xin Jin , Yalan Guo , Zhihui Yu , Shiqi Guo , Susan Morris-Natschke
c
c, d,*
b,*
a,*
,
Kuo-Hsiung Lee
, Hongrui Liu and Ying Chen
a
b
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, China
Department of Pharmacology and Biochemistry, School of Pharmacy, Fudan University,
Shanghai, China
c
Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of
North Carolina, Chapel Hill, NC 27599-7568, USA
d
Chinese Medicine Research and Development Center, China Medical University and Hospital,
Taichung 40402, Taiwan
§
Q. W. and X. J. are co-first authors and contributed equally to this work.
∗
Corresponding authors. Tel.: +1 919 962 0066; Fax: +1 919 966 3893 (K.H.L.);
+86-021-5198-0043 (H.R.L.); +86-021-5198-0116 (Y.C.)
E-mail addresses: khlee@unc.edu; liuhr@fudan.eud.cn; yingchen71@fudan.edu.cn
Abstract
Thirty-four seco-3'R,4'R-disubstituted-2',2'-dimethyldihydropyrano[2,3-f]chromone
(seco-DSP) derivatives were designed, synthesized and evaluated for chemo-reversal
activity when combined with paclitaxel or vincristine in P-gp overexpressing A2780/T
and KB-VIN drug-resistant cancer cell lines. Most of the compounds displayed
moderate to significant MDR reversal activities. Compound 7e showed the most
potent chemo-sensitization activity with more than 1471 reversal ratio at a
concentration of 10 μM, which was higher than verapamil (VRP) (212-fold).
Unexpectedly the newly synthesized compounds did not show chemosensitization
activities in a non-P-gp overexpressing cisplatin resistant human ovarian cancer cell
line (A2780/CDDP), implying that the MDR reversal effects might be associated with
P-gp overexpression. Moreover, the compounds did not exhibit significant
anti-proliferative activities against non-tumorigenic cell lines (HUVEC, HOSEC and
T29) compared to VRP at the tested concentration and might be safer than VRP. In

preliminary pharmacological mechanism studies, the compounds increased
accumulation of DOX and promoted P-gp ATPase activity in A2780/T cell lines.
Western blot analysis indicated they did not affect the expression level of P-gp in the
tested MDR cell lines. Thus, further studies on these seco-DSP derivatives are merited
with the goal of developing a desirable chemosensitizer drug candidate.
Keywords: Seco-DSP
•
MDR reversal activity
•
Chemosensitizer P-gp
•
1
. Introduction
Tumor cell resistance, especially multidrug resistance (MDR), is a major obstacle to
[
1]
cancer treatment . Statistically, 90% of failures in metastatic cancer chemotherapy
[
2]
are caused by multidrug resistance . Reasons for the production of MDR in tumor
cells include i) MDR gene expression and P-glycoprotein (P-gp)-mediated drug efflux
[
3]
;
ii) reduced tumor sensitivity resulting from decreased expression levels of
[
4]
intracellular DNA topoisomerase (Topo II) ; iii) apoptosis-related gene expression,
[
5]
such as bc1-2, mutant p53, etc. ; iv) decreased intracellular drug concentration
resulting from multidrug resistance protein (MRP) recognition and coupling of
[
6]
chemotherapeutic drugs ; v) enhanced drug pump function caused by intracellular
[
7]
protein kinase C (PKC) promoting phosphorylation of P-gp ; and vi) up-regulation
[
8]
of caveolin and some of its components in tumor multidrug-resistant cells . Since
overexpression of P-gp leading to MDR is very common, the development of P-gp
inhibitors is one of the most effective strategies to overcome MDR mediated by P-gp
overexpression.
Presently, three generations of P-gp inhibitors have been developed. Verapamil (VRP),
a representative first-generation P-gp inhibitor, enhances the intracellular
[
9]
accumulation of many anticancer drugs, e.g., doxorubicin (DOX) . However, due to
strong toxic side effects at its effective concentration, it has not been developed for
[
10-11]
use in cancer chemotherapy
. The second-generation P-gp inhibitors displayed
improved affinity for P-gp and enhanced activity compared to the first-generation
[
12]
inhibitors. However, strong CYP3A4 inhibitory activity remained
. Although the
third-generation P-gp inhibitors generally have stronger P-gp affinity and fewer toxic

[13]
side effects compared with the first- and second-generation inhibitors , currently,
no safe and effective P-gp inhibitor has been approved and marketed.
Wu, J. Y. et al. reported that (±)-praeruptorin A (PA, Figure 1), a pyranocoumarin
natural product isolated from Peucedanum Praeruptorum Dunn, exhibited multidrug
[
14]
resistance reversal activity
. Through structural modification, a series of
(±)-3'-O-4'-O-dicinnamoyl-cis-khellactone (DCK) compounds were synthesized as
non-competitive P-gp inhibitors. Notably, (±)-3'-O-4'-O-bis (3,4-dimethoxy-
cinnamoyl)-cis-khellactone (DMDCK) displayed remarkable activity with reversal
ratios ranging from 110- to 167-fold at a concentration of 4 μM when combined with
[
15]
different anticancer drugs in MDR HepG2/Dox cell lines
. Moreover,
3
'R,4'R-disubstituted-2',2'-dimethyldihydropyrano[2,3-f]chromone (DSP) derivatives,
obtained by scaffold hopping from DMDCK, also showed good MDR reversal
activity. Among them, the resistance reversal ratio of DSP-1 in vincristine
[
16]
(VCR)-resistant KB-VIN cells was more than 300-fold
.
Figure 1. Structures of PA and DMDCK.
In our previous research aimed at the development of a desirable P-gp inhibitor, we
opened the C-ring of 4-methyl-DMDCK to prepare seco-DMDCK derivatives
[
17]
(Figure 2)
. The synthesized compounds showed better multidrug resistance
reversal activity than 4-methyl-DMDCK. Especially, the drug resistance reversal
ratios of seco-DMDCK-1 in paclitaxel (PTX)-resistant A2780/T cells and
VCR-resistant KB-VIN cells were more than 700-folds, much better than those of
VRP.

Figure 2. Seco-C-ring strategy for 4-methyl-DMDCK.
Motivated by the above results with DSP-1 and seco-4-methyl-DMDCK compounds,
we further designed and synthesized 34 seco-DSP derivatives (Figure 3) based on
both modification strategies. The newly synthesized compounds with lower molecular
weights and fewer chiral centers might show maintained or even enhanced MDR
reversal activity but improved drug-like properties. Herein, the synthesis and
bioactivity evaluation of seco-DSPs containing 7-methoxy, -ethoxy or -isopropoxy
moieties and various acyl substituted 8-hydroxymethyl groups are reported.
Figure 3. Seco-C-ring strategy for new derivatives.
2
. Results and Discussion
2
.1 Chemistry
As shown in Scheme 1, Friedel-Crafts acylation of resorcinol with acetic acid using
ZnCl as a catalyst gave 2,4-dihydroxyacetophenone. The 4-hydroxy group was then
2
selectively protected to provide 1-(2-hydroxy-4-(methoxymethoxy)phenyl)ethanone,
which was condensed with ethyl propionate using NaH in THF to give intermediate 1.

Compound 4 was synthesized in three steps: cyclization of 1 with concentrated HCl in
EtOH to give 2, a Duff reaction to introduce an aldehyde group at C-8 to give 3, and
reduction of the aldehyde group using NaBH in MeOH. Then, the 7-hydroxyl of 4
4
was alkylated successively with iodomethane, bromoethane and 2-bromopropane in
the presence of K CO and KI to provide the three key intermediates 5a-c. Finally, 34
2
3
seco-DSP derivatives (6a-w, 7a-f and 8a-e) were prepared by esterification of 5a-c
with various aromatic acids catalyzed by 4-dimethylaminopyridine (DMAP) and
1
-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (EDCI) in yields
from 40.8% to 57.5%.
Scheme 1. Syntheses of seco-DSP derivatives (6a-w, 7a-f and 8a-e). Reagents and
conditions: (i) ZnCl , HOAc, reflux; (ii) MOMCl, K CO , acetone, rt; (iii) ethyl
2
2
3
propionate, NaH, THF, reflux; (iv) EtOH, conc. HCl, reflux; (v)
hexamethylenetetramine, HOAc, 95 °C, then HCl (conc.HCl:H O = 84:100 v/v),
2
7
0 °C; (vi) NaBH , MeOH, rt; (vii) iodomethane, bromoethane or 2-bromopropane,
4
K CO , KI, acetone, reflux; (viii) aromatic acids, EDCI, DMAP, CH Cl , 0 °C-r.t.
2
3
2
2

2
.2 Biological evaluation
.2.1. In vitro cytotoxicity
2
The 34 target compounds 6a-w, 7a-f and 8a-e were screened for cytotoxicity at a
concentration of 10 μM in an MTT assay against three non-tumorigenic cell lines
(HOSEC, HUVEC, and T29) and three drug-resistant cancer cell lines including PTX-
or cisplatin (CDDP)-resistant human ovarian (A2780/T and A2780/CDDP) and
VCR-resistant human oral epidermoid cancer cell lines (KB-VIN). VRP was used as a
positive control. As shown in Figure 4a-c, at 10 μM, only three compounds, 6e (R' =
4
4
-chloro-3-nitrobenzoyl), 6v (R' = (E)-3-(4-methoxyphenyl)acryloyl) and 6w (R' =
-cyanobenzoyl), were slightly toxic to two (HOSEC and T29) of the normal human
ovarian epithelial cell lines, the remaining 31 seco-DSP derivatives did not
significantly affect the proliferation of normal cells. The inhibition rates were lower
than that of VRP, suggesting that seco-DSP derivatives might be less toxic and safer
than VRP.

survival ratio of T29
100
90
80
70
60
50
40
30
20
10
0
compounds (C = 10 μM)
4b
As shown in Figure 4d, more than two thirds of seco-DSP derivatives had no
significant effect compared with VRP on the proliferation of A2780/T, while less than
one third of the compounds, including ones with 2-bromobenzoyl (6b),
4-propylbenzoyl (6l), 4-tert-butylbenzoyl (6n), 3,4-dimethoxybenzoyl (6q),
2-(3,4,5-trimethoxyphenyl)acetyl (6t), 4-methoxycinnamoyl (6v, 7e) and
4-cyanobenzoyl (7f, 8e) esters, were slightly more cytotoxic than VRP against
A2780/T.

2
.2.2. MDR reversal activity of compounds against A2780/T, KB-VIN, and
A2780/CDDP
[17]
In our previous research , the result of expression of P-gp in these MDR cell lines
A2780/CDDP, A2780/T and KB-VIN) has been detected by Western blot analysis (see in
supporting information) The result indicated P-gp overexpression in the A2780/T and
(
.
KB-VIN drug-resistant cell lines, but not in the cisplatin-resistant A2780/CDDP.
Furthermore, the newly synthesized compounds 6a-w, 7a-f and 8a-e at 10 μM were
co-administered with PTX at different concentrations to evaluate the MDR reversal
effects in PTX-resistant A2780/T cell lines. The reversal ratio (r.r.) was defined as
IC (PTX only) / IC (PTX with 10 μM synthesized compound), as a standard of the
5
0
50
evaluation. As shown in Table 1, 27 compounds exhibited moderate to significant
MDR reversal activity (r.r. > 50), and 15 compounds (6i, 6q-s, 6u-v, 7a-e and 8a-d)
showed greater reversal activity (r.r. 260-1471) than VRP (r.r. 211). The reversal ratios
of compounds bearing 3,4,5-trimethoxybenzoyl, 2-(3,4,5-trimethoxyphenyl) acetyl,
cinnamoyl, (E)-3-(4-methoxyphenyl)acryloyl esters at position-8 of the seco-DSP
core (6s, 6u-v, 7b-e and 8a-d) were over 400. Notably, compound 7e with an ethoxy
group at position-7 and an (E)-3-(4-methoxyphenyl)acryloyl ester at position-8
showed the most potent reversal effect (r.r. 1471), much higher than that of VRP.
Meanwhile compounds 6b, 6d-e, 6w, 7f and 8e with an electron-withdrawing group,
such as fluoro, chloro, bromo, nitro and cyano, on the benzene ring in the aromatic
ester at position-8 showed lower reversal activity (r.r. < 50). These results indicated
that an electron-donating group, such as alkoxy, on the aromatic ester was more
favorable for improved reversal activity. Also, the C-ring and chiral centers of the

DSP scaffold were not needed to produce chemosensitization. In addition, we further
tested the reversal activity of the best potency four compounds (7c, 7d, 7e and 8b) in
two lower doses of 1 µM and 200 nM (See the detailed data in the supporting
information). The results showed that in the concentration of 1 µM, the compounds 7c,
7
d, 7e and 8b had reversal ratios of 4.13, 4.24, 128 and 3.97 folds, respectively. And
in the concentration of 200 nM, their reversal activities hardly were observed with
reversal ratios from 1.05 to 1.11-fold in A2780/T. This result indicated that our newly
synthesized compounds still can keep a good reversal activity at lower a concentration
as 1 µM, especially the compound 7e had a reversal ratio of 128-fold. While the much
lower concentration as 200 nM was not favorable for the reversal activity of 7c, 7d,
7
e and 8b.The result suggested that the concentration of 10 µM might be a relatively
suitable dose for these compounds reversal MDR action.
Table 1 MDR reversal activity of 6a-w, 7a-f and 8a-e (10 µM) in A2780/T.
PTX
PTX
a
a
Compd
r.r.
Compd
r.r.
IC (μM)
IC (μM)
5
0
50
VRP
0.0188
0.0745
0.1695
0.0231
0.0934
0.2770
0.0701
0.0315
0.0225
0.0082
211
53
6r
6s
0.0140
0.0074
0.0735
0.0093
0.0096
0.4988
0.0087
0.0087
0.0059
0.0079
284
537
54
6
a
6b
23
6t
6c
172
43
6u
6v
6w
7a
7b
7c
7d
427
414
8
6d
6e
14
6f
57
457
457
677
503
6
g
126
177
484
6h
6i
<
0.00
>14
71
6j
0.0448
89
7e
2
7
6
k
0.0403
0.1091
0.0230
0.0252
99
36
7f
8a
8b
8c
0.2004
0.0079
0.0069
0.0058
20
503
576
685
6
l
6
m
173
158
6n

6
o
0.0747
0.0270
0.0153
53
147
260
8d
8e
0.0081
0.3993
3.972
490
10
1
6
p
q
6
PTX
a
reversal ratio (r.r.) defined as IC (PTX only) / IC (PTX with 10 µM synthesized
5
0
50
compound)
Next, compounds 6i, 6q-s, 6u-v, 7b-e and 8b-d, which displayed high MDR reversal
activity in A2780/T cell lines, were evaluated for antiproliferative and reversal
activities when administered alone and together with VCR in drug-resistant KB-VIN
cell lines. As observed in Figure 4e, all 13 compounds exhibited lower
antiproliferative activity than VRP against KB-VIN cells. However, most of the tested
compounds displayed significant MDR reversal activity (r.r 105−633) in the KB-VIN
cell line (Table 2). Among them, 6r, 7c, 7d and 8b with 2,4,5-trimethoxybenzoyl,
2
-(3,4,5-trimethoxyphenyl)acetyl (7c and 8b), and cinnamoyl esters, respectively, at
position-8 of the seco-DSP scaffold showed more potent reversal activity than VRP
r.r. 579, 628, 633, 604, and 418, respectively).
(
Table 2 MDR reversal activity of 6i, 6q-v, 7b-e and 8b-d (10 µM) in KB-VIN.
VCR
VCR
a
a
Compd
r.r.
Compd
r.r.
IC (nM)
IC (nM)
5
0
50

VRP
1.593
2.842
6.331
1.151
1.570
1.942
1.588
666.3
418
234
105
579
424
343
420
1
7b
7c
7d
7e
8b
8c
8d
2.692
1.061
1.053
2.228
1.103
1.429
1.947
248
628
633
299
604
466
342
6
i
6q
6r
6s
6u
6v
VCR
a
reversal ratio (r.r.) was defined as IC50 (VCR only) / IC50 (VCR with 10 µM
synthesized compound)
Interestingly, when co-administered with CDDP in the drug-resistant A2780/CDDP
cell line, neither VRP (r.r. 1.07) nor compounds 6i, 6s, 7a-e, 8a and 8c-d showed
reversal activity (r.r. 0.8-1.05) (Table 3). And in view of P-gp inhibitors being
developed for three generations, we chose a third generation P-gp inhibitor tariquidar,
which was at phase III clinic trial, as a positive control for the reversal activity assay
in A2780/CDDP. And the result indicated that at 10 µM, tariquidar only showed a
weak reversal ratio of 1.9 folds in A2780/CDDP. (See the detailed data in supporting
information). These results were consistent with our previously reported research;
both seco-DSPs and seco-4-methyl-DCK analogs were strong chemosensitizers in
A2780/T and KB-VIN cell lines but not in the A2780/CDDP cell line. The results of
western blot analysis suggested that overexpression of P-gp partially contributed to
MDR in the A2780/T and KB-VIN drug-resistant cell lines, but not in the
[
17]
CDDP-resistant A2780/CDDP cell lines
. Thus, we speculated that the MDR
reversal activity of our newly synthesized seco-DSPs might also depend on the
overexpression of P-gp in the relevant drug-resistant cancer cell lines.
Table 3 MDR reversal activity of 6i, 6s, 7a-e, 8a and 8c-d (10 µM) in A2780/CDDP.
CDDP
a
Compd
r.r.
IC (μM)
5
0
VRP
40.11
53.24
1.07
0.81
6i

6
s
53.17
48.86
41.13
44.25
47.06
48.95
43.59
53.02
0.81
0.88
1.05
0.97
0.92
0.88
0.99
0.81
7a
7b
7c
7d
7e
8a
8c
8
d
44.22
43.11
0.97
1
CDDP
a
reversal ratio (r.r.) was defined as IC (CDDP only) / IC (CDDP with 10 µM
5
0
50
synthesized compound)
2
.2.3. Structure-activity relationships
Some preliminary structure-activity relationship correlations are summarized as
follows. Electron withdrawing substituents, such as F, Cl, Br, NO , and CN, on a
2
benzoyl ester at position-8 were disadvantageous for the drug resistance reversal
activity. Notably, the introduction of a methoxy group on a benzoyl ester generally led
to improved reversal activity. The rank order of potency was 3-methoxy (6p, r.r. 147)
<
3,4-dimethoxy (6q, r.r. 260) < 2,4,5-trimethoxy (6r, r.r. 284) < 3,4,5-trimethoxy (6s,
r.r. 537). Electron-donating groups other than methoxy affected the reversal activity to
different extents; compound 6i with a 3-N,N-dimethylaminobenzoate on position-8 of
the seco-DSP had a good r.r of 484. Furthermore, except for 6t, compounds with
phenylacetyl, cinnamoyl, or phenylacryloyl esters rather than benzoyl esters (6u-v,
7
c-e, and 8b-d) exhibited good to remarkably improved potency (r.r. 414−1471).
Among them, compound 7e exhibited outstanding MDR reversal activity with a r.r.
greater than 1471 and completely reversed PTX resistance in A2780/T cells at a
concentration of 10 μM. Additionally, the potencies of 7-methoxy seco-DSPs were
only slightly lower than those of corresponding 7-ethoxy or isopropoxy substituted
derivatives; thus, the substituent at position-7 on the DSP A-ring affected the reversal
activity to a smaller degree compared with the diverse aroyl substituents at the
adjacent position-8. The type of ester groups might be key to maintain or improve the
drug resistance reversal activity.

2
.2.4. Effects on the expression of P-gp in A2780/T cells
The reversal of P-gp-mediated MDR can be achieved by inhibiting the function of
P-gp or suppressing its expression. Wu, J. Y. et al. reported that PA mainly affected
[
14]
cellular P-gp activity by suppressing the expression of P-gp
To determine whether
.
the reversal activity of the seco-DSP compounds was caused by inhibition of P-gp
expression, we incubated A2780/T cells with 7c, 7d, 7e and 8b at 10 μM and used
Western blot analysis to determine the protein expression level of P-gp. As shown in
Figure 5, the expression level neither increased nor decreased compared to the control
after exposure to the four compounds. Wang-Fun Fong reported that DCK did not
[18]
affect the P-gp expression , which is consistent with our experimental results.
Because the results indicated that the compounds did not interfere with P-gp genetic
expression and seco-DSP derivatives are derived from DCK/DSP compounds, they
likely inhibited the efflux function of P-gp.
Figure 5. (A) Western blot analysis shows the expression of P-gp after exposure to 10
μM 7c, 7d, 7e and 8b. Band intensity was analyzed by Quantity One software, and
protein expression was presented as the ratio of target protein’s band intensity to that
of β-tubulin. (B) Ratio of P-gp/GAPDH.
2
.2.4. Inhibition of DOX efflux mediated by P-gp in A2780/T
Chemotherapy of MDR phenotypes is often associated with decreased intracellular
drug concentration. An accumulation assay is commonly used to explore drug-efflux
mediated by P-gp. Hence, we used this method to investigate whether the reversal
effect of our seco-DSP compounds was also associated with a concomitant increase in
DOX accumulation. Due to the autofluorescence of DOX, its accumulation can be
determined by spectrofluorometry. The results are depicted in Figure 6. At 10 μM,

modulators 7c, 7d, 7e and 8b significantly enhanced DOX accumulation in A2780/T
cells. Moreover, these results indicate that 7c, 7d, 7e and 8b are potent P-gp inhibitors,
which are more potent than VRP in increasing accumulation of DOX in A2780/T
cells.
Figure 6. Intracellular accumulation of DOX in A2780/T cells. (A) Flow cytometry
analysis of the accumulation of DOX (10 μM) in A2780/T cells with various test
substances (10 μM) or VRP (10 μM), 0.1% DMSO as a negative control; (B)
Quantification of DOX in A2780/T cells by flow cytometry; (C) Fluorescence images
of DOX (red) under fluorescence microscopy. The results are presented as the mean ±
SD: *P < 0.005; **P < 0.001; ***P < 0.0005, compared to negative control.
2
.2.5. Effects on P-gp-ATPase activity
VRP is not only an inhibitor as well as a substrate of P-gp but also one of the best
[19]
stimulators of P-gp-ATPase ; thus, we determined the effects of 7c, 7d, 7e and 8b
on P-gp-ATPase activity. As shown in Figure 7, VRP visibly increased the
P-gp-ATPase activity over the basal level by 1.8-fold at 0.5 mM, while 7c, 7d, 7e and
8
b stimulated the activity by 1.2- to 2.3-fold at same tested concentration. These

findings suggest that the new compounds might function similarly to VRP toward
P-gp-ATPase.
Figure 7. Effects of 7c, 7d, 7e and 8b on P-gp-ATPase activity. P-gp ATPase was
measured in the absence (basal activity) or presence of P-gp modulators (VRP, 7c, 7d,
7
e and 8b). Results are presented as the mean ± SD of three independent experiments:
(∗∗∗) P < 0.001, (∗∗) P < 0.01, (∗) P < 0.05 relative to the basal activity.
3
. Conclusions
In summary, we synthesized 34 seco-DSPs with three alkoxy groups at position-7 and
various aryl-containing esters at position-8 and evaluated their ability to
chemosensitize PTX and VCR in A2780/T and KB-VIN cell lines. Most of the
compounds displayed lower cytotoxicity against HOSEC, HUVEC, T29, A2780/T,
A2780/CDDP and KB-VIN cell lines and greater MDR reversal activity compared
with VRP. Among them, compound 7e [(7-ethoxy-2-ethyl-4-oxo-4H-chromen-8-yl)
methyl (E)-3-(4-methoxyphenyl)acrylate] showed the most potent reversal activity
(r.r. >1471).
A
preliminary structure-activity relationships said that
electron-withdrawing group substitution on the benzoylbenzene ring was against the
MDR reversal activity, while methoxy as an electron-donating group was a favourable
groupto significantly improve the reversal activity. In addition, the activity also was
greatly increased by using cinnamoyl or p-methoxycinnamoyl insteady of benzoyl.
And the MDR reversal activity of the compounds with methoxyl group at position-7
was slight poor than that of ethoxyl and isopropoxyl groups substituted compounds.
Moreover, we explored the pharmacologic mechanism of four seco-DSP derivatives,
7
c, 7d, 7e and 8b. The four compounds significantly inhibited the transport activity of

P-gp, as shown by intracellular accumulation of DOX in resistant A2780/T cells.
However, Western blot analysis showed no significant difference between
compound-treated and untreated cells, suggesting that the MDR reversal of the four
compounds was likely due to the inhibition of P-gp efflux function rather than P-gp
protein expression. In addition, the new compounds clearly promoted P-gp ATPase
activity similarly to VRP. Overall, these results implied that C-ring of DSP is not
required for P-gp modulations and MDR reversal. Further studies on seco-DSP
derivatives are and merited to find and develop a promising P-gp-mediated MDR
reversal modulator candidate.
4
. Experimental section
4
.1 Chemistry
All starting materials, reagents, and solvents were obtained from commercial sources
and used without further purification unless otherwise indicated. Column
chromatography was performed on silica gel (100-200 mesh), monitored by thin layer
chromatography (TLC) on GF/UV 254 plates, and visualized using UV light at 254
and 365 nm. Melting points were measured on an SGW X-4 microscopy melting point
1
13
apparatus without correction. H and C NMR data were recorded with a Varian 400
MHz spectrometer at 303 K using TMS as an internal standard. Mass spectra were
recorded on Agilent Technologies 1260 infinity LC/MS instrument, and HRMS
spectra were recorded on an Agilent Technologies LC/MSD TOF instrument.
4
.2 Materials for biological studies
3
-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), doxorubicin
(DOX), verapamil (VRP) and dimethyl sulfoxide (DMSO) were purchased from
Sigma-Aldrich, USA. The antibodies including anti-GAPDH (ab181602) and anti-P
Glycoprotein (ab170904) were purchased from Abcam plc, USA (1:1000 dilutions).
Horseradish peroxidase (HRP)-conjugated secondary antibodies used for Western blot
were obtained from MR Biotech (Shanghai, China). Pgp-Glo assay systems were
purchased from Promega Corporation.

4
.3 Cell lines and cell culture
The A2780/CDDP, A2780/T and KB-VIN cell lines were kindly provided by
Shanghai Institute of Materia Medica Chinese Academy of Science. HUVEC, HOSEC
and T29 cells were kindly provided by Fudan University Shanghai Cancer Center.
HUVEC, HOSEC and T29 cell lines were cultured in RPMI-1640 medium
supplemented with 10% heat inactivated FBS in a humidified incubator at 37 °C with
5
% CO . A2780/CDDP, A2780/T and KB-VIN cell lines were cultured in DMEM/F12
2
medium supplemented with 10% heat inactivated FBS in a humidified incubator at
3
7 °C with 5% CO2.
4
.4 Growth inhibitory and MDR reversal assays
For MTT assay, the in vitro anti-proliferation of the chemical compounds was
[20]
3
measured by the MTT reagent, as described in the literature . Briefly, 3~5×10 cells
in 100 μL of medium per well were plated in 96-well plates. In cytotoxicity assays, after
being incubated for 24 h, the cells were treated with different concentrations of test
compound or DMSO (as negative control) for 48 h. In the MDR reversal assays, after
being incubated for 24 h, the compounds and paclitaxel/cisplatin were added together
to the 96-well plates or DMSO (as negative control) for 48 h. Then, the medium with
compound or DMSO was replaced with 200 μL of fresh medium containing 10%
MTT (5 mg/mL in PBS) in each well and incubation continued at 37 ºC for 4 h. Lastly,
the MTT-containing medium was removed and 150 μL of DMSO per well was added
to dissolve the newly formed formazan crystals. Absorbance of each well was
determined by a microplate reader (Synergy H4, Bio-Tek) at a 490 nm wavelength.
The inhibition rates of proliferation were calculated with the following equation:
Inhibition ratio = (ODDMSO-ODcompd.)/(ODDMSO-ODblank)×100%.
The concentrations of the compounds that inhibited cell growth by 50% (IC ) were
5
0
calculated using Graph Pad Prism version 6.0.
4
.5 Cellular doxorubicin accumulation
Experiments for the cellular doxorubicin accumulation were carried out with the
[21]
5
following modified method . Approximately 5×10 cells of A2780/T were grown in
six-well plates. After 24 h incubation, the cells were treated with 10 μM DOX and
various test substances (10 μM) or VRP (10 μM) at 37 °C for 4 h with 0.1% DMSO as
a negative control. Then, the cells were washed with ice-cold phosphate-buffered
saline (PBS) three times and resuspended in ice-cold PBS. Cellular doxorubicin
fluorescence level was monitored by CytoFLEX flow cytometer (Beckman Coulter,
CA) and confocal microscopy.

4
.6 Western blot analysis
5
Approximately 5×10 cells of A2780/T were grown in six-well plates. After 24 h
incubation, the cells were treated with various seco-DSP derivatives (10 μM) at 37 °C
for 48 h. Cells were incubated in ice-cold RIPA lysis buffer containing 1 mM PMSF
for 30 min and collected by centrifugation (12500 rpm) at 4 °C for 15 min. The
protein concentrations of supernatants were then subjected to BCA protein analysis.
Then, equal amounts of proteins (20 μg) were separated on 12% SDS-PAGE gel and
electro-transferred to PVDF membranes. Membranes were blocked with 3% bovine
serum albumin (BSA). The membranes were then incubated with the specific primary
antibodies (anti- P Glycoprotein antibody or GAPDH antibody, 1:1000 dilution) for
night at 4 °C, followed by horseradish–peroxidase-conjugated secondary antibodies
(1:10000 dilutions) for 1h at room temperature. Protein bands were visualized via
enhanced chemiluminescence. Band intensity was calculated with Image J software.
4
.7 P-gp ATPase assay
The effects of test substances on P-gp ATPase were detected by a P-gp-Glo assay
system. Initially, about 25 mg of human P-gp membrane fraction were added into
different wells: (1) “NT” well (20 μL of P-gp-Glo assay buffer), (2) “Na VO ” well
3
4
(
20 μL of 0.25 mM Na VO ), (3) “Ver” well (20 μL of 0.5 mM VRP), (4)
3 4
“test-substances” well (20 μL of 0.5 mM test compounds), respectively. The cells
were incubated at 37 °C for about 5 min. Secondly, reactions were initiated by adding
and mixing 10 μL of 25 mM MgATP to all wells, and then the cells were incubated
for 120 min at 37 °C. Thirdly, ATP detection reagent (50 μL) was added to all wells to
stop the reactions and initiate luminescence. Incubation was then continued at room
temperature for about 20 min. Finally, the luminescence signal was detected on a
Biotek multi-functional microplate reader (Molecular Devices, USA).
4
.8 Procedure for the synthesis of intermediate compound
2
,4-Dihydroxyacetophenone. After zinc chloride (92.80 g, 681.2 mmol) was
dissolved in 75 mL of acetic acid with heating, resorcinol (50.0 g, 454.1 mmol) was
added. The reaction mixture was heated to 130-140 °C. After reaction completion, the
mixture was cooled to room temperature, and the resulting solid was filtered. The
reaction mixture was poured into ice-water (500 mL) and extracted with EtOAc
(3×100 mL). The product was further purified by column chromatography to obtain
2
,4-dihydroxyacetophenone (35.71 g). Yield 51.1%, m.p. 143.1-145.0 °C, MS(ESI)
+
1
(m/z) 155.1 [M+H] . H NMR (400 MHz, CDCl ) δ 12.83 (s, 1H), 7.3-7.7 (m, 3 H),
3

6
.46 (s, 1 H), 2.72 (s, 3 H).
1
-(2-Hydroxy-4-(methoxymethoxy)phenyl)ethanone. To a stirred solution of
2
,4-dihydroxyacetophenone (20.0 g, 131.4 mmol) in acetone (500 mL) was added
anhydrous potassium carbonate (45.41 g, 328.5 mmol) at 0 °C. After stirring for 20
min at 0 °C, chloromethyl methyl ether (19.96 mL, 262.8 mmol) was added dropwise.
The reaction mixture was stirred at room temperature for 4 h and monitored by TLC.
After completion of the reaction, the reaction was quenched with water (300 mL).
EtOAc (300 mL) was added, the layers were separated, and the aqueous layer was
extracted with EtOAc (2×100 mL). The combined organic layer was dried over
anhydrous sodium sulfate. The residue was purified by silica gel column
chromatography to provide 1-(2-hydroxy-4-(methoxymethoxy)phenyl)ethanone (18.1
+
1
g) as a colorless liquid. Yield 84.4%, MS(ESI) (m/z) 197.2 [M+H] . H NMR (400
MHz, CDCl ) δ 12.77 (s, 1H), 7.54 (d, J = 8.8 Hz, 1H), 6.58 (d, J = 2.0 Hz, 1H), 6.55
3
(dd, J = 8.8, 2.0 Hz, 1H), 5.24 (s, 2H), 3.46 (s, 3H), 2.54 (s, 3H).
2
-Ethyl-7-hydroxy-4H-chromen-4-one (2). Sodium hydride (8.86 g, 222 mmol) was
added to a well-stirred suspension of 1-(2-hydroxy-4-(methoxymethoxy) phenyl)
ethanone (13.28 g, 67.69 mmol) in dry THF (100 mL) at 0 °C under N . After 30 min,
2
ethyl propionate (104.5 mL, 135.4 mmol) was added dropwise, and the reaction
mixture was heated to reflux for 2 h. The mixture was then cooled to room
temperature, and the excess sodium hydride was quenched with distilled water. The
reaction mixture was then poured on ice, followed by addition of 2M HCl, and the
products were extracted with EtOAc (4×50 mL). The organic layers were combined
and were washed with a saturated solution of sodium chloride (2×100 mL). The
EtOAc layer was dried over anhydrous sodium sulfate and then evaporated to obtain
the crude product 1. Compound 1 (16.22 g, 64.31 mmol) was dissolved in 500 mL of
EtOH, then conc. HCl (10 mL) was added. The reaction mixture was heated to reflux
for 30 min. After reaction completion, EtOH was evaporated to obtain the crude
product. The crude product was purified by silica gel column chromatography to
provide 2 (10.91 g). Yield 85.0% (two steps), m.p. 194.2-196.1 °C, MS(ESI) (m/z)
+
1
1
1
91.1 [M+H] . H NMR (400 MHz, (CD ) SO) δ 10.78 (s, 1H), 7.83 (d, J = 8.7 Hz,
3 2
H), 6.89 (d, J = 8.7 Hz, 1H), 6.84 (s, 1H), 6.07 (s, 1H), 2.62 (d, J = 7.4 Hz, 2H), 1.21
(t, J = 7.4 Hz, 3H).
2
-Ethyl-7-hydroxy-4-oxo-4H-chromene-8-carbaldehyde (3). A reaction mixture of
compound 2 (30.0 g, 157 mmol) and hexamethylenetetramine (154.8 g, 1104 mmol)
in HOAc (500 mL) was stirred for 5.5 h at 80-90 °C. Aqueous HCl (800 mL, conc.

HCl/H O 84:100, v/v) was then added, and the reaction mixture was stirred for 0.5 h
2
at 70 °C. After cooling, the reaction mixture was poured into ice-water (1.0 L) and
extracted with EtOAc (3×500 mL). The combined organic fraction was dried over
anhydrous sodium sulfate, and the solvent was removed in vacuo. The residue was
recrystallized from EtOAc to give 3 (8.65 g) as a light yellow solid. Yield 25.0%, m.p.
+
1
1
87.8-188.9 °C, MS(ESI) (m/z) 219.1 [M+H] . H NMR (400 MHz, CDCl ) δ 12.43
3
(s, 1H), 10.56 (s, 1H), 8.30 (d, J = 9.0 Hz, 1H), 6.98 (d, J = 9.0 Hz, 1H), 6.22 (s, 1H),
2
.72 (q, J = 7.5 Hz, 2H), 1.35 (t, J = 7.5 Hz, 3H).
2
-Ethyl-7-hydroxy-8-(hydroxymethyl)-4H-chromen-4-one (4). Sodium borohydride
(3.48 g, 92.1 mmol) was added to a well-stirred suspension of 3 (13.41 g, 61.4 mmol)
in MeOH (150 mL) at 0 °C. Then reaction mixture was stirred at room temperature.
After reaction completion, reaction mixture was poured on ice, followed by addition
of 2M HCl. The resulted solid was filtered to provide the crude product 4. The crude
product was purified by silica gel column chromatography to give 4 (12.85 g). Yield
+
1
9
5.1%, m.p. 164.0-166.1 °C, MS(ESI) (m/z) 221.1 [M+H] . H NMR (400 MHz,
(
CD ) SO) δ 10.70 (s, 1H), 7.78 (d, J = 8.7 Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H), 6.09 (s,
3
2
1
H), 4.83 (s, 1H), 4.67 (s, 2H), 2.66 (d, J = 7.3 Hz, 2H), 1.25 (d, J = 7.3 Hz, 3H).
2
7
2
-Ethyl-8-(hydroxymethyl)-7-methoxy-4H-chromen-4-one
(5a),
(5b),
-Ethoxy-2-ethyl-8-(hydroxymethyl)-4H-chromen-4-one
-Ethyl-8-(hydroxymethyl)-7-isopropoxy-4H-chromen-4-one (5c). Compound 4
(
0.50 g, 1.8 mmol) and potassium carbonate (0.63 g, 4.5 mmol) were added to stirred
DMF (5 mL). The temperature was raised to 80 °C, and iodomethane, bromoethane or
-bromopropane was added. The reaction mixture was stirred at 80 °C for 1 h and
2
then the insoluble solid was removed by filtration. The 30 mL of water were added to
the mixture solution, which was then extracted with EtOAc (3×10 mL). The combined
organic layers were washed with saturated sodium chloride solution (2×40 mL). The
organic layer was dried over anhydrous sodium sulfate. Then, the solvent was
removed under reduced pressure, and the residue was purified by column
chromatography to yield 5a-c. 5a: yield 85.0%; m.p. 138.0-140.3 °C; MS(ESI) (m/z)
+
1
2
35.1 [M+H] . H NMR (400 MHz, CDCl ) δ 8.16 (d, J = 8.9 Hz, 1H), 7.01 (d, J =
3
8
.9 Hz, 1H), 6.13 (s, 1H), 4.97 (s, 2H), 4.00 (s, 3H), 2.68 (q, J = 7.5 Hz, 2H), 1.32 (t,
+
J = 7.5 Hz, 3H). 5b: yield 80.2%; m.p. 107.1-109.2 °C; MS(ESI) (m/z) 249.1 [M+H] .
1
H NMR (400 MHz, CDCl ) δ 8.12 (d, J = 8.9 Hz, 1H), 6.98 (d, J = 8.9 Hz, 1H), 6.11
3
(s, 1H), 4.97 (s, 2H), 4.23 (q, J = 7.0 Hz, 2H), 2.68 (q, J = 7.5 Hz, 2H), 1.51 (t, J = 7.0
Hz, 3H), 1.32 (t, J = 7.5 Hz, 3H). 5c: yield 87.0%, m.p. 107.9-109.8 °C, MS(ESI)
+
1
(
m/z) 263.2 [M+H] . H NMR (400 MHz, CDCl ) δ 8.11 (d, J = 9.0 Hz, 1H), 6.99 (d,
3
J = 9.0 Hz, 1H), 6.11 (s, 1H), 4.95 (s, 2H), 4.79 (m, J = 6.0 Hz, 1H), 2.67 (q, J = 7.5

Hz, 2H), 1.43 (d, J = 6.0 Hz, 6H), 1.31 (t, J = 7.5 Hz, 3H).
4
.9 General procedure for the synthesis of compounds 6a-w, 7a-f and 8a-e
The corresponding aromatic acids (1.2 eq) and EDCI (1.2 eq) were dissolved and
stirred in CH Cl (10 mL) in an ice bath for 30 min, and then the corresponding
2
2
alcohol 5a-c (1.0 eq) was added to the reaction solution. The reaction mixture was
removed to room temperature and monitored by TLC. After reaction completion, 15
mL of water were added to the mixture solution. The organic layers were washed with
saturated sodium chloride solution (2×15 mL) and dried over anhydrous sodium
sulfate. Then, the solvent was removed under reduced pressure, and the residue was
purified by column chromatography to yield 6a-w, 7a-f and 8a-e.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 4-fluorobenzoate (6a). Yield:
+
+
4
5.1%. m.p. 147.8-149.4 °C. MS(ESI) (m/z) 357.1 [M+H] . ESI-HRMS (m/z) [M+H]
1
Calc.: 357.1133, Found: 357.1136. H NMR (400 MHz, CDCl ) δ 8.24 (d, J = 9.0 Hz,
3
1
H, 5-H), 8.03 (ddd, J = 8.3, 5.2, 2.4 Hz, 2H, (2',6'-Ph-H)), 7.12-7.03 (m, 2H,
3',5'-Ph-H)), 7.04 (d, J = 9.0 Hz, 1H, 6-H), 6.14 (s, 1H, 3-H), 5.64 (s, 2H, 8-CH2),
.99 (s, 3H, OCH ), 2.62 (q, J = 7.5 Hz, 2H, CH CH ), 1.25 (t, J = 7.5 Hz, 3H,
(
3
3
2
3
1
3
CH CH ). C NMR (100 MHz, CDCl ) δ 178.0, 170.5, 165.8, 165.7, 162.3, 156.3,
2
3
3
1
1
32.3, 132.2, 128.2, 126.4, 117.7, 115.6, 115.4, 111.2, 108.8, 108.6, 56.4, 55.5, 27.5,
0.9.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 2-bromobenzoate (6b). Yield:
+
+
5
0.2%. m.p. 145.3-147.8 °C. MS(ESI) (m/z) 417.0 [M+H] . ESI-HRMS (m/z) [M+H]
1
Calc.: 417.0332, Found: 417.0334. H NMR (400 MHz, CDCl ) δ 8.23 (d, J = 9.0 Hz,
3
1
H, 5-H), 7.75-7.62 (m, 2H, (3',6'-Ph-H)), 7.35-7.28 (m, 2H, (4',5'-Ph-H)), 7.04 (d, J
9.0 Hz, 1H, 6-H), 6.15 (s, 1H, 3-H), 5.68 (s, 2H, 8-CH ), 4.00 (s, 3H, OCH ), 2.66
=
2
3
13
(q, J = 7.5 Hz, 2H, CH CH ), 1.29 (t, J = 7.5 Hz, 3H, CH CH ). C NMR (100 MHz,
2 3 2 3
CDCl ) δ 178.0, 170.5, 166.2, 162.3, 156.2, 134.3, 132.5, 132.3, 131.3, 128.3, 127.1,
3
1
21.6, 117.6, 110.9, 108.7, 108.6, 56.4, 55.9, 27.5, 10.9.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 2-nitrobenzoate (6c). Yield:
+
+
5
6.0%. m.p. 144.0-145.3 °C. MS(ESI) (m/z) 384.1 [M+H] . ESI-HRMS (m/z) [M+H]
1
Calc.: 384.1078, Found: 384.1081. H NMR (400 MHz, CDCl ) δ 8.22 (d, J = 9.0 Hz,
3
1
2
4
H, 5-H), 7.91 (d, J = 7.7 Hz, 1H, 3'-Ph-H), 7.75-7.70 (m, 1H, 5'-Ph-H), 7.69-7.58 (m,
H, (4',6'-Ph-H)), 7.02 (d, J = 9.0 Hz, 1H, 6-H), 6.14 (s, 1H, 3-H), 5.68 (s, 2H, 8-CH2),
.00 (s, 3H, OCH ), 2.72 (q, J = 7.5 Hz, 2H, CH CH ), 1.32 (t, J = 7.5 Hz, 3H,
3
2
3

1
3
CH CH ). C NMR (100 MHz, CDCl ) δ 177.9, 170.7, 165.5, 162.4, 156.2, 148.1,
2
3
3
1
1
32.9, 131.6, 129.8, 128.5, 127.8, 123.9, 117.6, 110.2, 108.6, 108.5, 56.7, 56.4, 27.4,
0.9.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 2,6-difluorobenzoate (6d).
+
Yield: 43.4%. m.p. 139.0-141.9 °C. MS(ESI) (m/z) 375.1 [M+H] . ESI-HRMS (m/z)
+
1
[
M+H] Calc.: 375.1039, Found: 375.1042. H NMR (400 MHz, CDCl ) δ 8.22 (d, J
3
=
9.0 Hz, 1H, 5-H), 7.38 (tt, J = 8.3, 6.2 Hz, 1H, 4'-Ph-H), 7.03 (d, J = 9.0 Hz, 1H,
6
-H), 6.92 (t, J = 8.3 Hz, 2H, (3',5'-Ph-H)), 6.14 (s, 1H, 3-H), 5.70 (s, 2H, 8-CH2),
.99 (s, 3H, OCH ), 2.68 (q, J = 7.5 Hz, 2H, CH CH ), 1.30 (t, J = 7.5 Hz, 3H,
3
3
2
3
1
3
CH CH ). C NMR (100 MHz, CDCl ) δ 177.9, 170.6, 162.3, 161.6, 160.6, 160.5,
2
3
3
1
56.2, 132.6, 128.3, 117.6, 112.1, 111.8, 110.5, 108.7, 108.6, 56.4, 56.3, 27.4, 10.8.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 4-chloro-3-nitrobenzoate (6e).
+
Yield: 48.9%. m.p. 185.4-187.0 °C. MS(ESI) (m/z) 418.0 [M+H] . ESI-HRMS (m/z)
+
1
[
M+H] Calc.: 418.0688, Found: 418.0691 H NMR (400 MHz, CDCl ) δ 8.46 (d, J =
3
2
6
3
1
1
1
.0 Hz, 1H, 2'-Ph-H), 8.26 (d, J = 9.0 Hz, 1H, 5-H), 8.15 (dd, J = 8.5, 2.0 Hz, 1H,
'-Ph-H), 7.62 (d, J = 8.5 Hz, 1H, 5'-Ph-H), 7.06 (d, J = 9.0 Hz, 1H, 6-H), 6.15 (s, 1H,
-H), 5.69 (s, 2H, 8-CH ), 4.00 (s, 3H, OCH ), 2.64 (q, J = 7.3 Hz, 2H, CH CH ),
2
3
2
3
13
.27 (t, J = 7.3 Hz, 3H, CH CH ). C NMR (100 MHz, CDCl ) δ 177.8, 170.3, 163.7,
2
3
3
62.3, 156.2, 148.0, 133.6, 132.1, 131.7, 130.1, 128. 6, 126.6, 117.7, 110.4, 108.8,
08.7, 56.4, 27.5, 10.9.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 2-chloro-5-nitrobenzoate (6f).
+
Yield: 46.3%. m.p. 163.7-165.5 °C. MS(ESI) (m/z) 418.0 [M+H] . ESI-HRMS (m/z)
+
1
[
M+H] Calc.: 418.0688, Found: 418.0688. H NMR (400 MHz, CDCl ) δ 8.64 (d, J
3
=
2.7 Hz, 1H, 6'-Ph-H), 8.25 (dt, J = 8.8, 1.2 Hz, 2H, (3',4'-Ph-H)), 7.64 (d, J = 9.0 Hz,
1
H, 5-H), 7.06 (d, J = 9.0 Hz, 1H, 6-H), 6.16 (s, 1H, 3-H), 5.73 (s, 2H, 8-CH ), 4.03
2
(
s, 3H, OCH ), 2.69 (q, J = 7.6 Hz, 2H, CH CH ), 1.31 (t, J = 7.6 Hz, 3H, CH CH ).
3 2 3 2 3
1
3
C NMR (100 MHz, CDCl ) δ 177.8, 170.4, 163.5, 162.4, 156.2, 146.1, 140.6, 132.3,
3
1
31.4, 128.6, 126.8, 126.5, 117.7, 110.3, 108.8, 108.7, 56.7, 56.5, 27.5, 10.9.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 2,4-dichlorobenzoate (6g).
+
Yield: 47.8%. m.p. 132.0-134.5 °C. MS(ESI) (m/z) 407.0 [M+H] . ESI-HRMS (m/z)
+
1
[
M+H] Calc.: 407.0448, Found: 407.0447. H NMR (400 MHz, CDCl ) δ 8.23 (d, J
3
=
9.0 Hz, 1H, 5-H), 7.73 (d, J = 8.4 Hz, 1H, 6'-Ph-H), 7.46 (s, 1H, 3'-Ph-H), 7.25 (s,
1
H, 5'-Ph-H), 7.04 (d, J = 9.0 Hz, 1H, 6-H), 6.14 (s, 1H, 3-H), 5.66 (s, 2H, 8-CH2),

3
.99 (s, 3H, OCH ), 2.65 (q, J = 7.4 Hz, 2H, CH CH ), 1.28 (t, J = 7.4 Hz, 3H,
3 2 3
1
3
CH CH ). C NMR (100 MHz, CDCl ) δ 177.9, 170.4, 164.7, 162.3, 156.2, 138.3,
2
3
3
1
1
34.9, 132. 5, 131.0, 128.4, 128.3, 127.0, 117.7, 110.8, 108.7, 108.6, 56.4, 56.0, 27.5,
0.9.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 4-amino-3-methylbenzoate (6h).
+
Yield: 56.4%. m.p. 171.8-174.5 °C. MS(ESI) (m/z) 368.1 [M+H] . ESI-HRMS (m/z)
+
1
[
M+H] Calc.: 368.1492, Found: 368.1495. H NMR (400 MHz, CDCl ) δ 8.22 (d, J
3
=
9.0 Hz, 1H, 5-H), 7.73 (s, 2H, (2',6'-Ph-H)), 7.70 (s, 2H, NH ), 7.04 (d, J = 9.0 Hz,
2
1
3
=
H, 6-H), 6.63 (d, J = 9.0 Hz, 1H, 5'-Ph-H), 6.13 (s, 1H, 3-H), 5.59 (s, 2H, 8-CH2),
.98 (s, 3H, OCH ), 2.61 (q, J = 7.6 Hz, 2H, CH CH ), 2.16 (s, 3H, 2’-CH ), 1.24 (t, J
3
2
3
3
1
3
7.6 Hz, 3H, CH CH ). C NMR (100 MHz, CDCl ) δ 178.1, 170.6, 166.9, 162.3,
2
3
3
1
56.3, 149.2, 132.3, 129.4, 127.8, 121.0, 119.6, 117.7, 113.6, 111.9, 108.8, 108.5, 56.4,
4.8, 27.5, 17.1, 10.9.
5
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 3-(dimethylamino)benzoate (6i).
+
Yield: 54.2%. m.p. 165.2-167.0 °C. MS(ESI) (m/z) 382.2 [M+H] . ESI-HRMS (m/z)
+
1
[
M+H] Calc.: 382.1649, Found: 382.1650. H NMR (400 MHz, CDCl ) δ 8.22 (d, J
3
=
9.0 Hz, 1H, 5-H), 7.41 (s, 1H, 6'-Ph-H), 7.34 (s, 1H, 2'-Ph-H), 7.23 (s, 1H, 5'-Ph-H),
7
8
1
1
1
.04 (d, J = 9.0 Hz, 1H, 6-H), 6.92 (s, 1H, 4'-Ph-H), 6.13 (s, 1H, 3-H), 5.64 (s, 2H,
-CH ), 3.98 (s, 3H, OCH ), 2.97 (s, 6H, N(CH ) ), 2.62 (q, J = 7.5 Hz, 2H, CH CH ),
2
3
3 2
2
3
13
.26 (t, J = 7.5 Hz, 3H, CH CH ). C NMR (100 MHz, CDCl ) δ 178.0, 170.5, 167.2,
2
3
3
62.3, 156.3, 130.9, 129.0, 127.9, 117.7, 111.5, 108.8, 108.5, 56.4, 55.4, 40.8, 27.5,
0.9.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 2-(4-chlorophenyl)acetate (6j).
+
Yield: 48.3%. m.p. 94.4-96.5 °C. MS(ESI) (m/z) 387.1 [M+H] . ESI-HRMS (m/z)
+
1
[
M+H] Calc.: 387.0994, Found: 393.0997. H NMR (400 MHz, CDCl ) δ 8.21 (d, J
3
=
8.8 Hz, 1H, 5-H), 7.27-7.20 (m, 4H, (2',3',5',6'-Ph-H)), 7.01 (d, J = 8.8 Hz, 1H, 6-H),
6
2
.12 (s, 1H, 3-H), 5.42 (s, 2H, 8-CH ), 3.93 (s, 3H, OCH ), 3.59 (s, 2H, 1'-CH COO),
2
3
2
1
3
.52 (q, J = 7.4 Hz, 2H, CH CH ), 1.22 (d, J = 7.5 Hz, 3H, CH CH ). C NMR (100
2
3
2
3
MHz, CDCl ) δ 177.9, 171.0, 170.4, 162.2, 156.1, 133.1, 132.4, 130.6, 128.6, 128.2,
3
117.6, 111.0, 108.7, 108.5, 56.3, 55.4, 40.6, 27.3, 10.8.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 2-(o-tolyl)acetate (6k). Yield:
+
+
5
0.3%. m.p. 84.0-85.6 °C. MS(ESI) (m/z) 367.1 [M+H] . ESI-HRMS (m/z) [M+H]
1
Calc.: 367.1540, Found: 367.1544. H NMR (400 MHz, CDCl ) δ 8.20 (d, J = 8.9 Hz,
3

1
1
H, 5-H), 7.21-7.09 (m, 4H, (3',4',5',6'-Ph-H)), 7.01 (d, J = 8.9 Hz, 1H, 6-H), 6.12 (s,
H, 3-H), 5.43 (s, 2H, 8-CH ), 3.93 (s, 3H, OCH ), 3.64 (s, 2H, 1'-CH COO), 2.54 (q,
2
3
2
13
J = 7.5 Hz, 2H, CH CH ), 2.27 (s, 3H, 2'-CH ), 1.24 (t, J = 7.5 Hz, 3H, CH CH ). C
2
3
3
2
3
NMR (100 MHz, CDCl ) δ 177.9, 171.4, 170.4, 162.2, 156.1, 136.8, 132.8, 130.3,
3
1
1
30.1, 128.0, 127.4, 126.1, 117.6, 111.2, 108.7, 108.5, 56.3, 55.2, 39.1, 27.3, 19.5,
0.8.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 4-propylbenzoate (6l). Yield:
+
+
4
2.5%. m.p. 93.2-97.4 °C. MS(ESI) (m/z) 381.2 [M+H] . ESI-HRMS (m/z) [M+H]
1
Calc.: 381.1697, Found: 381.1700. H NMR (400 MHz, CDCl ) δ 8.22 (d, J = 9.0 Hz,
3
1
H, 5-H), 7.91 (d, J = 8.3 Hz, 2H, (2',6'-Ph-H)), 7.20 (d, J = 8.3 Hz, 2H, (3',5'-Ph-H)),
7
.04 (d, J = 9.0 Hz, 1H, 6-H), 6.13 (s, 1H, 3-H), 5.63 (s, 2H, 8-CH ), 3.98 (s, 3H,
2
OCH ), 2.65-2.58 (m, 4H, 2-CH CH , 4'-CH CH CH ), 1.64 (m, 2H, 4'-CH CH CH ),
3
2
3
2
2
3
2
2
3
3
1
1
.24 (t, J = 7.5 Hz, 3H, 2-CH CH ), 0.92 (t, J = 7.3 Hz, 3H, 4'-CH CH CH ). C
2
3
2
2
3
NMR (100 MHz, CDCl ) δ 178.0, 170.5, 166.7, 162.3, 156.3, 148.3, 129.7, 128.5,
3
1
28.0, 127.7, 117.7, 111.5, 108.8, 108.5, 56.4, 55.2, 38.0, 27.5, 24.3, 13.7, 10.9.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 4-pentylbenzoate (6m). Yield:
+
+
4
3.3%. m.p. 95.4-98.6 °C. MS(ESI) (m/z) 409.1 [M+H] . ESI-HRMS (m/z) [M+H]
1
Calc.: 409.2010, Found: 409.2009. H NMR (400 MHz, CDCl ) δ 8.22 (d, J = 9.0 Hz,
3
1
H, 5-H), 7.91 (d, J = 8.1 Hz, 2H, (2',6'-Ph-H)), 7.20 (d, J = 8.1 Hz, 2H, (3',5'-Ph-H)),
7
.04 (d, J = 9.0 Hz, 1H, 6-H), 6.13 (s, 1H, 3-H), 5.63 (s, 2H, 8-CH ), 3.98 (s, 3H,
2
OCH ), 2.61 (q, J = 8.3, 7.6 Hz, 4H, 2-CH CH , 4'-CH CH CH CH CH ), 1.61 – 1.58
3
2
3
2
2
2
2
3
(
m, 2H, 4'-CH CH CH CH CH ), 1.31-1.22 (m, 7H, 2-CH CH
4'
2
2
2
2
3
2
1
3,
3
CH CH CH CH CH ), 0.87 (t, J = 6.8 Hz, 3H, 4'-CH CH CH CH CH ). C NMR
2
2
2
2
3
2
2
2
2
3
(
100 MHz, CDCl ) δ 178.0, 170.5, 166.7, 162.3, 156.3, 148.6, 129.7, 128.4, 128.0,
3
1
27.6, 117.7, 111.6, 108.8, 108.5, 56.4, 55.2, 36.0, 31.4, 30.8, 27.5, 22.5, 14.0, 10.9.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 4-(tert-butyl)benzoate (6n).
+
Yield: 57.5%. m.p. 131.6-133.6 °C. MS(ESI) (m/z) 395.2 [M+H] . ESI-HRMS (m/z)
+
1
[
M+H] Calc.: 395.1489, Found: 395.1491. H NMR (400 MHz, CDCl ) δ 8.23 (d, J
3
=
9.0 Hz, 1H, 5-H), 7.94 (d, J = 8.4 Hz, 2H, (2',6'-Ph-H)), 7.42 (d, J = 8.4 Hz, 2H,
3',5'-Ph-H)), 7.05 (d, J = 9.0 Hz, 1H, 6-H), 6.14 (s, 1H, 3-H), 5.64 (s, 2H, 8-CH2),
.98 (s, 3H, OCH ), 2.62 (q, J = 7.5 Hz, 2H, CH CH ), 1.32 (s, 9H, C(CH ) ), 1.26 (t,
(
3
3
2
3
3 3
1
3
J = 7.5 Hz, 3H, CH CH ). C NMR (100 MHz, CDCl ) δ 178.0, 170.5, 166.6, 162.3,
2
3
3
1
3
56.6, 156.3, 129.5, 128.0, 127.4, 125.3, 117.7, 111.5, 108.8, 108.5, 56.4, 55.2, 35.1,
1.1, 27.5, 10.9.

(
(
(
(
E)-(2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 3-(4-chlorophenyl)acrylate
+
6o). Yield: 55.3%. m.p. 174.3-175.8 °C. MS(ESI) (m/z) 399.1 [M+H] . ESI-HRMS
+
1
m/z) [M+H] Calc.: 399.0994, Found: 399.0995. H NMR (400 MHz, CDCl ) δ 8.23
3
d, J = 9.0 Hz, 1H, 5-H), 7.65 (d, J = 16.0 Hz, 1H, CH=CHCOO), 7.43 (d, J = 8.5 Hz,
2
H, (2',6'-Ph-H)), 7.34 (d, J = 8.5 Hz, 2H, (3',5'-Ph-H)), 7.04 (d, J = 9.0 Hz, 1H, 6-H),
6
.41 (d, J = 16.0 Hz, 1H, CH=CHCOO), 6.14 (s, 1H, 3-H), 5.54 (s, 2H, 8-CH ), 3.99
2
(
s, 3H, OCH ), 2.65 (q, J = 7.6 Hz, 2H, CH CH ), 1.29 (t, J = 7.6 Hz, 3H, CH CH ).
3 2 3 2 3
1
3
C NMR (100 MHz, CDCl ) δ 177.9, 170.4, 166.7, 162.3, 156.2, 143.6, 136.3, 132.9,
3
1
29.2, 128.1, 118.5, 117.7, 111.3, 108.7, 108.6, 56.4, 55.1, 27.5, 10.9.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 3-methoxybenzoate (6p). Yield:
+
+
4
2.6%. m.p. 112.4-115.4 °C. MS(ESI) (m/z) 369.1 [M+H] . ESI-HRMS (m/z) [M+H]
1
Calc.: 369.1333, Found: 369.1336. H NMR (400 MHz, CDCl ) δ 8.23 (d, J = 9.0 Hz,
3
1
8
6
3
H, 5-H), 7.60 (d, J = 7.7 Hz, 1H, 6'-Ph-H), 7.56-7.52 (m, 1H, 5'-Ph-H), 7.31 (d, J =
.0 Hz, 1H, 4'-Ph-H), 7.08 (dd, J = 8.0, 1.9 Hz, 1H, 3'-Ph-H), 7.05 (d, J = 9.0 Hz, 1H,
-H), 6.13 (s, 1H, 3-H), 5.65 (s, 2H, 8-CH ), 3.99 (s, 3H, 7-OCH ), 3.83 (s, 3H,
2
3
1
3
'-OCH ), 2.62 (q, J = 7.5 Hz, 2H, CH CH ), 1.25 (t, J = 7.5 Hz, 3H, CH CH ). C
3
2
3
2
3
NMR (100 MHz, CDCl ) δ 178.0, 170.5, 166.5, 162.3, 159.5, 156.3, 131.5, 129.4,
3
1
28.1, 122.1, 119.2, 117.7, 114.3, 111.3, 108.8, 108.6, 56.4, 55.5, 55.5, 27.5, 10.9.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 3,4-dimethoxybenzoate (6q).
+
Yield: 50.8%，m.p. 178.4-179.3 °C，MS(ESI) (m/z) 399.2 [M+H] . ESI-HRMS (m/z)
+
1
[
M+H] Calc.: 399.1438, Found: 399.1440. H NMR (400 MHz, CDCl ) δ 8.23 (d, J
3
=
9.0 Hz, 1H, 5-H), 7.63 (dd, J = 8.4, 1.6 Hz, 1H, 6'-Ph-H), 7.52(d, J =1.6 Hz, 1H,
2
3
3
'-Ph-H), 7.05 (d, J = 9.0 Hz, 1H, 6-H), 6.84 (d, J = 8.4 Hz, 1H, 5'-Ph-H), 6.13 (s, 1H,
-H), 5.62 (s, 2H, CH OOC), 3.98 (s, 3H, 7-OCH ), 3.91 (s, 3H, 3'-OCH ), 3.90 (s,
2
3
3
H, 4'-OCH ), 2.62 (q, J = 7.5 Hz, 2H, CH CH ), 1.25 (t, J = 7.5 Hz, 3H, CH CH ).
3
2
3
2
3
1
3
C NMR (100 MHz, CDCl ) δ 178.0, 170.5, 166.4, 162.3, 156.3, 153.0, 148.6, 128.0,
3
1
23.7, 122.7, 117.7, 112.1, 111.6, 110.2, 108.8, 108.6, 56.4, 56.1, 56.0, 55.4, 27.5,
0.9.
1
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 2,4,5-trimethoxybenzoate (6r).
+
Yield: 46.3%. m.p. 165.1-167.1 °C. MS(ESI) (m/z) 429.2 [M+H] . ESI-HRMS (m/z)
+
1
[
M+H] Calc.: 429.1544, Found: 429.1547. H NMR (400 MHz, CDCl ) δ 8.22 (d, J
3
=
9.0 Hz, 1H, 5-H), 7.31 (s, 1H, 6'-Ph-H), 7.04 (d, J = 9.0 Hz, 1H, 6-H), 6.51 (s, 1H,
3
2
2
'-Ph-H), 6.13 (s, 1H, 3-H), 5.61 (s, 2H, 8-CH ), 3.99 (s, 3H, 7-OCH ), 3.92 (s, 3H,
2
3
'-OCH ), 3.82 (s, 3H, 5'-OCH ), 3.80 (s, 3H, 4'-OCH ), 2.64 (q, J = 7.6 Hz, 2H,
3
3
3
1
3
-CH CH ), 1.27 (t, J = 7.6 Hz, 3H, 2-CH CH ). C NMR (100 MHz, CDCl ) δ
2
3
2
3
3

1
78.1, 170.5, 165.5, 162.3, 156.3, 155.8, 153.6, 142.5, 127.8, 117.6, 114.5, 111.8,
110.9, 108.7, 108.5, 97.9, 57.1, 56.5, 56.3, 56.0, 55.1, 27.5, 10.9.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 3,4,5-trimethoxybenzoate (6s).
+
Yield: 42.6%. m.p. 147.9-150.4 °C. MS(ESI) (m/z) 429.1 [M+H] . ESI-HRMS (m/z)
+
1
[
M+H] Calc.: 429.1544, Found: 429.1547. H NMR (400 MHz, CDCl ) δ 8.24 (d, J
3
=
9.0 Hz, 1H, 5-H), 7.26 (s, 2H, (2',6'-Ph-H)), 7.06 (d, J = 9.0 Hz, 1H, 6-H), 6.15 (s,
1
6
3
1
1
H, 3-H), 5.64 (s, 2H, 8-CH ), 4.00 (s, 3H, 7-OCH ), 3.89 (s, 3H, 4'-OCH ), 3.87 (s,
2
3
3
H, 3'-OCH , 5'-OCH ), 2.64 (q, J = 7.5 Hz, 2H, 2-CH CH ), 1.27 (t, J = 7.5 Hz, 3H,
3
3
2
3
13
-CH CH ). C NMR (100 MHz, CDCl ) δ 177.9, 170.4, 166.3, 162.3, 156.3, 152.9,
2
3
3
42.3, 128.1, 125.2, 117.7, 111.4, 108.8, 108.6, 107.0, 60.9, 56.4, 56.3, 55.7, 27.5,
1.0.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl
2-(3,4,5-trimethoxyphenyl)
+
acetate (6t). Yield: 46.5%. m.p. 156.4-158.0 °C. MS(ESI) (m/z) 443.2 [M+H] .
+
ESI-HRMS (m/z) [M+H] Calc.: 443.1700, Found: 443.1702. ESI-HRMS (m/z)
+
1
[
M+H] Calc.: 443.1700, Found: 443.1697. H NMR (400 MHz, CDCl ) δ 8.21 (d, J
3
=
8.9 Hz, 1H, 5-H), 7.01 (d, J = 8.9 Hz, 1H, 6-H), 6.48 (s, 2H, (2',6'-Ph-H)), 6.11 (s,
1
6
1
1
5
H, 3-H), 5.44 (s, 2H, 8-CH ), 3.92 (s, 3H, 7-OCH ), 3.82 (s, 3H, 4'-OCH ), 3.78 (s,
2
3
3
H, 3'-OCH , 5'-OCH ), 3.55 (s, 2H, 1'-CH COO), 2.53 (q, J = 7.5 Hz, 2H, CH CH ),
3
3
2
2
3
13
.22 (t, J = 7.5 Hz, 3H, CH CH ). C NMR (100 MHz, CDCl ) δ 177.8, 171.3, 170.4,
2
3
3
62.2, 156.1, 153.2, 137.2, 129.5, 128.1, 117.6, 111.2, 108.7, 108.5, 106.4, 60.8, 56.3,
6.1, 55.3, 41.6, 27.3, 10.8.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl cinnamate (6u). Yield: 40.8%.
+
+
m.p. 140.2-140.9 °C. MS(ESI) (m/z) 365.2 [M+H] . ESI-HRMS (m/z) [M+H] Calc.:
1
3
5
7
1
65.1384, Found: 365.1386. H NMR (400 MHz, CDCl ) δ 8.22 (d, J = 9.0 Hz, 1H,
3
-H), 7.71 (d, J = 16.0 Hz, 1H, CH=CHCOO), 7.53-7.47 (m, 2H, (2',6'-Ph-H)),
.40-7.33 (m, 3H, (3',4',5'-Ph-H)), 7.04 (d, J = 9.0 Hz, 1H, 6-H), 6.45 (d, J = 16.0 Hz,
H, CH=CHCOO), 6.14 (s, 1H, 3-H), 5.55 (s, 2H, 8-CH ), 3.99 (s, 3H, OCH ), 2.66
2
3
13
(q, J = 7.8 Hz, 2H, CH CH ), 1.29 (t, J = 7.8 Hz, 3H, CH CH ). C NMR (100 MHz,
2 3 2 3
CDCl ) δ 178.0, 170.5, 166.9, 162.3, 156.3, 145.0, 134.4, 130.3, 128.9, 128.1, 117.9,
3
1
17.7, 111.4, 108.8, 108.6, 56.4, 55.0, 27.5, 10.9.
(
E)-(2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl
3-(4-methoxyphenyl)
+
acrylate (6v). Yield: 42.3%. m.p. 150.9-151.8 °C. MS(ESI) (m/z) 395.2 [M+H] .
+
1
ESI-HRMS (m/z) [M+H] Calc.: 395.1489, Found: 395.1491. H NMR (400 MHz,

CDCl ) δ 8.22 (d, J = 8.9 Hz, 1H, 5-H), 7.66 (d, J = 16.0 Hz, 1H, CH=CHCOO), 7.45
3
(d, J = 8.7 Hz, 2H, (2',6'-Ph-H)), 7.04 (d, J = 8.9 Hz, 1H, 6-H), 6.89 (d, J = 8.7 Hz,
2
H, (3',5'-Ph-H)), 6.31 (d, J = 16.0 Hz, 1H, CH=CHCOO), 6.14 (s, 1H, 3-H), 5.53 (s,
H, 8-CH ), 3.99 (s, 3H, OCH ), 3.83 (s, 3H, OCH ), 2.65 (q, J = 7.5 Hz, 2H,
2
2
3
3
1
3
CH CH ), 1.29 (t, J = 7.5 Hz, 3H, CH CH ). C NMR (100 MHz, CDCl ) δ 178.0,
2
3
2
3
3
1
70.5, 167.3, 162.2, 161.4, 156.2, 144.7, 129.7, 128.0, 127.1, 117.7, 115.3, 114.3,
111.5, 108.7, 108.5, 56.4, 55.4, 54.8, 27.5, 10.9.
(
2-Ethyl-7-methoxy-4-oxo-4H-chromen-8-yl)methyl 4-cyanobenzoate (6w). Yield:
+
+
4
5.6%. m.p. 197.1-198.2 °C. MS(ESI) (m/z) 364.1 [M+H] . ESI-HRMS (m/z) [M+H]
1
Calc.: 364.1179, Found: 364.1182. H NMR (400 MHz, CDCl ) δ 8.25 (d, J = 8.9 Hz,
3
1
H, 5-H), 8.12 (d, J = 8.2 Hz, 2H, (2',6'-Ph-H)), 7.72 (d, J = 8.2 Hz, 2H, (3',5'-Ph-H)),
7
.06 (d, J =8.9 Hz, 1H, 6-H), 6.14 (s, 1H, 3-H), 5.68 (s, 2H, 8-CH ), 3.99 (s, 3H,
2
1
3
OCH ), 2.62 (q, J = 7.6 Hz, 2H, CH CH ), 1.25 (t, J = 7.6 Hz, 3H, CH CH ). C
3
2
3
2
3
NMR (100 MHz, CDCl ) δ 177.8, 170.3, 164.9, 162.3, 156.2, 134.0, 132.2, 130.2,
3
1
28.4, 117.9, 117.7, 116.4, 110.7, 108.8, 108.7, 56.4, 56.2, 27.5, 10.9.
(
7-Ethoxy-2-ethyl-4-oxo-4H-chromen-8-yl)methyl 2,4,5-trimethoxybenzoate (7a).
+
Yield: 50.4%. m.p. 159.8-161.7 °C. MS(ESI) (m/z) 443.0 [M+H] . ESI-HRMS (m/z)
+
1
[
M+H] Calc.: 443.1700, Found: 443.1702. H NMR (400 MHz, CDCl ) δ 8.19 (d, J
3
=
8.9 Hz, 1H, 5-H), 7.32 (s, 1H, 6'-Ph-H), 7.01 (d, J = 8.9 Hz, 1H, 6-H), 6.51 (s, 1H,
3
7
2
7
1
5
'-Ph-H), 6.13 (s, 1H, 3-H), 5.62 (s, 2H, 8-CH ), 4.22 (q, J = 6.9 Hz, 2H,
2
-OCH CH ), 3.92 (s, 3H, 2'-OCH ), 3.82 (s, 3H, 5'-OCH ), 3.80 (s, 3H, 4'-OCH ),
2
3
3
3
3
.63 (q, J = 7.5 Hz, 2H, 2-CH CH ), 1.46 (t, J = 6.9 Hz, 3H, 7-OCH CH ), 1.26 (t, J =
2
3
2
3
13
.5 Hz, 3H, 2-CH CH ). C NMR (100 MHz, CDCl ) δ 178.1, 170.5, 165.5, 161.8,
2
3
3
56.3, 155.8, 153.6, 142.6, 127.7, 117.5, 114.6, 111.9, 111.0, 109.6, 108.5, 98.0, 64.8,
7.1, 56.5, 56.0, 55.2, 27.5, 14.7, 10.9.
(
7-Ethoxy-2-ethyl-4-oxo-4H-chromen-8-yl)methyl 3,4,5-trimethoxybenzoate (7b).
+
Yield: 52.6%. m.p. 162.0-164.1 °C. MS(ESI) (m/z) 443.0 [M+H] . ESI-HRMS (m/z)
+
1
[
M+H] Calc.: 443.1700, Found: 443.1704. H NMR (400 MHz, CDCl ) δ 8.21 (d, J
3
=
8.9 Hz, 1H, 5-H), 7.26 (s, 2H, (2',6'-Ph-H)), 7.03 (d, J = 8.9 Hz, 1H, 6-H), 6.14 (s,
1
4
H, 3-H), 5.66 (s, 2H, 8-CH ), 4.23 (q, J = 6.9 Hz, 2H, 7-OCH CH ), 3.89 (s, 3H,
2
2
3
'-OCH ), 3.87 (s, 6H, 3'-OCH , 5'-OCH ), 2.64 (q, J = 7.3 Hz, 2H, 2-CH CH ), 1.46
3
3
3
2
3
13
(t, J = 6.9 Hz, 3H, 7-OCH CH ), 1.27 (t, J = 7.3 Hz, 3H, 2-CH CH ). C NMR (100
2 3 2 3
MHz, CDCl ) δ 178.0, 170.4, 166.3, 161.8, 156.4, 153.0, 142.3, 128.0, 125.3, 117.6,
3
1
11.5, 109.7, 108.6, 107.0, 64.9, 60.9, 56.3, 55.8, 27.5, 14.8, 11.0.

(
7-Ethoxy-2-ethyl-4-oxo-4H-chromen-8-yl)methyl
2-(3,4,5-trimethoxyphenyl)
+
acetate (7c). Yield: 57.2%. m.p. 106.4-107.4 °C. MS(ESI) (m/z) 457.2 [M+H] .
+
1
ESI-HRMS (m/z) [M+H] Calc.: 457.1857, Found: 457.1860. H NMR (400 MHz,
CDCl ) δ 8.18 (d, J = 8.9 Hz, 1H, 5-H), 6.98 (d, J = 8.9 Hz, 1H, 6-H), 6.48 (s, 2H,
3
(
2',6'-Ph-H)), 6.12 (s, 1H, 3-H), 5.45 (s, 2H, 8-CH ), 4.15 (q, J = 6.9 Hz, 2H,
2
7
1
7
1
1
-OCH CH ), 3.82 (s, 3H, 4'-OCH ), 3.78 (s, 6H, 3'-OCH , 5'-OCH ), 3.56 (s, 2H,
2 3 3 3 3
'-CH COO), 2.53 (q, J = 7.6 Hz, 2H, 2-CH CH ), 1.39 (t, J = 6.9 Hz, 3H,
2
2
3
3
1
-OCH CH ), 1.23 (t, J = 7.6 Hz, 3H, 2-CH CH ). C NMR (100 MHz, CDCl ) δ
2
3
2
3
3
77.9, 171.3, 170.3, 161.6, 156.2, 153.2, 137.1, 129.6, 128.0, 117. 5, 111.3, 109.5,
08.5, 106.3, 64.9, 60.8, 56.0, 55.4, 41.7, 27.3, 14.6, 10.8.
(
7-Ethoxy-2-ethyl-4-oxo-4H-chromen-8-yl)methyl cinnamate (7d). Yield: 46.4%.
+
+
m.p. 128.7-130.7 °C. MS(ESI) (m/z) 379.2 [M+H] . ESI-HRMS (m/z) [M+H] Calc.:
1
3
5
7
1
7
7
1
79.1540, Found: 379.1542. H NMR (400 MHz, CDCl ) δ 8.20 (d, J = 9.0 Hz, 1H,
3
-H), 7.71 (d, J = 16.0 Hz, 1H, CH=CHCOO), 7.53-7.47 (m, 2H, (2',6'-Ph-H)),
.41-7.34 (m, 3H, (3',4',5'-Ph-H)), 7.02 (d, J = 9.0 Hz, 1H, 6-H), 6.45 (d, J = 16.0 Hz,
H, CH=CHCOO), 6.14 (s, 1H, 3-H), 5.56 (s, 2H, 8-CH ), 4.22 (q, J = 7.0 Hz, 2H,
2
-OCH CH ), 2.65 (q, J = 7.5 Hz, 2H, 2-CH CH ), 1.47 (t, J = 7.0 Hz, 3H,
2
3
2
3
13
-OCH CH ), 1.29 (t, J = 7.5 Hz, 3H, 2-CH CH ). C NMR (100 MHz, CDCl ) δ
2
3
2
3
3
78.0, 170.4, 166.9, 161.7, 156.3, 145.0, 134.4, 130.3, 128.9, 128.1, 127.9, 1179,
17.5, 111.4, 109.6, 108.6, 64.9, 55.1, 27.5, 14.7, 10.9.
1
(
(
(
(
E)-(7-Ethoxy-2-ethyl-4-oxo-4H-chromen-8-yl)methyl 3-(4-methoxyphenyl) acrylate
+
7e). Yield: 43.2%. m.p. 128.4-126.9 °C. MS(ESI) (m/z) 409.2 [M+H] . ESI-HRMS
+
1
m/z) [M+H] Calc.: 409.1646, Found: 409.1649. H NMR (400 MHz, CDCl ) δ 8.19
3
d, J = 9.0 Hz, 1H, 5-H), 7.67 (d, J = 15.9 Hz, 1H, CH=CHCOO), 7.46 (d, J = 8.7 Hz,
2
6
H, (2',6'-Ph-H)), 7.01 (d, J = 9.0 Hz, 1H, 6-H), 6.89 (d, J = 8.7 Hz, 2H, (3',5'-Ph-H)),
.32 (d, J = 15.9 Hz, 1H, CH=CHCOO), 6.14 (s, 1H, 3-H), 5.54 (s, 2H, 8-CH ), 4.22
2
(
q, J = 6.9 Hz, 2H, 7-OCH CH ), 3.83 (s, 3H, 4'-OCH ), 2.65 (q, J = 7.5 Hz, 2H,
2 3 3
2
-CH CH ), 1.47 (t, J = 6.9 Hz, 3H, 7-OCH CH ), 1.29 (t, J = 7.5 Hz, 3H, 2-CH CH ).
2 3 2 3 2 3
1
3
C NMR (100 MHz, CDCl ) δ 178.0, 170.4, 167.3, 161.7, 161.4, 156.3, 144.6, 129.7,
3
1
27.8, 127.1, 117.5, 115.3, 114.3, 111.6, 109.6, 108.5 , 64.9, 55.4, 54.9, 27.5, 14.7,
0.9.
1
(
7-Ethoxy-2-ethyl-4-oxo-4H-chromen-8-yl)methyl 4-cyanobenzoate (7f). Yield:
+
+
4
4.8%. m.p. 196.8-197.2 °C. MS(ESI) (m/z) 378.1 [M+H] . ESI-HRMS (m/z) [M+H]
1
Calc.: 378.1336, Found: 378.1337. H NMR (400 MHz, CDCl ) δ 8.22 (d, J = 8.9 Hz,
3
1
H, 5-H), 8.12 (d, J = 8.6 Hz, 2H, (2',6'-Ph-H)), 7.72 (d, J = 8.6 Hz, 2H, (3',5'-Ph-H)),