Extending the structure?activity relationship study of marine natural ningalin B analogues as P-glycoprotein inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.09.070

In the present study, a total of 25 novel ningalin B analogues were synthesized and evaluated for their P-gp modulating activity in a P-gp overexpressed breast cancer cell line LCC6MDR. Preliminary structure-activity study shows that A ring and its two methoxy groups are important pharmacophores for P-gp inhibiting activity. Among all derivatives, 23 is the most potent P-gp modulator with EC₅₀of 120–165?nM in reversing paclitaxel, DOX, vinblastine and vincristine resistance. It is relatively safe to use with selective index at least greater than 606 compared to verapamil. Mechanistic study demonstrates that compound 23 reverses P-gp mediated drug resistance by inhibiting transport activity of P-gp, thereby restoring intracellular drug accumulation. In summary, our study demonstrates that ningalin B analogue 23 is a non-cytotoxic and effective P-gp chemosensitizer that can be used in the future for reversing P-gp mediated clinical cancer drug resistance.

Full text of DOI:10.1016/j.ejmech.2016.09.070

Accepted Manuscript
Extending the structure−activity relationship study of marine natural ningalin B
analogues as P-glycoprotein inhibitors
Chao Yang, Iris L.K. Wong, Kai Peng, Zhen Liu, Peng Wang, Tingfu Jiang, Tao Jiang,
Larry M.C. Chow, Sheng Biao Wan
PII:
S0223-5234(16)30806-6
10.1016/j.ejmech.2016.09.070
EJMECH 8936
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 April 2016
Revised Date: 20 September 2016
Accepted Date: 21 September 2016
Please cite this article as: C. Yang, I.L.K. Wong, K. Peng, Z. Liu, P. Wang, T. Jiang, T. Jiang, L.M.C.
Chow, S.B. Wan, Extending the structure−activity relationship study of marine natural ningalin B
analogues as P-glycoprotein inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.09.070.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Extending the Structure−Activity Relationship study of Marine Natural Ningalin
B Analogues as P-glycoprotein Inhibitors
1
, 2†
3,4†
1, 2
1, 2
1, 2
Chao Yang , Iris L. K. Wong , Kai Peng , Zhen Liu , Peng Wang , Tingfu
1
,2
1,2
3,4*
1, 2*
Jiang , Tao Jiang , Larry M. C. Chow and Sheng Biao Wan
Rhodamine 123
.0X
8
2000
1500
1000
LCC6
LCC6MDR
4
.8X
N = 2-3 independent expts
2
.7X
500
0

ACCEPTED MANUSCRIPT
Graphical Abstract
Extending the Structure−Activity Relationship study of Marine Natural Ningalin
B Analogues as P-glycoprotein Inhibitors
1
, 2†
3,4†
1, 2
1, 2
1, 2
Chao Yang , Iris L. K. Wong , Kai Peng , Zhen Liu , Peng Wang , Tingfu
1
,2
1,2
3,4*
1, 2*
Jiang , Tao Jiang , Larry M. C. Chow and Sheng Biao Wan
Rhodamine 123
8.0X
2000
LCC6
LCC6MDR
1500
4
.8X
N = 2-3 independent expts
1000
2
.7X
500
0

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Extending the Structure−
Activ Relationship study of Marine Natural Ningalin
ACC Ei t yP TED MANUSCRIPT
B Analogues as P-glycoprotein Inhibitors
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, 2†
3,4†
1, 2
1, 2
1, 2
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Chao Yang , Iris L. K. Wong , Kai Peng , Zhen Liu , Peng Wang , Tingfu
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1,2
3,4*
1, 2*
Jiang , Tao Jiang , Larry M. C. Chow and Sheng Biao Wan
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Key Laboratory of Marine Drugs, Ministry of Education; Laboratory for Marine Drugs
and Bioproducts, Qingdao National Laboratory for Marine Science and Technology;
School of Medicine and Pharmacy, Ocean University of China, Qingdao, China.
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State Key Laboratory of Bioactive Substance and Function of Natural Medicines
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing, China.
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Department of Applied Biology and Chemical Technology, The Hong Kong
Polytechnic University, Hung Hom, Hong Kong SAR, China.
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State Key Laboratory for Chirosciences, The Hong Kong Polytechnic University,
Hong Kong SAR, China.
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†These two authors contribute equally to this work.
* Corresponding authors: Sheng Biao Wan and Larry M. C. Chow
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Larry M. C. Chow, Tel: 852-34008662; Fax: 852-23649932;
E-mail: larry.chow@polyu.edu.hk (L.M.C.C.).
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Sheng Biao Wan, Tel.: 86-532-82031087; Fax: 86-532-82033054;
E-mail: biaowan@ouc.edu.cn (S.B.W.).
Keywords
Ningalin B, Multidrug resistance (MDR); P-glycoprotein (P-gp); P-gp chemosensitizer,
ATP-binding cassette (ABC) transporter.
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Abstract
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In the present study, a total of 25 novel ningalin B analogues were synthesized and
evaluated for their P-gp modulating activity in a P-gp overexpressed breast cancer cell
line LCC6MDR. Preliminary structure-activity study shows that A ring and its two
methoxy groups are important pharmacophores for P-gp inhibiting activity. Among all
derivatives, 23 is the most potent P-gp modulator with EC of 120 to 165 nM in
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reversing paclitaxel, DOX, vinblastine and vincristine resistance. It is relatively safe to
use with selective index at least greater than 606 compared to verapamil. Mechanistic
study demonstrates that compound 23 reverses P-gp mediated drug resistance by
inhibiting transport activity of P-gp, thereby restoring intracellular drug accumulation.
In summary, our study demonstrates that ningalin B analogue 23 is a non-cytotoxic and
effective P-gp chemosensitizer that can be used in the future for reversing P-gp
mediated clinical cancer drug resistance.
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1. Introduction
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P-glycoprotein (P-gp or ABCB1) belongs to the ATP-Binding Cassette (ABC)
super-family of proteins[1]. Overexpression of P-gp in cancer patients has been
correlated with chemoresistance and poor prognosis. It can pump various anticancer
drugs out of cancer cells and reducing intracellular drug concentration below their
therapeutic levels[2-5]. P-gp has also been found cancer stem cells that are highly drug
resistant [6]. Considerable effort has been spent on developing P-gp inhibitors[7-14].
However, only a few non-toxic and specific P-gp inhibitors have been found[15-20]
and none of these inhibitors can be used clinically. Therefore, searching for novel P-gp
inhibitors with low toxicity and high potency remains an important approach to reverse
clinical multidrug resistance (MDR).
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Although P-gp was the most characterized ABC family member in terms of its
structure and mechanism of action[3, 21, 22], its exact binding sites of inhibitors
remains elusive. Recently, non-toxic natural products including curcumin[23],
kaempferol[24], quercetin[25], epigallocatechin gallate[26], lamallarine K, lamallarine
I, lamallarine O[27] and their derivatives or analogues such as quercetin pentamethyl
ether[28], permethyl lamallarine D, permethyl ningalin D and permethyl ningalin B
(shown in Figure 1)[29-31] have been discovered as a novel source for providing new
P-gp modulators[10]. We also have synthesized analogues of natural marine product
ningalin B and characterized their P-gp inhibitory activity. We found that four ningalin
B analogues had potent P-gp inhibitory activity, were safe to use and specific towards
P-gp (compounds 1-4 in Figure 1)[32-34]. Structure-activity relationship study
revealed that substituent at C-ring and the linkers between N atom of pyrroledione and
C-ring were important. Compounds 3 and 4 were the most potent and non-cytotoxic
lead compounds. In this report, we will study the effects of substituent in rings A and C
on P-gp modulating activity.
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Figure 1. Ningalin B analogues as P-gp inhibitors.
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2. Results and Discussion
2.1. Chemistry
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As shown in Scheme
N-substituted 3-arylpyrroledione derivatives were synthesized. Phthalimide was
reacted with
1, thr N-substituted phthalimide derivatives and two
ACC Ee eP TED MANUSCRIPT
3,4,5-trimethoxybenzylmethanesulfonate,2-bromo-1-(4-methoxyphenyl)ethanone, and
2-bromo-1-(4-benzoloxy-3-methoxyphenyl)ethanone in the presence of K CO in DMF
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3
to afford compounds 6, 7, and 8 respectively. Synthetic compound 9[35] was coupled
with 2-bromo-1-(4-benzoloxy-3-methoxyphenyl)ethanone in the presence of K CO in
2
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DMF to produce compound 11 which was then reduced to compound 12.
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Scheme 1. Synthetic route of compounds 6, 7, 8, 11, and 12.
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Synthetic route of compounds 15a-23 is shown in Scheme 2. The key
intermediates 13a-13f and 14a-14f were prepared as previously described[33, 34].
3,4,5-Trimethoxybenzyl methanesulfonate was reacted with 14a, 14b, 14c, or 14d in
the presence of K CO in DMF to afford compounds 15a, 15d, 15c, or 15d,
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3
respectively. Similarly, reaction of 4-benzoloxybenzyl methanesulfonate or
3-methoxy-4-benzoloxybenzyl methanesulfonate with 14e gave compounds 16e or 17e.
Catalyzed by K CO , coupling of 2-bromo-1-(4-methoxyphenyl)ethanone with 14a,
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14b, 14c,or 14d produced target compounds 18a, 18b, 18c, or 18d, respectively. Using
the same procedureꢀcompounds 19a, 19b, 19c, 19d, or 19f were obtained. 14e or 14f
was
coupled
with
4-(methylsulfonyl)benzyl
methanesulfonate,
or
2-bromo-1-(4-(methylsulfonyl)phenyl)ethanone,
2-bromo-1-(2-bromo-5-methoxy-4-((3,4,5-trimethoxybenzyl)oxy)phenyl)ethanone (22)
in the presence of K CO in DMF to provide compounds 20e, 20f, 21e, 21f, or 23 ,
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3
respectively.
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Scheme 2. Synthetic route of compounds 15a-23 .
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2.2. P-gp-modulating activity of ningalin B analogues
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P-gp-transfected breast cancer cell line MDA435/LCC6MDR was 87.1-fold more
resistant to paclitaxel (PTX) than its parental LCC6 cells (Table 1). Verapamil (RF =
4.0) and cyclosporine A (RF = 77.4) at 1 µM were positive controls. Based on our lead
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compounds 1-3[32-34], twenty five new ningalin B analogues were synthesized and
divided into 3 series according to their (1) type and number of substituent at rings A
and C and (2) type of linker between C1 of ring C and N atom of pyrrole-2,5-dione
(Table 1).
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The importance of substitution at rings A and C of ningalin B analogues on P-gp
modulating activity was studied. In series I, compound 1 (RF = 13.8)[34] with
methylene linker and di-methoxy groups on rings A and B and tri-methoxy groups on
ring C was the lead compound. Removal of phenyl rings A and B in compound 6 (RF =
1.2) completely abolished P-gp modulating activity, indicating that methylated
polyphenol structure is an important pharmacophore. Reducing the number of methoxy
group at phenyl ring A gradually diminished the potency to reverse P-gp-mediated
PTX resistance: di-methoxylated compound 1 (RF = 13.8) > mono-methoxylated 15a
(RF = 11.4) and 15b (RF = 5.1) and non-substituted 15d (RF = 7.1). Substitution
position at ring A is also important because mono-methoxylation at meta-position (15a
with RF = 11.4) displayed 2-fold higher RF value than that at para-position (15b with
RF = 5.1). Furthermore, the size of substituent is also important. Smaller para-methoxy
group at ring A in compound 1 (RF = 13.8) results in a higher activity than the bulkier
group of para-benzyloxy group in compound 15c (RF = 11.9). This result suggests that
smaller substituent is preferred. This effect of substituent size was also observed in ring
C among this series. At ring C when trimethoxy groups (compound 1 with RF = 13.8)
were replaced by methoxybenzyloxy, benzyloxy and methylsulfonyl substituents, there
was a reduction in P-gp modulating activity (compounds 16e, 17e, 20e and 20f with
RF = 9.3, 4.2, 3.4 and 3.0).
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Compounds of series II are analogues of lead compound 2. They have longer
carbonylmethylene linker than that of series I (methylene linker). They only have
mono-substitution at ring C instead of tri-substitution as in series I. Compound 2 (RF =
7.6) displayed the weakest P-gp modulating activity compared to the other two lead
compounds (1 with RF = 13.8 and 3 with RF = 39.8)[32,34]. Similar to series I, when
rings A and B are removed in compound 7 (RF = 1.7) in series II, the P-gp modulating
activity is completely lost. The influence of number and size of substituent on
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P-gp-modulating activity is investigated in series II. First, di-methoxylation at ring A (2
with RF = 7.6) displayed better activity than mono-methoxylation (18a and 18b with
RF = 3.5 and 2.7) or non-methoxylation (18d with RF = 2.6). Second, when the
substituent at ring A gradually increased in size from di-methoxy in 2 (RF = 7.6) to
benzyloxy in 18c (RF = 4.5), P-gp modulating activity was reduced. Similar to series I,
methylsulfonyl group in ring C was not preferred (21e with RF = 1.4 or 21f with RF =
2.0).
In series III, either removal of both A and B rings or A ring alone consistently
resulted in a complete loss of P-gp modulating activity (8, 11 and 12 with RF = 1.1, 1.4
and 1.4 respectively), indicating that both rings A and B were important
pharmacophores. Same as series I and II, di-substitution at ring A (3 with RF = 39.8)
displayed stronger activity than mono- (19a, 19b and 19f with RF = 8.8, 22.1 and 8.6)
and non-substituted analogues (19d with RF = 5.9). Contrary to series I,
mono-methoxylation at para-position (19b with RF = 22.1) yielded higher RF values
than that at meta-position (19a with RF = 8.8). Replacing the para-methoxy group of
compound 3 (RF = 39.8) at A ring by para-benzyloxy (19c with RF = 1.4) or replacing
para-methoxy group of 19b (RF = 22.1) at A ring by para-methyl group (19f with RF =
8.6) both caused a marked reduction in the P-gp modulating activity. Surprisingly,
additional modification at C ring of compound 3 (RF = 39.8) with a 2-bromo,
3-methoxy and 4-(3,4,5-trimethoxybenzloxy) groups resulted in a highly potent P-gp
modulator 23 (RF = 48.0). Further modification at C ring should be considered in the
future.
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Table 1. P-gp modulating activity, CLogP and PSA of ningalin B analogues
56 Ningalin B analogues are divided into 3 series according to their R and R groups at ring A, R
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57 group at ring C and types of linker used. Lead compounds 1, 2 and 3 of series I, II and III,
58 respectively have been reported previously and they were used as starting points for
59 modification[32,34]. P-gp-modulating activity was measured by determining IC50 towards
60 PTX in LCC6MDR cells in the absence or presence of 1.0 µM of modulator. At 1.0 µM, none
61 of the modulators displayed any cytotoxicity towards LCC6MDR cells was found. Relative
62 Fold (RF) reflects P-gp-modulating activity and is calculated as [IC50 of PTX without
63 modulator / IC with 1.0 µM modulator]. All modulators were dissolved in DMSO. Each
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65 as mean ± standard error of mean. These data have been reported previously[32,34].
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2.3. EC (nM) and selective index values of ningalin B analog 23
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We have chosen one potent ningalin analogue 23 for further characterization in terms of
its effective concentration (EC ) in reversing P-gp-mediated PTX, DOX, vinblastine and
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vincristine resistance and its selective index (Figure 2 and Table 2). Compound 23 was
non-cytotoxic towards L929 normal fibroblasts with IC greater than 100 µM (Table 2). Its
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EC for reversing P-gp mediated resistance towards PTX, DOX, vinblastine and vincristine
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ranged from 120 to 165 nM, which is about 2.0-fold to 3.7-fold more potent than verapamil
(EC ranged from 245 to 503 nM) (Figure 2 and Table 2). After considering toxicity,
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selective index of 23 ranged from >606 to >833 which is at least 2.3-fold higher than
verapamil (selective index ranged from 177 to 363). Overall, 23 is a non-cytotoxic and
effective P-gp chemosensitizer.
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Figure 2. Dose-response curves of 23 and verapamil on reversing drug resistance in
LCC6MDR cells. In 5-day cell proliferation assay, LCC6MDR cells were incubated with
different dosages of anticancer drugs (PTX, DOX, vinblastine or vincristine) with or without
defined concentration of modulators. In each concentration of modulators, the IC of each
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drug was determined using Prism software. Then, the dose-response curve of each modulator
in reducing IC of each drug by half was plotted using non-linear regression dose-response
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curve analysis. The values were presented as mean ± standard error of mean.
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Table 2. Effective concentration EC50 (nM) and selective index value of 23 in reversing
different drug resistance of LCC6MDR.
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EC50 values were presented as mean ± standard error of mean. N = 2-7 independent
experiments. Each experiment was done in triplicate. Verapamil was used as positive control.
Selective index is a preliminary indicator of safety of drug candidates. It is a ratio of
the dose of drug that causes cytotoxicity in normal cell lines divided by the dose that leads to
the desired P-gp modulating efficacy in cancer cell lines. Here, selective index = (IC of
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modulators towards L929 fibroblasts) / (EC of modulators for reversing PTX resistance in
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LCC6MDR cells).
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2.4. Ningalin B analogue 23 increases DOX and rhodamine 123 accumulation in LCC6MDR
cells
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Doxorubicin (DOX) and rhodamine 123 are known fluorescent P-gp substrates. We
found that LCC6 cells accumulated about 2.6- and 8.0-fold more DOX and rhodamine 123
than LCC6MDR cells (Figure 3A and 3B). Treatment of LCC6MDR cells with 2 µM of 23
or verapamil can inhibit P-gp and increase DOX accumulation by 2.4- or 2.3-fold to a level
similar to that of LCC6 cells (Figure 3A). Treating LCC6MDR cells with 2 µM of 23 or
verapamil partially restored rhodamine 123 accumulation by 4.8- and 2.7-fold, respectively
(Figure 3B). Compound 23 was 1.7-fold more potent than verapamil in inhibiting
P-gp-mediated transport of rhodamine 123. EC is a concentration of modulator at which the
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DOX accumulation level in LCC6MDR cells is increased to half of that of parental LCC6
cell. Compound 23 (EC = 78 nM) (Figure 4A) displayed 4.5-fold lower concentration than
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verapamil (EC = 350 nM) (Figure 4B) in increasing DOX accumulation, indicating that
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compound 23 is more promising than verapamil in inhibiting P-gp transport activity and then
restoring DOX intracellular accumulation.
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Figure 3. Effect of 23 on DOX and rhodamine 123 accumulation in LCC6MDR cells.
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Effect of compound 23 or verapamil on intracellular DOX and rhodamine 123 accumulation
was studied. LCC6 or LCC6MDR cells were incubated with 20 µM DOX (A) or 10 µg/mL
rhodamine 123 (B) with or without 2 µM of modulator for 150 minutes at 37 C. 0.2% of
DMSO was used as negative control. After the incubation period, cells were lysed and the
DOX level in supernatant was measured by spectrofluorometry. N = 2-4 independent
experiments and values were presented as mean ± standard error of mean.
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Figure 4. Dosage effect of 23 and verapamil on DOX accumulation in LCC6MDR cells.
Dosage effect of compound 23 or verapamil on intracellular DOX accumulation was studied.
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LCC6MDR cells were incubated with 20 µM DOX for 150 minutes at 37 C with 0, 78, 156,
313, 625, 1250, 2500 and 5000 nM of compound 23 (A) or verapamil (B) After the
incubation period, cells were lysed and the DOX level in supernatant was measured by
spectrofluorometry. N = 3-4 independent experiments and values were presented as mean ±
standard error of mean. An effective concentration (EC ) of modulator was determined at
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which DOX accumulation level in LCC6MDR cells is increased to a level which is half to the
parental LCC6 cells.
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3. Conclusion
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In the present study, a total of 25 novel ningalin B analogs were synthesized and
characterized for their P-gp modulating activity in a P-gp overexpressed breast cancer cell
line LCC6MDR. Several important pharmacophores for modulating P-gp were found
including (1) phenyl rings A and B, (2) di-methoxylation at rings A and (3) tri-substitution at
ring C with ortho-bromo, meta-methoxy and para-trimethoxybenzyloxy groups. Among all
ningalin B derivatives, 23 with dimethoxy groups at rings A and B and tri-substitution at ring
C with ortho-bromo, meta-methoxy, and para-trimethoxybenzyloxy groups is the most potent
P-gp inhibitor with EC ranged from 120 to 165 nM in reversing PTX, DOX, vinblastine and
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vincristine resistance (Figure 2 and Table 2). It is safe with selective index at least greater
than 606 compared to verapamil (Figure 2 and Table 2). The mechanism of 23 in reversing
P-gp mediated drug resistance is by virtue of inhibiting transport activity of P-gp and
restoring intracellular drug accumulation (Figure 3 and Figure 4). In summary, our study
demonstrates that ningalin B analogue 23 is non-cytotoxic and effective P-gp chemosensitizer
that can be used in future for reversing P-gp mediated clinical cancer drug resistance.
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4. Experimental Section
General. All non-aqueous reactions were carried out under nitrogen atmosphere in
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freshly distilled anhydrous solvents. Starting materials and reagents were reagent-grade and
were used without further purification unless otherwise stated. Solvents were dried according
to standard procedures. Analytical thin-layer chromatography (TLC) was performed on
pre-coated plates (silica gel 60 F ) purchased from Merck KGaA and they were visualized
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under short (254 nm) and long (365 nm) UV light. Column chromatography was carried out
using silica gel (200–300 mesh). Melting points were recorded on a micro melting point
apparatus MP-500D and were uncorrected. All NMR measurements were carried out at room
temperature and the chemical shifts are reported as parts per million (ppm) in δ units relative
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to the resonance of CDCl (7.26 ppm in the H, 77.0 ppm for the central line of the triplet in
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3
the C modes). Melting points were recorded on a micro melting point apparatus MP-500D
and were uncorrected. High-resolution (ESI) MS spectra were performed with a QTOF-2
Micromass spectrometer.
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2-(3,4,5-trimethoxybenzyl)isoindoline-1,3-dione (6)
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2
2
2
2
2
2
66
67
68
69
70
71
72
73
74
75
A mixture of compound 5 (100 mg, 0.68 mmol), 3,4,5-trimethoxybenzyl methanesulfonate
(227 mg, 0.82 mmol) and K CO (282 mg, 2.04 mmol) in anhydrous DMF (20 mL) was
2
3
stirred at room temperature for 6 h. The resulting solution was poured into water (100 mL),
extracted with CH Cl , washed with brine, dried over anhydrous MgSO , and the solvent was
2
2
4
removed under reduced pressure. The residue was purified by flash chromatography on silica
gel (EtOAc/PE = 1/2, v/v) to afford the desired compounds 6 (161 mg, 73%) as white solid;
1
mp114-116°C; H NMR (CDCl , 500 MHz) δ 7.84 (m, 2 H), 7.70 (m, 2 H), 6.70 (s, 2 H),
3
1
3
4.75 (s, 2 H), 3.86 (s, 6 H), 3.79 (s, 3 H); C NMR (CDCl , 126 MHz) δ 168.0, 153.3, 134.0,
3
+
132.0, 128.4, 106.0, 60.8, 56.1, 41.9; HRMS calcd for (C H O N + H) 328.1179, found
1
8
17
5
328.1185.
2-(2-(4-methoxyphenyl)-2-oxoethyl)isoindoline-1,3-dione (7)
2
76
2
2
2
2
2
2
77
78
79
80
81
82
7 was prepared as described for the synthesis of 6 using 5 (100 mg, 0.68 mmol),
2-bromo-1-(4-methoxyphenyl)ethanone (188 mg, 0.82 mmol) and K CO (282 mg, 2.04
2
3
1
mmol). Yield 78%; mp140-142°C; H NMR (CDCl , 500 MHz) δ 7.98 (d, J = 8.6 Hz, 2 H),
3
1
3
7.86 (m, 2 H), 7.74 (m, 2 H), 6.97 (d, J = 8.6 Hz, 2 H), 5.08 (s, 2 H), 3.88 (s, 3 H); C NMR
(CDCl , 126 MHz) δ 189.3, 167.9, 164.2, 134.3, 134.1, 132.6, 132.3, 130.4, 127.4, 123.5,
3
+
114.1, 55.5, 43.9, 29.7; HRMS calcd for (C H O N + H) 296.0917, found 296.0925.
1
7
13
4
1
0

ACCEPTED MANUSCRIPT
2
83
2-(2-(4-(benzyloxy)-3-methoxyphenyl)-2-oxoethyl)isoindoline-1,3-dione (8)
2
2
2
2
2
2
2
2
84
85
86
87
88
89
90
91
8 was prepared as described for the synthesis of 6 using 5 (100 mg, 0.68 mmol),
1-(4-(benzyloxy)-3-methoxyphenyl)-2-bromoethanone (275 mg, 0.82 mmol) and K CO (282
2
3
1
mg, 2.04 mmol); Yield 71%; mp146-148°C; H NMR (CDCl , 500 MHz) δ 7.90 (dd, J = 3.0,
3
5.0 Hz, 2 H), 7.75 (dd, J = 3.0, 5.0 Hz, 2 H), 7.58 (d, J = 8.4 Hz, 1 H), 7.54 (s, 1 H), 7.44 (d,
J = 7.4 Hz, 2 H), 7.39 (t, J = 7.4 Hz, 2 H), 7.33 (t, J = 7.2 Hz, 1 H), 6.94 (d, J = 8.4 Hz, 1 H),
1
3
5.26 (s, 2 H), 5.08 (s, 2 H), 3.93 (s, 3 H); C NMR (CDCl , 126 MHz) δ 189.5, 168.0, 153.1,
3
149.8, 136.0, 134.1, 132.3, 128.7, 128.2, 127.8, 127.2, 123.5, 122.5, 112.3, 110.7, 70.9, 56.1,
+
43.9, 29.7; HRMS calcd for (C H O N + H) 402.1336, found 402.1342.
2
4
19
5
2
2
92
93
1-(2-(4-(benzyloxy)-3-methoxyphenyl)-2-oxoethyl)-3-(3,4-dimethoxyphenyl)-4-(methylth
io)-1H-pyrrole-2,5-dione (11)
2
2
2
2
2
2
3
3
3
3
3
3
3
94
95
96
97
98
99
00
01
02
03
04
05
06
A
mixture of compound 3-(3,4-dimethoxyphenyl)-4-(methylthio)-1H-pyrrole-2,5
-dione 9 (300 mg, 1.07 mmol), 1-(4-(benzyloxy)-3-methoxyphenyl)-2-bromoethanone 10
(432 mg, 1.28 mmol) and K CO (443 mg, 3.21 mmol) in anhydrous DMF (40 mL) was
2
3
stirred at room temperature overnight. The resulting solution was poured into water (200 mL),
extracted with CH Cl , washed with brine, dried over anhydrous MgSO , and the solvent was
2
2
4
removed under reduced pressure. The residue was purified by flash chromatography on silica
gel (DCM/EtOAc/PE = 1/1/2, v/v/v) to afford the desired compounds 11 (354 mg, 62% yield)
1
as yellow solid; mp 125-127°C; H NMR (CDCl , 500 MHz) δ 7.54 (m, 2H), 7.40 (m, 6H),
3
7.32 (t, J = 7.2 Hz, 1H), 6.94 (dd, J = 12.7, 8.2 Hz, 2H), 5.24 (s, 2H), 4.95 (s, 2H), 3.93 (s, 3
13
H),3.92 (s, 3 H), 3.91 (s, 3 H) 2.72 (s, 3H); C NMR (CDCl , 126 MHz) δ189.8, 168.8,
3
167.8, 153.1, 150.2, 149.7, 148.5, 135.9, 135.7, 133.6, 128.7, 128.2, 127.6, 127.1, 123.6,
122.5, 121.8, 112.5, 112.2, 110.6, 110.5, 77.0, 76.7, 70.8, 56.0, 55.9, 55.8, 44.1, 15.8; HRMS
+
calcd for (C H O NS + H) 534.1581, found 534.1580.
2
9
27
7
3
3
07
08
1-(2-(4-(benzyloxy)-3-methoxyphenyl)-2-oxoethyl)-3-(3,4-dimethoxyphenyl)-1H-pyrrole-
2,5-dione (12)
3
3
3
3
3
3
09
10
11
12
13
14
To a solution of compounds 11 (300 mg, 0.56 mmol) in methanol (20 mL), acetic acid (20
mL) and chloroform (5 mL) was added Zn (109 mg, 1.68 mmol). The reaction mixture was
stirred for 8 h at room temperature, and then concentrated. Then, the residue was dissolved in
ethanol (30 mL) and triethylamine (2 mL) was added, after stirred at room temperature for 10
h, the reaction mixture was poured into water (100 mL), extracted with CH Cl , washed with
2
2
brine, dried over anhydrous MgSO , and the solvent was removed under reduced pressure. The
4
11

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
15
16
17
18
19
20
21
22
residue was purified by flash chromatography on silica gel (EtOAc/PE = 1/2, v/v) to afford
1
the desired compounds 12.Yield 38%; mp 148-150 °C; H NMR (CDCl , 500 MHz) δ7.54 (m,
3
2H),7.40 (m, 6H), 7.32 (t, J = 7.1 Hz, 1H), 6.94 (m, 2 H), 6.72 (s, 1 H), 5.25 (s, 2 H), 4.95 (s,
1
3
2 H), 3.94 (s, 3 H), 3.93 (s, 3 H), 3.92 (s, 3 H); C NMR (CDCl , 126 MHz) δ189.8, 168.8,
3
167.8, 153.1, 150.2, 149.7, 148.5, 135.9, 135.7, 133.6, 128.7, 128.2, 127.6, 127.1, 123.6,
122.5, 121.8, 112.5, 112.2, 110.6, 110.5, 77.0, 76.7, 70.8, 56.0, 55.9, 55.8, 44.1; HRMS calcd
+
for (C H O N + H) 488.1704, found 488.1700.
2
8
25
7
3-(3,4-dimethoxyphenyl)-4-(3-methoxyphenyl)-1H-pyrrole-2,5-dione (14a)
3
3
3
3
3
3
3
3
3
3
3
3
3
23
24
25
26
27
28
29
30
31
32
33
34
35
Under a N atmosphere, t-BuOK (2452 mg, 21.84 mmol) was added to a stirring solution
2
of compound 13a (2500 mg, 7.28 mmol) in t-BuOH (50 mL) at 0°C. Then the reaction was
allowed to slowly warm to room temperature. After 8-12 h, the reaction solution was exposed
to air. After 2 h, the resulting reaction solution was poured into ice-cold water (300 mL), and
then the pH was adjusted to 7 by adding 2 N hydrochloric acid to give a thick suspension.
The above suspension was filtered and purified by flash chromatography on silica gel to
+
afford compound 14a (963 mg, 2.84 mmol); mp 187-189 °C; ESI-MS m/z [M + H] 340.1;
1
H NMR (CDCl , 600 MHz) δ7.81 (s, 1H), 7.30 (d, J = 7.9 Hz, 1H), 7.28 (m, 1H), 7.04 (d, J
3
= 8.1 Hz, 1H), 7.03(m, 1H), 6.98 (d, J = 1.9 Hz, 1H), 6.93 (dd, J = 8.3, 2.6 Hz, 1H), 6.85 (d, J
1
3
= 8.5 Hz, 1H), 3.90 (s, 3H), 3.74 (s, 3H), 3.64 (s, 3H); C NMR (CDCl , 150 MHz) δ170.7,
3
170.5, 159.6, 150.8, 148.7, 136.8, 135.1, 130.1, 129.8, 124.0, 122.2, 120.8, 115.7, 115.0,
112.7, 111.0, 77.3, 77.1, 76.9, 56.0, 55.7, 55.3.
3-(3,4-dimethoxyphenyl)-4-(4-methoxyphenyl)-1H-pyrrole-2,5-dione (14b)
3
3
3
3
3
3
3
36
37
38
39
40
41
42
Following the procedure for the preparation of compound 14a, but using compound 13b as
the starting material, the desired compound 14b was obtained: yield 41%; mp 182-184 °C;
+
1
ESI-MS m/z [M + H] 340.1; H NMR (CDCl , 600 MHz) δ8.11 (s, 1H), 7.48 (d, J = 9.0 Hz,
3
2H), 7.21 (dd, J = 8.5, 2.0 Hz, 1H), 6.98 (d, J = 2.0 Hz, 1H), 6.88 (d, J = 9.0 Hz, 2H), 6.86 (d,
1
3
J = 8.5 Hz, 1H), 3.90 (s, 3H), 3.82 (s, 3H), 3.68 (s, 3H); C NMR (CDCl , 150 MHz) δ171.0,
3
160.7, 150.4, 148.7, 135.0, 134.8, 131.5, 123.5, 121.2, 121.0, 114.0, 112.4, 111.0, 55.7, 55.3.
3-(4-(benzyloxy)-3-methoxyphenyl)-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2,5-dione (14c)
Following the procedure for the preparation of compound 14a, but using compound 13c as
3
43
1
2

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
44
45
46
47
48
49
50
the starting material, the desired compound 14c was obtained: yield 44%; mp 188-190 °C;
+
1
ESI-MS m/z [M + H] 446.1; H NMR (CDCl , 500 MHz)δ7.86 (s, 1H), 7.41 (d, J = 7.5 Hz,
3
2H), 7.36 (t, J = 7.5 Hz, 2H), 7.30 (t, J = 7.2 Hz, 1H), 7.22 (dd, J = 8.4, 1.4 Hz, 1H), 7.12 (dd,
J = 8.4, 1.4 Hz, 1H), 7.06 (s, 1H), 6.99 (s, 1H), 6.86 (dd, J = 8.4, 5.3 Hz, 2H), 5.18 (s, 2H),
1
3
3.90 (s, 3H), 3.73 (s, 3H), 3.65 (s, 3H); C NMR (CDCl , 126 MHz) δ170.8, 150.4, 149.5,
3
149.2, 148.6, 136.4, 135.0, 134.9, 128.6, 128.0, 127.1, 123.6, 123.3, 121.5, 121.1, 113.2,
113.0, 112.4, 110.9, 70.7, 55.9, 55.7.
3
51
3-(3,4-dimethoxyphenyl)-4-phenyl-1H-pyrrole-2,5-dione (14d)
3
3
3
3
3
3
52
53
54
55
56
57
Following the procedure for the preparation of compound 14a, but using compound 13d as
the starting material, the desired compound 14d was obtained: yield 42%; mp 207-209 °C;
+
1
ESI-MS m/z [M + H] 309.1; H NMR (CDCl , 500 MHz) δ 7.89 (s, 1H), 7.51 (m, 2H), 7.38
3
(d, J = 3.1 Hz, 3H), 7.27 (s, 1H), 6.94 (s, 1H), 6.85 (d, J = 8.5 Hz, 1H), 3.90 (s, 3H), 3.62 (s,
1
3
3H); C NMR (CDCl , 126 MHz) δ 170.7, 170.6, 150.7, 148.6, 136.6, 135.2, 129.8, 129.7,
3
128.8, 128.6, 123.8, 120.8, 112.5, 110.9, 55.8, 55.5.
3
58
3-(3,4-dimethoxyphenyl)-4-(p-tolyl)-1H-pyrrole-2,5-dione (14f)
3
3
3
3
3
3
3
59
60
61
62
63
64
65
Following the procedure for the preparation of compound 14a, but using compound 13f as
the starting material, the desired compound 14f was obtained: yield 40%; mp 205-207 °C;
+
1
ESI-MS m/z [M + H] 324.1; H NMR (CDCl , 500 MHz) δ 7.65 (s, 1H), 7.39 (d, J = 8.0 Hz,
3
2H), 7.23 (dd, J = 8.5, 1.9 Hz, 1H), 7.19 (d, J = 8.0 Hz, 2H), 6.98 (d, J = 1.9 Hz, 1H), 6.85 (d,
1
3
J = 8.5 Hz, 1H), 3.90 (s, 3H), 3.66 (s, 3H), 2.37 (s, 3H); C NMR (CDCl , 126 MHz) δ 170.7,
3
170.6, 150.5, 148.6, 140.0, 135.8, 135.4, 129.7, 129.3, 125.8, 123.6, 121.0, 112.5, 110.9, 55.8,
55.6, 29.6, 21.4.
3
3
3
3
3
3
3
3
3
66
67
68
69
70
71
72
73
74
3-(3,4-dimethoxyphenyl)-4-(3-methoxyphenyl)-1-(3,4,5-trimethoxybenzyl)-1H-pyrrole-2,
5-dione (15a)
To a solution of compound 14a (100mg, 0.29 mmol) and 3,4,5-trimethoxybenzyl
methanesulfonate (96 mg, 0.35 mmol) in dry DMF (20 mL) was added K CO (120 mg, 0.87
2
3
mmol). The reaction mixture was heated to 40 °C for 5-6 h and then poured into water (100
mL), extracted with CH Cl , washed with brine, dried over anhydrous MgSO , and the solvent
2
2
4
was removed under reduced pressure. The residue was purified by flash chromatography on
silica gel (DCM/EtOAc/PE = 1/1/2, v/v/v) to afford the desired compounds 15a (108 mg,
1
72%); mp 115-117°C; H NMR (CDCl , 500 MHz) δ 7.28 (m, 2 H), 7.04 (d, J = 7.8 Hz, 2 H),
3
1
3

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
6.99 (s, 1 H), 6.92 (d, J = 7.8 Hz, 2 H), 6.84 (d, J = 8.4 Hz, 1 H), 6.72 (s, 2 H), 4.71 (s, 2 H),
13
3.89 (s, 3 H), 3.87 (s, 3 H), 3.83 (s, 3 H), 3.73 (s, 3 H), 3.64 (s, 3 H); C NMR (CDCl , 126
3
MHz) δ 170.6, 170.5, 159.6, 153.3, 150.7, 148.6, 137.7, 135.9, 134.2, 132.1, 130.3, 129.7,
123.8, 122.2, 121.2, 121.0, 115.6, 114.9, 112.7, 110.9, 106.2, 60.8, 56.2, 55.9, 55.6, 55.3, 42.3,
+
29.7; HRMS calcd for (C H O N + H) 520.1966, found 520.1981.
2
9
29
8
3-(3,4-dimethoxyphenyl)-4-(4-methoxyphenyl)-1-(3,4,5-trimethoxybenzyl)-1H-pyrrole-2,
5-dione (15b)
Following the procedure for the preparation of compound 15a, but using compound 14b as
1
the starting material, the desired compound 15b was obtained: yield 69%; mp 59-61°C; H
NMR (CDCl , 500 MHz) δ 7.48 (d, J = 8.5 Hz, 2 H), 7.20 (d, J = 8.4 Hz, 1 H), 6.99 (s, 1 H),
3
6.88 (d, J = 8.5 Hz, 2 H), 6.85 (d, J = 8.4 Hz, 1 H), 6.72 (s, 2 H), 4.69 (s, 2 H), 3.88 (s, 3 H),
1
3
3.86 (s, 6 H), 3.83 (s, 6 H), 3.67 (s, 3 H); C NMR (CDCl , 126 MHz) δ 170.9, 160.7, 153.3,
3
150.4, 148.7, 134.3, 134.1, 132.2, 131.5, 123.5, 121.4, 121.2, 114.0, 112.5, 111.0, 106.2, 60.8,
+
56.2, 55.8, 55.3, 42.2, 29.7; HRMS calcd for (C H O N + H) 520.1966, found 520.1972.
2
9
29
8
3-(4-(benzyloxy)-3-methoxyphenyl)-4-(3,4-dimethoxyphenyl)-1-(3,4,5-tri
methoxybenzyl)-1H-pyrrole-2,5-dione(15c)
Following the procedure for the preparation of compound 15a, but using compound 14c as
1
the starting material, the desired compound 15c was obtained: yield 70%; mp 59-61°C; H
NMR (CDCl , 500 MHz) δ 7.41 (d, J = 7.3 Hz, 2 H), 7.35 (t, J = 7.4 Hz, 2 H), 7.29 (t, J = 7.2
3
Hz, 1 H), 7.22 (dd, J = 1.8, 8.4 Hz, 1 H), 7.12 (dd, J = 1.8, 8.4 Hz, 1 H), 7.06 (d, J = 1.8 Hz,
1 H), 7.00 (d, J = 1.8 Hz, 1 H), 6.85 (dd, J = 5.6, 8.4 Hz, 2 H), 6.71 (s, 2 H), 5.17 (s, 2 H),
1
3
4.69 (s, 2 H), 3.89 (s, 3 H), 3.85 (s, 6 H), 3.82 (s, 3 H), 3.72 (s, 3 H), 3.64 (s, 3 H); C NMR
(CDCl , 126 MHz) δ 170.8, 153.3, 150.4, 149.5, 149.2, 148.6, 137.6, 136.5, 134.2, 132.2,
3
128.6, 128.1, 127.2, 123.6, 123.3, 121.7, 121.3, 113.3, 113.1, 112.5, 110.9, 106.1, 70.7, 60.8,
+
56.2, 55.8, 42.2; HRMS calcd for (C H O N + Na) 648.2204, found 648.2215.
3
6
35
9
4
4
00
01
3-(3,4-dimethoxyphenyl)-4-phenyl-1-(3,4,5-trimethoxybenzyl)-1H-pyrrole-2,5-dione
(15d)
4
4
4
02
03
04
Following the procedure for the preparation of compound 15a, but using compound 14d as
1
the starting material, the desired compound 15d was obtained: yield 69%; mp 65-66°C; H
NMR (CDCl , 500 MHz) δ 7.46 (s, 2 H), 7.36 (s, 3 H), 7.24 (s, 1 H), 6.95 (s, 1 H), 6.83 (d, J
3
1
4

ACCEPTED MANUSCRIPT
4
4
4
4
05
06
07
08
= 8.5 Hz, 1 H), 6.72 (s, 2 H), 4.70 (s, 2 H), 3.88 (s, 3 H), 3.86 (s, 6 H), 3.82 (s, 3 H), 3.60 (s,
1
3
3 H); C NMR (CDCl , 126 MHz) δ 170.6, 153.3, 150.7, 148.6, 137.6, 135.8, 134.4, 132.1,
3
129.7, 129.1, 128.6, 123.8, 121.0, 112.6, 110.9, 106.2, 60.8, 56.2, 55.9, 55.6, 42.3; HRMS
+
calcd for (C H O N + H) 490.1860, found 490.1870.
2
8
27
7
4
4
09
10
1-(4-(benzyloxy)-3-methoxybenzyl)-3,4-bis(3,4-dimethoxyphenyl)-1H-pyrrole-
2,5-dione (16e)
4
4
4
4
4
4
4
4
4
4
4
4
11
12
13
14
15
16
17
18
19
20
21
22
To a solution of compound 14e (100mg, 0.27 mmol) and 4-(benzyloxy)-3-methoxybenzyl
methanesulfonate (105 mg, 0.32 mmol) in dry DMF (20 mL) was added K CO (112 mg,
2
3
0.81 mmol). The reaction mixture was heated to 40 °C for 5-6 h and then poured into water
(100mL), extracted with CH Cl , washed with brine, dried over anhydrous MgSO , and the
2
2
4
solvent was removed under reduced pressure. The residue was purified by flash
chromatography on silica gel (DCM/EtOAc/PE = 1/1/3, v/v/v) to afford the desired
1
compounds 16e (125 mg, 78%); mp 116-118°C; H NMR (CDCl , 500 MHz) δ 7.41 (d, J =
3
7.2 Hz, 2 H), 7.34 (t, J = 7.2 Hz, 2 H), 7.29 (d, J = 7.0 Hz, 1 H), 7.19 (d, J = 8.4 Hz, 2 H),
7.03 (s, 3 H), 6.95 (d, J = 8.1 Hz, 1 H),6.83 (t, J = 8.9 Hz, 3 H), 5.13 (s, 2 H), 4.70 (s, 2 H),
1
3
3.89 (s, 9 H), 3.70 (s, 6 H); C NMR (CDCl , 126 MHz) δ 170.8, 150.4, 149.6, 148.7, 137.1,
3
134.1, 129.7, 128.5, 127.8, 127.2, 123.6, 121.4, 113.8, 112.8, 112.6, 110.9, 70.9, 56.1, 55.8,
+
41.7, 29.7; HRMS calcd for (C H O N + H) 596.2279, found 596.2296.
3
5
33
8
4
23
1-(4-(benzyloxy)benzyl)-3,4-bis(3,4-dimethoxyphenyl)-1H-pyrrole-2,5-dione (17e)
Following the procedure for the preparation of compound 16e, but using compound
4
4
4
4
4
4
4
4
24
25
26
27
28
29
30
31
4-(benzyloxy)benzyl methanesulfonate as the starting material, the desired compound 17e
1
was obtained: yield 66%; mp 131-133°C; H NMR (CDCl , 500 MHz) δ7.40 (m, 5H), 7.30
3
(m, 1H), 7.19 (d, J = 8.4 Hz, 2H), 7.04 (s, 2H), 6.93 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.4 Hz,
13
2H), 5.04 (s, 2H), 4.72 (s, 2H), 3.89 (s, 6H), 3.70 (s, 6H); C NMR (CDCl , 126 MHz)
3
δ170.8, 158.4, 150.3, 148.6, 136.8, 134.1, 130.2, 129.1, 128.5, 127.9, 127.4, 123.5, 121.4,
114.8, 112.5, 110.9, 77.2, 77.0, 76.7, 69.9, 55.8, 55.7, 41.2; HRMS calcd for (C H O N +
3
4
31
7
+
H) 566.2173, found 566.2189.
4
4
4
4
32
33
34
35
3-(3,4-dimethoxyphenyl)-4-(3-methoxyphenyl)-1-(2-(4-methoxyphenyl)-2-oxoethyl)-1H-
pyrrole-2,5-dione (18a)
To
a
solution
of
compound
14a
(100mg,
0.29
mmol)
and
2-bromo-1-(4-methoxyphenyl)ethanone (80 mg, 0.35 mmol) in dry DMF (20 mL) was added
1
5

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
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K CO (120 mg, 0.87 mmol). The reaction mixture was heated to 40 °C for 5-6 h and then
2
3
poured into water (100 mL), extracted with CH Cl , washed with brine, dried over anhydrous
2
2
MgSO , and the solvent was removed under reduced pressure. The residue was purified by
4
flash chromatography on silica gel (DCM/EtOAc/PE = 1/1/3, v/v/v) to afford the desired
1
compounds 18a (102 mg, 72%); mp 130-132°C; H NMR (CDCl , 500 MHz) δ 7.98 (d, J =
3
8.6 Hz, 2 H), 7.29 (d, J = 6.8 Hz, 2 H), 7.08 (m, 3 H), 6.97 (d, J = 8.6 Hz, 2 H), 6.92 (d, J =
8.1 Hz, 1 H), 6.84 (d, J = 8.4 Hz, 1 H), 5.03 (m, 2 H), 3.90 (m, 3 H), 3.88 (m, 3 H), 3.74 (m,
1
3
3 H), 3.66 (m, 3 H); C NMR (CDCl , 126 MHz) δ 189.8, 170.7, 170.4, 164.2, 159.5, 150.6,
3
148.6, 136.4, 134.7, 130.4, 129.6, 127.4, 123.9, 122.3, 121.1, 115.8, 114.8, 114.1, 112.8,
+
110.9, 55.9, 55.6, 55.3, 44.2, 29.7; HRMS calcd for (C H O N + H) 488.1704, found
2
8
25
7
488.1716.
3-(3,4-dimethoxyphenyl)-4-(4-methoxyphenyl)-1-(2-(4-methoxyphenyl)-2-oxoethyl)-1H-
pyrrole-2,5-dione (18b)
Following the procedure for the preparation of compound 18a, but using compound 14b as
1
the starting material, the desired compound 18b was obtained: yield 77%; mp 99-101°C; H
NMR (CDCl , 500 MHz) δ 7.97 (d, J = 8.6 Hz, 2 H), 7.53 (d, J = 8.6 Hz, 2 H), 7.23 (d, J =
3
8.4 Hz, 1 H), 7.06 (s, 1 H), 6.96 (d, J = 8.6 Hz, 2 H), 6.88 (d, J = 8.6 Hz, 2 H), 6.85 (d, J =
1
3
8.4 Hz, 1 H), 5.02 (s, 2 H), 3.89 (s, 3 H), 3.87 (s, 3 H), 3.82 (s, 3 H), 3.69 (s, 3 H); C NMR
(CDCl , 126 MHz) δ 189.9, 170.9, 164.1, 160.7, 150.4, 148.7, 134.7, 134.5, 131.6, 130.4,
3
127.5, 123.5, 121.5, 114.0, 112.6, 111.0, 55.8, 55.5, 55.3, 44.1; HRMS calcd for (C H O N
2
8
25
7
+
+ H) 488.1704, found 488.1717.
3-(4-(benzyloxy)-3-methoxyphenyl)-4-(3,4-dimethoxyphenyl)-1-(2-(4-methoxy-
phenyl)-2-oxoethyl)-1H-pyrrole-2,5-dione (18c)
Following the procedure for the preparation of compound 18a, but using compound 14c as
1
the starting material, the desired compound 18c was obtained: yield 71%; mp 66-68°C; H
NMR (CDCl , 500 MHz) δ 7.98 (d, J = 8.9 Hz, 2 H), 7.42 (d, J = 7.3 Hz, 2 H), 7.36 (t, J =
3
7.4 Hz, 2 H), 7.30 (t, J = 7.3 Hz, 1 H), 7.25 (m, 1 H), 7.15 (dd, J = 2.0, 8.4 Hz, 1 H), 7.12 (d,
J = 1.9 Hz, 1 H), 7.06 (d, J = 1.9 Hz, 1 H), 6.97 (d, J = 8.9 Hz, 2 H), 6.86 (dd, J = 4.7, 8.4 Hz,
1
3
2 H), 5.18 (s, 2 H), 5.02 (s, 2 H), 3.89 (s, 3 H), 3.74 (s, 3 H), 3.66 (s, 3 H); C NMR (CDCl ,
3
126 MHz) δ 189.8, 170.8, 164.1, 150.3, 149.4, 149.2, 148.6, 136.6, 134.6, 130.4, 128.6,
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128.0, 127.4, 127.2, 123.7, 123.4, 121.8, 121.4, 114.1, 113.2, 112.6, 110.9, 70.7, 55.8, 55.6,
+
44.1; HRMS calcd for (C H O N + H) 594.2122, found 594.2132.
3
5
31
8
3-(3,4-dimethoxyphenyl)-1-(2-(4-methoxyphenyl)-2-oxoethyl)-4-phenyl-1H-
pyrrole-2,5-dione (18d)
Following the procedure for the preparation of compound 18a, but using compound 14d as
1
the starting material, the desired compound 18d was obtained: yield 80%; mp 167-169°C; H
NMR (CDCl , 500 MHz) δ 7.99 (d, J = 8.6 Hz, 2 H), 7.53 (d, J = 3.4 Hz, 2 H), 7.38 (s, 3 H),
3
7.29 (d, J = 8.4 Hz, 1 H), 7.02 (s, 1 H), 6.98 (d, J = 8.6 Hz, 2 H), 6.85 (d, J = 8.4 Hz, 1 H),
1
3
5.04 (s, 2 H), 3.89 (s, 6 H), 3.63 (s, 3 H); C NMR (CDCl , 126 MHz) δ 189.8, 170.7, 170.5,
3
164.1, 150.6, 148.6, 136.2, 134.8, 130.4, 129.9, 129.6, 129.1, 128.5, 127.5, 123.8, 121.1,
+
114.1, 112.7, 110.9, 55.9, 55.6, 44.2; HRMS calcd for (C H O N + H) 458.1598, found
2
7
23
6
458.1609.
1-(2-(4-(benzyloxy)-3-methoxyphenyl)-2-oxoethyl)-3-(3,4-dimethoxyphenyl)-4-(3-methox
yphenyl)-1H-pyrrole-2,5-dione (19a)
To
a
solution
of
compound
14a
(100mg,
0.29
mmol)
and
1-(4-(benzyloxy)-3-methoxyphenyl)-2-bromoethanone (117 mg, 0.35 mmol) in dry DMF (20
mL) was added K CO (120 mg, 0.87 mmol). The reaction mixture was heated to 40 °C for
2
3
5-6 h and then poured into water (100 mL), extracted with CH Cl , washed with brine, dried
2
2
over anhydrous MgSO , and the solvent was removed under reduced pressure. The residue was
4
purified by flash chromatography on silica gel (DCM/EtOAc/PE = 1/1/3, v/v/v) to afford the
1
desired compounds 18a (143 mg, 83%); mp 130-132°C; H NMR (CDCl , 500 MHz) δ 7.56
3
(m, 2 H), 7.44 (d, J = 7.5 Hz, 2 H), 7.39 (t, J = 7.4 Hz, 2 H), 7.34 (d, J = 7.2 Hz, 1 H), 7.29
(m, 2 H), 7.09 (m, 2 H), 7.05 (s, 1 H), 6.93 (m, 2 H), 6.85 (d, J = 8.5 Hz, 1 H), 5.25 (s, 2 H),
13
5.03 (s, 2 H), 3.94 (s, 3 H), 3.90 (s, 3 H), 3.74 (s, 3 H), 3.66 (s, 3 H); C NMR (CDCl , 126
3
MHz) δ 189.9, 170.7, 170.4, 159.5, 153.1, 150.7, 149.8, 148.6, 136.4, 136.0, 134.7, 130.3,
129.6, 128.7, 128.2, 127.8, 127.2, 123.9, 122.5, 122.3, 121.1, 115.8, 114.8, 112.8, 112.3,
+
110.9, 110.7, 70.8, 56.1, 55.9, 55.6, 55.3, 44.2; HRMS calcd for (C H O N + H) 594.2122,
35
31
8
found 594.2137.
1-(2-(4-(benzyloxy)-3-methoxyphenyl)-2-oxoethyl)-3-(3,4-dimethoxyphenyl)-4-(4-methox
yphenyl)-1H-pyrrole-2,5-dione (19b)
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Following the procedure for the preparation of compound 19a, but using compound 14b as
1
the starting material, the desired compound 19b was obtained: yield 80%; mp 67-69°C; H
NMR (CDCl , 500 MHz) δ 7.54 (m, 4 H), 7.43 (d, J = 7.3 Hz, 2 H), 7.38 (t, J = 7.4 Hz, 2 H),
3
7.32 (t, J = 7.2 Hz, 1 H), 7.23 (dd, J = 8.4, 1.8 Hz, 2 H), 7.05 (d, J = 1.6 Hz, 1 H), 6.93 (d, J =
8.4 Hz, 1 H), 6.88 (d, J = 8.8 Hz, 2 H), 6.84 (d, J = 8.4 Hz, 1 H), 5.24 (s, 2 H), 5.01 (s, 2 H),
1
3
3.92 (s, 3 H), 3.89 (s, 3 H), 3.82 (s, 3 H), 3.68 (s, 3 H); C NMR (CDCl , 126 MHz) δ 190.1,
3
170.9, 160.7, 153.1, 150.4, 149.7, 148.7, 136.0, 134.7, 134.5, 131.6, 128.7, 128.2, 127.9,
123.5, 122.5, 121.5, 121.3, 114.0, 112.6, 112.3, 111.0, 110.7, 70.9, 56.1, 55.8, 55.3, 44.1;
+
HRMS calcd for (C H O N + H) 594.2122, found 594.2133.
3
5
31
8
3-(4-(benzyloxy)-3-methoxyphenyl)-1-(2-(4-(benzyloxy)-3-methoxyphenyl)-2
-oxoethyl)-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2,5-dione (19c)
Following the procedure for the preparation of compound 19a, but using compound 14c as
1
the starting material, the desired compound 19c was obtained: yield 77%; mp 140-142°C; H
NMR (CDCl , 500 MHz) δ 7.55 (m, 2 H), 7.36 (m, 10 H), 7.24 (s, 1 H), 7.15 (d, J = 8.5 Hz, 1
3
H), 7.12 (s, 1 H), 7.06 (s, 1 H), 6.93 (d, J = 8.3 Hz, 1 H), 6.86 (m, 2 H), 5.24 (s, 2 H), 5.18 (s,
1
3
2 H), 5.01 (s, 2 H), 3.93 (s, 3 H), 3.89 (s, 3 H), 3.73 (s, 3 H), 3.66 (s, 3 H); C NMR (CDCl ,
3
126 MHz) δ 190.0, 170.8, 153.1, 150.4, 149.7, 149.5, 149.2, 148.6, 136.6, 136.0, 134.6,
128.7, 128.2, 128.0, 127.8, 127.2, 123.7, 123.4, 122.5, 121.8, 121.4, 113.2, 112.6, 112.3,
+
110.9, 110.6, 70.8, 56.1, 55.8, 44.1; HRMS calcd for (C H O N + Na) 722.2361, found
4
2
37
9
722.2376.
1-(2-(4-(benzyloxy)-3-methoxyphenyl)-2-oxoethyl)-3-(3,4-dimethoxyphenyl)-4-phenyl-1
H-pyrrole-2,5-dione (19d)
Following the procedure for the preparation of compound 19a, but using compound 14d as
1
the starting material, the desired compound 19d was obtained: yield 82%; mp 116-118°C; H
NMR (CDCl , 500 MHz) δ 7.54 (m, 4 H), 7.43 (d, J = 7.4 Hz, 2 H), 7.38 (m, 5 H), 7.32 (t, J
3
= 7.2 Hz, 1 H), 7.28 (dd, J = 1.5, 8.4 Hz, 1 H), 7.01 (d, J = 1.3 Hz, 1 H), 6.93 (d, J = 8.4 Hz,
1 H), 6.84 (d, J = 8.4 Hz, 1 H), 5.25 (s, 2 H), 5.03 (s, 2 H), 3.93 (s, 3 H), 3.89 (s, 3 H), 3.62 (s,
1
3
3 H); C NMR (CDCl , 126 MHz) δ 189.9, 170.7, 153.1, 150.6, 149.7, 148.6, 136.0, 134.8,
3
129.9, 129.6, 128.7, 128.3, 127.8, 127.2, 123.8, 122.5, 121.1, 112.7, 112.3,110.8, 70.8, 55.9,
+
55.8, 55.6, 44.2; HRMS calcd for (C H O N + H) 564.2017, found 564.2023.
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1-(2-(4-(benzyloxy)-3-methoxyphenyl)-2-oxoethyl)-3-(3,4-dimethoxyphenyl)-4-(p-tolyl)-1
H-pyrrole-2,5-dione (19f)
Following the procedure for the preparation of compound 19a, but using compound 14f as
1
the starting material, the desired compound 19f was obtained: yield 81%; mp 71-73°C; H
NMR (CDCl , 500 MHz) δ 7.54 (m, 2 H), 7.43 (d, J = 8.0 Hz, 4 H), 7.37 (t, J = 7.5 Hz, 2 H),
3
7.31 (t, J = 7.2 Hz, 1 H), 7.24 (d, J = 1.5 Hz, 1 H), 7.17 (d, J = 8.0 Hz, 2 H), 7.05 (s, 1 H),
6.92 (d, J = 8.3 Hz, 1 H), 6.83 (d, J = 8.5 Hz, 1 H), 5.23 (s, 2 H), 5.01 (s, 2 H), 3.91 (s, 3 H),
1
3
3.88 (s, 3 H), 3.71 (s, 3 H), 2.35 (s, 3 H); C NMR (CDCl , 126 MHz) δ 190.1, 170.8, 153.1,
3
150.5, 149.7, 148.6, 139.9, 136.0, 135.5, 135.0, 129.8, 129.3, 128.7, 128.2, 127.8, 127.2,
126.1, 123.7, 122.5, 121.3, 112.7, 112.2, 110.9, 110.5, 70.8, 56.0, 55.9, 55.7, 44.1, 21.5;
+
HRMS calcd for (C H O N + H) 578.2173, found 578.2190.
3
5
31
7
3,4-bis(3,4-dimethoxyphenyl)-1-(4-(methylsulfonyl)benzyl)-1H-pyrrole-2,5-dione (20e)
To a solution of compound 14e (100mg, 0.27 mmol) and 4-(methylsulfonyl)benzyl
methanesulfonate (85 mg, 0.32 mmol) in dry DMF (20 mL) was added K CO (112 mg, 0.81
2
3
mmol). The reaction mixture was heated to 40 °C for 5-6 h and then poured into water (100
mL), extracted with CH Cl , washed with brine, dried over anhydrous MgSO , and the solvent
2
2
4
was removed under reduced pressure. The residue was purified by flash chromatography on
silica gel (DCM/EtOAc/PE = 1/1/3, v/v/v) to afford the desired compounds 20e (100 mg,
1
69%); mp 158-160°C; H NMR (CDCl , 500 MHz) δ 7.91 (d, J = 8.2 Hz, 2 H), 7.63 (d, J =
3
8.2 Hz, 2 H), 7.21 (dd, J = 1.7, 8.4 Hz, 2 H), 7.04 (d, J = 1.6 Hz, 2 H), 6.85 (d, J = 8.4 Hz, 2
13
H), 4.86 (s, 2 H), 3.90 (s, 6 H), 3.70 (s, 6 H), 3.03 (s, 3 H); C NMR (CDCl , 126 MHz) δ
3
170.6, 150.6, 148.7, 142.5, 140.0, 134.1, 129.5, 127.9, 123.6, 121.1, 112.5, 110.9, 55.9, 44.5,
+
41.3; HRMS calcd for (C H O NS + H) 538.1530, found 538.1530.
2
8
27
8
3-(3,4-dimethoxyphenyl)-1-(4-(methylsulfonyl)benzyl)-4-(p-tolyl)-1H-pyrrole-2,5-dione
(20f)
Following the procedure for the preparation of compound 20e, but using compound 14f as
1
the starting material, the desired compound 20f was obtained: yield 73%; mp 166-168°C; H
NMR (CDCl , 500 MHz) δ 7.91 (d, J = 8.2 Hz, 2 H), 7.63 (d, J = 8.2 Hz, 2 H), 7.39 (d, J =
3
8.0 Hz, 2 H), 7.23 (dd, J = 1.5, 8.4 Hz, 1 H), 7.18 (d, J = 8.0 Hz, 2 H), 7.01 (d, J = 1.4 Hz, 1
H), 6.83 (d, J = 8.4 Hz, 1 H), 4.86 (s, 2 H), 3.89 (s, 3 H), 3.65 (s, 3 H), 3.03 (s, 3 H), 2.36 (s,
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3 H); C NMR (CDCl , 126 MHz) δ 170.6, 150.7, 148.7, 142.5, 140.2, 140.0, 135.1, 134.5,
3
129.7, 129.5, 129.3, 127.9, 125.8, 123.6, 120.9, 112.5, 110.9, 55.9, 55.7, 44.5, 41.3, 21.5;
+
HRMS calcd for (C H O NS + Na) 514.1295, found 514.1301.
2
7
25
6
5
5
60
61
3,4-bis(3,4-dimethoxyphenyl)-1-(2-(4-(methylsulfonyl)phenyl)-2-oxoethyl)-1H-pyrrole-2,
5-dione (21e)
5
5
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To
a
solution
of
compound
14e
(100mg,
0.27
mmol)
and
2-bromo-1-(4-(methylsulfonyl)phenyl)ethanone (89 mg, 0.32 mmol) in dry DMF (20 mL)
was added K CO (112 mg, 0.81 mmol). The reaction mixture was heated to 40 °C for 5-6 h
2
3
and then poured into water (100 mL), extracted with CH Cl , washed with brine, dried over
2
2
anhydrous MgSO , and the solvent was removed under reduced pressure. The residue was
4
purified by flash chromatography on silica gel (DCM/EtOAc/PE = 1/1/3, v/v/v) to afford the
1
desired compounds 21e (119 mg, 78%); mp151-153°C; H NMR (CDCl , 500 MHz) δ 8.18
3
(d, J = 8.0 Hz, 2 H), 8.11 (d, J = 8.0 Hz, 2 H), 7.24 (s, 2 H), 7.08 (s, 2 H),6.87 (d, J = 8.4 Hz,
1
3
2 H), 5.08 (s, 2 H), 3.91 (s, 6 H), 3.71 (s, 6 H), 3.10 (s, 3 H); C NMR (CDCl , 126 MHz) δ
3
190.8, 170.5, 150.5, 148.7, 145.0, 138.3, 134.6, 129.0, 128.1, 123.7, 121.2, 112.6, 110.9, 55.9,
+
44.7, 44.3, 29.7; HRMS calcd for (C H O NS + H) 566.1479, found 566.1485.
2
9
27
9
5
5
5
5
5
5
5
5
5
5
5
5
5
73
74
75
76
77
78
79
80
81
82
83
84
85
3-(3,4-dimethoxyphenyl)-1-(2-(4-(methylsulfonyl)phenyl)-2-oxoethyl)-4-(p-tolyl)-1H-pyr
role-2,5-dione (21f)
Following the procedure for the preparation of compound 21e, but using compound 14f as
1
the starting material, the desired compound 21f was obtained: yield 81%; mp 143-145°C; H
NMR (CDCl , 500 MHz) δ 8.19 (d, J = 8.2 Hz, 2 H), 8.11 (d, J = 8.2 Hz, 2 H), 7.43 (d, J =
3
7.9 Hz, 2 H), 7.27 (s, 1 H), 7.19 (d, J = 7.9 Hz, 2 H), 7.04 (s, 1 H), 6.85 (d, J = 8.4 Hz, 1 H),
1
3
5.09 (s, 2 H), 3.91 (s, 3 H), 3.67 (s, 3 H),3.10 (s, 3 H), 2.37 (s, 3 H); C NMR (CDCl , 126
3
MHz) δ 190.7, 170.5, 150.6, 148.6, 145.0, 140.1, 138.4, 135.6, 135.0, 129.8, 129.4, 129.0,
128.1, 125.9, 123.7, 121.1, 112.6, 110.9, 55.9, 55.7, 44.7, 44.3, 29.7, 21.5; HRMS calcd for
+
(C H O NS + H) 520.1424, found 520.1429.
2
8
25
7
2-bromo-1-(4-((2-bromo-3,4,5-trimethoxybenzyl)oxy)-3-methoxyphenyl) ethanone (22)
To a stirred solution of 1-(3-methoxy-4-((3,4,5-trimethoxybenzyl)oxy)phenyl)ethanone (200
mg, 0.57 mmol) in chloroform (30 mL) at 0 ℃ , a solution of bromine (182 mg, 1.14 mmol) in
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chloroform (5 mL) was added slowly. After 5 h, the resulting reaction solution was poured
into ice-cold water (100 mL), extracted with CH Cl , washed with brine, dried over
2
2
anhydrous MgSO4, and the solvent was removed under reduced pressure. The residue was
purified by flash chromatography on silica gel (EtOAc/PE = 1/3, v/v) to afford the desired
compounds 22 (92 mg, 32% yield) as white solid; mp 140-142 °C; ESI-MS m/z [M + H]+
505.1; 1H NMR (CDCl , 500 MHz) δ 7.56 (s, 1 H), 7.52 (d, J = 8.4 Hz, 1 H), 6.94 (s, 1 H),
3
6.88 (d, J = 8.4 Hz, 1 H), 5.23 (s, 2 H), 5.03 (s, 2 H), 3.95 (s, 3 H), 3.91 (s, 3 H), 3.87 (s, 3 H),
3.80 (d, 3 H); 13C NMR (CDCl , 126 MHz) δ 196.7, 153.1, 152.0, 150.8, 149.4, 142.7, 131.0,
3
123.1, 112.4, 110.6, 108.1, 107.4, 70.3, 61.0, 61.0, 56.1.
1-(2-(2-bromo-5-methoxy-4-((3,4,5-trimethoxybenzyl)oxy)phenyl)-2-oxoethyl)-3,4-bis(3,
4-dimethoxyphenyl)-1H-pyrrole-2,5-dione (23)
To a solution of compound 14e (100mg, 0.27 mmol) and 22 (161 mg, 0.32 mmol) in dry
DMF (20 mL) was added K CO (112 mg, 0.81 mmol). The reaction mixture was heated to
2
3
40 °C for 6 h and then poured into water (100 mL), extracted with CH Cl , washed with brine,
2
2
dried over anhydrous MgSO , and the solvent was removed under reduced pressure. The
4
residue was purified by flash chromatography on silica gel (DCM/EtOAc/PE = 1/1/3, v/v/v)
1
to afford the desired compounds 23 (141 mg, 66%); mp 158-160 °C; H NMR (CDCl , 500
3
MHz) δ 7.61 (dd, J = 8.4, 1.9 Hz, 1H), 7.57 (d, J = 1.9 Hz, 1H), 7.24 (dd, J = 8.4, 1.9 Hz, 2H),
7.09 (d, J = 1.9 Hz, 2H), 6.93 (t, J = 4.2 Hz, 2H), 6.86 (d, J = 8.5 Hz, 2H), 5.26 (s, 2H), 5.04
13
(s, 2H), 3.95 (s, 3H), 3.92 (s, 3H), 3.90 (s, 6H), 3.88 (s, 3H), 3.82 (s, 3H), 3.72 (s, 6H); C
NMR (CDCl , 126 MHz) δ 190.0, 170.8, 153.1, 152.7, 150.8, 150.3, 149.7, 148.6, 142.7,
3
134.6, 130.8, 128.1, 123.6, 122.6, 121.4, 112.6, 112.5, 110.9, 110.7, 108.1, 107.3, 70.3, 61.1,
+
61.0, 56.1, 56.1, 55.8, 55.7, 44.1, 29.6; HRMS calcd for (C H O NBr + H) 794.1630,
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found 794.1652.
Materials for biological studies.
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DMSO, verapamil, cyclosporine A, doxorubicin (DOX), paclitaxel (PTX), vinblastine,
vincristine and rhodamine 123 were purchased from Sigma-Aldrich. Dulbecco’s modified
Eagle’s medium (DMEM), trypsin-ethylenediaminetetracetic acid (EDTA), and
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penicillin/streptomycin were from Gibco BRL. Fetal bovine serum (FBS) was from Hyclone
Laboratories.
2-(4,5-Dimethylthiazol-2-yl-)-5-[3-(carboxymethoxy)phenyl]-
2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosulfate (PMS) were purchased
from Promega. Human breast cancer cell lines MDA435/LCC6 and MDA435/LCC6MDR
were kindly provided by Dr. Robert Clarke (Georgetown University, Washington, DC).
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Cell culture.
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MDA435/LCC6, MDA435/LCC6MDR and L929 cell lines were cultured in
supplemented DMEM media with 10% heat inactivated FBS and 100 U/mL penicillin and
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100 ꢀg/mL of streptomycin. They were maintained at 37 C in a humidified atmosphere with
5% CO . The cells were split constantly after a confluent monolayer has been formed. To
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split cells, the plate was washed briefly with phosphate-buffered saline (PBS), treated with
0.05% trypsin-EDTA and harvested by centrifugation.
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Cell proliferation assay.
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6,000 cells of LCC6 or LCC6MDR were mixed with PTX (400, 133, 44, 15, 5, 1.6 or 0
nM) with or without 1 µM modulators to a final volume of 200 ꢀL in each well of a 96-well
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plate. The plates were then incubated for 5 days at 37 C. The cell viability was determined
using the Cell Titer 96 A Queous Assay (Promega) as reported previously[36-38].
IC values of LCC6MDR was determined using non-linear regression dose-response curve
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analysis of Prism software.
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Cytotoxicity assay.
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10,000 cells of L929 were mixed with different concentrations (100, 33.3, 11.1, 3.7, 1.2,
0.4 and 0 µM) of modulators to a final volume of 100 ꢀL in each well of a 96-well plate. The
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plates were then incubated for 3 days at 37 C. The percentage of survival was determined
using MTS assay. IC50 of modulators was determined using non-linear regression
dose-response curve analysis of Prism software.
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Determination of effective concentration (EC ) and selective index of modulator
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In 200 µL supplemented media, 6,000 cells of LCC6MDR were mixed with a ranged
concentration of PTX, DOX, vinblastine or vincristine and with 0, 31.3, 62.5, 125, 250, 500,
1000 and 2000 nM of modulator in each well of a 96-well plate. The plates were then
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incubated for 5 days at 37 C. The cell viability was determined using the Cell Titer 96 A
Queous Assay (Promega) as reported previously[36-38]. IC values (PTX, DOX, vinblastine
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or vincristine) of LCC6MDR at different doses of modulator was determined using non-linear
regression dose-response curve analysis of Prism software. Effective concentration (EC ) is
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a concentration of modulator at which it can reduce the IC values of drug of LCC6MDR
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cells by half. EC50 value of modulator was also determined using non-linear regression
dose-response curve. Selective index is a preliminary indicator of safety of drug candidates. It
is a ratio of the dose of drug that causes cytotoxicity in normal cell lines divided by the dose
that leads to the desired P-gp modulating efficacy in cancer cell lines. Here, selective index =
(IC50 of modulators towards L929 fibroblasts) / (EC50 of modulators for reversing PTX
resistance in LCC6MDR cells).
Intracellular DOX and rhodamine 123 accumulation.
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1 x 10 cells of LCC6 or LCC6MDR cells were mixed with 20 µM DOX or 10 µg/mL
rhodamine 123 in the presence of 2 µM of modulator or 0.2 % DMSO (as a negative control).
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Cells were incubated at 37 C for 150 min. Cells were spinned down and washed with cold
PBS, pH7.4 and lysed with lysis buffer. Supernatant was saved and kept in a 96-well black
plate with flat bottom. Fluorescence level of DOX was determined by fluorescence
spectrophotometer (BMG Technologies) using an excitation and an emission wavelength of
460 nm and 610 nm. Rhodamine 123 level was determined at wavelength of 480 nm for
excitation and 520 nm for emission[33, 36, 37].
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Dosage effect of modulator on DOX accumulation.
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1 x 10 cells of LCC6MDR cells were mixed with 20 µM DOX and different concentrations
of modulator including 0, 78, 156, 313, 625, 1250, 2500 and 5000 nM. Cells were incubated
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at 37 C for 150 min. Fluorescence level of intracellular DOX was determined as described
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previously.
Acknowledgment
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This project was funded by NSFC-Shandong Joint Fund for Marine Science Research Centers
(U1406402), National Natural Science of China (NSFC 81172926), Project of Strategic
Importance (1-ZE22) of the Hong Kong Polytechnic University and the General Research Fund
(PolyU 5606/11M) of the Research Grant Council of Hong Kong.
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