Design and syntheses of permethyl ningalin B analogues: Potent multidrug resistance (MDR) reversal agents of cancer cells

Article abstract of DOI:10.1021/jm100035c

A series of novel N-arylalkyl-3,4-diaryl-substituted pyrrole-2,5-diones were synthesized. They exhibited promising P-gp modulating activity in a P-gp overexpressing breast cancer cell line (LCC6MDR). Compound 6 (with three methoxy groups at D-ring) displayed the highest P-gp modulating activity. 6 at 1 μM can sensitize LCC6MDR cells toward paclitaxel by 18.2-fold. Interestingly, a synergy on modulating P-gp was noted when 6 and 25 were used together (fractional inhibitory concentration index FICI = 0.42). Combination of 6 (0.5 μM) and 25 (0.5 μM) resulted in a 66-fold sensitization of LCC6MDR cells toward paclitaxel. They also reversed P-gp mediated doxorubicin (DOX) and vincristine resistance. Kinetic characterization suggests that permethyl ningalin B analogues likely act as a noncompetitive inhibitor of P-gp-mediated DOX transport (K_i = 5.4-5.8 μM). The present study demonstrates that synthetic analogues of permethyl ningalin B can be employed as effective and safe modulators of P-gp-mediated drug resistance in cancer cells.

Full text of DOI:10.1021/jm100035c

5108 J. Med. Chem. 2010, 53, 5108–5120
DOI: 10.1021/jm100035c
Design and Syntheses of Permethyl Ningalin B Analogues: Potent Multidrug Resistance (MDR)
Reversal Agents of Cancer Cells
Pu Yong Zhang,†, Iris L. K. Wong,‡,§, Clare S. W. Yan,‡,§ Xiao Yu Zhang,† Tao Jiang,† Larry M. C. Chow,*,‡,§ and
Sheng Biao Wan*,†
†Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao,
China, ‡Department of Applied Biology and Chemical Technology and the State Key Laboratory for Chirosciences, The Hong Kong Polytechnic
University, Hung Hom, Hong Kong SAR, China, and §State Key Laboratory in Chinese Medicine and Molecular Pharmacology, Shenzhen, China.
These two authors contribute equally to this work.
Received January 11, 2010
A series of novel N-arylalkyl-3,4-diaryl-substituted pyrrole-2,5-diones were synthesized. They exhibited
promising P-gp modulating activity in a P-gp overexpressing breast cancer cell line (LCC6MDR).
Compound 6 (with three methoxy groups at D-ring) displayed the highest P-gp modulating activity. 6 at
1 μM can sensitize LCC6MDR cells toward paclitaxel by 18.2-fold. Interestingly, a synergy on
modulating P-gp was noted when 6 and 25 were used together (fractional inhibitory concentration
index FICI = 0.42). Combination of 6 (0.5 μM) and 25 (0.5 μM) resulted in a 66-fold sensitization of
LCC6MDR cells toward paclitaxel. They also reversed P-gp mediated doxorubicin (DOX) and
vincristine resistance. Kinetic characterization suggests that permethyl ningalin B analogues likely act
as a noncompetitive inhibitor of P-gp-mediated DOX transport (Ki = 5.4-5.8 μM). The present study
demonstrates that synthetic analogues of permethyl ningalin B can be employed as effective and safe
modulators of P-gp-mediated drug resistance in cancer cells.
Introduction
blockers,12 calmodulin antagonists,13 indole alkaloids,14 cyclos-
porine A,15 quinoline,16 and steroid17 are classified as first-gene-
ration MDR modulators.2,18 However, their unacceptable
toxicity precluded them from clinical use. Although second-
generation MDR modulators including 6-[(2S,4R,6E)-4-methyl-
2-(methylamino)-3-oxo-6-octenoic acid]-7-L-valine-cyclosporin
A (valspodar PSC833),19 1,7-di(pyridin-3-yl)heptan-4-yl-1-[2-
oxo-2-(3,4,5-trimethoxyphenyl)acetyl]piperidine 2-carboxylate
(biricodar, VX-710),20 dexverapamil,21 and dexniguldipine22
have lower toxicity and higher MDR-reversal activity, their uses
were limited by their unpredictable pharmacokinetic interactions
with the coadministered anticancer drug.2,18 Third-generation
MDR modulators,2,23,24 developed by structure-activity rela-
tionship studies and combinatorial chemistry approaches,
included (2R)-1-{4-[(1aR,6r,10bS)-1,1-difluoro-1,1a,6,10b-tetra-
hydrodibenzo[a,e]cyclopropa[c]cyclohepten-6-yl]pipera- zin-1-
yl}-3-(quinolin-5-yloxy)propan-2-ol (zosuquidar, LY-335979),
N-[2-[[4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-
ethyl]phenyl]carbamoyl]-4,5-dimethoxyphenyl]quinoline-3-car-
boxamide (tariquidar, XR-9576), and N-[4-[2-[4-[3-ethoxy-
1-(E)-propenyl]phenyl]-4-[4-(isopropylamino)phenyl]-1H-
imidazol-5-yl]phenyl]-N-isopropylamine (OC144-093). Tari-
quidar, one of the most potent P-gp inhibitors, is in phase III
trial in breast cancer.
Chemotherapy is one of the most important treatment
methods for cancers. Several anticancer drugs including pacli-
taxel, doxorubicin, and vinblastine have been used clinically to
treat breast, lung, stomach, prostate, colon, and pancreatic
tumor and other solid tumors. Extensive multidrug resistance
(MDRa) in cancer cells has been a major obstacle to successful
cancer chemotherapy.1 The major resistance mechanism is
through increased expression of the ABCB1 (also known as
mdr1) gene which encodes for a membrane permeability glyco-
protein (P-gp).2,3 P-gp belongs to a family of ATP-binding
cassette (ABC) transporters.4-8 Most ABC transporters consist
of two transmembrane domains and two highly conserved
nucleotide binding domains. P-gp is probably the best charac-
terized ABC transporter. It pumps a variety of hydrophobic
anticancer drugs out of the cell, resulting in a lowered intracel-
lular drug accumulation.4-8 Recent determination of three-
dimensional structure of P-gp by electron crystallography9
could allow a better understanding of molecular mechanism
of P-gp and the development of MDR inhibitors.
Great efforts have been made to discover effective and safe
MDR inhibitors over the past 2 decades. Verapamil, a calcium
channel blocker, was the first MDR reversal agent that was
found to inhibit P-gp-mediated drug efflux.10,11 Verapamil and
other more effective P-gp inhibitors including calcium channel
Marine natural products are important sources for devel-
opment of new MDR modulators. Some pyrrole-derived
alkaloids including permethyl ningalin B (Figure 1), per-
methyl ningalin D, permethyl lamallarine D, lamallarine K,
lamallarine I, lamallarine O, lukianol A, and permethyl
storniamideAhavebeen reported toactasnontoxicinhibitors
of MDR in various cancer cell lines.25-33
*To whom correspondence should be addressed. For L.M.C.C.: phone,
852-34008662; fax, 852-23649932; e-mail, bclchow@polyu.edu.hk. For
S.B.W.: phone, 86-532-82032712; fax, 86-532-82033054; e-mail, biaowan@
ouc.edu.cn.
a Abbreviations: MDR, multidrug resistance; ABC, ATP-binding
cassette; P-gp, P-glycoprotein; RF, relative fold; DOX, doxorubicin.
r
pubs.acs.org/jmc
Published on Web 06/18/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5109
Figure 1. Structures of ningalin B compounds and target compounds.
Ningalin B (Figure 1) is the second member of this newly
described family of marine natural products. It was first isolated
by Fenical’s group from an ascidian of the genus Didemnum
collected in Western Australia near Ningaloo Reef in 1997.30
Total synthesis of ningalin B, permethyl ningalin B (1), andtheir
derivatives including 2 and 3 (Figure 1) were performed by
Boger and Gupton.28,32,33 These compounds, containing a
common 3,4-diaryl-substituted pyrrole nucleus bearing 2- or
2,5-carboxylates, can reverse MDR more effectively than ve-
rapamil by inhibiting the P-gp-mediated drug efflux at a non-
cytotoxic concentration. We are interested in improving the
MDR-reversing activity of permethyl ningalin B by modifying
its pyrrole nucleus. In this study, we have replaced the nucleus of
pyrrole 2- or 2,5-carboxylate of Boger’s compounds 2 and 3
with pyrrole-2,5-dione. 3,4-Diaryl-substituted pyrrole-2,5-
dione has been reported to be more stable and easier to prepare
than 3,4-diaryl-substituted pyrrole-2,5-carboxylates.31 The tar-
get molecules were formed by linking 3,4-diaryl-substituted
pyrrole-2,5-dione with a benzene ring (D-ring) containing
various alkyl linkers. These novel permethyl ningalin B analo-
gues were assessed for their MDR-reversing activity in P-gp
overexpressing cancer cell line.
compound 10 with a yield of 66%. With the same synthesis
strategy, compounds 4, 5, 6, 7, 8, 9, 11, and 12 with one to
three carbon linkers and various numbers of methoxy groups
at the D-ring were obtained.
Coupling of 18 with (E)-3-(3,4,5-trimethoxyphenyl)prop-
2-en-1-ol under Mitsunobu reaction conditions provided
compound 21 (Scheme 2). Catalyzed by K2CO3 and in DMF,
compounds 24, 25, and 26 were obtained by N-alkylation of 18
with 2-bromo-1-phenylethanone, 2-bromo-1-(4-methoxyphe-
nyl)ethanone, and 2-bromo-1-(3,4-dimethoxyphenyl)ethanone,
respectively (Scheme 2). No products were obtained when
intermediate 17 reacted with (3,4,5-trimethoxyphenyl)methanol
(27) using the above two coupling methods. When compounds
17 and 27 were treated with NaH in DMF, byproduct 28 was
isolated from the reaction mixture (Scheme 2). Compound 28
was characterized by 1H NMR and 13C NMR. The two methy-
lene groups of compound 28 were further confirmed by 13C
NMR-DEPT and HMQC.
Dimers 30, 31, and 32 were prepared respectively by
treating intermediate 18 with dibromomethane, 1,2-dibro-
moethane, and 1,3-dibromopropane catalyzed by K2CO3 in
DMF (Scheme 3). The Mitsunobu reaction was tried but
found to be inefficient in the synthesis of dimers. Compound
29 was obtained when intermediate 18 was treated with
ethane-1,2-diol under Mitsunobu reaction conditions
(Scheme 3). Additionally, compounds 7 and 11 were refluxed
with Lawesson’s reagent in toluene to provide compounds 33
and 34, respectively (Scheme 3).
Biological Studies. Effect of Permethyl Ningalin B Analo-
gues on Reversing Paclitaxel Resistance of MDA435/
LCC6MDR Cells. We employed two cell lines in this study,
namely, MDA435/LCC6 cell line (an estrogen-independent
breast cancer cell line) and its ABCB1-transfected derivative
(MDA435/LCC6MDR).36 Paclitaxel is one of the drugs for
treating breast cancer,37 and it has been demonstrated that
paclitaxel resistance is mediated by overexpression of P-gp.38-40
We found that LCC6MDR cells were about 70.5-fold more
resistant to paclitaxel (IC50 = 183.2 ( 6.3 nM) than its parental
LCC6 cells (IC50 = 2.6 ( 0.5 nM) (Table 1). None of the
permethyl ningalin B analogues showed any cytotoxicity to-
ward LCC6, LCC6MDR, or normal mouse connective tissue
fibroblast (L929) even when used at a very high concentration
(100 μM) except for compound 9 (Table 2). MDR reversal
activity of permethyl ningalin B analogues was compared by
measuring the relative fold (RF), defined as a ratio of IC50
without modulator to IC50 with modulator. A relatively low
Results
Synthesis of Analogues of Permethyl Ningalin B. A concise
synthetic method was used in the preparation of key intermedi-
ate 3,4-bis(3,4-dimethoxyphenyl)-1H-pyrrole-2,5-dione (18)
(Scheme 1).34 Ammoniation of 2-bromo-1-(3,4-dimethoxyphe-
nyl)ethanone (13) with hexamethylenetetramine, followed by
addition of concentrated hydrobromic acid in absolute metha-
nol, provided 2-amino-1-(3,4-dimethoxyphenyl)ethanone hy-
drobromide (15). Coupling of compound 15 with 2-(3,4-
dimethoxyphenyl)acetic acid, catalyzed by EDCI and HOBt,
resulted in compound 16. Subsequent cyclization of 16, cata-
lyzed by t-BuOK in t-BuOH under nitrogen protection, yielded
3,4-bis(3,4-dimethoxyphenyl)-1H-pyrrole-2-one (17). The key
intermediate 3,4-bis(3,4-dimethoxyphenyl)-1H-pyrrole-
2,5-dione (18) was obtained from cyclization of 16 under
air with a yield of 45%. Following reported methods,35 trace
amounts of 17 and 18 were derived from the cyclization of 16
catalyzed by K2CO3 in aqueous ethanol or DBU in acetoni-
trile. The Mitsunobu method, used in the preparation of
ningalin B derivative 29, was also adopted in the synthesis of
target compound 10 because of the NH acidity of 18. Inter-
mediate 18 was coupled with 2-(3,4-dimethoxyphenyl)-
ethanol in the presence of PPh3 and DIAD to produce

5110 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Scheme 1. Synthesis of Compounds 4-12a
Zhang et al.
a Reagents and conditions: (a) hexamethylenetetramine, CHCl3, room temp, 4 h; (b) HBr, CH3OH, 0 °C to room temp, 12 h; (c) 2-(3,4-
dimethoxyphenyl)acetic acid, HOBt, DCC, Et3N, CH2Cl2, 0 °C to room temp, N2, 18 h; (d) t-BuOK, t-BuOH, 30 °C, air, 2 h; (e) t-BuOK, t-BuOH,
30 °C, N2, 2 h; (f) PPh3, DIAD, THF, 0 °C to room temp.
and noncytotoxic concentration of permethyl ningalin B
analogues (1 μM) was used in this study. Permethyl ningalin
B analogues are grouped into seven series according to types
of substituents and linkers used. Among all permethyl ninga-
lin B analogues, compound 6 displayed the highest modulat-
ing activity with a relative fold (RF) of 18.2 (Table 1). Well-
known P-gp modulators including verapamil, cyclosporine
A, and PSC833 were employed as positive controls. Verapa-
mil displayed a moderate activity with a RF of 5.6 (Table 1).
In contrast, both cyclosporine A and PSC833 exhibited the
strongest reversal activity with a RF of 91.6 and 101.8,
respectively (Table 1).
Among series 1, 2, and 5, it was found that permethyl
ningalin B analogues containing methoxy substituent(s) on
the benzyl group gave higher RF values than one without
methoxy group(s) (Table 1), suggesting that the presence of
methoxy substituent(s) is needed for inhibiting the function-
ality of P-gp. Moreover, the number of methoxy substituents
on the benzyl group is also an important factor for determin-
ing the P-gp modulating activity. In series 1, compound 6
with trimethoxy substituents displayed better activity than
the di- and monomethoxy analogues (compounds 4 and 5)
(Table 1). In series 2, the analogue with a monomethoxy
substituent (compound 9) showed stronger activity than the
analogues with di- and trimethoxy substituents (compounds
10 and 11). On the contrary, analogues with either mono- or
dimethoxy substituent(s) (compounds 25 and 26) displayed
similar activity in series 5. In addition to the methoxy sub-
stitution, the length and the nature of linker also play an
important role in resensitizing LCC6MDR to paclitaxel. The
analogue with the shortest linker displayed a stronger P-gp
modulating activity than other analogues with longer linker
(Table 1). Compound 7 with methylene linker (RF = 7.9)
displayed about 2-fold higher modulating activity than
compound 8 with bismethylene (RF=3.6) and compound
24 with carbonylmethylene (RF=3.9), suggesting that shor-
ter linker in permethyl ningalin B analogues is crucial for
modulating P-gp. When these two requirements are put toge-
ther, the permethyl ningalin B analogue 6 containing the
shortest linker and three methoxy substituents displayed the
highest P-gp modulating activity with a RF of 18.2 (Table 1).
Among the dimeric analogues, only compound 30 (RF =
8.0) and compound 32 (RF = 5.2) gave moderate reversal
activities, indicating that the dimeric analogues have poorer
reversal activities than monomeric analogues.
Synergistic Effect of Ningalin B Analogues on Reversing
Paclitaxel Resistance of MDA435/LCC6MDR Cells. Com-
pound 6 (RF = 18.2) is the strongest P-gp modulator among
all permethyl ningalin B analogues (Table 1). We mixed
compound 6 with equimolar concentrations of other mod-
erate modulators including 5, 9, 25, or 26 and tested for their
P-gp modulating activity. We found that a combination of
0.5 μM 6 and 0.5 μM 25 resulted in a 66-fold resensitization
of LCC6MDR toward paclitaxel (IC50=2.8 nM, Table 3).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5111
Scheme 2. Synthesis of Compounds 21, 24-26, and 28a
a Reagents and conditions: (a) PPh3, DIAD, THF, 0 °C to room temp, N2; (b) K2CO3, DMF, room temp, N2; (c) NaH, DMF, 0 °C to room temp, -80 °C,
12 h, N2.
Scheme 3. Synthesis of Compounds 29-34a
a Reagents and conditions: (a) PPh3, DIAD, THF, 0 °C to room tempt, N2; (b) K2CO3, DMF, 90 °C, N2; (c) Lawesson’s reagent, toluene, reflux, 8 h, N2.
This level is similar to that of parental LCC6 (IC50 = 2.6 nM,
Table 1). Moreover, this combination almost displayed com-
parable paclitaxel resistance reversal activity as the standard
P-gp modulators such as cyclosporine A (IC50 = 2.0 nM)
and PSC833 (IC50=1.8 nM) (Table 1). On the basis of the
RF values, the combination of 6 and 25 is about 3.6-fold

5112 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Zhang et al.
Table 1. P-gp Modulating Activity of Permethyl Ningalin B Analoguesa
compd
(1 μM)
no. of methoxy
on benzyl group
types of
linker
mean IC50 of
paclitaxel (nM)
series
1
RF
7
methylene
methylene
23.1 ( 4.2
19.6 ( 2.5
14.7 ( 1.9
10.1 ( 0.6
51.1 ( 7.3
17.1 ( 1.3
26.6 ( 4.3
40.7 ( 4.5
39.0 ( 2.7
29.4 ( 1.2
47.3 ( 12.3
18.4 ( 1.1
22.4 ( 1.6
99.0 ( 24.1
115.9 ( 10.1
23.0 ( 1.5
131.3 ( 2.3
35.0 ( 3.8
54.4 ( 11.5
66.5 ( 5.1
32.9 ( 3.5
2.0 ( 0.2
7.9
9.3
4
mono
di
tri
5
methylene
methylene
12.4
18.2
3.6
6
2
8
bismethylene
bismethylene
bismethylene
bismethylene
trismethylene
vinylmethylene
carbonylmethylene
carbonylmethylene
carbonylmethylene
hydroxyethyl
methylene
9
mono
di
tri
10.7
6.9
10
11
12
21
24
25
26
29
28
30
31
32
33
34
4.5
4.7
3
4
5
tri
tri
6.2
3.9
mono
di
9.9
8.2
6
7
1.8
1.6
tri
di
di
di
methylene
8.0
1.4
bismethylene
trismethylene
methylene
5.2
3.4
tri
bismethylene
2.8
5.6
verapamil
cyclosporine A
PSC833
LCC6MDRb
LCC6c
91.6
101.8
1.0
1.8 ( 0.3
183.2 ( 6.3
2.6 ( 0.5
70.5
a IC50 values were determined for LCC6MDR cells after they were treated with a series of concentrations of paclitaxel in the presence of 1 μM of
different permethyl ningalin B analogues, verapamil, cyclosporine A, and PSC833. Relative fold (RF) represents fold-change in drug sensitivity. RF =
(IC50 without modulator)/(IC50 with modulator). N = 2-18 independent experiments were performed, and averaged IC50 values are presented as the
mean ( standard error of mean. b No modulator was used in LCC6MDR cells. IC50 value from this control is used for normalization (RF = 1.0). c LCC6
cells were used without addition of modulator.
Table 2. In Vitro Cytotoxicity of Permethyl Ningalin B Analogues to
including 6 and 5 (RF = 13.8), 6 and 9 (RF = 18.0), and 6
and 26 (RF = 12.9) did not display any synergy (Table 3).
LCC6, LCC6MDR, and L929 Cellsa
IC50 (μM)
To quantitatively measure the synergistic effect among dif-
ferent combinations of modulators, we used the fractional
inhibitory concentration index (FICI) approach modified from
Chou and Talalay41 (see Experimental Section for details). We
can determine the fractional IC50 (FIC) for each combination of
modulators. The sum of two FICs will give the FICI value for
that combination. A FICI value lower than or equal to 0.5
indicates synergy. As shown in Table 3, the synergistic effect was
observed only when 0.5 μM of 6 was used in combination with
0.5 μM 25 (FICI = 0.42). Other combinations were found to
give an additive effect because their FICI values were between
1.6 and 2.2. A different approach using a plot of different frac-
tions affected (Fa) against FICI also demonstrated that there
was a synergy between 6 and 25 but not in 6 and 5 or 6 and 9 or 6
and 26 combinations in modulating paclitaxel resistance (see
Supporting Information).
Effect of Permethyl Ningalin B Analogues on Reversing
DOX and Vincristine Resistance of MDA435/LCC6MDR
Cells. We found that permethyl ningalin B analogues also
potentiated the action of other anticancer drugs including DOX
and vincristine in LCC6MDR cells. Both DOX and vincristine
are known P-gp substrates. For DOX cytotoxicity, compound 6
(RF = 9.5) exhibited a higher modulating activity than 25
(RF=5.2) (Table 4). The combination of 6 and 25 (RF=12.0)
displayed a similar modulating activity as compound 6 alone,
and the FICI value was greater than 0.5 (FICI=1.26) (Table 4),
suggesting that there was no synergy in modulating P-gp.
For vincristine cytotoxicity, compound 6 (RF=14.6) was better
than compound 25 (RF = 9.3) in resensitizing LCC6MDR
(Table 4). Interestingly, the combination of them (RF=26.9)
compd
LCC6
>100
LCC6MDR
L929
>100
4
>100
>100
>100
>100
>100
18.0 ( 2.1
>100
>100
>100
>100
>100
90.0 ( 0.0
>100
84
5
>100
>100
>100
ND
>100
>100
>100
>100
21.6 ( 5.9
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
ND
6
7
8
9
32.5 ( 2.5
>100
>100
>100
>100
ND
10
11
12
21
24
25
26
28
29
30
31
32
33
34
>100
>100
ND
>100
>100
ND
>100
>100
>100
>100
>100
ND
>100
ND
ND
a The IC50 values were determined after exposure to a series of
concentrations of permethyl ningalin analogues using LCC6,
B
LCC6MDR, and L929. L929 is a mouse connective tissue fibroblast
cell line. Each experiment has been repeated one to three times with the
data presented as the mean ( standard error of mean. Many of
permethyl ningalin B analogues did not show cytotoxicity at the highest
concentration used (100 μM). ND = not determined.
(P< 0.0001) and 6.7-fold (P< 0.0001) better than 6 and 25
alone, respectively, suggesting that 6 and 25 can act synergisti-
cally to modulate P-gp. On the contrary, other combinations

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5113
Table 3. In Vitro Synergistic Interaction of Permethyl Ningalin B Analogues on Reversing Paclitaxel Resistance of LCC6MDR Cellsa
mean IC50 of
paclitaxel (nM)
compd
RF
FIC6
FIC5
0.90
FIC9
0.60
FIC25
FIC26
FICI
drug interaction
183.2 ( 6.3
13.3 ( 3.5
10.2 ( 1.4
2.8 ( 0.5b
14.3 ( 2.5
10.1 ( 0.6b
14.7 ( 1.9
17.1 ( 1.3
18.4 ( 1.1b
22.4 ( 1.6
1.0
0.5 μM 6 þ 0.5 μM 5
0.5 μM 6 þ 0.5 μM 9
0.5 μM 6 þ 0.5 μM 25
0.5 μM 6 þ 0.5 μM 26
1 μM 6
13.8
18.0
66.0
12.9
18.2
12.4
10.7
9.9
1.31
1.01
0.27
1.41
1.00
2.21
1.61
0.42
2.05
1.00
1.00
1.00
1.00
1.00
additivec
additived
synergistice
additivef
0.15
0.64
1.00
1 μM 5
1 μM 9
1.00
1.00
1 μM 25
1 μM 26
1.00
8.2
a The IC50 values were displayed asthe mean ( standard error of mean, and the IC50 values of the combination of two modulators were from two to four
independent experiments. The Pvalue of IC50 of the combination of 0.5 μM6and 0.5 μM25 compared to 6alone or 25 alone was determined by Student’s ttest.
b P < 0.0001. FIC values were calculated using IC50 values. Interaction is classified as synergistic (FICI e 0.5), additive (FICI between 0.5 and 4), or
antagonistic (FICI > 4). c Drug interaction was relative to 1 μM6alone or 1 μM5alone. d Drug interaction was relative to 1 μM6alone or 1 μM9alone. e Drug
interaction was relative to 1 μM 6 alone or 1 μM 25 alone. f Drug interaction was relative to 1 μM of 6 alone or 1 μM of 26 alone.
Table 4. In Vitro Additive Interaction of Permethyl Ningalin B Analo-
treated with 6 alone (3.0-fold higher than the LCC6MDR’s
gues on Reversing DOX and Vincristine Resistance of LCC6MDR
Cellsa
control) (Figure 2A).
We determined the dosage effect of 6 and 25 on increasing
anticancer
drugs
drug
FICI interaction
DOX accumulation. We found that restoration of DOX
accumulation in LCC6MDR cells by 6 or 25 was dosage-
dependent up to 3 μM (Figure 2B and Figure 2C). Further
increase in concentration of 6 or 25 resulted in a slight drop in
DOX accumulation (Figure 2B and Figure 2C). Intracellular
DOX accumulation in LCC6MDR, however, cannot be
completely restored to LCC6’s level (Figure 2A). Surpris-
ingly, the combination of 6 and 25 displayed a dosage-
dependent restoration of DOX accumulation in LCC6MDR
(Figure 2D). Intracellular DOX accumulation can be re-
stored to the parental LCC6 level when a combination of
8 μM 6 and 8 μM 25 was applied (Figure 2D).
compd
RF
DOX
1 μM 6
1 μM 25
9.5 ( 0.8
5.2 ( 0.6
1.00
1.00
0.5 μM 6 þ 0.5 μM 25 12.0 ( 1.5 1.26
additiveb
additiveb
vincristine 1 μM 6
1 μM 25
14.6 ( 3.3 1.00
9.3 ( 0.1 1.00
0.5 μM 6 þ 0.5 μM 25 26.9 ( 4.8 0.89
a Relative fold (RF) represents fold-change in drug sensitivity. RF =
(IC50 without modulator)/(IC50 with modulator). RF values were pre-
sented as the mean ( standard error of mean. N = 2-5 independent
experiments. The IC50 values were used to calculate the FICI value.
Interaction is classified as synergistic (FICI e 0.5), additive (FICI
between 0.5 and 4), or antagonistic (FICI > 4). b Drug interaction was
relative to 1 μM 6 alone or 1 μM 25 alone.
Dixon Plot Suggests a Noncompetitive Relationship be-
tween Permethyl Ningalin B Analogues and DOX in Binding
P-gp. We have employed Dixon analysis to further charac-
terize the inhibition mechanism of permethyl ningalin B analo-
gues on P-gp’s DOX transport activity. The increase in DOX
retention in LCC6MDR cells was generally dependent on the
concentration of permethyl ningalin B analogues. The Dixon
plot is a common method to determine inhibition constants (Ki)
of competitive inhibitors in which the reciprocal of the velocity
(1/v) is plotted against the inhibitor concentration at two or more
substrate concentrations.42 The intersection point in the Dixon
plot provides a measure of Ki. Linear regression line obtained
from the replot of slopes of Dixon plot versus the reciprocal of
substrate concentration can distinguish between a competitive
and noncompetitive or partially competitive relationship. A
competitive inhibitor will give a regression line passing through
the origin, whereas a noncompetitive inhibitor would not.43,44
The Ki value was determined to be 5.5 μM for 6 (Figure 3A),
5.4 μM for 25 (Figure 3B), and 5.8 μM for the combination of 6
and 25 (Figure 3C), respectively. Linear regression line does not
coincide with the origin for 6 (inset of Figure 3A), 25 (inset of
Figure 3B), and the combination of 6 and 25 (inset of
Figure 3C), suggesting that permethyl ningalin B analogues
likely act as a noncompetitive inhibitor of P-gp-mediated DOX
transport. The Ki value of the combination of 6 and 25 was
similar to that of compound 6or 25 alone, indicating that there is
no synergy for 6 and 25 in inhibiting the P-gp-mediated DOX
transport and that these data are in good agreement with the in
vitro DOX cytotoxicity assay in which an additive effect was
found when combining 6 and 25 (Table 4).
resulted in a higher P-gp modulating activity, which was about
1.8-fold and 2.9-fold higher than compound 6 alone (RF=14.6)
and compound 25 alone (RF = 9.3) (Table 4), respectively.
However, there was no synergistic activity of 6 and 25 on
reversing vincristine resistance of LCC6MDR because the FICI
value was greater than 0.5 (FICI = 0.89) (Table 4).
Dosage Effect of Permethyl Ningalin B Analogues on
Intracellular Accumulation of DOX in MDA435/LCC6MDR
Cells. The above results showed that the permethyl ningalin
B analogues are promising P-gp modulators. We are inter-
ested in studying whether modulation of P-gp-mediated drug
resistance is associated with an increase in drug accumula-
tion. DOX is a fluorescent P-gp substrate that can be used to
monitor intracellular drug accumulation. As shown in
Figure 2A, the level of DOX accumulation in parental
LCC6cellswas about 3.3-foldhigherthan thatof LCC6MDR
cells in the absence of modulators. This is because P-gp can
pump DOX out of the cells, thereby lowering intracellular
DOX level. Treatment of LCC6 cells with 3 μM 6, 25, or
combination of 6 and 25 did not increase DOX accumulation.
In contrast, treatment of LCC6MDR cells with 3 μM 6, 25, or
combination of 6 and 25 resulted in 3.0-, 2.1-, or 2.8-fold
increase in intracellular DOX accumulation, respectively
(Figure 2A). This result suggests that the mechanism of
reversal activity of permethyl ningalin B analogues is by
inhibiting the transport activity of P-gp. The combination of
6 and 25 (2.8-fold higher than the LCC6MDR’s control)
restored the DOX accumulation to a level similar to that

5114 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Zhang et al.
Figure 2. Effect of permethyl ningalin B analogues added at different concentrations on intracellular DOX accumulation in LCC6 and
LCC6MDR cells. (A) LCC6 or LCC6MDR cells were incubated with 20 μM DOX for 150 min at 37 °C with or without 6, 25, or combination of
6 and 25. After the incubation period, we lysed the cells and used the supernatant to measure the DOX level by spectrofluorometry.
Experiments were performed in duplicate and repeated twice. The results were presented as the mean ( standard error of mean. The dosage
effect of permethyl ningalin B analogues on intracellular DOX accumulation was also studied by incubating LCC6MDR cells with different
concentrations of modulators including 6 (B), 25 (C), combination of 6 and 25 (D). DMSO (0.4%) was used as negative control.
Discussion
suggested that the mechanism of synthetic permethyl ningalin
B analogues for reversing MDR is by increasing the intracel-
lular drug concentration, thereby sensitizing LCC6MDR cells
toward anticancer drugs.
Overexpression of P-gp is one of the major obstacles for suc-
cessful treatment of cancer. Numerous agents have been devel-
oped to reverse P-gp-mediated MDR; however, none of them
could be used clinically because of lack of potency or high
cytotoxicity at an effective concentration. Herein, synthetic
permethyl ningalin B analogues were found to be noncytotoxic
(IC50 > 100 μM) to L929 mouse fibroblast cell lines and cancer
cell lines even when used at a very high concentration (Table 2).
While lacking inherent cytotoxicity, synthetic permethyl ninga-
lin B analogues exhibited a promising P-gp modulating activity
at a low concentration (1 μM) and were found to be more effec-
tive than other traditional modulators like verapamil (Table 1).
As all permethyl ningalin B analogues exerted no cytotoxicity
toward fibroblast even at 100 μM (except 9), we estimate that
the therapeutic index for permethyl ningalin B analogues to be
used as P-gp modulator will be at least 100 (IC50 of permethyl
ningalin B analogues toward fibroblast/concentration of per-
methyl ningalin B analogues used).
We found that methoxy substituents on benzyl group and
the type of linkers used are two key structural features for
determining P-gp modulating activity of synthetic permethyl
ningalin B analogues. Compound 6 with trimethoxy substit-
uents and the shortest linker displayed the strongest P-gp
modulating activity, and it caused about 18.2-fold resensitiza-
tion of LCC6MDR toward paclitaxel (Table 1). It is possible
that permethyl ningalin B with methoxy substituents and the
shortest linkerwould bind more specificallytoP-gpthanother
analogues with longer linker and nonmethoxy substituents.
Intracellular DOX accumulation data shown in Figure 2
Surprisingly, a synergistic activity on reversing paclitaxel
resistance was noted when the combination of compounds 6
and 25 was used. This synergy resulted in a 66-fold (IC50=
2.8 nM) sensitization of LCC6MDR to paclitaxel. This po-
tent modulating activity can completely reverse the IC50 of
LCC6MDR (IC50 =183.2 nM, Tables 1 and 3) to that of
parental LCC6 (IC50=2.6 nM, Table 1), and its potency is
comparable to other standard P-gp modulators such as
cyclosporine A (IC50=2.0 nM) and PSC833 (IC50=1.8 nM)
(Table 1). The above data suggest that a cocktail che-
motherapy including two modulators acting synergistically
and an anticancer drug could likely be a novel, safe, and
highly potent way to treat MDR cancer when other tradi-
tional treatment methods fail.
Synergism of 6 and 25 on modulating P-gp suggests that
they might have slightly different mechanisms of action,
possibly by binding to different regions of P-gp, although
there is no experimental proof at this point. No synergy was
noted in DOX and vincristine cytotoxicity when combining 6
and 25, suggesting that the synergism by 6 and 25 on reversing
drug resistance is drug/substrate specific. It is possible that 6
and 25 could work more cooperatively to prevent paclitaxel
from being pumped out from the cell by P-gp than DOX and
vincristine. One possible mechanism is that 6 and 25 could
share more overlapping binding sites with paclitaxel on P-gp
than DOX or vincristine, thereby inhibiting paclitaxel from
binding onto P-gp to a larger extent.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5115
permethyl ningalin B analogues for modulating MDR is by
virtue of increasing intracellular drug accumulation. Surpris-
ingly, a synergy (FICI = 0.42) on reversing paclitaxel resis-
tance was noted for the combinationof 6 and 25, which caused
about66-foldresensitizationofLCC6MDR towardpaclitaxel
and completely reversed the IC50 of LCC6MDR to the
parental LCC6’s level. Kinetic analysis suggests that per-
methyl ningalin B analogues likely act as a noncompetitive
inhibitor of DOX transport. The present study demonstrates
that the synthetic permethyl ningalin B analogues can be
employed as safe and effective modulators of P-gp-mediated
drug resistance in cancer cells.
Experimental Section
General. The starting materials and reagents, purchased from
commercial suppliers, were used without further purification.
Literature procedures were used for the preparation of 2-bro-
mo-1-(3,4-dimethoxyphenyl)ethanone (13),45 2-bromo-1-phenyl-
ethanone (22),45 2-bromo-1-(4-methoxyphenyl)ethanone (23),45
(4-methoxyphenyl)methanol (19a),46,47 (3,4-dimethoxyphenyl)-
methanol (19b),46,47 (3,4,5-trimethoxyphenyl)methanol (19c),46,47
2-(4-methoxyphenyl)ethanol (19f),48,49 2-(3,4-dimethoxyphenyl)-
ethanol (19g),48,49 2-(3,4,5-trimethoxyphenyl)ethanol (19h),48,49
3-(3,4,5-trimethoxyphenyl)propan-1-ol (19i),48,50 (E)-3-(3,4,5-tri-
methoxyphenyl)prop-2-en-1-ol (20),51 3,4,5-trimethoxybenzyl-
methyl sulfate (27).28
All reactions were monitored by thin-layer chromatography
(TLC) on aluminum sheets (silica gel 60-F254, E. Merck).
Compounds were visualized by UV light. Anhydrous THF
was distilled under nitrogen from sodium benzophenone ketyl.
Anhydrous methylene chloride was distilled under nitrogen
from CaH2. Anhydrous DMF was distilled under vacuum from
CaH2. Reaction flasks were flame-dried under a stream of N2.
Flash chromatography was carried out using silica gel 60
(200-300 mesh). Silica gel chromatography solvents were of
analytical grade. Melting points were recorded on a micromelt-
ing point apparatus MP-500D and were uncorrected. 1H NMR
and 13C NMR (600 or 400 MHz) spectra were measured with
TMS as an internal standard when CDCl3 was used as a solvent.
Chemical shifts are expressed in δ (ppm) and coupling constants
(J) in Hz. High-resolution (ESI) mass spectra were recorded
using a QTOF-2 Micromass spectrometer.
2-Hexamethylenetetramino-1-(3,4-dimethoxyphenyl)ethanone-
2-ium Bromide (14). To a stirred solution of hexamethylenete-
tramine (3.57 g, 25.5 mmol) in chloroform (60 mL) at room
temperature, a solution of 13 (5.5 g, 21.2 mmol) in chloroform
(5 mL) was added quickly. After 4 h, the resulting reaction
suspension was filtered, washed with chloroform, dried in
vacuo, and recrystallized in methanol to afford the desired
compound 14 (8.5 g, 99% yield): mp 110-112 °C.
Figure 3. Kinetic study of relationship between permethyl ningalin
B analogues and DOX using Dixon plot. LCC6MDR cells were
incubated with different concentration of DOX (1.5, 4.4, 13.3, and
40 μM) in the presence of four concentrations (0, 0.5, 1, and 2 μM) of
compound 6 (A), 25 (B), or combination of 6 and 25 (C), respec-
tively. The reciprocal of DOX retention rate is plotted against the
concentration of modulator. The intersection point represents the
Ki value. The slope of the Dixon plot is plotted against reciprocal of
DOX concentration to determine their relationship (insets of parts
A, B, and C). Each data point represents two to four independent
experiments and is presented as the mean ( standard error mean.
Kinetic analysis suggests that permethyl ningalin B analo-
gues likely act as a noncompetitive inhibitor of DOX trans-
port with Ki values ranging from 5.4 to 5.8 μM (Figure 3). The
permethyl ningalin B analogues probably bind to P-gp at a
binding site(s) different from the binding site(s) of DOX.
Binding of permethyl ningalin B analogues to P-gp would
reduce the affinity of DOX for its substrate binding site,
diminish the amount of DOX being pumped out by P-gp,
and eventually chemosensitize LCC6MDR cells to DOX.
In summary, we demonstrated that synthetic permethyl
ningalin B analogues exhibited promising MDR reversal
activity without any inherent cytotoxicity to cancer cell lines
or normal mouse fibroblast cell lines. The methoxy substitu-
entsand types oflinker used are two key structural features for
determining MDR reversal potency of permethyl ningalin B
analogues. Compound 6 containing trimethoxy substituents
and the shortest linker displayed the strongest modulating
activity and caused about 18.2-fold hypersensitization of
LCC6MDR toward paclitaxel. The mechanism of synthetic
2-Amino-1-(3,4-dimethoxyphenyl)ethanone Hydrobromic Acid
(15). A solution of 14 (15.5 g, 37.5 mmol) in absolute methanol
(20 mL) was added to a stirring solution of concentrated
hydrobromic acid (40 mL) in absolute methanol (200 mL) at
0 °C. The clear reaction solution gradually changed into a thick
suspension. The above mixture was allowed to warm to room
temperature naturally. After 12 h, the resulting suspension was
filtered, washed with ethyl ether and acetone, and dried in vacuo
to afford the desired compound 15 (9.8 g, 95% yield): mp
236-238 °C; m/z [M þ H]þ 196.1.
2-(3,4-Dimethoxyphenyl)-N-(2-(3,4-dimethoxyphenyl)-2-etha-
none)acetamide (16). To a stirred solution of 2-(3,4-dimethoxy-
phenyl)acetic acid (3.75 g, 19 mmol) and HOBt (2.56 g,
19 mmol) in dry CH2Cl2 (30 mL) at 0 °C under N2 atmosphere
was added DCC (3.9 g, 19 mmol) in batch over 10 min. After
30 min, to the above reaction solution was added 15 (3.3 g,
12 mmol) and triethylamine (1.57 g, 15.5 mmol). The reaction
mixture was stirred for 18 h while the reaction temperature rose

5116 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Zhang et al.
to room temperature naturally. Solid dicyclohexylurea in the
above reaction mixture was removed by filtration. The filter
cake was washed with CH2Cl2. The filtrate was evaporated
under reduced pressure and purified by flash chromatography
on silica gel (EtOAc/PE = 3/1, v/v) to afford the desired com-
pound 16 (5.45 g, 77% yield): mp 138-140 °C; ESI-MS; m/z
[M þ H]þ 374.2; 1H NMR (CDCl3, 600 MHz) δ 7.59-7.57 (dd,
J = 8.4, 1.8 Hz, 1 H), 7.46 (d, J = 1.8 Hz, 1 H), 6.91-6.85 (m, 4
H), 6.65 (s, 1 H), 4.70-4.69 (d, J = 4.3 Hz, 2 H), 3.95-3.89 (m,
12H), 3.62 (s, 2 H); 13C NMR (CDCl3, 600 MHz) δ 192.38,
171.5, 154.0, 149.2, 149.1, 148.3, 127.3, 126.8, 122.5, 121.5,
112.3, 111.4, 110.2, 109.7, 56.1, 55.8, 45.9, 43.1.
3,4-Bis(3,4-dimethoxyphenyl)-1H-pyrrole-2,5-dione (18). t-BuOK
(250 mg, 2.25 mmol) was added to a stirred solution of 16 (280 mg,
0.75 mmol) in t-BuOH (20 mL) at 35 °C under air. After 2 h, the
resulting reaction solution was poured into ice-cold water (100 mL),
and then the pH was adjusted to 7-8 by adding 2 N hydrochloric
acid to give a yellow thick suspension. The above yellow thick
suspension was filtered and purified by flash chromatography on
silica gel (EtOAc/PE = 2/1, v/v) to afford the desired compound 18
(120 mg, 45% yield): mp 238-240 °C; 1HNMR(CDCl3, 600MHz)
δ 7.63 (s, 1 H), 7.22-7.20 (dd, J = 8.4, 2.2 Hz, 2 H), 7.03-7.02 (d,
J = 2.2 Hz, 2 H), 6.88-6.86 (d, J = 8.4 Hz, 2 H), 3.91 (s, 6 H), 3.71
(s, 6 H); 13C NMR (CDCl3, 600 MHz) δ 170.7, 150.4, 148.7, 134.9,
123.5, 121.1, 112.4, 110.9, 55.8, 55.7; HRMS calcd for (C20H19-
NO6 þ H)þ 370.1290, found 370.1284.
(yield, 77-81%). The physical and spectral data for 30-32 are
listed below.
1-((3,4-Bis(3,4-dimethoxyphenyl)-2,5-dioxo-2H-pyrrol-1(5H)-
yl)methyl)-3,4-bis(3,4-dimethoxyphenyl)-1H-pyrrole-2,5-dione
(30). Yield 81%; mp 187-188 °C; 1H NMR (CDCl3, 600 MHz)
δ 7.21 (dd, J = 1.8, 8.7 Hz, 4 H), 7.05 (d, J = 1.8 Hz, 4H), 6.82
(d, J = 8.7 Hz, 4 H), 5.53 (s, 2 H), 3.89 (s, 12 H), 3.70 (s, 12 H);
13C NMR (CDCl3, 600 MHz) δ 169.8, 150.6, 148.8, 134.3, 123.8,
121.2, 112.7, 111.0, 56.0, 55.9, 40.7; HRMS calcd for (C41H38-
N2O12 þ Na)þ 773.2322, found 773.2311.
1-(2-(3,4-Bis(3,4-dimethoxyphenyl)-2,5-dioxo-2H-pyrrol-1(5H)-
yl)ethyl)-3,4-bis(3,4-dimethoxyphenyl)-1H-pyrrole-2,5-dione (31).
Yield 80%; mp 232-234 °C; 1H NMR (CDCl3, 600 MHz) δ 7.10
(dd, J = 1.8, 8.3 Hz, 4 H), 6.96 (d, J = 1.8 Hz, 4 H), 6.78 (d, J =
8.3 Hz, 4 H), 3.94 (s, 4 H), 3.88 (s, 12 H), 3.63 (s, 12 H); 13C NMR
(CDCl3, 600 MHz) δ 171.2, 150.4, 148.7, 134.5, 123.6, 121.5,
112.6, 110.9, 55.9, 55.8, 37.1; HRMS calcd for (C42H40N2O12
H)þ 765.2659, found 765.2652.
þ
1-(3-(3,4-Bis(3,4-dimethoxyphenyl)-2,5-dioxo-2H-pyrrol-1(5H)-
yl)propyl)-3,4-bis(3,4-dimethoxyphenyl)-1H-pyrrole-2,5-dione (32).
Yield 77%; mp 76-79 °C; 1H NMR (CDCl3, 600 MHz) δ
7.21-7.16 (m, 4 H), 7.03 (m, 4 H), 6.82 (m, 4 H), 3.89 (d, J =
3.6 Hz, 12 H), 3.73-3.69 (m, 16 H), 2.02 (m, 2 H); 13C NMR
(CDCl3, 600 MHz) δ 171.2, 150.5, 148.7, 134.2, 123.7, 121.5,
112.7, 111.0, 55.9, 55.8, 36.1, 31.5; HRMS calcd for (C43H42-
N2O12 þ H)þ 779.2816, found 779.2825.
3,4-Bis(3,4-dimethoxyphenyl)-1H-pyrrol-2(5H)-one(17).t-BuOK
(250 mg, 2.25 mmol) was added to a stirring solution of 16 (280 mg,
0.75 mmol) in t-BuOH (20 mL) at 35 °C under N2 atmosphere.
After 2 h, the pH of the resulting reaction solution was adjusted to
7-8 by adding 2 N hydrochloric acid under N2 atmosphere. Then
80 mL of water was poured into the above solution to give yellow
thick suspension. The suspension was then filtered and purified by
flash chromatography on silica gel (EtOAc/PE = 1/1, v/v) to yield
the desired compound 17 (140 mg, 50% yield): mp 170-172 °C; 1H
NMR (CDCl3, 600 MHz) δ 7.70 (brs, 1 H), 7.01 (dd, J = 2.2, 8.2
Hz, 1H), 6.95-6.89 (m, 3 H), 6.83 (d, J = 2.2 Hz, 1H), 6.80 (d, J =
8.3 Hz, 1 H), 4.35 (s, 2 H), 3.87 (d, J = 5.5 Hz, 6 H), 3.78 (s, 3 H),
3.56 (s, 3 H); 13C NMR (CDCl3, 600 MHz) δ 174.9, 150.1, 149.5,
149.0, 148.7, 130.7,125.9, 124.8, 122.4, 120.3, 112.7, 111.5, 111.0,
55.9, 5.7, 48.1; HRMS calcd for (C20H21NO5 þ H)þ 356.1498,
found 356.1491.
3,4-Bis(3,4-dimethoxyphenyl)-1-(2-hydroxyethyl)-1H-pyrrole-
2,5-dione (29). DIAD (548 mg, 2.71 mmol) was added dropwise
to a stirring solution of glycol (1 mL), PPh3 (710 mg, 2.71 mmol),
and 18 (200 mg, 0.54 mmol) in dry THF (40 mL) at 0 °C under
N2 atmosphere. The reaction mixture was allowed to warm to
room temperature and stirred for 12 h until TLC showed the
reaction has completed. The resulting reaction mixture was
poured into water (60 mL), extracted with EtOAc, washed with
brine, dried over anhydrous MgSO4, and the solvent was
removed under reduced pressure. The residue was purified by
flash chromatography on silica gel (EtOAc/PE = 1/2, v/v) to
yield the desired compound 29 (158 mg, 71% yield): mp
152-154 °C; 1H NMR (CDCl3, 400 MHz) δ 7.21 (dd, J =
1.9, 8.4 Hz, 2 H), 7.05 (d, J = 1.9 Hz, 2 H), 6.86 (d, J = 8.4 Hz,
2 H), 3.90 (s, 6 H), 3.86 (m, 4 H), 3.71 (s, 6 H); 13C NMR (CDCl3,
400 MHz) δ 172.5, 151.3, 149.5, 135.1, 124.4, 122.1, 113.4, 111.8,
62.1, 56.7, 56.6, 42.0; HRMS calcd for (C22H23NO7 þ H)þ
414.1553, found 414.1561.
1-(2,3,4,5-Tetramethoxybenzyl)-2-(3,4,5-trimethoxybenzyl-
oxy)-3,4-bis(3,4-dimethoxyphenyl)-1H-pyrrole (28). NaH (300 mg,
50-65%, moistened in oil) was added to dry DMF (20 mL) at
0 °C under N2 atmosphere. To the above stirring suspension, 17
(200 mg, 0.56 mmol) in DMF (20 mL) was added drop by drop.
The mixture was stirred at room temperature for 30 min, followed
by the addition of a solution of 27 (200 mg, 0.72 mmol) in DMF
(5 mL). The reaction mixture was heated at 80 °C for 12 h until
TLC showed the reaction has completed. The reaction mixture was
then cooled to room temperature and quenched with methanol
(2 mL). The resulting solution was poured into water (100 mL),
extracted with EtOAc, washed with brine, dried over anhydrous
MgSO4, and the solvent was removed under reduced pressure. The
residue was purified by flash chromatography on silica gel (EtOAc/
PE = 1/1, v/v) to yield the desired compound 28 (75% yield): mp
78-79 °C; 1H NMR (CDCl3, 600 MHz) δ 6.99 (dd, J = 1.8, 8.2
Hz, 1 H), 6.87 (s, 1 H), 6.79 (d, J = 8.7 Hz, 1 H), 6.65 (d, J = 8.2
Hz, 1 H), 6.51 (dd, J = 1.8, 8.2 Hz, 1 H), 6.48 (s, 1 H), 6.46 (s, 1 H),
6.28 (s, 2 H), 6.03 (s, 2 H), 4.88 (d, J = 14.6 Hz, 1 H), 3.87 (d, J =
14.6 Hz, 1 H), 3.81 (s, 3 H), 3.78 (d, J = 12.8 Hz, 1 H), 3.75 (s, 3 H),
3.73 (s, 3 H), 3.72 (s, 3 H), 3.69 (s, 3 H), 3.66 (s, 6 H), 3.60 (s, 3 H),
3.51 (s, 6 H), 3.23 (d, J = 12.8 Hz, 1 H); 13C NMR-DEPT (CDCl3,
600 MHz) δ 153.5, 152.3, 149.4, 148.9, 148.7, 147.9, 137.5, 136.3,
132.1, 131.8, 126.5, 126.1, 125.1, 118.9, 118.1, 111.5, 111.3, 110.0,
108.5, 106.5, 104.8, 60.8, 60.7, 56.1, 55.9, 55.7, 45.8 (CH2), 39.9
(CH2); HRMS calcd for (C40H45NO11 þ H)þ 716.3071, found
716.3076.
General Procedure for Compounds 24-26. Appropriate sub-
stituted 2-bromo-1-phenylethanone 13, 22, or 23 (1 mmol) was
added to a suspension of 18 (1 mmol) and K2CO3 (2 mmol) in
DMF (10 mL) at room temperature under an N2 atmosphere.
The suspension was stirred for 1 h until TLC showed the
reaction has completed. The resulting solution was poured into
water (40 mL), extracted with EtOAc, washed with brine, dried
over anhydrous MgSO4, and the solvent was removed under
reduced pressure. The residue was purified by flash chromatog-
raphy on silica gel (EtOAc/PE = 1/1, v/v) to afford the desired
compounds 24-26 (yields 88-91%). The physical and spectral
data for 24-26 are listed below.
General Procedure for Compounds 30-32. An appropriate
dibromide (0.5 mmol) was added to a suspension of 18 (1 mmol)
and K2CO3 (2 mmol) in DMF (10 mL) at 90 °C under N2
atmosphere. The suspension was stirred for 1 h until TLC
showed the reaction has completed. The resulting solution was
cooled to room temperature and poured into water (40 mL),
extracted with EtOAc, washed with brine, dried over anhydrous
MgSO4, and the solvent was removed under reduced pressure.
The residue was purified by flash chromatography on silica gel
(EtOAc/PE = 1/1, v/v) to afford the desired compounds 30-32
3,4-Bis(3,4-dimethoxyphenyl)-1-[2-phenyl-2-oxoethyl]-1H-
1
pyrrole-2,5-dione (24). Yield 88%; mp 175-177 °C; H NMR
(CDCl3, 600 MHz) δ 8.00 (dd, J=1.3, 7.8 Hz, 2 H), 7.63 (m, 1 H),
7.51 (t, J=7.8 Hz, 2 H), 7.23 (dd, J=1.8, 8.7 Hz, 2 H), 7.09 (d,
J=1.8 Hz, 2 H), 6.86 (d, J=8.7 Hz, 2 H), 5.08 (s, 2H), 3.90

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5117
(s, 6 H), 3.71 (s, 6 H); 13C NMR (CDCl3, 600 MHz) δ 191.6,
170.8, 150.5, 148.7, 134.7, 134.5, 134.1, 129.0, 128.2, 123.7,
121.5, 112.7, 111.0, 56.0, 55.9, 44.5; HRMS calcd for (C28H25-
NO7 þ H)þ 488.1709, found 488.1704.
2 H), 6.86-6.84 (m, 4 H), 3.89 (s, 6 H), 3.83 (t, J = 7.7 Hz, 2 H),
3.78 (s, 3 H), 3.72 (s, 6 H), 2.90 (t, J = 7.7 Hz, 2 H); 13C NMR
(CDCl3, 600 MHz) δ 171.1, 158.4, 150.4, 148.8, 134.2, 130.3,
129.9, 123.6, 121.6, 114.1, 112.6, 111.0, 56.0, 55.9, 55.3, 39.9,
33.9; HRMS calcd for (C29H29NO7 þ H)þ 504.2022, found
504.2028.
1-(3,4-Dimethoxyphenethyl)-3,4-bis(3,4-dimethoxyphenyl)-
1H-pyrrole-2,5-dione (10). Yield 66%; mp 178-180 °C; 1H
NMR (CDCl3, 600 MHz) δ 7.18 (dd, J = 8.2, 2.2 Hz, 2 H),
7.03 (d, J=2.2 Hz, 2 H), 6.86 (d, J=8.2 Hz, 2 H), 6.82 (s, 2 H),
6.77 (s, 1H), 3.91 (s, 6 H), 3.87-3.84 (m, 8 H), 3.72 (s, 6 H), 2.93
(t, J=7.6 Hz, 2 H); 13C NMR (CDCl3, 400 MHz) δ 171.8, 151.2,
149.7, 149.5, 148.5, 134.9, 131.5, 124.3, 122.2, 121.7, 113.4,
112.8, 112.1, 111.8, 56.7, 56.6, 40.5, 35.0; HRMS calcd for
(C30H31NO8 þ H)þ 534.2128, found 534.2133.
1-[2-(4-Methoxyphenyl)-2-oxoethyl]-3,4-bis(3,4-dimethoxyphe-
nyl)-1H-pyrrole-2,5-dione (25). Yield 90%; mp 170-172 °C; 1H
NMR (CDCl3, 600 MHz) δ 7.98 (d, J = 8.7 Hz, 2 H), 7.24 (dd,
J = 1.8, 8.2 Hz, 2 H), 7.09 (d, J = 1.8 Hz, 2 H), 6.98 (d, J = 8.7
Hz, 2 H), 6.86 (d, J = 8.7 Hz,2 H), 5.03 (s, 2 H), 3.90 (s, 6 H), 3.88
(s, 3 H), 3.71 (s, 6 H); 13C NMR (CDCl3, 600 MHz) δ 189.9,
170.9, 164.2, 150.4, 148.7, 134.7, 130.5, 127.5, 123.7, 121.6,
114.2, 112.7, 111.0, 56.0, 55.9, 55.6, 44.2; HRMS calcd for
(C29H27NO8 þ H)þ 518.1815, found 518.1823.
1-[2-(3,4-Dimethoxyphenethyl)-2-oxoethyl]-3,4-bis(3,4-dietho-
xyphenyl)-1H-pyrrole-2,5-dione (26). Yield 91%; mp 91-94 °C;
1H NMR (CDCl3, 600 MHz) δ 7.65 (dd, J = 1.8, 8.7 Hz, 1 H),
7.52 (d, J = 1.4 Hz, 1 H), 7.24 (dd, J = 1.8, 8.7 Hz, 2 H), 7.09 (s,
2 H), 6.93 (d, J = 8.7 Hz, 1 H), 6.86 (d, J = 8.7 Hz, 2 H), 5.05 (s,
3,4-Bis(3,4-dimethoxyphenyl)-1-(3-(3,4,5-trimethoxyphenyl)-
propyl)-1H-pyrrole-2,5-dione (12). Yield 61%; mp 59-60 °C; 1H
NMR (CDCl3, 600 MHz) δ 7.16 (dd, J = 1.8, 8.3 Hz, 2 H), 7.00
(d, J = 1.8 Hz, 2 H), 6.84 (d, J = 8.3 Hz, 2 H), 6.41 (s, 2 H), 3.89
(s, 6 H), 3.79 (s, 6 H), 3.77 (s, 3 H), 3.71-3.69 (m, 8 H), 2.64 (t,
J =7.3 Hz, 2 H), 2.03 (quintet, J = 7.3 Hz, 2 H); 13C NMR
(CDCl3, 600 MHz) δ 171.3, 153.1, 150.4, 148.8, 136.9, 134.1,
123.6, 121.5, 112.7, 111.1, 105.2, 60.8, 56.0, 55.9, 38.2, 33.6, 29.4;
HRMS calcd for (C32H35NO9 þ H)þ 578.2390, found 578.2381.
1-Benzyl-3,4-bis(3,4-dimethoxyphenyl)-1H-pyrrole-2,5-dione
(7). Yield 70%; mp 162-165 °C; ESI-MS (m/z) [M þ H]þ 460.1;
1H NMR (CDCl3, 600 MHz) δ 7.46 (m, 2 H), 7.33 (m, 2 H), 7.28
(m, 1 H), 7.20 (dd, J = 8.2, 2.2 Hz, 2 H), 7.04 (d, J = 2.2 Hz,
2 H), 6.84 (d, J = 8.2 Hz, 2 H), 4.79 (s, 2 H), 3.89 (s, 6 H), 3.70
(s, 6 H); 13C NMR (CDCl3, 400 MHz) δ 171.6, 151.2, 149.4,
137.4, 134.9, 129.5, 128.6, 124.4, 122.2, 113.4, 111.7, 56.7, 56.6,
42.6; HRMS calcd for (C27H25NO6 þ H)þ 460.1760, found
460.1768.
2 H), 3.96 (s, 3 H), 3.92 (s, 3 H), 3.89 (s, 6 H), 3.71 (s, 6 H); 13
C
NMR (CDCl3, 600 MHz) δ 190.1, 170.9, 154.1, 150.5, 149.3,
148.7, 134.7, 127.7, 123.7, 122.8, 121.5, 112.7, 111.0, 110.3, 56.2,
56.1, 56.0, 55.9, 44.2; HRMS calcd for (C30H29NO9 þ H)þ
548.1920, found 548.1912.
General Procedure for Compounds 4-12. DIAD (5 mmol) was
added dropwise to a stirred solution of the appropriate alcohol
19a-i (1.05 mmol), PPh3 (5 mmol), and 18 (1 mmol) in dry THF
(40 mL) at 0 °C under N2 atmosphere. The reaction mixture was
allowed to warm to room temperature and stirred for 12 h until
TLC showed that the reaction has completed. The resulting reaction
mixture was poured into water (100 mL), extracted with EtOAc,
washed with brine, dried over anhydrous MgSO4, and the solvent
was removed under reduced pressure. The residue was purified by
flash chromatography on silica gel (EtOAc/PE = 1/4 to 1/1, v/v) to
afford the desired compounds 4-12 (yield 61-71%). The physical
and spectral data for 4-12 are listed below.
1-(3,4,5-Trimethoxyphenethyl)-3,4-bis(3,4-dimethoxyphenyl)-
1H-pyrrole-2,5-dione (11). Yield 63%; mp 126-127 °C; 1H
NMR (CDCl3, 600 MHz) δ 7.16 (d, J=7.7 Hz, 2 H), 7.01 (s, 2
H), 6.84 (d, J=7.7 Hz, 2 H), 6.46 (s, 2 H), 3.89 (s, 6 H), 3.85 (t,
J=7.7 Hz, 2 H), 3.83 (s, 6 H), 3.79 (s, 3 H), 3.70 (s, 6 H), 2.92 (t,
J=7.7 Hz, 2H); 13C NMR (CDCl3, 600 MHz) δ 171.1, 153.3,
150.5, 148.8, 136.7, 134.2, 133.8, 123.6, 121.4, 112.6, 111.1,
105.8, 60.9, 56.1, 56.0, 55.9, 39.5, 35.0; HRMS calcd for
(C31H33NO9 þ H)þ 564.2233, found 564.2225.
1-(4-Methoxybenzyl)-3,4-bis(3,4-dimethoxyphenyl)-1H-pyr-
role-2,5-dione (4). Yield 71%; mp 125-126 °C; 1H NMR (CD-
Cl3, 600 MHz) δ 7.40 (dd, J = 7.8, 2.2 Hz, 2 H), 7.19 (dd, J =
8.2, 2.2 Hz, 2 H), 7.03 (d, J = 2.2 Hz, 2 H), 6.86-6.83 (m, 4 H),
4.72 (s, 2 H), 3.89 (s, 6 H), 3.78 (s, 3 H), 3.70 (s, 6 H); 13C NMR
(CDCl3, 400 MHz) δ 171.6, 160.0, 151.1, 149.4, 134.9, 131.0,
129.7, 124.4, 122.3, 114.6, 113.4, 111.7, 56.7, 56.6, 56.0, 42.1;
HRMS calcd for (C28H27NO7 þ H)þ 490.1866, found 490.1861.
1-(3,4-Dimethoxybenzyl)-3,4-bis(3,4-dimethoxyphenyl)-1H-
(E)-1-(3,4,5-Trimethoxycinnamyl)-3,4-bis(3,4-dimethoxyphe-
nyl)-1H-pyrrole-2,5-dione (21). Yield 61%; mp 129-130 °C; 1H
NMR (CDCl3, 600 MHz) δ 7.18 (dd, J = 1.8, 8.7 Hz, 2 H), 7.02
(d, J=1.8 Hz, 2 H), 6.83 (d, J=8.7 Hz, 2 H), 6.75 (s, 2 H), 6.61
(dt, J=7.2, 10.1, 17.5 Hz, 1 H), 5.78 (d, J=7.2 Hz, 1 H), 5.34 (dd,
J=10.1, 17.8 Hz, 2 H), 3.89 (s, 6 H), 3.85 (s, 6 H), 3.81 (s, 3 H),
3.69 (s, 6 H); 13C NMR (CDCl3, 600 MHz) δ 170.6, 153.3, 150.5,
148.7, 137.6, 134.6, 134.5, 134.1, 128.7, 123.7, 121.4, 112.7,
111.0, 105.5, 60.9, 57.5, 56.3, 56.0, 55.9; HRMS calcd for
(C32H33NO9 þ H)þ 576.2233, found 576.2239.
1
pyrrole-2,5-dione (5). Yield 68%; mp 122-124 °C; H NMR
(CDCl3, 600 MHz) δ 7.18 (dd, J = 8.2, 1.6 Hz, 2 H), 7.03-7.01
(m, 4 H), 6.84 (d, J = 8.2 Hz, 2 H), 6.81 (d, J = 8.2 Hz, 1 H), 4.71
(s, 2 H), 3.88 (s, 6 H), 3.87 (s, 3 H), 3.84 (s, 3 H), 3.69 (s, 6 H) ; 13
C
NMR (CDCl3, 400 MHz) δ 171.7, 151.2, 149.7, 149.5, 135.0,
130.0, 124.4, 122.2, 113.4, 113.0, 111.8, 56.7, 56.6, 42.5; HRMS
calcd for (C29H29NO8 þ H)þ 520.1971, found 520.1965.
1-(3,4,5-Trimethoxybenzyl)-3,4-bis(3,4-dimethoxyphenyl)-
1H-pyrrole-2,5-dione (6). Yield 67%; mp 154-155 °C; 1H NMR
(CDCl3, 600 MHz) δ 7.20 (dd, J = 8.2, 2.2 Hz, 2 H), 7.03 (d, J =
2.2 Hz, 2 H), 6.86 (d, J = 8.2 Hz, 2 H), 6.71 (s, 2 H), 4.71 (s, 2 H),
3.89 (s, 6 H), 3.86 (s, 6 H), 3.82 (s, 3 H), 3.69 (s, 6 H); 13C NMR
(CDCl3, 400 MHz) δ 171.6, 154.1, 151.2, 149.5, 138.4, 135.0, 133.0,
124.4, 122.2, 113.4, 111.8, 107.0, 61.6, 57.0, 56.7, 56.6, 43.0; HRMS
calcd for (C30H31NO9 þ H)þ 550.2077, found 550.2072.
General Procedure for Compounds 33 and 34. 2,4-Bis-
(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane 2,4-disulfide
(Lawesson reagent) (1.05 mmol) was added to a stirring solution
of 7 or 11 (1 mmol) in dehydrated toluene (30 mL) at room
temperature under an N2 atmosphere. The mixture was refluxed
for 8 h until TLC showed that the reaction has completed. The
reaction mixture was concentrated and the residue was purified
by flash chromatography on silica gel (AcOEt/n-hexane1 = 1/3,
v/v) to afford the desired compounds 33 and 34. The physical
and spectral data for 33 and 34 are listed below.
3,4-Bis(3,4-dimethoxyphenyl)-1-phenethyl-1H-pyrrole-2,5-
1
dione (8). Yield 67%; mp 128-131 °C; H NMR (CDCl3, 600
MHz) δ 7.31-7.27 (m, 5 H), 7.17 (dd, J = 1.8, 8.2 Hz, 2 H), 7.03
(d, J = 1.8 Hz, 2 H), 6.85 (d, J = 8.2 Hz, 2 H), 3.9 (s, 6 H), 3.85 (t,
1-Benzyl-3,4-bis(3,4-dimethoxyphenyl)-5-thioxo-1H-pyrrol-
2(5H)-one (33). Yield 68%; mp 177-178 °C; 1H NMR (CDCl3,
600 MHz) δ 7.46 (d, J=7.3 Hz, 2 H), 7.32 (m, 2 H), 7.29-7.24
(m, 2 H), 7.04 (s, 1 H), 6.90 (d, J=8.2 Hz, 1 H), 6.87 (d, J = 8.2
Hz, 1 H), 6.83 (s, 1 H), 6.80 (d, J=8.2 Hz, 1 H), 5.19 (s, 2 H), 3.90
(s, 3 H), 3.88 (s, 3 H), 3.74 (s, 3 H), 3.63 (s, 3 H); 13C NMR
(CDCl3, 600 MHz) δ 199.9, 173.1, 150.4, 149.8, 148.6, 139.0,
136.5, 129.8, 128.9, 128.6, 127.8, 124.2, 123.9, 123.6, 121.5,
J = 7.8 Hz, 2 H), 3.72 (s, 6 H), 2.97 (t, J = 7.8 Hz, 2 H); 13
C
NMR (CDCl3, 600 MHz) δ 171.1, 150.4, 148.8, 138.3, 134.2,
128.9, 128.6, 123.6, 121.5, 112.7, 111.1, 56.0, 55.9, 39.7, 34.9;
HRMS calcd for (C28H27NO6 þ H)þ 474.1916, found 474.1922.
1-(4-Methoxyphenethyl)-3,4-bis(3,4-dimethoxyphenyl)-1H-
1
pyrrole-2,5-dione (9). Yield 65%; mp 108-110 °C; H NMR
(CDCl3, 600 MHz) δ 7.19-7.17 (m, 4 H), 7.03 (d, J = 1.8 Hz,

5118 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Zhang et al.
of 6 and 25 divided by IC50 of paclitaxel with 6 alone; FIC25
113.8, 112.8, 110.9, 110.8, 56.0, 55.9, 55.7, 44.8; HRMS calcd for
(C27H25NO5S þ H)þ 476.1531, found 476.1541.
=
IC50 of paclitaxel with the combination of 6 and 25 divided by
IC50 of paclitaxel with 25 alone). FICI values were used to
classify the overall nature of drug interaction. A FICI greater
than 0.5 to 4.0 represents additivity; less than or equal to 0.5
represents synergy or potentiation, and greater than 4.0 means
antagonism.53 The modulator interaction on reversing drug
resistance is further confirmed by a Fa-FICI plot54 which is a
plot with FICI against Fa value.53
1-(3,4,5-Trimethoxyphenethyl)-3,4-bis(3,4-dimethoxyphenyl)-
5-thioxo-1H-pyrrol-2(5H)-one (34). Yield 60%; mp 60-62 °C;
1H NMR (CDCl3, 600 MHz) δ 7.19 (d, J = 8.7 Hz, 1 H), 7.01 (s,
1 H), 6.89 (s, 2 H), 6.81-6.80 (m, 2 H), 6.48 (s, 2 H), 4.21 (t, J =
7.8 Hz, 2 H), 3.91 (s, 3 H), 3.88 (s, 3 H), 3.85 (s, 6 H), 3.82 (s, 3 H),
3.75 (s, 3 H), 3.64 (s, 3 H), 2.95 (t, J = 7.8 Hz, 2 H); 13C NMR
(CDCl3, 600 MHz) δ 200.1, 172.9, 153.3, 150.4, 149.8, 148.7,
148.6, 138.9, 136.6, 133.9, 129.8, 124.1, 123.8, 123.5, 121.5,
113.8, 112.7, 111.0, 110.9, 105.9, 60.9, 56.2, 55.9, 55.7, 42.7,
34.6; HRMS calcd for (C31H33NO8S þ H)þ 580.2005, found
580.2013.
Intracellular DOX Accumulation. The 1 ꢀ 106 cells of LCC6
or LCC6MDR cells were added to an Eppendorf tube contain-
ing 20 μM DOX and different concentrations of ningalin B
analogues at 37 °C for 150 min. Then 0.4% DMSO in place of
ningalin B analogues was used as a negative control. After
incubation, the cells were spinned down and washed with cold
PBS, pH7.4, and lysed with lysis buffer (0.75 M HCl, 0.2%
Triton-X100 in isopropanol). The lysate was spinned down and
the supernatant was saved. The fluorescence level of DOX was
determined by fluorescence spectrophotometer (BMG Tech-
nologies) using an excitation and an emission wavelength of
460 and 610 nm.52
Kinetic Characterization of P-gp Inhibition by Modulator.
Kinetic parameters for P-gp inhibition by modulators were
determined by incubating 1 ꢀ 106 LCC6MDR cells with various
concentrations of DOX (1.5, 4.4, 13.3, and 40 μM) in the
presence of four concentrations (0, 0.5, 1, and 2 μM) of
compounds 6 and 25 or the combination of 6 and 25 for 150
min at 37 °C. The cell lysis and fluorescence intensity were
carried out as described previously. The relationship between
DOX and ningalin analogue for P-gp inhibition was analyzed by
Dixon plot. In the Dixon plot, the Ki was estimated from the
linear regression of reciprocal plot of DOX retention rate versus
modulator concentration. The slope of Dixon plot was plotted
against the reciprocal of DOX concentration to define the
relationship between the modulator and DOX.
Materials for Biological Studies. DMSO, verapamil, cyclos-
porine A, doxorubicin (DOX), paclitaxel, and vincristine were
purchased from Sigma-Aldrich. PSC833 is a generous gift of
Novartis pharmaceutical company. Dulbecco’s modified Eagle’s
medium (DMEM), trypsin-ethylenediaminetetracetic acid (ED-
TA), and 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-sulfo-
phenyl)-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). L929, a mouse connective tissue fibroblast,
was purchased from ATCC.
Cell Culture. MDA435/LCC6, MDA435/LCC6MDR, and
L929 cells were maintained in DMEM media supplemented with
10% (v/v) heat-inactivated FBS and 100 units/mL penicillin and
100 μg/mL streptomycin. Cells were cultured at 37 °C in a
humidified atmosphere with 5% CO2. When the cells reached
confluence, they were detached by 0.05% trypsin-EDTA.18
P-gp Modulating Activity. Six-thousand cells of LCC6 or
LCC6MDR and anticancer drug paclitaxel, DOX, or vincristine
were mixed with or without modulators to a final volume of
200 μL in each well of 96-well plates. The plates were then
incubated for 5 days at 37 °C. The CellTiter 96 AQueous assay
(Promega) was used to measure the cell proliferation according
to the manufacturer’s instructions. MTS (2 mg/mL) and PMS
(0.92 mg/mL) were mixed in a ratio of 20:1. An aliquot (10 μL) of
the freshly prepared MTS/PMS mixture was added to each well,
and the plate was incubated for 2 h at 37 °C. Optical absorbance
at 490 nm was then recorded with a microplate absorbance
reader (Bio-Rad). IC50 values were calculated from the dose-
response curves of MTS assays (Prism 4.0). All experiments were
performed in triplicate and repeated at least thrice, and the
results were represented as mean ( standard error of mean.52
Cytotoxicity Assay. Ten-thousand cells of LCC6 or LCC6-
MDR or L929 were mixed with a series of concentrations of
ningalin B analogues to a final volume of 100 μL in each well of
96-well plates. The plates were then incubated for 3 days at
37 °C. Then 50% inhibitory concentration (IC50) of ningalin B
analogues was determined using MTS proliferation assay as
described previously.
Acknowledgment. This research was supported by Key Inter-
national S&T Cooperation Projects (Grant 2008DFA31040)
of Ministry of Science and Technology of the People’s Re-
public of China. I.L.K.W. is supported by a Postdoctoral
Fellowship of the Hong Kong Polytechnic University. This
project is also partly funded by the General Research Fund
(Grant B-Q16G) of the Research Grant Council of Hong
Kong. We thank Professor Tak-Hang Chan for his good
advice on this project.
Supporting Information Available: 1H NMR and 13C NMR
spectra for new compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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