1-(3,4,5-Trimethoxyphenyl)ethane-1,2-diyl esters, a novel compound class with potent chemoreversal activity

Article abstract of DOI:10.1016/j.bmcl.2012.09.096

1-(3,4,5-Trimethoxyphenyl)ethane-1,2-diyl esters, which share a fragment from (±)-3′-O-4′-O-bis(3,4-dimethoxycinnamoyl)-cis- khellactone (DMDCK) and 3′R,4′R-disubstituted-2′,2′- dimethyldihydropyrano[2,3-f]chromone (DSP), exhibited remarkable chemoreversa

Full text of DOI:10.1016/j.bmcl.2012.09.096

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7726–7729
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
1-(3,4,5-Trimethoxyphenyl)ethane-1,2-diyl esters, a novel compound class
with potent chemoreversal activity
Hsin-Yi Hung a,c, Emika Ohkoshi a, Masuo Goto a, Kyoko Nakagawa-Goto a,b, Kuo-Hsiung Lee a,c,
⇑
a Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7568, USA
b Division of Pharmaceutical Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan
c Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung, Taiwan 401, Republic of China
a r t i c l e i n f o
a b s t r a c t
1-(3,4,5-Trimethoxyphenyl)ethane-1,2-diyl esters, which share a fragment from ( )-30-O-40-O-bis(3,4-
dimethoxycinnamoyl)-cis-khellactone (DMDCK) and 30R,40R-disubstituted-20,20-dimethyldihydropyr-
ano[2,3-f]chromone (DSP), exhibited remarkable chemoreversal activity on multidrug resistant human
nasopharyngeal carcinoma (KB) when combined with three anticancer drugs, paclitaxel, vincristine
and doxorubicin. Among 15 novel synthesized analogs, bis-trimethoxybenzoyl derivative 15 was the
most active (340-fold more active than verapamil when used with vincristine) followed by two
di-cinnamoyl derivatives, 10 and 11, and then di-cyclohexanecarbonyl derivative 9. All aliphatic chain
derivatives, 3–5, showed no activity. Structure-activity relationship study indicated that a di-ester
structure was critical to enhance the activity resulting from the maintenance of the spatial arrangement
proposed by the pharmacophore based on the verapamil-binding site. Further mechanism of action study
showed 15 inhibited mainly P-glycoprotein efflux pump function, while 13 exhibited an additional
multidrug resistance-associated protein efflux pump function.
Article history:
Received 16 July 2012
Revised 21 September 2012
Accepted 25 September 2012
Available online 13 October 2012
Keywords:
1-(3,4,5-Trimethoxyphenyl)ethane-1,2-diyl
esters
Chemoreversal activity
KB cell line
Ó 2012 Elsevier Ltd. All rights reserved.
Chemotherapy is an important strategy for cancer treatment.
However, it is difficult to eliminate all tumor cells, resulting in re-
lapse with resistant cancer cells. Emerging evidence suggests that
epithelial-mesenchymal transition (EMT)-type cells and cancer
stem cells (CSCs) or initiating cells are more resistant to conven-
tional chemotherapy and are linked to anticancer drug resistance
leading to failure of chemotherapy.1 Another issue of chemothera-
peutic resistance is the tumor microenvironment, in which cancer
cells are surrounded by various sets of non-transformed cells and a
heterogenous stromal compartment.2 The interaction between tu-
mor cells and normal cells in this tumor microenvironment occurs
via secreted and surface-bound proteins, and is critical for tumor
progression.2
In addition to the tumor microenvironment, cancer cells also
adapt other mechanisms to deal with chemotherapeutic agents.3
Over-expression of drug efflux transporters such as P-glycoprotein
(P-gp) and multidrug resistance-associated proteins (MRPs) is the
primary cause leading to multidrug resistance. Numerous studies
have explored chemosensitizer development. However, clinical
drugs are still unavailable.
positive control in various chemosensitizing research efforts.
Before the structure of P-glycoprotein (P-gp) was resolved, a phar-
macophore model based on the verapamil-binding site was pro-
posed.4 In this model, the pharmacophore for P-gp substrates or
inhibitors consists of two hydrophobic planes, three optional
hydrogen bond (HB) acceptor points, and one optional HB donor
point spatially arranged like a butterfly shape.4 Based on this
model,
( )-30-O-40-O-bis-(3,4-dimethoxycinnamoyl)-cis-khellac-
tone (DMDCK) was found to be a hit and was also confirmed to
exhibit chemosensitizing ability.5 Moreover, 30R,40R-disubsti-
tuted-20,20-dimethyldihydropyrano[2,3-f]chromone (DSP) analogs
sharing similar structures to DMDCK have also been reported to
overcome multidrug resistance (MDR) in an acquired MDR human
nasopharyngeal carcinoma (KB) cell line.6 With inspiration from
the above results,
a simplified scaffold, 1-(3,4,5-trimethoxy-
phenyl)ethane-1,2-diyl ester, was designed (Fig. 1) to provide an
ethane-1,2-dilyl ester partial structure mimicking a fragment in
both DSP and DMDCK. By esterifying different substituents, the
importance of the spatial arrangement mentioned in the pharma-
cophore model can be evaluated. According to results based on an-
other model, P-gp inhibitors should show high hydrophobicity,
with two or more aromatic rings, a methoxy group (HB acceptor)
on the aromatic ring, and one or two protonatable nitrogens.7
Verapamil also contains two methoxy group on an aromatic ring.
In addition, previous study of DDB substitutions also showed
Verapamil (VRP), the first generation chemosensitizer, was pre-
cluded from clinical use due to its high toxicity, but it is used as a
⇑
Corresponding author. Tel.: +1 919 962 0066; fax: +1 919 966 3893.
E-mail address: khlee@unc.edu (K.-H. Lee).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.096

H.-Y. Hung et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7726–7729
7727
O
OCH3
OCH3
H3CO
X
O
O
O
O
O
O
OR
O
O
OR
O
OR
OR'
DSP
OCH3
OCH3
Proposed structure
NC
H3CO
H3CO
N
H3CO
OCH3
OCH3
OCH3
(+/-)DMDCK
Verapamil
Figure 1. Structures of ( ) DMDCK, DSP, and verapamil and proposed structure for this study
increasing activity along with an increased number of methoxy
substituents.8 Therefore, a trimethoxy-substituted aromatic ring
was designed as the basic scaffold in this study.
but not on KB cells in the presence of paclitaxel. This result indi-
cates that these compounds are specifically toxic for MDR KB cells
when combined with the current anticancer drug. Brief SAR corre-
lations were observed as follows. Among the alkyl esters, acetyl (3),
butyryl (4) and dodecanoyl (5) groups showed no chemoreversal
activity. In the case of short fatty acid esters (6 and 7), moderate
reversal effects were observed, indicating a conjugated system
may contribute to chemoreversal activity. Interestingly, compound
9 with bulky di-cyclohexanecarbonyl esters displayed remarkable
reversal activity, while mono-cyclohexanecarbonyl ester (8) totally
lost activity. This fact suggested that di-esters would be better than
mono-esters, probably due to higher hydrophobicity, a critical fea-
ture for drug efflux pump inhibitors or the importance of the but-
terfly shape proposed in the pharmacophore model.4 The ester
derivatives containing an aromatic ring showed relatively better
reversal ability than the aliphatic ester derivatives. Especially, cin-
namoyl ester 10 and 2-iodo-3,4-dimethoxycinnamoyl ester 11,
with the same side chain as DMDCK, totally reversed the cytotox-
icity of paclitaxel in the KBvin cell line. Similarly, 2-iodo-
mono-3,4,5-trimethoxybenzoyl (14) as well as 2-iodo-di-3,4,
5-trimethoxybenzoyl (15) esters displayed high chemoreversal ef-
fects. These four compounds resulted in 0% of KBvin cell survival at
1-(3,4,5-Trimethoxyphenyl)ethane-1,2-diol (1),
a
common
intermediate for all synthesized compounds, was obtained through
Wittig reaction of 3,4,5-trimethoxybenzaldehyde following
dihydroxylation with osmium tetroxide and N-methylmorpholine
N-oxide (Scheme 1). Various acyl groups, including linear alkyl
(3–5), unsaturated fatty acid (8–9), cyclic alkyl (6–7), and aromatic
(10–15) side chains, were selected for the structure-activity rela-
tionship (SAR) study. Both mono- and di-esters were also synthe-
sized by controlling the equivalents of the acyl chloride to
investigate the importance of the spatial arrangement and hydro-
phobicity. Diesterification of diol was achieved with excess acyl
chloride under basic conditions to generate the related diesters,
3, 5–7, 9, and 10. The bulky acyl chlorides, such as butyryl chloride
and cyclohexanecarbonyl chloride, were selectively introduced on
only the primary alcohol to produce mono-ester derivatives, 4 and
8. Monobenzoylester 13 was prepared through the esterification of
the primary alcohol using 1.1 equiv of benzoyl chloride, following
silylation of the secondary alcohol. Halogenated compounds are
abundant among drug candidates. Therefore, iodinated analogs,
2, 11, 12, 14, and 15, were also designed and synthesized through
iodination of 3,4,5-trimethoxybenzaldehyde following the same
reaction sequence as mentioned above (Scheme 1). Newly synthe-
sized derivatives, 1–15 (Table 1), were screened for cytotoxicity
and chemoreversal effects on KB and MDR KB cells in the absence
(ꢀ) or presence (+) of paclitaxel.
10 lM. The comparison of 10 and 11, as well as 12 and 13, sug-
gested that iodination of the C-2 position is not very important
for the activity. In the case of benzoyl ester, similar effects were
shown regardless of mono-ester (14) or di-ester (15). In a compar-
ison of 12 and 14, the silyl group led to reduced chemoreversal
when an iodo group was present at the C-2 position and also
increased cytotoxicity in the presence of paclitaxel. Among the
potent derivatives 9–15, compound 12 displayed cytotoxicity.
Therefore, only 9–11 and 13–15, mainly cinnamoyl and trim-
ethoxybenzoyl derivatives, were selected for further evaluation
with three anticancer drugs, paclitaxel, vincristine and doxorubicin
(Table 3).
Most of the analogs exhibited no significant cytotoxicity at
10 lM against four tumor cell lines, A549, DU145, KB and KBvin
(Supplementary data, Table S1). Reversal activity of 1-(3,4,5-trime-
thoxyphenyl)ethane-1,2-diyl esters using paclitaxel is shown in
Table 2. In the absence of paclitaxel, most compounds had no cyto-
toxic effect, while chemoreversal effects were seen on KBvin cells
OH
OR
O
H3CO
H3CO
OH
H3CO
H3CO
OR'
H3CO
H3CO
H3CO
H3CO
H
d
b
c
X
OCH3
X
X
a
X
OCH3
OCH3
X = H
X = I
OCH3
3, 5-7, 9, 10: X = H, R = R' = Acyl
4, 8: X = H, R = Acyl, R' = H
11, 15: X = I, R = R' = Acyl
1: X = H
2: X = I
X= H or I
12: X = I, R = TBS, R' = 3,4,5-triOMe-Bz
13: X = H, R = TBS, R' = 3,4,5-triOMe-Bz
14: X = I, R = H, R' = 3,4,5-triOMe-Bz
e
Reagents and conditions: (a) trifluoroacetate, iodine; (b) Ph3PCH3Br, tBuOK, toluene; (c) OsO4, NMO, THF, 55°C;
(d) RCOCl, Et3N, CH2Cl2; (e) 2,6-lutidine, TBSOTf, CH2Cl2, -78 °C
Scheme 1. Synthetic route to 1-(3,4,5-trimethoxyphenyl)ethane-1,2-diyl esters

7728
H.-Y. Hung et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7726–7729
Table 1
Table 2
Structures of 1-(3,4,5-trimethoxyphenyl)ethane-1,2-diyl esters
Screening of reversal activity of 1-(3,4,5-trimethoxyphenyl)ethane-1,2-diyl esters
using paclitaxel
OR
Compd Conc. (
l
M) Cell viability (%) SDa
KB
H3CO
H3CO
OR'
KBvin
X
(ꢀ)
(+)
(ꢀ)
(+)
99.0 10.5
OCH3
1
2
3
4
5
6
7
8
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
101.4 4.2 96.8 4.4
95.1 0.8 97.0 0.9
98.0 3.6 96.0 2.0
99.3 4.3 95.1 3.2
92.9 3.8 85.5 6.7
91.1 0.9 83.9 6.3
91.2 3.9 76.8 9.1
92.4 17.8
90.0 22.0
91.5 19.4
90.0 14.0
85.7 8.5
79.9 10.2
81.5 9.9
91.8 11.6
92.0 9.1
90.4 10.1
81.4 6.6
44.0 15.1
31.3 11.8
102.8 8.6
6.2 14.4
No
x
R
R0
1
2
H
I
H
H
H
H
O
O
O
3
4
5
H
H
H
98.9 7.8 96.7 5.0 109.9 8.9
9
91.1 7.4 88.1 5.4
82.2 2.2 73.8 4.9
85.9 2.7 76.5 4.0
64.2 5.5 58.0 9.3
83.9 1.0 70.3 6.8
77.0 4.1 75.7 3.2
84.2 4.8 73.7 8.7
89.3 6.5
74.3 7.0
95.3 2.0
41.0 5.5
74.3 3.2
83.3 12.9
87.1 6.9
H
10
11
12
13
14
15
0
0
5.7
4.4
9
19.6 5.1
12.3 11.9
O
O
O
O
0
0
6.0
6.9
6
7
H
H
9
a
Cell viability was measured by SRB method in the absence (ꢀ) or presence (+) of
non-toxic concentration of paclitaxel. (1 nM for KB cell and 100 nM for KBvin cell)
O
O
O
Table 3
8
9
H
H
H
Reversal activity of selected 1-(3,4,5-trimethoxyphenyl)ethane-1,2-diyl esters
Compd Conc. Paclitaxel (nM)
Vincristine (nM)
Foldb IC50 SD
Fold IC50 SD
215 106.5 13.8
Doxorubicin (nM)
(lM)
IC50 SD
Fold
O
9
10
10
10
10
10
10
10
43.1 7.33
30
9.49 1.22
3.89 0.83
3.29 0.28
14
37
23
79
51
O
O
10
11
13
14
15
VRPa
11.0 5.65 116
7.66 1.54 166
5.91 0.69 216
7.79 0.55 164
9.04 4.55 141
524
620
39.0 1.58
61.9 11.1
18.4 7.99
28.4 9.42
9.29 2.71 156
6
OMe
OMe
OMe
OMe
1.21 0.03 1684
2.56 0.42 798
0.89 0.25 2285
21.0 4.63
10
11
H
I
O
O
28.47 8.64
72
97 249.4 78.5
a
VRP: verapamil.
O
b
The reversal fold values were calculated as: reversal fold=IC50 (anticancer drug
OMe
OMe
alone)/IC50 (anticancer drug + test compound). The IC50 values of paclitaxel, vin-
cristine, and doxorubicin against KBvin cells were determined in the presence of
10 lM compound, and expressed with mean SD of three independent
12
13
14
15
I
TBS
TBS
H
O
experiments.
OMe
OMe
OMe
H
I
O
O
O
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
I
O
Figure 2. Inhibitory effects of compounds on P-gp function in KBvin Cells. The
KBvin cells were pretreated with the compounds (10 lM) followed by addition of
Among the six compounds, trimethoxybenzoyl analogs, 13–15,
generally had better chemoreversal ability than cinnamoyl deriva-
tives (10 and 11) when combined with the three anticancer drugs.
The reversal activity was similar between 10 and 11. Among com-
pounds 13–15 with mono- or di-3,4,5-trimethoxylbenzoyl modifi-
cation, di-ester 15 was the most active showing 340-fold more
potent than verapamil when used with vincristine. Compound 13
with a bulky and hydrophobic tert-butyldimethylsilyl group also
showed significant activity. Cyclohexanoyl ester 9 displayed less
activity than the other five compounds. These results indicated
calcein-AM. The cellular accumulation of calcein is represented by fluorescent units
(FLU). Cellular accumulation of calcein demonstrates inhibition of efflux activity of
P-gp. Data with mean SD of three independent experiments are shown in
Supplementary Table S2.
that hydrophobicity, higher electron density on aromatic group,
as well as the maintenance of the spatial arrangement are critical
for better chemoreversal activity.
All of the active compounds (9–11and 13–15) were further
evaluated for effects on P-gp efflux function in KBvin cells using

H.-Y. Hung et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7726–7729
7729
Figure 3. Effect of 13 on MRP function in KBvin Cells. KB (a) or KBvin (b, c, d) cells were pre-treated with DMSO (a, b), 13 (c), or 15 (d) for 1 h before addition of Fluo-3 AM for
2 h. Cellular accumulation of Fluo-3, a fluorescence MRPs substrate, was observed in KB cells (a), and effluxed from KBvin cells. (b). Cellular retention of Fluo-3 fluorescence
was observed in KBvin cells treated with 13. (c) yet undetectable in cells treated with 15. (d). (Bar, 50 lm.)
fluorescent P-gp substrate calcein-AM (Fig. 2).8 Cellular accumula-
tion of calcein was observed in the presence of 10 M compounds,
accumulation of anticancer drugs, such as paclitaxel, vincristine,
and doxorubicin. Moreover, compound 13 showed additional
inhibitory activity against MRPs. Further modifications and more
detailed SAR will be reported in the near future.
l
demonstrating that the compounds prevent calcein-AM efflux
from KBvin cells by inhibiting P-gp. Compound 15 exhibited two-
fold more calcein accumulation than verapamil at the same con-
centration, indicating 15 is more potent than verapamil at inhibit-
ing P-gp function. Although 13 showed strong reversal activity
compared with verapamil, its inhibitory effect on the P-gp efflux
pump was relatively weaker than verapamil. Hence, 13 may inhibit
other drug transporters, such as multidrug resistance-associated
proteins (MRPs). The inhibitory effect of 13 on MRPs can be deter-
mined by using a fluorogenic MRP substrate Fluo-3 AM, which is
accumulated in KB cells and pumped out from KBvin cells
(Fig. 3). Although no significant Fluo-3 fluororescence was seen
in KBvin cells pre-treated with 15, cellular accumulation of Fluo-
3 was observed in KBvin cells pre-treated with 13. These results
demonstrate that 13 inhibits efflux pump function of MRPs,
whereas 15 does not affect MRPs. Taken together, 15 selectively
inhibits P-gp and 13 can interfere with both P-gp and MRPs.
In this study, we demonstrated that a simplified scaffold con-
taining the core of DMDCK and DSP exhibited excellent chemore-
versal activity. Fifteen ester analogs were designed, synthesized,
and evaluated for chemoreversal activity. Based on the results,
di-ester modification is critical for better activity, indicating the
importance of maintaining the spatial arrangement of the
compound. SAR study indicates that di-trimethoxylbenzoyl substi-
tution resulted in the most activity, followed by di-3,4-dimeth-
oxylcinnamoyl and then di-cycloheanecarbonyl substitutions.
Aliphatic groups, such as acetyl or butyryl, led to no activity.
Mechanism of action study showed that these compounds inhib-
ited the drug efflux function of P-gp, which increases the cellular
Acknowledgement
This study was supported by grant CA-17625-32 from the Na-
tional Cancer Institute, NIH, awarded to K. H. Lee. Support in part
was also due to the Cancer Research Center of Excellence (CRC)
(DOH-100-TD-C-111-005). We would like to thank Dr. Mitch Eddy
(NIEHS/NIH) for providing a fluorescence microscope, and also Dr.
Susan Morris-Natschke for her valuable suggestions and help in
preparation of this manuscript.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
09.096.
References and notes
1. Singh, A.; Settleman, J. Oncogene 2010, 29, 4741.
2. Gilbert, L. A.; Hemann, M. T. Cancer Res. 2011, 71, 5062.
3. Fodale, V.; Pierobon, M.; Liotta, L.; Petricoin, E. Cancer J. 2011, 17, 89.
4. Pajeva, I. K.; Wiese, M. J. Med. Chem. 2002, 45, 5671.
5. Fong, W. F.; Shen, X. L.; Globisch, C.; Wiese, M.; Chen, G. Y.; Zhu, G. Y.; Yu, Z. L.;
Tse, A. K.; Hu, Y. J. Bioorg. Med. Chem. Lett. 2008, 16, 3694.
6. Zhou, T.; Shi, Q.; Bastow, K. F.; Lee, K. H. J. Med. Chem. 2010, 53, 8700.
7. Pajeva, I. K.; Globisch, C.; Wiese, M. ChemMedChem 1883, 2009, 4.
8. Hung, H. Y.; Ohkoshi, E.; Goto, M.; Bastow, K. F.; Nakagawa-Goto, K.; Lee, K. H. J.
Med. Chem. 2012, 14, 5413.