Synthesis and multidrug resistance reversal activity of dihydroptychantol A and its novel derivatives

Article abstract of DOI:10.1016/j.bmc.2009.05.077

The macrocyclic bisbibenzyl dihydroptychantol A (DHA), previously isolated from Asterella angusta, was synthesized and showed significant multidrug resistance (MDR) reverting activity in chemoresistant cancer cells. In an attempt to discover more potent M

Full text of DOI:10.1016/j.bmc.2009.05.077

Bioorganic & Medicinal Chemistry 17 (2009) 4981–4989
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and multidrug resistance reversal activity of dihydroptychantol
A and its novel derivatives
a,
Bin Sun a, Hui-qing Yuan b, Guang-min Xi b, Yu-dao Ma c, , Hong-xiang Lou
*
*
a School of Pharmaceutical Sciences, Shandong University, Jinan 200012, PR China
b School of Medicine, Shandong University, Jinan 250012, PR China
c School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The macrocyclic bisbibenzyl dihydroptychantol A (DHA), previously isolated from Asterella angusta, was
synthesized and showed significant multidrug resistance (MDR) reverting activity in chemoresistant can-
cer cells. In an attempt to discover more potent MDR reversal agents for efficient cancer chemotherapy,
DHA derivatives with thiazole rings (19–22) were synthesized, and their cytotoxicities and MDR reversal
activities were evaluated in adriamycin-resistant K562/A02, vincristine-resistant KB/VCR and in their
parental cells by MTT assays. In response to treatment with each compound, the K562 cell line was
the most sensitive, and the vincristine-resistant KB/VCR cell line was the most resistant. Marked
decreases in K562 and K562/A02 cell viability were detectable after treatment with the synthesized
derivatives of DHA, while less inhibitory effects on cell growth were observed in chemical-resistant
KB/VCR and KB cells. Moreover, among the tested compounds, the intermediate 17 and the analogues
19, 20, and 21 showed potent MDR reversal activities and increased vincristine cytotoxicity in KB/VCR
Received 2 May 2009
Revised 28 May 2009
Accepted 29 May 2009
Available online 6 June 2009
Keywords:
Dihydroprychantol A
Derivatives
Multidrug resistance
P-Glycoprotein
cells, with the reversal fold ranges from 10.54 to 13.81 (10
the natural product DHA.
lM), which is 3.2–4.3-fold stronger than
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
and the natural products are gaining much more attention for their
anti-tumor activities.
Multidrug resistance (MDR), defined as the ability of tumor cells
to show resistance to a wide variety of structurally and function-
ally unrelated compounds, is still one of the most serious threats
to successful chemotherapy for a majority of cancer patients.1,2
In mammals, tumors, which are initially sensitive to cytotoxic
agents, often develop resistance to a broad spectrum of structurally
different drugs.3 The most significant mechanism of MDR is the
overexpression of the ATP binding cassette (ABC) transporter
proteins which work as energy-dependent pumps for chemothera-
peutic agents and prevent drugs from reaching effective concentra-
tions within the cells.4 To date, the proteins of ABC family such as
the P-glycoprotein (P-gp), multidrug resistance associated protein
(MRP1), the lung resistance protein (LRP), and the breast cancer
resistance protein (BCRP) have been discovered. The most and
best-studied one is P-gp, which is characterized by a broad
substrate spectrum.5–8 Although a large number of compounds
with a wide spectrum of chemical structures have been found to
inhibit P-gp and reverse MDR, there are currently no reversal
agents available clinically. Thus, the discovery and development
of novel and potent P-gp inhibitory agents are still much desired,
Macrocyclic bisbibenzyls are a series of phenolic natural prod-
ucts that are found exclusively in bryophytes. These natural
products have a variety of biological activities, including 5-lipoxy-
genase, cyclooxygenase and calmodulin inhibitory effects, and
antifungal, anti-HIV, antimicrobial, antioxidative, muscle-relaxing,
LXRa
-activating, FXR-activating, and cytotoxic activities.9–18
A
recent research indicated that they also have
a
significant
antiproliferative activity due to the inhibition of microtubule
polymerization.19,20
In our previous reports, we found that the macrocyclic bisbib-
enzyl plagiochin E, which was isolated from Marchantia polymor-
pha, exhibits excellent MDR reversal activity.21 Recently, our
group also found that the macrocyclic bisbibenzyl dihydroptychan-
tol A (DHA) (Fig. 1), isolated from liverwort Asterella angusta as an
* Corresponding authors. Tel.: +86 531 88364778; fax: +86 531 88382019.
E-mail addresses: ydma@sdu.edu.cn (Y. Ma), louhongxiang@sdu.edu.cn (H. Lou).
Figure 1. Chemical structures of DHA and dendroamide A.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.077

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B. Sun et al. / Bioorg. Med. Chem. 17 (2009) 4981–4989
antifungal natural product,22 exhibits more potent MDR reversal
activity by increasing the adriamycin cytotoxicity toward K562/
A02 cells and vincristine cytotoxicity toward KB/VCR cells. DHA’s
mechanism of MDR reversal activity is through inhibition of P-gp
function and its expression, which prevents efflux of drugs.23
The encouraging biological results motivated us to synthesize
additional DHA derivatives in order to improve activity and to dis-
cover more potent MDR reversal agents. It has been reported that
compounds with the thiazole ring, such as the cyclic hexapeptide
dendroamide A (Fig. 1), exhibit MDR reversal activity24–27 and
some heterocyclic derivatives of combretastatin A-4, with the
structure containing bibenzyl moiety, are also cytotoxic to MDR
cell lines.28 In light of these results, we were interested in the effect
of the thiazole group on the macrocyclic bisbibenzyl system.
Accordingly, some DHA derivatives with thiazole moieties were
synthesized and their biological activities were evaluated. In this
study, we report the synthetic details for DHA and its thiazole
derivatives, as well as their MDR reversal activities toward the
multidrug resistant cancer cell lines K562/A02 and KB/VCR. Molec-
ular docking analyses were also used to elucidate the binding
modes of the analogues to P-gp.
and 40 lM with 58.92%, 79.26%, and 94.33% repression, respec-
tively. Whereas, except for derivative 22, no obvious inhibition of
cell growth was oberved in the KB and KB/VCR cells.
According to our previous report, DHA and its synthetic inter-
mediates (13–15) significantly increased the sensitivity toward
adriamycin in K562/A02 cells.23 However, when coadministered
with adriamycin, each derivative compound (17, 19–21) did not
remarkably improve adriamycin’s cytotoxicity in K562/A02 cells
at 10 lM, and their reversal fold ranged from 1.65 to 2.68, which
were no better than that of DHA (5.22). These results imply that
K562/A02 cells are not sensitive to thiazole derivatives. We then
tested the MDR reversal effect of these four compounds on KB/
VCR cells at the same concentration. To our surprise, when coad-
ministered with each derivative compound, there was a great in-
crease in cytotoxicity of vincristine toward KB/VCR cells, and as
shown in Table 2, its ID50 ranged from 0.135 to 0.177 lM, and
the MDR reversal fold of these compounds ranged from 10.58 to
13.81. It is obvious that their MDR reversal effects are much more
potent than that of DHA (3.25).23
Based on the test results, we can find that the MDR reversal po-
tency of DHA has been improved greatly by the introduction of a
thiazole ring to the macrocyclic bisbibenzyl system, and the KB/
VCR cells are more sensitive to DHA derivatives than K562/A02
cells.
2. Results and discussion
The total synthesis of the natural product DHA was achieved in
13 steps as shown in Scheme 1. The synthetic route began with the
Ullmann coupling of the protected 3-hydroxybenzaldehyde (1)29
with commercially available 2-bromo-5-methoxybenzaldehyde,
resulting in the formation of the diphenyl ether 2. The phospho-
nium salt 7, as the second diphenyl ether building block, was
synthesized in a conventional sequence starting with SNAr dis-
placement of methyl 4-bromobenzoate by 3-hydroxy-4-methoxy-
benzaldehyde (3) to give the diphenyl ether 4. Compound 4 was
then reduced with sodium borohydride to give the benzyl alcohol
5, followed by bromination and reaction with triphenylphosphine,
affording 7 in four steps. The building blocks 2 and 7 were com-
bined by the Wittig reaction in the presence of potassium carbon-
ate and 18-crown-6,30 and the stilbene 8 (obtained as E/Z mixture)
was hydrogenated over Pd/C to give the bibenzyl 9. Next, the
carboxylic ester group of 9 was reduced with lithium aluminum
hydride, followed by deprotection with HCl/H2O to yield com-
pound 10. Again the bromination of 10 by carbon tetrabromide
and subsequent reaction with triphenylphosphine afforded com-
pound 12. Cyclization of 12 by means of an intramolecular Wittig
reaction was achieved with sodium methoxide, leading to key
macrocyclic intermediate 13.31 Finally, 13 could be demethylated
by boron tribromide at ꢀ78 °C to give phenol 14 or hydrogenated
to give dimethyl DHA (15), followed by demethylation to afford
the natural product DHA (16).
This excellent bioactivity encouraged us to investigate the pos-
sible mechanism of action at the molecular level, specifically, the
binding mode of bisbibenzyls to P-glycoprotein. Recently, Chang
et al.35 solved the crystal structure of murine P-gp (which has
87% sequence identity to the human protein) in complex with li-
gands. According to their research, there are three binding sites in
the protein. In order to figure out the best binding site for DHA
and its analogues, the compounds 15, 17, 19–21 and DHA were
docked into all three potential binding sites of Chang’s structure
using the program GOLD (Genetic Optimization for Ligand Dock-
ing).36–38According to our docking results, we found that the top
10 ranked conformations of each compound are almost the same
and the best ranked conformation of each compound overlays
very well with each other in the binding site of Chang’s ligand
cyclic-tris-(R)-valineselenazole (QZ59-RRR).35 Based on this result,
it is likely that our compounds bind in the same site as QZ59-RRR
and Figure
compound.
3 shows the top-ranked GOLD pose for each
The binding mode of the representative compound 19 has been
studied further by energy minimization. In this minimized docking
mode for compound 19, the amine group of the thiazole forms a
hydrogen bond with the backbone carbonyl group of Ser725. Also,
the thiazole ring may form a p-p interaction with the phenyl ring
of Phe728, (Figs. 4 and 5A). We believe that these interactions are
responsible for enhancing the P-gp inhibitory activity of thiazole
derivatives. It is also possible that the methoxyl group of ring C
forms a hydrogen bond with the amide group of Gln 721, and a
As shown in Scheme 2, the preparation of derivatives began
with compound 13, which reacted with NBS in a 1:1 mixture of
THF and water at 45 °C and selectively afforded compound 17.32
As shown in Figure 2, the substitution pattern of the hydroxyl
group and bromine atom on the fragment CH2(7)–CH2(8) was con-
firmed by analysis of its HMBC. After the hydroxyl group was oxi-
CH-
exist as well. As shown in Figure 5A and B, rings A and C exhibit
potential interactions with phenyl groups of Phe 724 and
Phe 974, respectively, and ring A may also create a CH- interac-
p
interaction39 between this group and Tyr 303 (Fig. 5A) may
p-p
p
dized to
a
ketone under Swern conditions,33 the afforded
tion with methyl group of Val 978. We were able to observe that
the methoxyl group of ring D could fit into the hydrophobic pocket
formed by Leu 64, Met 68, and Ile 336 as well (Figs. 5A and 6).
compound 18 reacted with thiourea and thiosemicarbazide,
respectively, to yield the thiazoles 19 and 21.34 After the demeth-
ylation of 19 and 21 by boron tribromide, compounds 20 and 22,
respectively, were prepared.
To discover a promising initial candidate for MDR reversal
agents, we first assayed the effect of compounds 17, 19, 20, 21,
and 22 on KB, KB/VCR, K562, and K562/A02 cells growth at a con-
centration of 10 lM. As shown in Table 1, treatment with bisbi-
benzyls resulted in cell growth decreases in the K562 and K562/
A02 cells and cell viability was significantly reduced at 20, 30,
These combined p–p interactions, CH–p interaction, and hydrogen
bonds could play a crucial role in P-gp inhibitory activity of the
macrocyclic bisbibenzyl compounds. In addition, as shown in Fig-
ure 3, DHA and compound 20, with the phenolic hydroxyl group
on C ring, may create stronger hydrogen bonds with Gln 721,
and this could contribute to their higher potency than the com-
pounds with methoxyl groups. The loss of the hydroxyl and amine
groups (as in compound 15) attenuates these hydrogen bond inter-

B. Sun et al. / Bioorg. Med. Chem. 17 (2009) 4981–4989
4983
Scheme 1. Synthesis of DHA. Reagents and conditions: (a) 2-bromo-5-methoxybenzaldehyde, K2CO3, CuO, pyridine, reflux; (b) 4-bromobenzoate, K2CO3, pyridine, CuO,
reflux; (c) NaBH4, THF, rt; (d) CBr4, PPh3, CH2Cl2, 0 °C; (e) PPh3, toluene, reflux; (f) K2CO3, 18-crown-6, CH2Cl2, reflux; (g) H2, 10% Pd/C, Et3N, AcOEt, rt; (h) (1) LiAlH4, THF,
30 °C; (2) H+/H2O, rt; (i) NaOMe, CH2Cl2, rt; (j) H2, 10% Pd/C, AcOEt, rt; (k) BBr3, CH2Cl2, ꢀ78 °C.
actions and may be responsible for the lower activity of 15. Overall,
the docking studies revealed that bisbibenzyl and its analogues
interacted with P-gp in the same binding site as QZ59-RRR, and
also supported that the macrocyclic bisbibenzyl skeleton is the
basic structure for the MDR reversal activity, and the increased
inhibitory effect of derivatives 19, 20, and 21 is due to their hydro-
gen bonds and
p–p interactions with active site amino acid resi-
dues of P-gp.

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B. Sun et al. / Bioorg. Med. Chem. 17 (2009) 4981–4989
Figure 3. Overlap of the best-docked conformations of compounds 15 (yellow), 17
(red), 19 (blue), 20 (cyan), 21 (green) and DHA (orange) in the binding site, and the
hydrogen bonds are labeled as yellow broken lines.
Scheme 2. Synthesis of DHA’s derivatives. Reagents and conditions: (l) NBS, THF/
H2O (1:1), 45 °C; (m) DMSO, TFAA, CH2Cl2, ꢀ78 °C; (n) thiourea, DMF, 60 °C; (o)
BBr3, CH2Cl2; ꢀ78 °C; (p) thiosemicarbazide, DMF, 75 °C; (q) thioacetamide, K2CO3,
DMF, 80 °C.
Table 1
Inhibitory effect of DHA’s derivatives on cellsa
Compound
K562
K562/A02
KB
KB/VCR
% of inhibition
% of inhibition
% of inhibition
% of inhibition
17
19
20
21
22
38.55
58.67
11.56
32.07
37.15
11.86
26.81
10.11
18.76
13.69
2.82
0
0
0
0
0
0
0
51.15
19.42
a
After treatment of cancer cells with 10 lM of each compound for 48 h, the
inhibitory rate (%) was calculated by using the MTT assay.
Table 2
Multidrug resistance (MDR) reversal activity of DHA’s derivatives against KB/VCR
cellsa
b
Treatment
ID50
(lM) (KB/VCR)
RF (KB/VCR)
VCR
1.864 0.702
0.177 0.012
0.152 0.003
0.135 0.009
0.176 0.016
VCR + 17 (10
VCR + 19 (10
VCR + 20 (10
VCR + 21 (10
l
l
l
l
M)
M)
M)
M)
10.54
12.26
13.81
10.58
Figure 2. The substitution of hydroxyl group and bromine atom on the fragment
CH2(7)–CH2(8) was confirmed by the following long-range correlations (Fig. 1): H-3
and H-5 with C-7, H-10 and H-14 with C-8, H-30 and H-50 with C-70, H-100 and H-140
with C-80 by use of HMBC.
Bold numbers emphasize the excellent biological activity.
KB/VCR cells were seeded at a density of 6 ꢁ 104/ml in 24-well plates and co-
a
treated with various concentrations of vincristine in the presence of 17, 19, 20, 21,
3. Conclusions
22, or 23 at the concentration of 10 lM. Cell viability was determined using the
MTT assay.
b
As shown in Schemes 1 and 2, and Tables 1 and 2, we have
achieved the total synthesis of DHA, prepared a series of deriva-
tives (19, 20, 21, and 22) with the thiazole ring system, and tested
their MDR reversal activity toward K562/A02 and KB/VCR cells.
Among the tested compounds, 20 showed the most potent MDR
reversal activity toward KB/VCR cells. While compounds 17, 19,
and 21 also exhibited excellent bioactivity. In addition, molecular
docking analyses were also used to elucidate the binding models
of the analogues to P-gp. From the study of a preliminary struc-
ture–activity relationship, it was considered that the macrocyclic
bisbibenzyl skeleton is the basic structure for the MDR reversal
activity, and the phenolic hydroxyl groups, amine group, and thia-
zole ring could play essential roles in enhancing the P-gp inhibitory
activity of bisbibenzyls. In conclusion, the macrocyclic bisbibenzyl
system is a promising scaffold for the development of novel MDR
reversal agents for cancer chemotherapy and merits further inves-
tigation. In order to discover novel MDR reversal agents for
Data are expressed as means standard deviation from three independent
experiments. *P < 0.05.
efficient cancer chemotherapy, we will screen additional natural
products with different macrocyclic bisbibenzyl skeletons and di-
rect our efforts toward rational modifications of the promising
candidates.
4. Experimental
4.1. General procedures
Column chromatography was carried out on silica gel or alu-
mina (200–300 mesh). Reactions were monitored by thin-layer
chromatography, using Merck plates with fluorescent indicator.
Melting points were determined on an X-6 melting-point appara-
tus and are uncorrected. The 1H NMR spectra were recorded on a

B. Sun et al. / Bioorg. Med. Chem. 17 (2009) 4981–4989
4985
Figure 4. Ribbon diagram illustrating the crystal structure of murine P-gp with
compound 19 bound to the active site, and the hydrogen bonds are labeled as
yellow broken lines.
Figure 6. Top-ranked, minimized docking pose compound 19 in murine P-gp active
site.
Reagents were used as purchased without further purification. Sol-
vents (THF, DMF, CH2Cl2, DMSO, and toluene) were dried and
freshly distilled before use according to the procedures reported
in the literature.
4.2. Syntheses
4.2.1. 2-(3-(1,3-Dioxan-2-yl)phenoxy)-5-methoxybenzaldehyde
(2)
A mixture of 3-(1,3-dioxan-2-yl)phenol (1,23 8.81 g, 48.12 mmol),
2-bromo-5-methoxybenzaldehyde (10.27 g, 48.12 mmol), potas-
sium carbonate (13.24 g, 96.33 mmol), and cupric oxide (0.38 g,
4.82 mmol) in pyridine (50 mL) was stirred under reflux for 12 h.
The pyridine was distilled off in vacuo and the residue was extracted
with ethyl acetate (200 mL). The solution was concentrated and the
residue was purified by flash column chromatograph (Al2O3), eluting
with a 2:1 solution of petroleum ether–CH2Cl2 to afford 2 (8.92 g,
62%) as a yellow solid; mp 66–67 °C. 1H NMR (300 MHz, CDCl3) d
10.38 (s, 1H), 7.40 (d, J = 3.3 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.23 (s,
1H), 7.16 (s, 1H), 7.12 (dd, J = 9.1, 3.1 Hz, 1H), 6.97–6.91 (m, 2H),
5.47 (s, 1H), 4.26 (dd, J = 10.7, 5.1 Hz, 2H), 3.97 (dt, J = 12, 2.4 Hz,
2H), 3.85 (s, 3H), 2.28–2.15 (m, 1H), 1.45 (d, J = 13.5 Hz, 1H); MS
(ESI) 315 (M+H)+.
4.2.2. Methyl 4-(5-formyl-2-methoxyphenoxy) benzoate (4)
This compound was prepared from methyl 4-bromobenzoate
(15.05 g, 69.98 mmol) and 3-hydroxy-4-methoxybenzaldehyde
(10.63 g, 69.98 mmol) by means of a procedure similar to that used
for 2. After concentration of the solution, the residue was purified
by flash column chromatography (SiO2), eluting with a 3:2 solution
of petroleum ether–CH2Cl2 to afford 4 (13.01 g, 65%) as an orange
solid; mp 118–120 °C. 1H NMR (300 MHz, CDCl3) d 9.87 (s, 1H),
8.02–7.99 (m, 2H), 7.76 (dd, J = 8.2, 2.1 Hz, 1H), 7.59 (d, J = 2.1 Hz,
1H), 7.14 (d, J = 8.3 Hz, 1H), 6.94 (d, J = 8.7 Hz, 2H), 3.91 (s, 3H),
3.90 (s, 3H); MS (ESI) 287 (M+H)+.
4.2.3. Methyl 4-(5-(hydroxymethyl)-2-methoxyphenoxy)
benzoate (5)
Figure 5. Potential interactions between compound 19 and Leu64, Met 68, Tyr 303,
Ile 336, Gln 721, Ser725, Phe728, Phe 974, Val 978 (A), and Phe 724 (B) in the
murine P-gp active site, and the hydrogen bonds are labeled as yellow broken lines.
Sodium borohydride (0.68 g, 18.32 mmol) was added to a solu-
tion of 4 (12.87 g, 45.73 mmol) in THF (40 mL) over 15 min at 0 °C.
The reaction mixture was then stirred at room temperature for 3 h.
Water (10 mL) and 1 M HCl (18 mL) were added and the THF was
evaporated in vacuo. The resulting mixture was extracted with
CH2Cl2 (20 mL), washed with satd aq NaCl (10 mL ꢁ 3), and dried
over sodium sulfate. The solution was concentrated to obtain a
crude oil that was purified by flash column chromatography
(SiO2), eluting with CH2Cl2 to afford 5 (11.41 g, 88%) as a white so-
lid; mp 77–79 °C. 1H NMR (300 MHz, CDCl3) d 7.97 (d, J = 8.7 Hz,
Bruker Spectospin spectrometer at 300 or 600 MHz, using TMS as
an internal standard. The chemical shifts are reported in parts
per million (ppm d) referenced to the residual 1H resonance of
the solvent (CDCl3, 7.26 ppm). Abbreviations used in the splitting
pattern were as follows: s = singlet, d = doublet, t = triplet,
quin = quintet, m = multiplet, and br = broad. All HRMS spectra
(ESI) were obtained on
a LTQ Orbitrap mass spectrometer.

4986
B. Sun et al. / Bioorg. Med. Chem. 17 (2009) 4981–4989
2H), 7.21 (dd, J = 8.1, 1.8 Hz, 1H), 7.09 (d, J = 1.8 Hz, 1H), 7.00 (d,
J = 8.1 Hz, 1H), 6.92 (d, J = 8.7 Hz, 2H), 4.63 (s, 2H), 3.89 (s, 3H),
3.80 (s, 3H), 1.67 (br s, 1H); MS (ESI) 289 (M+H)+.
(200 mL), washed with satd aq NaCl (25 mL ꢁ 3), and dried over so-
dium sulfate. The solution was concentrated to yield 10 (9.64 g,
87%) as colorless oil. 1H NMR (300 MHz, CDCl3) d 9.85 (s, 1H),
7.49 (d, J = 7.5 Hz, 1H), 7.40 (t, J = 7.9 Hz, 1H), 7.27–7.25 (m, 3H),
7.11 (dd, J = 8.1, 1.8 Hz, 1H), 6.90–6.81 (m, 5H), 6.76–6.72 (m,
2H), 6.66 (d, J = 1.36 Hz, 1H), 4.62 (s, 2H), 3.77 (s, 6H), 2.75 (s,
4H), 1.79 (br s, 1H); MS (ESI) 483 (M+H)+.
4.2.4. (4-Methoxy-3-(4-(methoxycarbonyl)phenoxy)benzyl)-
triphenylphosphonium bromide (7)
Carbon tetrabromide (25.91 g, 78.43 mmol) and triphenylphos-
phine (11.24 g, 43.12 mmol) were successively added to a stirred
solution of
5
(11.23 g, 39.23 mmol) in CH2Cl2 (50 mL) over
4.2.8. (4-(5-(2-(3-Formylphenoxy)-5-methoxyphenethyl)-2-
methoxyphenoxy)benzyl) triphenylphosphonium bromide (12)
Carbon tetrabromide (13.28 g, 40.02 mmol) and triphenylphos-
phine (5.76 g, 22.39 mmol) were successively added to a stirred
solution of 10 (9.64 g, 20.67 mmol) in CH2Cl2 (50 mL) over
20 min at 0 °C. The resulting reaction mixture was then stirred at
0 °C for 2.5 h. Removal of solvent in vacuo afforded an orange oil
which was purified by flash column chromatography (SiO2), elut-
ing with a 2:1 solution of petroleum ether–CH2Cl2, to yield 11
30 min at 0 °C. The resulting reaction mixture was then stirred at
0 °C for 2.5 h. The solution was concentrated to obtain an orange
oil that was purified by flash column chromatography (SiO2), elut-
ing with a 2:1 solution of petroleum ether–CH2Cl2, to afford 6
(12.04 g, 88%) as
a white solid. The compound 6 (11.9 g,
34.43 mmol) was added subsequently to a solution of triphenyl-
phosphine (9.8 g, 37.32 mmol) in anhydrous toluene, and the reac-
tion mixture was then stirred under reflux for 3 h. The reaction
mixture was allowed to cool to room temperature, and the precip-
itate was filtered and washed with hexane to provide 7 (20.43 g,
98%) as a white solid; mp 217–219 °C. 1H NMR (300 MHz, CDCl3)
d 7.90 (d, J = 9.0 Hz, 2H), 7.80–7.73 (m, 9H), 7.65–7.58 (m, 6H),
7.27 (s, 1H), 6.84 (d, J = 8.4 Hz, 1H), 6.69 (d, J = 8.7 Hz, 2H), 6.51
(s, 1H), 5.46 (d, J = 14 Hz, 2H), 3.90 (s, 3H), 3.73 (s, 3H); MS (ESI)
533 (MꢀBr)+.
(8.92 g, 79%) as
a white solid. The compound 11 (8.92 g,
16.3 mmol) was then added subsequently to a solution of triphen-
ylphosphine (4.7 g, 18 mmol) in toluene, and the reaction mixture
was heated at reflux for 3 h. The reaction mixture was allowed to
cool to room temperature, and the precipitate was filtered and
washed with hexane to provide 12 (12.9 g, 98%) as a white solid;
mp 233–235 °C. 1H NMR (300 MHz, CDCl3) d 9.90 (s, 1H), 7.78–
7.71 (m, 9H), 7.65–7.59 (m, 6H), 7.53–7.39 (m, 2H), 7.25–7.10
(m, 2H), 7.02 (d, J = 6.3 Hz, 2H), 6.88–6.63 (m, 8H), 5.40 (d,
J = 13.8 Hz, 2H), 3.77 (s, 3H), 3.75 (s, 3H), 2.75 (s, 4H); MS (ESI)
729 (MꢀBr)+.
4.2.5. (E/Z)-Methyl 4-(5-(2-(3-(1,3-dioxan-2-yl)phenoxy)-5-
methoxystyryl)-2-methoxy- phenoxy) benzoate (8)
Potassium carbonate (7.73 g, 56.09 mmol) and a small amount
of 18-crown-6 were added to a solution of 2 (8.79 g, 28.03 mmol)
and 7 (18 g, 29.30 mmol) in anhydrous CH2Cl2 (50 mL), and the
resulting mixture was stirred under reflux for 24 h. The insoluble
material was then filtered off and the filtrate was concentrated
to provide the orange oil that was purified by flash column chro-
matography (Al2O3), eluting with a 2:1 solution of petroleum
ether–CH2Cl2 to afford 8 (13.68 g, 86%) as a yellow oil.
4.2.9. Dimethyl ether of isoptychantol A (13)
A solution of 12 (1.13 g, 1.41 mmol) in anhydrous CH2Cl2
(150 mL) was added dropwise to a stirred suspension of sodium
methoxide (152 mg, 2.82 mmol) in anhydrous CH2Cl2 (200 mL)
over 7 h and the reaction mixture was stirred for 5 h at room tem-
perature. The reaction mixture was filtered, concentrated, and the
residue was purified by flash column chromatography (SiO2), elu-
ating with CH2Cl2 to yield 13 (0.49 g, 78%) as a white solid; mp
170–171 °C. 1H NMR (600 MHz, CDCl3) d 7.18–7.13 (m, 3H), 7.08
(d, J = 8.4 Hz, 2H), 6.92 (d, J = 9 Hz, 2H), 6.86 (t, J = 8.7 Hz, 2H),
6.82 (d, J = 2.4 Hz, 1H), 6.80 (d, J = 1.8 Hz, 1H), 6.75–6.72 (m, 2H),
6.69 (s, 1H), 6.42 (d, J = 1.8 Hz, 1H), 6.40 (d, J = 1.8 Hz, 1H), 3.95
(s, 3H), 3.82 (s, 3H), 2.60–2.58 (m, 2H), 2.56–2.54 (m, 2H); MS
(ESI) 451 (M+H)+.
4.2.6. Methyl 4-(5-(2-(3-(1,3-dioxan-2-yl)phenoxy)-5-
methoxyphenethyl)-2-methoxy- phenoxy) benzoate (9)
Pd/C 10% (1 g) and triethylamine (27 mL) were added to a solu-
tion of 8 (13.63 g, 24.06 mmol) in ethyl acetate (150 mL). The sus-
pension was stirred under H2 for 24 h at room temperature. The
mixture was filtered, and concentration provided a crude yellow
solid that was purified by precipitating from ethyl ether–petro-
leum ether to afford 9 (13.4 g, 98%) as a white solid; mp 103–
104 °C. 1H NMR (300 MHz, CDCl3) d 7.95 (d, J = 9 Hz, 2H), 7.22 (d,
J = 7.8 Hz, 1H), 7.12 (d, J = 8.3 Hz, 1H), 7.02 (s, 1H), 6.93–6.80 (m,
7H), 6.72–6.67 (m, 2H), 5.42 (s, 1H), 4.23 (dd, J = 10.5, 5.1 Hz,
2H), 3.96 (dd, J = 12.3, 2.7 Hz, 2H), 3.88 (s, 3H), 3.76 (s, 3H), 3.75
(s, 3H), 2.80 (s, 4H), 2.27–2.11 (m, 1H), 1.42 (d, J = 13.5 Hz, 1H);
MS (ESI) 571 (M+H)+.
4.2.10. Isoptychantol A (14)
A solution of boron tribromide (1.95 g, 7.83 mmol) in anhy-
drous CH2Cl2 (10 mL) was added dropwise to a stirred solution of
13 (0.44 g, 0.98 mmol) in anhydrous CH2Cl2 (10 mL) at ꢀ78 °C.
The reaction mixture was stirred at ꢀ78 °C for 3 h, and then was
allowed to warm up to room temperature within 12 h. The ice-cold
water was added, and the reaction mixture was stirred vigorously
for 1 h. The solution was then diluted with CH2Cl2 (50 mL), washed
with satd aq NaCl (20 mL ꢁ 3), and dried over sodium sulfate. The
solution was concentrated, and the residue was purified by flash
column chromatography (SiO2), eluting with CH2Cl2 to yield 14
(0.3 g, 73%) as a grey solid. mp 93–95 °C. 1H NMR (300 MHz, CDCl3)
d 7.18–7.11 (m, 3H), 6.97 (dd, J = 8.5, 1.8 Hz, 2H), 6.88–6.85 (m,
2H), 6.75–6.71 (m, 4H), 6.66–6.63 (m, 3H), 6.41 (dd, J = 8.3,
1.9 Hz, 1H), 6.35 (d, J = 1.9 Hz, 1H), 5.54 (br s, 1H), 4.57 (br s,
1H), 2.59–2.53 (m, 2H), 2.52–2.49 (m, 2H); MS (ESI) 423 (M+H)+.
4.2.7. 3-(2-(3-(4-(Hydroxymethyl)phenoxy)-4-
methoxyphenethyl)-4-methoxyphenoxy) benzaldehyde (10)
A solution of 9 (13.11 g, 23.72 mmol) in anhydrous THF (25 mL)
was added dropwise to a stirred suspension of lithium aluminum
hydride (1.75 g, 46.07 mmol) in anhydrous THF (30 mL). The
resulting mixture was stirred at room temperature for 2.5 h and
carefully hydrolyzed with satd aq ammonium chloride (10 mL).
THF was removed in vacuo and the resulting mixture was diluted
with CH2Cl2 (100 mL), washed with satd aq NaCl (20 mL ꢁ 3),
and dried over sodium sulfate. The solution was concentrated to
obtain a crude oil that was subsequently dissolved in a solution
of ethanol (100 mL) and 10% aq HCl (20 mL). The resulting mixture
was then stirred at room temperature for 12 h. Satd aq sodium
bicarbonate (150 mL) was added and the ethanol was removed in
vacuo. The resulting mixture was extracted with CH2Cl2
4.2.11. Dimethyl ether of dihydroptychantol A (15)
Pd/C 10% (250 mg) was added to a solution of 13 (2.25 g,
5.34 mmol) in ethyl acetate (150 mL). The suspension was stirred
under H2 for 24 h at room temperature. The reaction mixture
was filtered, and the solution was concentrated to provide the

B. Sun et al. / Bioorg. Med. Chem. 17 (2009) 4981–4989
4987
crude product that was recrystallized from ethanol to yield 15
(2.2 g, 96%) as a white solid; mp 202–203 °C. 1H NMR (300 MHz,
CDCl3) d 7.10 (t, J = 7.8 Hz, 1H), 6.92–6.85 (m, 6H), 6.82–6.70 (m,
4H), 6.33–6.21 (m, 3H), 3.93 (s, 3H), 3.80 (s, 3H), 2.97 (s, 4H),
2.64–2.57 (m, 2H), 2.53–2.46 (m, 2H); MS (ESI) 453 (M+H)+.
2.52 (m, 2H), 2.49–2.37 (m, 2H); 13C NMR (75 MHz, CDCl3) d
191.1, 159.7, 159.3, 157.2, 149.2, 147.4, 145.0, 139.3, 135.5,
134.2, 131.8, 131.2, 130.5, 130.4, 123.1, 123.0, 122.3, 122.1,
121.7, 117.2, 115.5, 115.4, 112.5, 112.3, 112.2, 56.93, 56.2, 55.6,
35.5, 31.6; HRMS (ESI) calcd for C30H25O5Br 545.0961, found:
+
545.0963 (M)+; MS (ESI) 545 (M)
.
4.2.12. Dihydroptychantol A (16)
A solution of boron tribromide (2.65 g, 10.61 mmol) in anhy-
drous CH2Cl2 (15 mL) was added dropwise to a stirred solution of
15 (0.6 g, 1.33 mmol) in anhydrous CH2Cl2 (15 mL) at ꢀ78 °C. The
reaction mixture was stirred at ꢀ78 °C for 3 h, and then was al-
lowed to warm up to room temperature within 12 h. The ice-cold
water was added and the reaction mixture was stirred for 1 h.
The solution was then diluted with CH2Cl2 (50 mL), washed with
satd aq NaCl (20 mL ꢁ 3), and dried over sodium sulfate. The solu-
tion was concentrated, and the residue was purified by flash col-
umn chromatography (SiO2), eluting with CH2Cl2 to yield 16
(0.3 g, 73%) as a grey solid; mp 165–167 °C. 1H NMR (300 MHz,
CDCl3) d 7.10 (t, J = 8.0 Hz, 1H), 6.94–6.88 (m, 5H), 6.86 (d,
J = 8.4 Hz, 1H), 6.80 (d, J = 8.6 Hz, 1H), 6.73 (d, J = 3 Hz, 1H), 6.70
(dd, J = 8.0, 1.9 Hz, 1H), 6.64 (dd, J = 8.6, 3.0 Hz, 1H), 6.31–6.26
(m, 2H), 6.16 (d, J = 1.9 Hz, 1H), 5.56 (br s, 1H), 4.59 (br s, 1H),
3.00–2.94 (m, 4H), 2.61–2.56 (m, 2H), 2.50–2.44 (m, 2H); MS
(ESI) 425 (M+H)+.
4.2.15. 2-Amino-thiazole derivative of dimethyl ether of
dihydroptychantol A (19)
A solution of 18 (80 mg, 0.15 mmol) and thiourea (17 mg,
0.23 mmol) in anhydrous DMF (15 mL) was stirred at 65 °C for
4 h. The reaction mixture was then diluted with CH2Cl2 (30 mL),
washed with water (20 mL ꢁ 3), and dried over sodium sulfate.
The solution was concentrated to provide a crude product that
was purified by precipitating from CH2Cl2–hexane to afford 19
(63 mg, 83%) as
a
yellow solid. mp 270–272 °C; 1H NMR
(300 MHz, CDCl3) d 7.39 (d, J = 8.7 Hz, 2H), 7.18–7.01 (m, 4H),
6.93 (d, J = 8.7 Hz, 1H), 6.88–6.79 (m, 3H), 6.76–6.70 (m, 2H),
6.45–6.41 (m, 2H), 5.17 (br s, 2H), 3.95 (s, 3H), 3.82 (s, 3H), 2.60–
2.56 (m, 2H), 2.55–2.51 (m, 2H); 13C NMR (75 MHz, CDCl3) d
165.7, 158.8, 156.5, 155.8, 149.5, 149.4, 147.5, 145.4, 135.7,
134.8, 134.1, 131.0, 129.5, 122.4, 122.1, 122.0, 121.9, 121.3,
119.0, 116.4, 115.7, 112.4, 112.0, 111.9, 111.8, 56.2, 55.6, 37.7,
34.3; HRMS (ESI) calcd for C31H27O4N2S 523.1686, found:
+
523.1673 (M+H)+; MS (ESI) 523 (M+H)
.
4.2.13. 7-Hydroxyl-8-bromo derivative of demethyl ether of
dihydroptychantol A (17)
4.2.16. 2-Amino-thiazole derivative of dihydroptychantol A
(20)
N-Bromosuccinimide (149 mg, 0.83 mmol) was added to a sus-
pension of 13 (250 mg, 0.56 mmol) in THF-H2O (1:1) (12 mL) in
three portions over 15 min at 0 °C. The reaction mixture was stir-
red vigorously at 0 °C for 0.5 h and then was allowed to warm up
to 50 °C. After 5 h, the mixture was poured into 5% aq Na2S2O3
(10 mL) and extracted with CH2Cl2 (25 mL ꢁ 3). The combined or-
ganic layer was then dried over sodium sulfate. The solution was
concentrated, and the residue was purified by flash column chro-
matography (SiO2), eluting with a 1:1 solution of petroleum
ether–CH2Cl2 to afford 17 (186 mg, 61%) as a white solid; mp
216–218 °C. 1H NMR (300 MHz, CDCl3) d 7.68 (d, J = 8.4 Hz, 1H),
7.28 (d, J = 8 Hz, 1H), 7.23–7.17 (m, 3H), 7.05 (s, 1H), 6.87–6.85
(m, 2H), 6.75 (dd, J = 8.8, 2.8 Hz, 1H), 6.66–6.58 (m, 2H), 6.46 (s,
1H), 6.29 (dd, J = 8.2, 2.0 Hz, 1H), 6.07 (s, 1H), 5.07 (d, J = 9.7 Hz,
1H), 5.00 (d, J = 9.7 Hz, 1H), 3.91 (s, 3H), 3.82 (s, 3H), 3.14 (br s,
1H), 2.71 (t, J = 8.3 Hz, 2H), 2.51–2.35 (m, 2H); 13C NMR (75 MHz,
CDCl3) d 158.5, 156.8, 155.4, 149.5, 147.2, 145.9, 139.9, 135.9,
134.7, 134.6, 130.2, 129.9, 126.8, 123.0, 122.5, 121.6, 121.4,
121.2, 117.2, 115.7, 115.1, 112.8, 112.5, 111.7, 79.0, 63.9, 56.1,
A solution of boron tribromide (213 mg, 0.88 mmol) in anhy-
drous CH2Cl2 (5 mL) was added dropwise to a stirred suspension
of 19 (60 mg, 0.11 mmol) in anhydrous CH2Cl2 (10 mL) at ꢀ78 °C.
The reaction mixture was stirred at ꢀ78 °C for 3 h, and the mixture
was allowed to warm up to room temperature within 12 h. The ice-
cold water was added, and the solution was stirred for 1 h. The
solution was then diluted with CH2Cl2 (20 mL), washed with satd
aq NaCl (20 mL ꢁ 3), and dried over sodium sulfate. The solution
was concentrated to provide the crude product that was recrystal-
lized from ethanol–H2O to afford 20 (42 mg, 76%) as a yellow solid;
mp 211–213 °C. 1H NMR (600 MHz, DMSO-d6) d 9.37 (s, 1H), 9.28
(s, 1H), 7.31 (d, J = 8.4 Hz, 2H), 7.24 (s, 2H), 7.20 (t, J = 8.4 Hz,
1H), 7.03 (d, J = 8.4 Hz, 2H), 7.00 (dd, J = 7.2, 1.2 Hz, 1H), 6.83 (d,
J = 8.4 Hz, 1H), 6.78 (d, J = 7.8 Hz, 1H), 6.73 (t, J = 3.0 Hz, 1H), 6.72
(d, J = 1.8 Hz, 1H), 6.64 (dd, J = 8.4, 3.0 Hz, 1H), 6.48 (t, J = 1.8 Hz,
1H), 6.30 (dd, J = 8.4, 2.4 Hz, 1H), 6.26 (d, J = 1.8 Hz, 1H), 2.44–
2.42 (m, 2 H), 2.37–2.34 (m, 2H); 13C NMR (150 MHz, DMSO-d6)
d 166.5, 158.3, 155.0, 154.1, 147.5, 146.0, 144.6, 142.9, 134.9,
134.5, 132.3, 131.6, 130.7, 129.7, 122.2, 121.8, 121.6, 121.2,
118.2, 117.4, 116.5, 116.1, 115.7, 113.8, 110.6, 37.1, 33.4; HRMS
55.6, 37.1, 34.8; HRMS (ESI) calcd for C30H27O5Br 547.1115, found:
+
547.1106 (M)+; MS (ESI) 529 (MꢀH2O)
.
(ESI) calcd for C29H23O4N2S 495.1373, found: 495.1377 (M+H)+;
+
4.2.14. 7-Keto-8-bromo derivative of demethyl ether of
dihydroptychantol A (18)
MS (ESI) 495 (M+H)
.
A solution of anhydrous DMSO (47
(5 mL) was added to a stirred solution of trifluoroacetic anhydride
(69
l
L, 0.66 mmol) in CH2Cl2
4.2.17. 2-Hydrazino-thiazole derivative of dimethyl ether of
dihydroptychantol A (21)
l
L, 0.5 mmol) in CH2Cl2 (10 mL) at ꢀ55 °C. After 30 min, a solu-
A solution of 18 (100 mg, 0.19 mmol) and thiosemicarbazide
(26 mg, 0.29 mmol) in anhydrous DMF (20 mL) was stirred at
65 °C for 4 h. The reaction mixture was then diluted with CH2Cl2
(30 mL), washed with water (20 mL ꢁ 3), and dried over sodium
sulfate. The solution was concentrated to provide a crude product
that was purified by precipitating from CH2Cl2- hexane to afford 21
(57 mg, 56%) as a grey solid; mp 286–288 °C. 1H NMR (300 MHz,
DMSO-d6) d 7.26 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 7.2 Hz, 1H), 7.15
(d, J = 7.8 Hz, 1H), 7.09 (d, J = 8.4 Hz, 2H), 7.00–6.93 (m, 3H), 6.86
(dd, J = 8.1, 1.8 Hz, 1H), 6.82 (dd, J = 8.7, 3.0 Hz, 1H), 6.33 (s, 1H),
6.29–6.25 (m, 2H), 2.48–2.41 (m, 4H); 13C NMR (75 MHz, DMSO-
d6) d 158.6, 156.5, 155.3, 149.5, 147.5, 147.4, 145.1, 145.0, 136.1,
135.5, 134.5, 134.6, 131.2, 130.1, 122.6, 122.5, 122.3, 122.1,
tion of 17 (180 mg, 0.33 mmol) in dry CH2Cl2 (15 mL) was added
dropwise. The reaction mixture was stirred at ꢀ50 °C for 2 h. Tri-
ethylamine (143 lL, 0.99 mmol) was added slowly and the reac-
tion mixture was then allowed to warm up to room temperature.
After 0.5 h, the CH2Cl2 layer was washed with water (20 mL ꢁ 3)
and dried over sodium sulfate. The solution was concentrated to
provide a crude product that was purified by precipitating from
CH2Cl2–hexane to afford 18 (167 mg, 93%) as a white solid; mp
244–246 °C. 1H NMR (300 MHz, CDCl3) d 7.89 (d, J = 8.4 Hz, 1H),
7.66 (d, J = 8.7 Hz, 1H), 7.15–7.00 (m, 4H), 6.94 (d, J = 8.7 Hz, 1H),
6.88–6.82 (m, 4H), 6.77 (dd, J = 8.7, 3.0 Hz, 1H), 6.40 (s, 1H), 6.25
(d, J = 7.8 Hz, 1H), 5.83 (s, 1H), 3.93 (s, 3H), 3.82 (s, 3H), 2.70–

4988
B. Sun et al. / Bioorg. Med. Chem. 17 (2009) 4981–4989
122.0, 118.4, 116.1, 115.8, 115.7, 113.2, 113.1, 113.0, 110.0, 56.2,
55.8, 37.6, 34.0; HRMS (ESI) calcd for C31H24NO4S 506.1420
(M+HꢀNH2NH2)+, found: 506.1416 (M+HꢀNH2NH2)+; MS (ESI)
506 (M+HꢀNH2NH2)+.
growth response to the chemicals was detected by measuring the
absorbance of formazan crystals produced by living cultured cells
at 490 nm. Three replicates were used for each treatment and the
assay was repeated at least thrice.
The multidrug resistance reversal ability of these compounds to
potentiate adriamycin and vincristine cytotoxicity was evaluated
in K562/A02 cells and in KB/VCR cells, respectively, by the MTT as-
say described above. The cells were treated with vehicle and tested
compound combined with desired concentrations of chemicals.
ID50 values for all tested compounds were calculated from plotted
results using untreated cells as 100%. The reversal fold (RF) values,
as potency of reversal, were obtained from fitting the data to
RF = ID50 of cytotoxic drug alone/ID50 of cytotoxic drug in the pres-
ence of the test drugs.
4.2.18. 2-Hydrazino-thiazole derivative of dihydroptychantol A
(22)
This compound was prepared from 21 by means of a procedure
similar to that used for 20. Yield 71%; mp 225–227 °C; 1H NMR
(600 MHz, DMSO-d6) d 7.25 (d, J = 8.4 Hz, 2H), 7.21 (t, J = 8.4 Hz,
1H), 7.14 (d, J = 8.4 Hz, 1H), 7.10 (d, J = 8.4 Hz, 2H), 6.81 (d,
J = 9.0 Hz, 1H), 6.78 (d, J = 7.8 Hz, 1H), 6.74–6.72 (m, 2 H), 6.64
(dd, J = 8.4, 3.0 Hz, 1H), 6.31 (t, J = 1.8 Hz, 1H), 6.25 (dd, J = 8.4,
2.4 Hz, 1H), 6.22 (d, J = 1.8 Hz, 1H), 2.44–2.40 (m, 2 H), 2.39–2.34
(m, 2H); 13C NMR (150 MHz, DMSO-d6) d 158.4, 155.2, 154.1,
150.9, 147.8, 146.1, 144.6, 143.1, 139.9, 134.9, 132.4, 131.1,
130.5, 129.5, 122.2, 122.0, 121.6, 121.4, 121.3, 117.6, 117.4,
116.6, 116.1, 115.6, 113.8, 112.9, 111.5, 37.2, 33.3; HRMS (ESI)
calcd for C29H20NO4S 478.1107 (M+HꢀNH2NH2)+, found:
478.1102 (M+HꢀNH2NH2)+; MS (ESI) 510 (M+H)+ and 478
(M+HꢀNH2NH2)+.
Acknowledgments
This work was supported by grant from the National Natural Sci-
ence Foundation (NNSF) of China (No. 30730109). S.B. wishes to
thank Dr. Mark Cushman and his research group (Purdue Univer-
sity, USA) for assistance and insight.
4.3. Molecular modeling
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4.4.1. Cell culture
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