3′-O, 4′-O-aromatic acyl substituted 7,8-pyranocoumarins: A new class of P-glycoprotein modulators

Article abstract of DOI:10.1111/j.2042-7158.2011.01378.x

Objectives P-glycoprotein (Pgp) overexpression in tumour cells leads to multidrug resistance (MDR) and causes failure in cancer chemotherapy. We have previously identified (±)-praeruptorin A (PA) as a potential lead compound for Pgp modulators. In this study we investigated the MDR-reversing activities of PA derivatives. Methods Series 7,8-pyranocoumarins with various C-3′ and C-4′ side chains had been semi-synthesized and their MDR-reversing activity was investigated in Pgp-overexpressing MDR tumour cell line HepG2/Dox and in a KB V1 xenograft animal model. Key findings All 7,8-pyranocoumarins exhibited equal or higher activity in modulating Pgp. DCK (12), DMDCK (15), 16, 21, 23 and 24 at 4 μm achieved 91%～99% decrease in IC50 value (concentration inhibiting cell growth by 50%) of anticancer agents vinblastine, doxorubicin, puromycin and paclitaxel, and were more active than others. DMDCK also remarkably enhanced the growth inhibitory effect of paclitaxel on KB V1 xenografts (P < 0.05), showing a potency required for clinical usage. Mechanistic studies suggested that these 7,8-pyranocoumarins might reverse Pgp-MDR through directly binding to substrate binding site(s) or allosteric site(s) on Pgp therefore impairing Pgp-mediated drug transport. Conclusions Results from the study suggested that 3′-O, 4′-O-aromatic acyl substituted 7,8-pyranocoumarins could serve as a new class of Pgp modulator. Acyls play an important role in maintaining and enhancing the Pgp-modulating ability of pyranocoumarins. 3,4-Dimethoxyl substituted aromatic acyls, bearing a methoxy that might interact with Pgp as hydrogen bond accepter, were shown to be the most potent for reversing MDR. 2011 The Authors. JPP

Full text of DOI:10.1111/j.2042-7158.2011.01378.x

Research Paper
Journal of Pharmacy
And Pharmacology
3Ј-O, 4Ј-O-aromatic acyl substituted 7,8-pyranocoumarins:
a new class of P-glycoprotein modulatorsjphp_1378 90..100
Xiaoling Shena, Guangying Chenb, Guoyuan Zhuc, Jiazhong Caia, Lu Wanga, Yingjie Hua and
Wang-Fun Fongc
aTropical Medicine Institute, Guangzhou University of Chinese Medicine, Guangzhou, bDepartment of Chemistry, Hainan Normal University, Haikou
and cResearch & Development Division, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China
Keywords
(Ϯ)-Praeruptorin A; 7, 8-pyranocoumarin;
Abstract
Objectives P-glycoprotein (Pgp) overexpression in tumour cells leads to multidrug
resistance (MDR) and causes failure in cancer chemotherapy. We have previously
identified (Ϯ)-praeruptorin A (PA) as a potential lead compound for Pgp modula-
tors. In this study we investigated the MDR-reversing activities of PA derivatives.
Methods Series 7,8-pyranocoumarins with various C-3′ and C-4′ side chains had
been semi-synthesized and their MDR-reversing activity was investigated in Pgp-
overexpressing MDR tumour cell line HepG2/Dox and in a KB V1 xenograft animal
model.
multidrug resistance; P-glycoprotein
Correspondence
Wang-Fn Fong, RDD, SCM, Hong Kong Baptist
University, Hong Kong SAR, China.
E-mail: wffong@hkbu.edu.hk
Received February 15, 2011
Accepted September 22, 2011
Key findings All 7,8-pyranocoumarins exhibited equal or higher activity in modu-
lating Pgp. DCK (12), DMDCK (15), 16, 21, 23 and 24 at 4 mm achieved 91%~99%
decrease in IC50 value (concentration inhibiting cell growth by 50%) of anticancer
agents vinblastine, doxorubicin, puromycin and paclitaxel, and were more active
than others. DMDCK also remarkably enhanced the growth inhibitory effect of
paclitaxel on KB V1 xenografts (P < 0.05), showing a potency required for clinical
usage. Mechanistic studies suggested that these 7,8-pyranocoumarins might reverse
Pgp-MDR through directly binding to substrate binding site(s) or allosteric site(s)
on Pgp therefore impairing Pgp-mediated drug transport.
doi: 10.1111/j.2042-7158.2011.01378.x
Conclusions Results from the study suggested that 3′-O, 4′-O-aromatic acyl substi-
tuted 7,8-pyranocoumarins could serve as a new class of Pgp modulator. Acyls play
an important role in maintaining and enhancing the Pgp-modulating ability of
pyranocoumarins. 3,4-Dimethoxyl substituted aromatic acyls, bearing a methoxy
that might interact with Pgp as hydrogen bond accepter, were shown to be the most
potent for reversing MDR.
Introduction
P-glycoprotein (Pgp) is an ATP-dependent membrane
transporter protein encoded by the MDR1 gene.Pgp plays the
role of bodyguard by extruding xenotoxics out of cells thus
protecting living organs and tissues from damage. However,
overexpression of Pgp in tumour cells confers multidrug
resistance (MDR).[1–4] In Pgp overexpressing cells, Pgp
decreases the cellular concentration of anticancer drugs that
are Pgp substrates by actively transporting them out. Pgp
inhibitors (or modulators) restore the chemosensitivity of
MDR cells via impairing Pgp-directed drug transport. Since
verapamil was found to have MDR-reversing activity in
1981,[5] thousands of natural and synthesized compounds
have been studied for their Pgp-modulating activity. Hun-
dreds of compounds with Pgp-inhibiting properties were
investigated in vitro or in vivo, which led to the discovery of
highly active Pgp inhbitors, including valspodar (PSC 833,
a ciclosporin A derivative), dexverapamil (verapamil deri-
vative), tariquidar (XR9576), zosuquidar (LY335979) and
biricodar (VX-710).[6,7] Though some Pgp inhibitors are
undergoing phase II or III clinical study, there are now cur-
rently no reversal agents clinically available.
Identification of target sites on Pgp has been challenging
because of the difficulty in obtaining crystallized Pgp as well
as the structural diversity of molecules that Pgp recognizes
and transports. Therefore, great effort has been made for
many years to identify common characteristics on Pgp sub-
strates and inhibitors by structure–activity relationship
(SAR) studies. Some commonly shared structural features
were rapidly recognized, such as the amphiphilic character of
the molecules, the presence of aromatic rings, hydrogen bond
© 2011 The Authors. JPP © 2011
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 90–100
90

Xiaoling Shen et al.
Praeruptorin A derivatives as P-gp modulators
accepter or electron donor (O or N atom), and hydrogen
bond donor (OH or NH group) et al., in certain spatial
arrangements.[8–11]
werepurchasedfromSigma/AldrichCo.(StLouis,MO,USA).
RPMI1640medium,MEMmedium,fetalbovineserum(FBS)
and antibiotic–antimycotic (100 ¥) were Gibco products
(Brooklyn, NY, USA). Anti-MDR1 monoclonal UIC2 (UIC2
mouse monoclonal anti-human MDR1) was from Immu-
notech (Marseilles, France). Goat anti-mouse IgG2a-PE was
fromSantaCruzBiotechnology(SantaCruz,CA,USA).
NMR spectra were recorded on a Varian NMR-300 MHz
spectrometer in CDCl3. Optical rotations were measured on a
PE343 polarimeter. MS were carried out on a LCQ Advantage
mass spectrometer.
Wehaveshownthat(Ϯ)-praeruptorinA(PA),anatural7,8-
pyranocoumarin from Peucedanum praeruptorum Dunn.,
reverses Pgp-mediated MDR in cancer cells.[12] To find more
potent Pgp modulators, a number of PA derivatives were pre-
pared. These compounds had the same 7,8-pyranocoumarin
core structure but different C-3′-O and C-4′-O acyls in cis- or
trans-configuration. We have previously reported that one of
these new semi-synthetic 7,8-pyranocoumarins (Ϯ)-3′-O,4′-
O-dicynnamoyl-cis-khellactone (DCK,or 12),is more potent
than PA or verapamil in reversing Pgp-MDR.[13] Further
studies revealed that 3,4-dimethoxylation on cinnamoyl
remarkably enhanced the MDR-reversing activity of DCK.[14]
It was found in our study that the type of 3′-acyl and 4′-acyl
seemstoplayaveryimportantroleinkeepingorenhancingthe
ability of 7,8-pyranocoumarins in modulating Pgp. Side
chains altered the drug–Pgp interaction mode. To explore the
role of acyls, the structure–activity relationship of these pyra-
nocoumarinswasstudiedbycomparingtheirabilitytoreverse
Pgp-mediated drug resistance in human tumours. The mode
of drug–Pgp interaction was also investigated. DMDCK,[15]
which shows the highest MDR-reversing ability in vitro, was
further studied in vivo, for its potential medical usage as a
chemo-sensitizerof paclitaxel.
Semi-synthesis of 7,8-pyranocoumarins
7,8-Pyranocoumarins were prepared in a two-step reaction as
described previously with minor modification.[13–16] Firstly
the basic hydrolysis of PA provided intermediates 2, 3 and 4
(Figure 1) and secondly acylation of 2, 3 and 4 provided the
target compounds (Figures 2–4).
Basic hydrolysis was carried out by adding 200 ml of 0.5 m
KOH to 15.5 mmol PA (6.0 g in 500 ml of dioxane) and the
mixture was stirred at 60°C for 20 min. After cooling, the pH
was adjusted to 2~3 by 10% H2SO4 and the mixture was
stirred for 2 h at room temperature and extracted three times
with CHCl3 (300 ml, 200 ml, 200 ml). The combined CHCl3
fraction was washed with 300 ml of saturated NaHCO3 fol-
lowed by 300 ml of water and dried over Na2SO4.After solvent
removal, the residue was separated by repeated flash chroma-
tography on silica gel (CHCl3–EtOAc, 3:1) and fractions were
monitored by thin-layer chromatography (TLC) at 365 nm.
Yield of 2, 3 and 4 was 400 mg (7%), 1.0 g (22%) and 900 mg
(20%) respectively.
Materials and Methods
Doxorubicin, vinblastine, puromycin, paclitaxel, verapamil,
sulforhodamine B (SRB), 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) and other chemicals
O
O
O
5
4
OH
O
O
O
O
3
O
6
O
2
O
OH
OH
O
7
O
O
2'
OH
8
OH
O
4'
a,b
3'
OAc
+
+
O
O
O
O
O
O
O
O
O
O
O
OH
OH
OH
OH
OH
O
1 (PA)
O
2
3
4
Figure 1 Scheme for preparation of intermediates 2, 3 and 4. Reagents and conditions: (a) 0.5 M KOH, dioxane, 60°C; (b) 10% H2SO4, pH 2~3, rt.
© 2011 The Authors. JPP © 2011
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 90–100
91

Praeruptorin A derivatives as P-gp modulators
Xiaoling Shen et al.
O
O
O
O
O
O
O
O
O
O
O
O
a
O
OH
O
O
O
OH
O
O
OR1
OR1
O
O
2
O
O
6
R1=
7
R1=
5
R1=
O
N
O
O
8
R1=
9
R1=
MeO
Figure 2 Scheme for preparation of 5~9. Reagents and conditions: (a) CH2Cl2, DCC, DMAP, RnOH, reflux.
Acylation of 2 (Figure 2) was carried out by adding, sepa-
rately, 1.2 mmol benzoic acid (145 mg), 1.2 mmol benzene-
acetic acid (160 mg), 1.2 mmol picolinic acid (145 mg),
1.2 mmol 4-methoxybenzoic acid (180 mg) and 1.2 mmol
cinnamic acid (180 mg) to a mixture of 2 (80 mg or
0.233 mmol) in dichloromethane (CHCl2) 5 ml, N,N’-
dicyclohexylcarbodiimide (DCC) 206 mg or 1 mmol, and
4-dimethylaminopyridine (DMAP) 3 mg or 0.025 mmol.
The mixture was stirred/refluxed for 3 h, cooled to room
temperature, filtered and the filtrate was separated and puri-
fied by repeated flash silica gel 60 column chromatography
(petroleum ether–EtOAc, 4:1). Fractions were monitored by
TLC under 365 nm and purified 5 (40 mg, 38%), 6 (49 mg,
46%), 7 (35 mg, 33%), 8 (46 mg, 41%), 9 (54 mg, 49%) were
obtained, respectively.
10 (46 mg, 32%), 11 (52 mg, 34%), 12 (also DCK, 81 mg,
50%),[13] 13 (62 mg, 36%), 14 (also MMDCK, 17 mg, 7%),[14]
15 (also DMDCK, 22 mg, 11%),[14] 16 (46 mg, 25%) and 17
(19 mg, 12%). The same treatment of 4 (80 mg) separately
with 15 mmol of benzoic acid, benzeneacetic acid, cinnamic
acid,4-methoxybenzeneaceticacid,4-methoxycinnamicacid,
3,4-dimethoxycynnamicacidand3,4-dimethoxybenzoicacid
gave 18 (38 mg, 26%), 19 (43 mg, 28%), 20 (51 mg), 21
(41 mg, 24%), 22 (20 mg, 11%), 23(30 mg, 15%) and 24
(13 mg,7%),respectively.
Structural determination of above compounds was based
on their MS data and 1H NMR data, and on comparison with
data reported in the literature.[15,16]
Cell lines and cell culture
For the preparation of 10~24 (Figures 3 and 4), 1.5 mmol
benzoic acid (180 mg), 1.5 mmol benzeneacetic acid
(200 mg), 1.5 mmol cinnamic acid (220 mg), 1.5 mmol
Pgp-overexpressing human tumour cell lines HepG2/Dox
(hepatocarcinoma), K562/Dox (leukaemia) and KB V1 (epi-
demoid carcinoma) were generously provided by Dr Judy
Chan of the Chinese University of Hong Kong, Dr Morjani of
the University of Reims Champagne-Ardenne, France and Dr
Michael Gottesman of the National Institute of Health,
Bethesda, USA, respectively. HepG2/Dox cells and K562/Dox
cells were routinely cultured at 37°C, 5% CO2, in RPMI-1640
medium supplemented with 10% FBS and 100 U antibiotics.
KB V1 cells were maintained in MEM containing 10%
FBS and 100 U antibiotics at 37°C in a humidified 5% CO2
4-methoxybenzeneacetic
acid
(250 mg),
1.5 mmol
4-methoxycinnamic acid (270 mg), 1.5 mmol 3,4-
dimethoxycynnamic acid (310 mg), 1.5 mmol 3,4-
dimethoxybenzoic acid (270 mg) and 1.5 mmol 4-
methoxybenzoic acid (230 mg) were separately added to a
mixture of 3 (80 mg or 0.31 mmol) in CHCl2 5 ml, DCC
206 mg and DMAP 4 mg, and stirred under refluxing condi-
tion for 3 h. The mixture was then treated by following the
preparation steps for compounds 5~9 described above to give
© 2011 The Authors. JPP © 2011
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 90–100
92

Xiaoling Shen et al.
Praeruptorin A derivatives as P-gp modulators
a
O
O
O
O
O
O
O
O
O
O
O
O
OR2
OR2
OH
OH
OR2
OH
OH
OR2
3
O
O
MeO
MeO
13 R2=
16 R2=
17 R2=
O
10 R2=
11 R2=
MeO
MeO
O
O
O
MeO
14 R2=
15 R2=
O
O
MeO
MeO
Figure 3 Scheme for preparation of 10~17. Reagents and conditions: (a) CH2Cl2, DCC, DMAP, RnOH, reflux.
12 R2=
a
O
O
O
O
O
O
O
O
O
O
O
O
OH
OH
OR2
OR2
OH
OH
OR2
OR2
4
O
O
MeO
MeO
18 R2=
23 R2=
24 R2=
21 R2=
O
MeO
O
19 R2=
20 R2=
O
MeO
MeO
O
O
22 R2=
MeO
Figure 4 Scheme for preparation of 18~24. Reagents and conditions: (a) CH2Cl2, DCC, DMAP, RnOH, reflux.
incubator. Into a medium of HepG2/Dox, K562/Dox and KB
V1 cells, 1.2 mm doxorubicin, 0.1 mm doxorubicin and
500 ng/ml vinblastine were added separately to maintain the
drug resistance phenotype. All MDR cells were grown in
drug-free medium for at least seven days before testing.
Growth inhibitory assay
Approximately 5000 cells per well were seeded in 96-well
plates and incubated overnight. Cells were then treated with
various concentrations of drug for 72 h. Cell growth inhibi-
© 2011 The Authors. JPP © 2011
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 90–100
93

Praeruptorin A derivatives as P-gp modulators
Xiaoling Shen et al.
tory effects of drugs were determined by SRB assay and were
evaluated according to their respective IC50 values (concen-
tration of compound inhibiting 50% of cell growth) as
previously described.[13] Each experiment was performed
independently at least three times and results were expressed
as mean Ϯ standard deviation (SD).Solvents and media were
included as blank control.
Pgp was assayed based on phosphate-release catalysed by the
membrane vesicle with all other major membrane ATPases
inhibited as described previously.[13] Baseline controls con-
tained 100 mm Na3VO4, pH 10.0 and/or ethanol (vehicle) at a
maximum final concentration of 2% v/v. All experiments
were repeated three times.
Animals studies
Assessment of Pgp-multidrug resistance
reversing activity
BALB/c nude mice (female, 4–5 weeks old) were provided
by Experiment Animal Center of Guangzhou University of
TCM. All animal experiments were approved by the Animal
Ethics Committee at Guangzhou University of Chinese
Medicine (Document No. syxk(Yue)2008-0001) and per-
formed following Animal Care and Use guidelines set by
NIH (National Institute of Health, USA). After one week of
acclimatization to laboratory conditions, mice were subcu-
taneously injected with 3 ¥ 106 of KB V1 cells on the back to
establish tumour xenografts. Two days after tumour cell
injection, mice were randomized into four groups and were
treated by intraperitoneal injection of 10 mg/kg of paclitaxel
every other day for five doses (group B, n = 7), or 50 mg/kg
of DMDCK daily for ten doses (group D, n = 8), or a combi-
nation of five doses of 10 mg/kg of paclitaxel with ten doses
of 50 mg/kg of DMDCK (group C, n = 8). Mice treated with
vehicle (8.6% of Cremophor EL, 8% of EtOH and 5% of
DMSO in 0.9% saline, 10 ml/kg) were used as drug
negative control (group A, n = 7). Tumour size was moni-
tored by measuring two perpendicular diameters with a
caliper. Tumour volume (V) was calculated from: V = 1/
2(L ¥ W2), where L is the length and W is the width in
millimeters.
IC50 values of Pgp substrate anticancer agents vinblastine,
doxorubicin, puromycin and paclitaxel were tested in the
presence or absence of a Pgp modulator separately. The
ability of a pyranocoumarin in modulating Pgp was evalu-
ated by percentage reduction of IC50 value of an anticancer
agent achieved by 4 mm of the pyranocoumarin and was
expressed as R% = [1 - IC50(absence)/IC50(presence)] ¥ 100. The
more potent a Pgp modulator, the closer the %R is to 100%.
Verapamil was used as a positive control.
Drug accumulation assay
Approximately 1 ¥ 106 K562/Dox cells were suspended in
1 ml of medium containing 10 mm doxorubicin and various
concentrations of test substances and incubated at 37°C for
1 h. Cells were then washed with ice-cold PBS twice and
resuspended in 1 ml of ice-cold PBS. Cellular fluorescence of
doxorubicin was measured on a FACSCalibur flow cytometer
(Becton Dickinson, Mountain View, CA, USA). Verapamil
was the positive control.
MDR1 reactivity shift assay
Statistical analysis
Pgp reactivity to the conformation-sensitive mAb UIC2 was
performed as described previously.[13] HepG2/Dox cells were
pretreated with 5 mm ciclosporin (Pgp substrate), 1 mm
sodium vanadate (ATP inhibitor) or 5 mm pyranocoumarin
at 37°C for 10 min. UIC2 was then added to the mixture
and reacted at 37°C for another 10 min. After removal of the
unconjugated UIC2 molecules by washing and centrifuging,
UIC2 molecules that bound to Pgp were labelled with
goat anti-mouse IgG2a-PE and detected by using a FACS-
Calibur flow cytometer. Normal mouse IgG2a served as a
negative control. Pgp reactivity to UIC2 was evaluated by
variation in fluorescence intensity compared to modulator-
free control.
All results were expressed as mean Ϯ SD unless otherwise
noted. Effects of various treatments were analysed by using
the Kruskal–Wallis test. Bonferroni procedure was then used
to evaluate individual differences between treatments. The
Bonferroni procedure-adjusted P-values < 0.05 were consid-
ered statistically significant.
Results
Structural identification
Compounds 5–24 were enantiomers with zero specific
optical rotation value. Structural determination of pyrano-
coumarins was based on the MS and 1H NMR data, and on
comparing the NMR data with that of compounds reported
in the literature.[15,16] MS data and NMR data of newly syn-
thesized compounds 13, 16, 21, 23 and 24 are presented as
follow.
Pgp-ATPase activity assay
Pgp-enriched membrane vesicles were prepared from Pgp-
overexpressing K5652/Dox cells. The effect of 15, 16, 23 and
24 on both basal and substrate-stimulated ATPase activity of
© 2011 The Authors. JPP © 2011
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 90–100
94

Xiaoling Shen et al.
Praeruptorin A derivatives as P-gp modulators
13. C32H30O9. MS (ESI): 581 [M + Na]+. H NMR dppm
(CDCl3-300 MHz): 7.61 (1H, d, J = 9.6 Hz, 4-H), 7.36
(1H, d, J = 8.4 Hz, 5-H), 7.31~7.16 (10H, m, Ar-H), 6.78
(1H, d, J = 8.4 Hz, 6-H), 6.56 (1H, d, J = 4.8 Hz, 4′-H),
6.25 (1H, d, J = 9.6 Hz, 3-H), 5.23(1H, d, J = 4.8 Hz,
3′-H), 3.76, 3.75 (3H each, s, 2¥Ar-OCH3), 3.65, 3.50,
3.43 and 3.35 (1H each, d, J = 15.2 Hz, 2¥Ar-CH2-CO),
1.27 and 1.24 [3H each, s, 2′-(Me)2].
1
Table 1 Growth inhibitory effect of pyranocooumarins against HepG2/
Dox cells
Compound
No.
Compound
No.
IC50 (mM)
IC50 (mM)
PA (1)
5
6
7
8
10.84 Ϯ 1.58
15.01 Ϯ 3.60
18.57 Ϯ 2.48
29.23 Ϯ 6.81
22.02 Ϯ 5.19
20.11 Ϯ 4.29
19.87 Ϯ 10.46
18.73 Ϯ 1.49
26.31 Ϯ 1.41
19.87 Ϯ 10.46
2.48 Ϯ 0.52
14
15
16
17
18
19
20
21
22
23
24
72.62 Ϯ 0.30
76.33 Ϯ 2.22
58.36 Ϯ 2.81
45.69 Ϯ 7.28
46.35 Ϯ 3.83
16.48 Ϯ 0.06
28.66 Ϯ 2.02
49.54 Ϯ 10.45
62.54 Ϯ 4.17
86.38 Ϯ 2.48
72.29 Ϯ 5.56
1
16. C32H30O11. MS (ESI): 613 [M + Na]+. H NMR dppm
9
(CDCl3-300 MHz): 7.71 (2H, d, J = 8.5 Hz, 2¥Ar-6-H),
7.57 (1H, d, J = 9.6 Hz, 4-H), 7.54 (2H, s, 2¥Ar-2-H),
7.46 (1H, d, J = 8.5 Hz, 5-H), 6.90 (1H, d, J = 4.9 Hz,
4′-H), 6.87 (2H, d, J = 8.5 Hz, 2¥Ar-5-H), 6.85 (1H, d,
J = 8.5 Hz, 6-H), 6.14 (1H, d, J = 9.6 Hz, 3-H), 5.61 (1H,
d, J = 4.9 Hz, 3′-H), 3.90 (3H, s, Ar-OCH3), 3.89 (3H, s,
Ar-OCH3), 3.84(6H, s, 2¥Ar-OCH3), 1.62 and 1.47 [3H
each, s, 2′-(CH3)2].
10
11
12
10
13
IC50, concentration inhibiting cell growth by 50%. Cells were exposed to
drugs for 72 h. Values are means Ϯ SD of three experiments.
1
21. C32H30O9. MS (ESI): 581 [M + Na]+. H NMR dppm
(CDCl3-300 MHz): 7.61 (1H, d, J = 9.4 Hz, 4-H), 7.36
(1H, d, J = 8.5 Hz, 5-H), 7.20 and 7.12 (2H each, d,
J = 8.0 Hz, Ar-H), 6.82~6.76 (4H, m, Ar-H), 6.78 (1H, d,
J = 8.5 Hz, 6-H), 6.25 (1H, d, J = 4.8 Hz, 4′-H), 6.24 (1H,
d, J = 9.4 Hz, 3-H), 5.22 (1H, d, J = 4.8 Hz, 3′-H), 3.78
(6H, s, 2¥Ar-OCH3), 3.68 and 3.55 (1H each, d,
J = 15.4 Hz,Ar-CH2-CO), 3.50 (2H, s,Ar-CH2-CO), 1.21
[6H, s, 2′-(CH3)2].
marins to reduce Pgp-mediated drug resistance in MDR cells
was evaluated in HepG2/Dox cells by their ability to decrease
the IC50 value of anti-tumour agents that are Pgp substrates
and the results re presented in Table 2. PA was the least active
Pgp modulator tested.R% values of vinblastine,doxorubicin,
puromycin and paclitaxel achieved by 4 mM of PA were
30.9%, 15.5%, 37.5% and 68.8%, respectively. Verapamil, a
known Pgp modulator, showed activity higher than PA and
the corresponding R% values achieved by 4 mm of verapamil
were 60.6%, 70.9%, 87.7% and 79.6%, respectively. In
general, all semi-synthesized pyranocoumarins exhibited
MDR-reversing ability higher than, or equivalent to, PA.
Compounds 12, 15, 16, 23 and 24 achieved R%s of all four
anticancer agents by more than 90%, and were shown to be
the most active group of pyranocoumarins. Among these,
compounds 15 and 16 were the most active because they
achieved R%s of all four anticancer agents that were not only
significantly greater than PA (P < 0.001), but also signifi-
cantly greater than verapamil (P < 0.01). Compounds 10, 17
and 21, though less active than the above five, also exhibited
remarkable ability to reduce resistance of all four anticancer
drugs when compared with PA. Compounds 7, 19 and 20
were the least active pyranocoumarins because they exhibited
equivalent activity to PA.
1
23. C36H34O11. MS (ESI): 665 [M + Na]+. H NMR dppm
(CDCl3-300 MHz): 7.66 (2H, d, J = 15.9 Hz,
2¥-OCOCH=),7.61 (1H,d,J = 9.6 Hz,4-H),7.41 (1H,d,
J = 8.4 Hz, 5-H), 7.06 (2H, d, J = 8.4 Hz, 2¥Ar-6-H), 7.04
and 7.00 (1H each, s, 2¥Ar-2-H), 6.86 (1H, d, J = 8.4 Hz,
6-H), 6.82 and 6.81 (1H each, d, J = 8.4 Hz, 2¥Ar-5-H),
6.41 (1H, d, J = 3.9 Hz, 4′-H), 6.30 (2H, d, J = 15.9 Hz,
2¥Ar-CH=), 6.22 (1H, d, J = 9.6 Hz, 3-H), 5.51 (1H, d,
J = 3.9 Hz, 3′-H), 3.90, 3.88, 3.87, 3.86 (3H each, s, 4¥Ar-
OCH3), 1.53 and 1.43 [3H each, s, 2′-(CH3)2].
1
24. C32H30O11. MS (ESI): 613 [M + Na]+. H NMR dppm
(CDCl3-300 MHz): 7.76 (2H, d, J = 8.4 Hz, 2¥Ar-6-H),
7.57 (2H, s, 2¥Ar-2-H), 7.54 (1H, d, J = 9.6 Hz, 4-H),
7.42 (1H, d, J = 8.1 Hz, 5-H), 6.90 (2H, d, J = 8.4 Hz,
2¥Ar-5-H), 6.80 (1H, d, J = 8.1 Hz, 6-H), 6.58 (1H, d,
J = 3.6 Hz, 4′-H), 6.19 (1H, d, J = 9.6 Hz, 3-H), 5.62 (1H,
d, J = 3.6 Hz, 3′-H), 3.93 (9H, s, 3¥Ar-OCH3), 3.88 (3H,
s, OCH3), 1.58 and 1.47 [3H each, s, 2′-(CH3)2].
3Ј-O, 4Ј-O-aromatic acyl substituted 7,
8-pyranocoumarins increased doxorubicin
accumulation within K562/Dox cells
Pgp-multidrug resistance was reversed by
aromatic acyl substituted 7,
8-pyranocoumarins
We had reported previously that DCK[12] and DMDCK (15)
might increase cellular drug accumulation and slow down
Pgp-mediated drug efflux within tumour cells by directly
interacting with Pgp.[13,14] In this study, the effect of PA and
10–24 on doxorubicin accumulation within K562/Dox cells
was investigated and compared. Cells were incubated with
The effects of 5–24 on cell proliferation were tested firstly in
HepG2/Dox cells (Table 1). Compound 13 was then excluded
from follow-up study because it strongly inhibited tumour
cell growth (IC50 < 5 mm). The ability of other pyranocou-
© 2011 The Authors. JPP © 2011
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 90–100
95

Praeruptorin A derivatives as P-gp modulators
Xiaoling Shen et al.
Table 2 Percentage reduction of IC50 values of anticancer drugs in HepG2/Dox cells
Compound
Vinblastine
Doxorubicin
Puromycin
Paclitaxel
PA (1)a
5
6
7
8
9
10
11
12a
14a
15a
16
17
18
30.9 Ϯ 12.8
77.6 Ϯ 3.6*
76.7 Ϯ 5.8*
69.9 Ϯ 14.3
70.3 Ϯ 9.0
15.5 Ϯ 2.8
46.3 Ϯ 8.7
68.6 Ϯ 2.2
52.6 Ϯ 12.5
33.3 Ϯ 22.5
49.5 Ϯ 8.0
51.8 Ϯ 13.0
58.4 Ϯ 9.5
71.1 Ϯ 18.9
67.5 Ϯ 32.0
79.6 Ϯ 16.4
77.4 Ϯ 6.7
56.9 Ϯ 4.4
69.1 Ϯ 3.0***
57.9 Ϯ 2.2*
86.1 Ϯ 5.8***
75.5 Ϯ 6.6***
93.8 Ϯ 0.9***
94.6 Ϯ 1.9***###
99.4 Ϯ 0.0***###
99.1 Ϯ 0.1***###
66.1 Ϯ 4.9
74.5 Ϯ 12.9*
88.1 Ϯ 4.1***
77.8 Ϯ 8.5**
78.0 Ϯ 0.6***
78.1 Ϯ 4.9***
77.5 Ϯ 6.2***
94.3 Ϯ 0.6***
91.0 Ϯ 1.6***
99.2 Ϯ 0.3***###
85.8 Ϯ 5.7
95.5 Ϯ 1.5*** #
91.4 Ϯ 2.5*
95.9 Ϯ 3.3*** ##
87.3 Ϯ 3.8
##
96.2 Ϯ 1.6***###
##
79.6 Ϯ 5.4**
99.3 Ϯ 0.2***###
98.7 Ϯ 0.2***###
99.3 Ϯ 0.2*** ###
98.4 Ϯ 0.1*** ###
91.9 Ϯ 1.2**
85.7 Ϯ 2.4
##
98.6 Ϯ 0.5***
#
##
84.61 Ϯ 7.1***
92.1 Ϯ 3.9***
83.7 Ϯ 1.8***
56.6 Ϯ 10.0
70.7 Ϯ 9.2
71.8 Ϯ 6.3***
39.8 Ϯ 5.6
19
40.4 Ϯ 20.5
37.5 Ϯ 10.9
69.7 Ϯ 7.3
20
21
22
23
39.4 Ϯ 11.0
41.2 Ϯ 15.6
64.6 Ϯ 7.4
66.2 Ϯ 7.9
96.0 Ϯ 2.8***###
87.6 Ϯ 2.3***
85.9 Ϯ 2.8***
98.2 Ϯ 0.6***###
96.1 Ϯ 4.1***###
70.9 Ϯ 5.2***
96.1 Ϯ 1.2***
83.4 Ϯ 8.3***
96.1 Ϯ 0.8***
96.5 Ϯ 0.0***
86.1 Ϯ 2.3***
99.6 Ϯ 0.1*** ###
65.5 Ϯ 3.2
74.9 Ϯ 5.3
91.7 Ϯ 3.8***###
93.9 Ϯ 2.7***###
60.6 Ϯ 7.0
96.2 Ϯ 2.0*** ##
97.8 Ϯ 0.3*** ##
79.6 Ϯ 2.5
24
Verapamil
R% = [1 - IC50(absence)/IC50(presence)] ¥ 100, in which IC50 (presence) is IC50 (concentration inhibiting cell growth by 50%) of a drug in the presence of 4 mM of
a Pgp modulator, IC50 (absence) is IC50 of a drug in the absence of any Pgp modulator. Values are means Ϯ SD of three experiments. *P < 0.05, **P < 0.01,
or ***P < 0.001, compared with PA; #P < 0.05, ##P < 0.01, and ###P < 0.001, compared with verapamil. aData derived from Fong et al.[14]
2 μ
M
M
5 μ
120
100
80
60
40
20
0
10 μM
10
11
12
14
15
16
17
18
19
20
21
22
23
24
VRP
Compound
Figure 5 Increased cellular doxorubicin accumulation by (Ϯ)-praeruptorin A (PA) derivatives or verapamil in K562/Dox cells. Cells were incubated with
10 mM doxorubicin with or without addition of 2 mM, 5 mM or 10 mM PA derivative or verapamil at 37°C for 1 h. The effect of PA derivatives and verapamil
on cellular doxorubicin accumulation was analysed by flow cytometry. Increase of cellular fluorescence is shown as a percentage. Results are means Ϯ SD
values of three experiments.
10 mm doxorubicin in the presence or absence of 2 mm, 5 mm
or 10 mm of pyranocoumarin or verapamil at 37°C for 1 h.
Fluorescence intensity of cellular doxorubicin was analysed.
Figure 5 shows that all tested pyranocoumarins increased cel-
lular fluorescence in a dose-dependent manner. On the
whole, the ability of these compounds to increase cellular
doxorubicin accumulation was positively correlated with
their ability to reverse doxorubicin resistance in K562/Dox
cells.For example,15 or 16 at 5 mm increased cellular doxoru-
bicin fluorescence by more than 70%, whereas the corre-
sponding increase achieved by 18, 19 or 20 was less than 20%.
The above result suggested that these pyranocoumarins
© 2011 The Authors. JPP © 2011
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 90–100
96

Xiaoling Shen et al.
Praeruptorin A derivatives as P-gp modulators
might restore the drug sensitivity of Pgp-MDR cells through
increasing cellular drug accumulation.
(a)
12
Na3VO4
Ciclosporin
Control
IgG2a
15
16
3Ј-O, 4Ј-O-aromatic acyl substituted 7,
8-pyranocoumarins changed Pgp
reactivity to UIC2
24
23
Direct Pgp modulators may interact with Pgp on substrate
site(s) and thus decrease the binding opportunity of a trans-
port drug. They may also interact with Pgp on allosteric
site(s) that causes conformational change of Pgp that is unfa-
vorable for drug binding and transportation. The mode of
interaction between Pgp and its modulator can be detected by
MDR1 reactivity shift assay using conformationally sensitive
mAbUIC2. UIC2 preferably binds to Pgp that is associated
with a substrate or a competitive inhibitor. Binding of a com-
pound such as ciclosporin to the substrate-binding site(s) on
Pgp increases Pgp reactivity to UIC2 whereas binding of a
compound such as sodium vanadate to ATP site(s) on Pgp
decreases the reactivity.[17–19] We have previously reported that
DCK[12] may bind to Pgp allosterically and decrease UIC2
binding whereas DMDCK[15] may bind to the Pgp substrate
site and therefore increase UIC2 binding. In this study, the
effect of other pyranocoumarins on UIC2 binding to Pgp was
also studied and the results are shown in Figure 6. Com-
pounds 16 and 24 increased UIC2 binding to Pgp as did 15,
exhibiting substrate-like activity, whereas the others, includ-
ingPA,decreasedUIC2binding(Figure 6)asdid 12,inferring
an allosteric binding to Pgp.
100
101
102
103
104
24
(b)
FL2-H
120
80
Ciclosporin
16
15
40
0
23
22
–40
–80
14
1
19
21
20
17
18
101112
5 6
8 9
7
Na3VO4
Figure 6 Effect of (Ϯ)-praeruptorin A (PA) derivatives on Pgp reactivity
to mAb UIC2. HepG2/Dox cells were reacted with mAb UIC2 at 37°C, in
the presence or in the absence (control) of 1 mM Na3VO4, 5 mM
ciclosporin, or 5 mM PA derivative. Normal IgG2a was used as negative
control. UIC2 binding affinity to Pgp was detected by labelling with a
fluorescent secondary antibody and analysed by flow cytometry. (a) Pic-
tures from a typical experiment showed the cellular fluorescence varia-
tion in HepG2/Dox cells when cells were pretreated with 1 mM sodium
vanadate, or 5 mM 12, 15, 16, 23 and 24 or 5 mM ciclosporin. (b) Changes
in cellular fluorescence intensity caused by 5 mM ciclosporin, 1 mM NaVO4
or 5 mM PA derivative. Results are means Ϯ SD values of three
experiments.
3Ј-O, 4Ј-O-aromatic acyl substituted 7,
8-pyranocoumarins affected Pgp
ATPase activity
Compound 15 enhanced paclitaxel toxicity in
KB V1 xenograft model
As an ATP-dependent membrane transporter, Pgp-
mediated drug transport is accompanied by increased ATP
hydrolysis, therefore Pgp-catalysed ATP hydrolysis indirectly
reflects transport function of Pgp. Our previous study
showed that DCK[12] and DMDCK[15] inhibited substrate-
stimulated ATP hydrolysis of Pgp.[13,14] In this study, 16, 23
and 24 had also been studied for their effects on Pgp-
ATPase activity in membrane fractions prepared from Pgp-
overexpressing K562/Dox cells, with activities of other
major membrane ATPases suppressed. As shown in
Figure 7b, verapamil, a standard Pgp substrate, stimulated
ATP hydrolysis in a dose-dependent manner. In the pres-
ence of 1 mm of 16, 23 and 24, verapamil-stimulated Pgp-
ATPase activity was inhibited with notably reduced Vmax
but relatively stable Km, suggesting a primarily non-
competitive inhibition. Pyranocoumarins acted differently
on basal Pgp-ATPase activity. Figure 7a shows that 15 inhib-
ited Pgp-catalysed ATP hydrolysis and 24 stimulated
Pgp-catalysed ATP hydrolysis, both dose-dependently.
An in-vivo experiment was performed to evaluate the ability
of DMDCK[15] to enhance paclitaxel toxicity against tumour
growth in nude mice bearing KB V1 xenografts. Figure 8
shows average tumour volumes in different treatment groups
during a 12-day experimental period. Compared with the
vehicle-treated group (A), treatment with five doses of
10 mg/kg paclitaxel only slightly decreased tumour volume
(B).Treatment withtendoses of 50 mg/kg15didnotdecrease
tumour volume at all (D).However,treatment with 10 mg/kg
of paclitaxel every other day, together with 50 mg/kg/d of 15,
significantly decreased tumour volume (C); as shown in
Figure 8, six days of treatment decreased tumour volume by
48% whereas ten days of treatment decreased tumour volume
by 56% (P < 0.05).
Discussion
In our study, 7,8-pyranocoumarins bearing C-3′ and/or
C-4′ aromatic acyl(s) exhibited an ability higher than, or
© 2011 The Authors. JPP © 2011
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 90–100
97

Praeruptorin A derivatives as P-gp modulators
Xiaoling Shen et al.
(a)
1000
800
600
400
200
0
A
B
C
D
35
24 16
23 15
30
25
20
15
*
(μM)
*
9
0
5
10
0
15
20
10
3
5
7
11
Days from the start of drug treatment
Vmax
Km(μM)
36.93 1.08 0.70 0.13
(b)
15 1μM 22.39 0.98 0.89 0.22
Figure 8 DMDCK enhanced growth inhibitory effect of paclitaxel on
KB-3-1 tumour xenografts. Tumour-bearing mice were treated with
vehicle (a), paclitaxel (b) or DMDCK (d), or the combination of paclitaxel
and DMDCK (c). Tumour size was monitored by measuring two perpen-
dicular diameters with a caliper. Tumour volume (V) was calculated from
equation: V = 1/2(L ¥ W2), where L is the length and W is the width in mil-
limetres. Data were expressed as means Ϯ SEM. *P < 0.05, compared
with group A.
×
16 1μM
29.21 0.71 0.33 0.07
23 1μM 23.59 0.36 0.48 0.06
24 1μM 31.05 0.62 0.33 0.06
50
40
30
20
10
0
20
40
60
80
Verapamil concentration (μM)
and 3,4-dimethoxybenzolyoxy > cinnamoyloxy and 4-
methoxybenzeneacetyloxy > 4-methoxybenzoyloxy and 4-
methoxycinamoyloxy > benzoyloxy and benzeneacetyloxy.
Thirdly, the configuration of acyls may also affect the
MDR-reversing ability of pyranocoumarins. Compounds
10, 11, 12 and 14, bearing two identical aromatic acyls in
3′,4′-cis-configuration, are relatively more active than
their trans-isomers 18, 19, 20 and 22. Compounds 5–9,
having one aromatic acyl and one aliphatic acyl in
3′,4′-trans-configuration, are less active than verapamil
(Table 2).
Pgp modulators have diverse structures but common char-
acteristics such as hydrophobic points and hydrogen bond
accepter points in certain special arrangements. Results
from a pharmacophore search for DCK,[12] MMDCK[14]
and DMDCK,[15] using a verapamil-based pharmacophore
template, had been performed previously and the results
might give an explanation for the role of aromatic acyls.[14]
DMDCK[15] has four functional groups (two aromatic rings
and two hydrogen bond accepters) simultaneously involved
in interaction with Pgp in different binding modes, and
therefore,are the most active group of pyranocoumarins.The
3-methoxy on cinnamoyloxy may serve as a hydrogen bond
accepter whereas aromatic acyls may serve as hydrophobic
points to interact with Pgp.DCK,bearing no methoxy on cin-
namoyloxy, has only three groups simultaneously involved.
MMDCK, though containing a methoxy on cinnamoyloxy
but at a different site (4-methoxy), partially matches the
pharmacophore with four functional groups. Therefore
DCK and MMDCK are relatively less active than DMDCK.
Compounds 16, 23 and 24 have a 3-methoxy and 4-methoxy
on aromatic acyls as has DMDCK, may also use 3-methoxy as
Figure 7 Effect of pyranocoumarins on basal and substrate-stimulated
ATPase activity of Pgp. Membrane vesicles were prepared from Pgp-
overexpressing K562/Dox cells. Pgp-ATPase activity was measured with
other major membrane ATPases suppressed. (a) Inhibitory effect of 15,
16, 23 or 24 on verapamil-stimulated Pgp-ATPase activity. (b) Inhibitory
effect of 15, 16, 23 or 24 on verapamil-stimulated Pgp-ATPase activity.
equivalent to, PA to reverse Pgp-MDR in HepG2/Dox cells
(Table 2). Among the compounds, DCK,[12] DMDCK,[15] 16,
23 and 24 showed higher activity than other pyranocou-
marins and verapamil: as a matter of fact, they belong to the
most active group of pyranocoumarins. When used alone at
4 mm, they did not affect cell proliferation, but significantly
restored the chemo-sensitivity of HepG2/Dox cells to anti-
cancer agents that are Pgp substrates (Table 2). In addition,
DMDCK remarkably enhanced paclitaxel toxicity to KB-3-1
tumour growth in vivo, showing its potential for medical
usage as a chemosensitizer for treatment of drug-resistant
tumours.
Structure–activity relationship studies reveal that side
chains at the C-3′ and C-4′ site play a very important role in
keeping or enhancing MDR-reversal ability of a 7,8-
pyranocoumarin. Firstly, aromatic acyls are more active
than aliphatic acyls. PA has only two aliphatic acyls and is
the least active whereas the other pyranocoumarins bearing
aromatic acyl(s) are more or less more potent than PA. Sec-
ondly, a methoxy group on aromatic acyls also contributes
to the activity. Based on data presented in Table 2, the
contribution of aromatic acyloxy to MDR-reversing
ability follows the order: 3,4-dimethoxycinnamoyloxy
© 2011 The Authors. JPP © 2011
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 90–100
98

Xiaoling Shen et al.
Praeruptorin A derivatives as P-gp modulators
a hydrogen bond accepter to interact with Pgp, and thus
belong to the most active of pyranocomarins. Compounds 17
and 21 just have a 4-methoxy on the aromatic acyl as does
MMDCK[14] and thus have comparable ability to 14.
Conclusions
We show here that 3′, 4′-aromatic acyloxy substituted 7,8-
pyranoucoumarins could serve as a new class of Pgp modu-
lator. They may reverse Pgp-MDR through directly binding
to Pgp and therefore hinder the binding and transporta-
tion of substrate drug. As Pgp modulators with a 7,8-
pyranocoumarin core structure, their MDR-reversal activity
is closely related to the type and configuration of C-3′ and
C-4′ side chains. 3,4-Dimethoxy substituted aromatic acyls
are shown to be more active than other acyls in enhancing
the MDR-reversal activity of pyranocoumarin. DMDCK[15]
bears two 3,4-dimethoxycynamoylxoys at the C-3′ site and
C-4′ site of 7,8-pyranocoumarin in cis-configuration, exhib-
ited remarkable MDR-reversing activity both in vitro
and in vivo and thus exhibited promising prospect in
medical usage. Three newly synthesized compounds 16, 23
and 24, all bearing methoxy substituted aromatic acyls at
the C-3′ site and C-4′ site of pyranocoumarins, also showed
exciting Pgp-modulating ability and are worth further
investigation.
Pyranocoumarins dose-dependently increased doxorubi-
cin accumulation within Pgp-overexpressing K562/Dox cells.
Further studies revealed that they may directly interact with
Pgp in different manners. Results from the MDR1 reactivity
shift assay, which investigated drug–Pgp binding modes,
suggested that 15, 16 and 24 might bind to Pgp substrate
site(s) whereas the others may bind to Pgp allosteric site(s)
and cause Pgp conformational change. Assay for basal Pgp-
ATPase activity provided additional information about the
drug–Pgp interaction (Figure 7a). DMDCK[15] inhibited
basal Pgp-ATPase activity, implying that it may be an inhibi-
tor of Pgp that occupies the transport substrate binding site of
Pgp but can not be transported. Compound 24 dose-
dependently stimulated basal Pgp-ATPase activity, acting as a
typical Pgp transport substrate. Compound 16 stimulated
basal Pgp-ATPase activity at a concentration less than 2 mm,
but with increase in drug concentration, Pgp-catalysed ATP
hydrolysis slowed down. This suggested that 16 is partly a
transport substrate of Pgp. Compound 23 moderately stimu-
lated ATP hydrolysis at a concentration lower than 1.5 mm
and slowed down ATP hydrolysis at higher concentration,
suggesting that 23 might be a Pgp transport substrate but at
the same time also modifies Pgp-substrate intermediate com-
plexes to a less active form. All four compounds exhibited an
inhibitory effect on verapamil-stimulated ATP hydrolysis,
with decreased Vmax and relatively constant Km, suggesting
a non-competitive mode of inhibition. This means that
pyranocoumarins may simultaneously bind with substrate
drugs to Pgp but at different sites and therefore impair Pgp-
mediated drug transportation.
Declarations
Conflict of interest
The Author(s) declare(s) that they have no conflicts of
interest to disclose.
Funding
This work was partly supported by a Faculty Research Grant
of Hong Kong Baptist University (FRG/07-08/II24).
reversal using modulators of MDR and
the role of MDR modulators in influ-
against cancer? J Natl Cancer Inst 2003;
95: 255–257.
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