Seco-4-methyl-DCK derivatives as potent chemosensitizers

Article abstract of DOI:10.1016/j.bmcl.2018.11.023

Twenty-five seco-4-methyl-DCK derivatives were designed, synthesized and evaluated for chemoreversal activity when combined with paclitaxel or vincristine in two drug-resistant cancer cell lines (A2780/T and KB-V) respectively. Most of the new compounds displayed moderate to significant MDR reversal activities in the P-gp overexpressing A2780/T and KB-V cells. Especially, compounds 7o and 7y showed the most potent chemosensitization activities with more than 496 and 735 reversal ratios at a concentration of 10 μM. Unexpectedly the newly synthesized compounds did not show chemosensitization activities observed in a non-P-gp overexpressing cisplatin resistant human ovarian cancer cell line (A2780/CDDP), implying that the MDR reversal effects might be associated with P-gp overexpression. Moreover, these compounds did not exhibit significant antiproliferative activities against nontumorigenic cell lines (HUVEC, HOSEC and T29) compared to the positive control verapamil at the tested concentration, which suggested better safety than verapamil. The pharmacological actions of the compounds will be studied further to explore their merit for development as novel candidates to overcome P-gp mediated MDR cancer.

Full text of DOI:10.1016/j.bmcl.2018.11.023

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Seco-4-methyl-DCK derivatives as potent chemosensitizers
a,e
a,e
a,d
a
a
b
Yalan Guo , Ke Wang , Xiaoyu Chen , Haihong Li , Qi Wan , Susan Morris-Natschke ,
b,c,⁎
a,⁎
Kuo-Hsiung Lee
b
c
, Ying Chen
a
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, China
Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, USA
Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
Department of Materials Science & Engineering, Faculty of Engineering, Monash University, Clayton, VIC 3800, Australia
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
Twenty-five seco-4-methyl-DCK derivatives were designed, synthesized and evaluated for chemoreversal activity
when combined with paclitaxel or vincristine in two drug-resistant cancer cell lines (A2780/T and KB-V) re-
spectively. Most of the new compounds displayed moderate to significant MDR reversal activities in the P-gp
overexpressing A2780/T and KB-V cells. Especially, compounds 7o and 7y showed the most potent chemo-
sensitization activities with more than 496 and 735 reversal ratios at a concentration of 10 μM. Unexpectedly the
newly synthesized compounds did not show chemosensitization activities observed in a non-P-gp overexpressing
cisplatin resistant human ovarian cancer cell line (A2780/CDDP), implying that the MDR reversal effects might
be associated with P-gp overexpression. Moreover, these compounds did not exhibit significant antiproliferative
activities against nontumorigenic cell lines (HUVEC, HOSEC and T29) compared to the positive control ver-
apamil at the tested concentration, which suggested better safety than verapamil. The pharmacological actions
of the compounds will be studied further to explore their merit for development as novel candidates to overcome
P-gp mediated MDR cancer.
Seco-4-methyl-DCK
MDR reversal activity
Chemosensitizer
P-gp
Multidrug resistance (MDR), described first by Biedler and Riehm in
970, leads to over 90% failure in the treatment of metastatic cancer,
as chemotherapy sensitizers.
1
Fong et al. reported that ( ± )-praeruptorin A (PA), a naturally oc-
curring pyranocoumarin, reversed P-gp mediated MDR, while its 3′,4′-
because the cancer cells resist several structurally unrelated drugs si-
1
–3
multaneously.
While several factors can lead to MDR, the over-
modified
khellactone
analog,
( ± )-3′-O,4′-O-bis(3,4-dimethox-
expression of ABC (ATP Binding Cassette) transporters is one of the
most probable. Among these transporters, the most prevalent is P-gly-
ycinnamoyl)-cis-khellactone (DMDCK) (Fig. 1), was even more potent
with reversal ratios ranging from 110 − 167 fold when combined at a
concentration of 4 μM with different anticancer drugs in MDR HepG2/
4
coprotein (P-gp, also known as MDR1). Using the energy of ATP hy-
1
3,14
drolysis, P-gp transports hundreds of substrates, including various an-
ticancer drugs, out of cells. The resultant downregulation in the
Dox cell lines.
To potentially improve the druggability, we designed
a series of seco-4-methyl-DCK derivatives (Fig. 2), which lacked the C-
ring and two chiral centers of PA and its khellactone analogs. Ad-
ditionally, because the prior study indicated that 3′,4′-acyl groups were
important to maintain reversal activity, we also introduced variously
substituted benzoyl and cinnamoyl acyl groups at the 8-position of the
coumarin, as well as including an ethyl or isopropyl ether at the 7-
position. The 25 new synthetic coumarin derivatives (7a–y) were
screened as chemosensitizers in comparison with the first-generation P-
gp inhibitor verapamil used as a positive control.
5
–8
intracellular concentration of anticancer drugs can lead to MDR.
Therefore, the development of P-gp inhibitors is one of the most ef-
fective strategies to overcome MDR mediated by P-gp overexpression.
Presently, although three generations of small molecule P-gp inhibitors
9
have been identified, none have been approved as a drug for clinical
use. Unfortunately, a phase III clinical trial with tariquidar, a third-
generation P-gp inhibitor, was terminated due to an undesirable ben-
1
0,11
efit/risk ratio.
Although another third-generation P-gp inhibitor
11,12
HM30181 is currently in Phase I-II clinical trials,
efforts continue to
As shown in Scheme 1, a Duff reaction of 1 gave 2, and the gen-
erated aldehyde was protected to produce 3. The 7-hydroxyl of 3 was
identify and develop highly effective P-gp inhibitors with low toxicity
⁎
Corresponding authors at: Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-
568, USA (K.-H. Lee). Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203, China (Y. C.).
E-mail addresses: khlee@unc.edu (K.-H. Lee), yingchen71@fudan.edu.cn (Y. Chen).
7
e
Y.-L.G. and K.W. are co-first-authors and contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2018.11.023
Received 15 August 2018; Received in revised form 8 November 2018; Accepted 11 November 2018
0960-894X/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Guo, Y., Bioorganic & Medicinal Chemistry Letters, https://doi.org/10.1016/j.bmcl.2018.11.023

Y. Guo et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Table 1
Cytotoxicity in non-tumorigenic and cancer cell lines.a
Compound
Survival Ratio (%)
HOSEC
HUVEC
T29
A2780/T
KB-V
A2780/CDDP
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
97.24
78.26
78.25
50.10
73.45
68.42
72.01
78.15
86.71
64.65
76.03
79.79
92.23
90.70
84.88
75.96
70.48
106.85
86.31
61.73
79.80
87.51
90.64
105.03
93.59
80.18
82.82
24.04
96.31
87.23
86.33
81.02
85.01
79.76
83.36
88.78
99.71
77.12
78.44
76.00
84.72
101.03
93.77
84.78
79.66
84.17
72.67
73.08
75.63
82.25
83.70
95.86
98.90
84.66
82.07
20.98
106.35
88.58
94.62
72.61
91.34
83.38
86.14
89.51
103.19
86.09
97.25
87.07
98.67
105.99
98.91
90.75
85.32
101.12
90.94
81.53
91.21
92.68
96.04
104.38
90.40
88.57
81.85
10.80
97.05
93.41
100.64
78.72
101.38
76.50
83.07
84.07
97.63
95.81
84.02
95.14
98.94
102.49
83.57
102.17
102.01
101.86
105.70
94.58
95.16
100.55
104.30
102.74
98.09
94.64
85.34
ND
103.72
ND
117.41
ND
ND
ND
ND
ND
ND
ND
ND
ND
g
h
i
ND
ND
ND
ND
Fig. 1. Structures of PA and DMDCK.
ND
ND
j
ND
ND
k
l
ND
ND
ND
ND
m
n
o
p
q
r
ND
ND
ND
ND
104.60
ND
122.78
ND
ND
ND
ND
ND
s
ND
ND
t
96.14
ND
65.55
ND
Fig. 2. Seco-C-ring strategy for new derivatives.
u
v
w
x
y
ND
ND
ND
ND
reacted with bromoethane or 2-bromopropane to give 4a and 4b, re-
spectively. Deprotection regenerated the 8-aldehydes of 5a–b. Key in-
termediates 6a–b were synthesized by reduction of the aldehyde to a
hydroxymethyl group. Finally, 25 seco-4-methyl-DCK derivatives
ND
ND
108.46
ND
131.10
ND
6b
Verapamil
Cisplatin
75.58
ND
113.84
ND
(
7a–y) were prepared via a esterification reaction between 6a–b and
various aromatic acids catalyzed by 4-dimethylaminopyridine (DMAP)
and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
a
The survival ratio was determined in the presence of 10 μM compound and
expressed as the mean of three independent experiments. ND: Not determined.
(
EDCI).
Target compounds 7a–y were screened for cytotoxicity at a con-
their MDR reversal effects in the paclitaxel-resistant A2780/T cell line.
The reversal ratio (r.r.) was defined as IC50(drug only)/IC50(drug with syn-
thesized compound), as a standard of the evaluation. As shown in Table 2,
centration of 10 μM in an MTT assay against three non-tumorigenic cell
lines (HOSEC, HUVEC, and T29), paclitaxel- or cisplatin-resistant
human ovarian cancer cell lines (A2780/T and A2780/CDDP) and a
vincristine-resistant human oral epidermoid cancer cell line (KB-V).
Verapamil was used as a positive control. As shown in Table 1, survival
ratios of the above six cell lines ranged from 50% to 131% after
treatment with 10 μM of 7a–y. Most test compounds produced higher
survival ratios than those with verapamil (76–114%). The data imply
that some seco-4-methyl-DCK derivatives might have lower toxicity and
higher safety than verapamil.
1
8 compounds exhibited moderate to significant MDR reversal activity
(
r.r. > 50), and four compounds (7a, 7o, 7t, and 7y) exhibited greater
reversal activity (r.r. 224–735) than verapamil (r.r. = 211). Notably,
compounds 7o and 7y with an (E)-3-(4-methoxyphenyl)acryloyl group
at the 8-position showed the most potent reversal effects with 735- and
4
96-fold reversal ratios. In other words, the IC50 of paclitaxel against
A2780/T was 3.97 μM, while those of paclitaxel when combined with
o or 7y were 5.4 and 8.0 nM, respectively. Thus, the C-ring and chiral
centers of the 4-methyl DCK scaffold were not necessary to produce
7
Next, the newly synthesized compounds 7a − y at 10 μM were co-
administered with different concentrations of paclitaxel to evaluate
Scheme 1. Syntheses of seco-4-methyl-DCK derivatives (7a–y). Reagents and conditions: (i) hexamethylenetetramine (2.5 equiv), HOAc, 95 °C, 4.5 h; then HCl (conc.
HCl:H
2
O = 84:100 v/v), 70 °C, 1 h, 22%; (ii) ethylene glycol (2 equiv), p-toluenesulfonic acid (catalytic amount), benzene, circumfluence to repel water, 2 h, 48%;
(
iii) bromoethane or 2-bromopropane (1.2 equiv), K
2
CO (3 equiv), KI (0.1 equiv), acetone, reflux, 5 h, 81–93%; (iv) 2 N HCl (30 mL/mmol compound 4a or 4b), rt,
3
2
4 h, 85–90%; (v) NaBH
4
(1.4 equiv), MeOH, rt, 2 h, 95–99%; (vi) aromatic acids (1.1 equiv), EDCI (1.2 equiv), DMAP (0.5 equiv), DCM, 2 h, 0 °C ∼ rt, 39–82%.
2

Y. Guo et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Table 2
the double bond of the acryloyl moiety was saturated; analog 7e with a
4-methoxyphenylpropanoyl ester at the 8-position was the least potent
compound.
MDR reversal activity of 7a–y (10 µM) against MDR cell lines.
Compd
A2780/T
A2780/CDDP
KB-V
Paclitaxel (nM)
Cisplatin (µM)
VCR (nM)
Next, compounds 7a, 7o, 7t, and 7y, which displayed high MDR
reversal activity in the A2780/T cell line, were further evaluated in two
additional drug-resistant cell lines KB-V and A2780/CDDP, when co-
administered with vincristine (VCR) and cisplatin, respectively. To our
surprise, the four compounds showed strong drug-resistant reversal
activity with high reversal ratios in KB-V cells, but no significant effects
in A2780/CDDP cells. The r.r. values of 7o and 7y were 793 and 1257,
higher than that of verapamil (5 0 9) in KB-V, but only 0.76 and 0.78,
slightly lower than that of verapamil (1.07) in A2780/CDDP cells. The
IC50 values of VCR against the KB-V cell line decreased dramatically
from 666.30 nM alone to 0.84 nM and 0.53 nM when co-administered
with 7o or 7y, respectively. The results were consistent with those for
paclitaxel in A2780/T cells. In contrast, no reversal effects were seen in
A2780/CDDP cells.
IC50
r.r.
IC50
r.r.
IC50
r.r.
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
17.7
64.7
54.9
129.2
668.6
71.2
39.2
83.8
77.4
155
224
61
51.97
0.83
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.76
ND
ND
ND
ND
0.86
ND
ND
ND
ND
0.78
1.07
ND
2.12
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.84
ND
ND
ND
ND
4.99
ND
ND
ND
ND
0.53
1.31
ND
ND
666.3
314
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
793
ND
ND
ND
ND
134
ND
ND
ND
ND
1257
509
ND
ND
3173
a
ND
72
ND
31
ND
6
ND
56
ND
g
h
i
101
47
ND
ND
51
ND
j
26
ND
k
l
74.2
25.1
40.8
288.4
5.4
54
ND
158
97
ND
m
n
o
p
q
r
ND
14
ND
P-gp over-expression readily causes MDR in many cancer cell lines.
Therefore, we measured the P-gp expression in the five tested cancer
cell lines, two drug-sensitive (A2780 and KB) and three drug-resistant
735
86
56.59
ND
46.4
42.9
20.2
24.6
11.2
41.8
88.6
55.5
124.7
8
93
ND
(
A2780/T, A2780/CDDP and KB-V), using Western blot analysis. As
197
161
354
95
ND
s
ND
shown in Fig. 3, we observed P-gp overexpression in the A2780/T and
KB-V drug-resistant cell lines, but not in the cisplatin-resistant A2780/
CDDP or A2780 and KB drug-sensitive cell lines. This outcome sup-
ported our above findings that the seco-C khellactone analogs were
active as chemosensitizers in A2780/T and KB-V but not A2780/CDDP
cell lines. A close relationship exists between the chemoreversal activity
and P-gp over-expression in the drug-resistant cancer cell lines. We are
performing studies on the detailed pharmacological mechanism and
will report our findings in the future.
t
50.13
ND
u
v
w
x
y
45
ND
72
ND
32
ND
496
211
1418
ND
ND
54.98
40.11
ND
Verapamil
Paclitaxel
Cisplatin
VCR
18.8
3970
ND
b
c
43.11
ND
17
d
ND
ND
In brief, we designed and synthesized 25 seco-4-methyl-DCK deri-
vatives and evaluated their ability to chemosensitize A2780/T and KB-
V cell lines toward paclitaxel and vincristine. None of the compounds
displayed significant cytotoxicity in HOSEC, HUVEC, T29, A2780/T,
A2780/CDDP and KB-V cell lines, and some of them had stronger MDR
reversal activity compared with verapamil. Among them, compounds
a
b
ND: Not determined.
Drug resistance ratio of A2780/T, defined as IC50 (A2780/T)/IC50 (A2780) of
paclitaxel = 3970 nM/2.8 nM.
c
Drug resistance ratio of A2780/CDDP, defined as IC50 (A2780/CDDP)/IC50
(
A2780) of cisplatin = 43.11 µM/2.55 µM.
d
Drug resistance ratio of KB-V, defined as IC50 (KB-V)/IC50 (KB) of
7
o and 7y showed the most potent reversal activity. When paclitaxel
VCR = 666.3 nM/0.21 nM.
and vincristine were administrated together with 10 μM of 7o or 7y, the
reversal ratios (735 and 496 in A2780/T; 793 and 1257 in KB-V) were
higher than those of verapamil (211 and 509). A preliminary structure-
activity relationship (SAR) analysis indicated that MDR reversal activity
was maintained with an appropriately substituted bicyclic coumarin
core. Additionally, various benzoyl and cinnamoyl aromatic esters,
particularly (E)-3-(4-methoxyphenyl)acryloyl, at the 8-position were
favorable to the reversal ability. Interestingly, these compounds did not
reverse resistance to cisplatin in the non-P-gp overexpressing A2780/
CDDP cell line, indicating a close relationship between the MDR re-
versal activity of these newly synthesized compounds and the P-gp
expression level of drug-resistant cancer cell lines. Further studies will
be carried out to investigate the detailed mechanistic and pharmaco-
kinetic properties of these seco-4-methy-DCK derivatives as potential
chemosensitizer.
Fig. 3. P-gp expression in drug-sensitive and coupled drug-resistant cancer cell
lines.
chemosensitization. The MDR reversal activity was not affected sig-
nificantly or consistently by the choice of ethoxy or isopropoxy at the 7-
position of the coumarin. As mentioned above, the most potent com-
pounds contained methoxy-substituted cinnamoyl esters at the cou-
marin 8-position (7o and 7y), while the next most potent compounds
contained 3,4,5-trimethoxybenzoyl and 3-(dimethylamino)benzoyl es-
ters (7t and 7a, respectively). Introduction of electron-donating moi-
eties on the aromatic ring of the ester group generally improved the
reversal activity, while electron-withdrawing groups decreased the ac-
tivity [e.g., 7g (3,4-dimethoxybenzoyl) > 7d (4-chloro-3-nitrobenzyl);
Acknowledgments
Many thanks to the grant from “Major New Drugs Innovation and
Development” of National Science and Technology Major Projects
(
2018ZX09711002-007-005) for supporting this study.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2018.11.023.
7
y
(p-methoxycinnamoyl) > 7w
(cinnamoyl) > 7x
(p-chlor-
ocinnamoyl)]. In addition, the potency decreased dramatically when
3

Y. Guo et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
References
screening: a perspective from bioavailability enhancement. Pharmacol Res.
2
003;48:347–359.
9
.
Yang K, Wu J, Li X. Recent advances in research on P-glycoprotein inhibitors. Biosci
Trends. 2008;2:137–146.
1
.
Biedler JL, Riehm H. Cellular resistance to actinomycin D in Chinese hamster cells in-
vitro: cross-resistance, radiographic, and cytogenetic studies. Cancer Res.
1
0. Pusztai L, Wagner P, Ibrahim N, et al. Phase II study of tariquidar, a selective P-
glycoprotein inhibitor, in patients with chemotherapy-resistant, advanced breast
carcinoma. Cancer. 2005;104:682–691.
1
970;30:1174–1184.
2
3
4
5
6
7
8
.
.
.
.
.
.
.
Longley DB, Johnston PG. Molecular mechanisms of drug resistance. J Pathol.
2
005;205:275–292.
1
1
1. Clinical trials. https://www.clinicaltrials.gov/.
Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-de-
pendent transporters. Nat Rev Cancer. 2002;2:48–58.
2. Kwak JO, Lee SH, Lee GS, et al. Selective inhibition of MDR1 (ABCB1) by HM30181
increases oral bioavailability and therapeutic efficacy of taxol. Eur J Pharmacol.
Hayes JD, Wolf CR. Molecular mechanisms of drug-resistance. Biochem J.
2
010;627:92–98.
1
990;272:281–295.
1
1
3. Fong WF, Shen XL, Globisch C, et al. Methoxylation of 3',4'-aromatic side chains
improves P-glycoprotein inhibitory and multidrug resistance reversal activities of
Loo TW, Clarke DM. Recent progress in understanding the mechanism of P-glyco-
protein-mediated drug efflux. J Membr Biol. 2005;206:173–185.
7
,8-pyranocoumarin against cancer cells. Bioorg Med Chem. 2008;16:3694–3703.
Fodale V, Pierobon M, Liotta L, Petricoin E. Mechanism of cell adaptation when and
how do cancer cells develop chemoresistance? Cancer J. 2011;17:89–95.
Aller SG, Yu J, Ward A, et al. Structure of P-glycoprotein reveals a molecular basis for
poly-specific drug binding. Science. 2009;323:1718–1722.
4. Wu JY-C, Fong WF, Zhang JX, et al. Reversal of multidrug resistance in cancer cells
by pyranocoumarins isolated from Radix Peucedani. Eur J Pharmacol.
2
003;473:9–17.
Varma MV-S, Ashokraj Y, Dey CS, Panchagnula R. P-glycoprotein inhibitors and their
4