Structure-activity relationship and molecular mechanisms of ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H -chromene-3- carboxylate (CXL017) and its analogues

Article abstract of DOI:10.1021/jm200764t

Multidrug resistance (MDR) in cancer is a phenomenon in which administration of a single chemotherapeutic agent causes cross-resistance of cancer cells to a variety of therapies even with different mechanisms of action. Development of MDR against standard therapies is a major challenge in the treatment of cancer. Previously we have demonstrated a unique ability of CXL017 (5) to selectively target MDR cancer cells and synergize with mitoxantrone (MX) in HL60/MX2 MDR cells. Here we expand its scope and demonstrate that 5 can synergize with both vincristine and paclitaxel in three different MDR cell lines (HL60/DNR, K562/HHT300, and CCRF-CEM/VLB100). We also demonstrate that 5 has potent cytotoxicity in the NCI-60 panel of cell lines with an average IC ₅₀ of 1.04 μM. In addition, 5 has a unique mechanism of action in comparison with standard agents in the NCI database based on COMPARE analysis. Further structure-activity relationship study led to the development of a more potent analogue, compound 7d, with an IC₅₀ of 640 nM in HL60/MX2. Additionally, one enantiomer of 5 is 13-fold more active than the less active enantiomer. Taken together, our study has led to the discovery of a series of analogues that selectively target drug-resistant cancer cells with the potential for the treatment of drug-resistant cancers.

Full text of DOI:10.1021/jm200764t

ARTICLE
pubs.acs.org/jmc
StructureꢀActivity Relationship and Molecular Mechanisms of Ethyl
2-Amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-
chromene-3-carboxylate (CXL017) and Its Analogues
||
Sonia G. Das,† Balasubramanian Srinivasan,†, David L. Hermanson,† Nicholas P. Bleeker,† Jignesh M. Doshi,†
Ruoping Tang,‡ William T. Beck,§ and Chengguo Xing*,†
†Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, 308 Harvard Street SE, Minneapolis,
Minnesota 55455, United States
‡Universitꢀe Pierre et Marie Curie, UMRs 872, Equipe 18, 1 Place du Parvis de Notre-Dame, 75181 Paris Cedex 04, France
§Department of Biopharmaceutical Sciences and Cancer Center, University of Illinois at Chicago, 833 S. Wood Street, Chicago,
Illinois 60605, United States
S Supporting Information
b
ABSTRACT:
Multidrug resistance (MDR) in cancer is a phenomenon in which administration of a single chemotherapeutic agent causes cross-
resistance of cancer cells to a variety of therapies even with different mechanisms of action. Development of MDR against standard
therapies is a major challenge in the treatment of cancer. Previously we have demonstrated a unique ability of CXL017 (5) to
selectively target MDR cancer cells and synergize with mitoxantrone (MX) in HL60/MX2 MDR cells. Here we expand its scope
and demonstrate that 5 can synergize with both vincristine and paclitaxel in three different MDR cell lines (HL60/DNR, K562/
HHT300, and CCRF-CEM/VLB100). We also demonstrate that 5 has potent cytotoxicity in the NCI-60 panel of cell lines with an
average IC50 of 1.04 μM. In addition, 5 has a unique mechanism of action in comparison with standard agents in the NCI database
based on COMPARE analysis. Further structureꢀactivity relationship study led to the development of a more potent analogue,
compound 7d, with an IC50 of 640 nM in HL60/MX2. Additionally, one enantiomer of 5 is 13-fold more active than the less active
enantiomer. Taken together, our study has led to the discovery of a series of analogues that selectively target drug-resistant cancer
cells with the potential for the treatment of drug-resistant cancers.
’ INTRODUCTION
cell death mechanism.7 Antiapoptotic members, such as Bcl-2,
Bcl-XL, and Mcl-1, and proapoptotic members, including Bax,
Bak, and Bad, antagonize each other to maintain the balance
between cell survival and death.8 Overexpression of the anti-
apoptotic Bcl-2 family proteins in cancer cells disrupts this
balance and prevents cell death.9 Indeed, over 60ꢀ90% of all
cancers reveal increased expression of the antiapoptotic Bcl-2
family proteins.10,11 Therefore, the antiapoptotic Bcl-2 family
proteins are promising targets for the development of new
therapies to treat drug-resistant malignancies.12
Over the years, tremendous progress has been made in
designing inhibitors for the antiapoptotic Bcl-2 family proteins.13
Many of these inhibitors are currently in clinical trials for the
treatment of various cancers.14 Our research has focused on a
putative inhibitor of the antiapoptotic Bcl-2 family proteins
Cancer is a disease that has been known to humans for
centuries. Although there has been a substantial improvement in
the survival rate and patient care, finding a complete cure for it
has been a challenging task. One primary cause of treatment
failure is the emergence of resistance in cancer. For example,
acute myeloid leukemia (AML) patients have a remission rate of
60ꢀ80% but only ∼20% of these patients survive more than
5 years.1ꢀ3 The remaining patients relapse with residual diseases
that are typically resistant to standard chemotherapies. There-
fore, there is an unmet clinical need for new therapies targeting
drug-resistant malignancies.
Cancer cells utilize multiple mechanisms for development of
drug resistance, such as alteration in drug transport, protein
targets, or cellular repair mechanisms.4,5 One of the major mech-
anisms is the alteration of the apoptotic pathway through over-
expression of the antiapoptotic Bcl-2 family proteins.6 Bcl-2
family proteins are the key regulators of apoptosis, a programmed
Received: June 14, 2011
Published: July 22, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
ARTICLE
Figure 1. Structures of compounds. The asterisk (/) represents chiral centers.
Table 1. Cross-Resistance (Ratio of IC50 in Resistant Cell Lines Relative to That in Parent Cell Lines) of MDR Cancer Cell Lines
for Standard Therapies and 5a
cross-resistance
resistant cell line
doxorubicin
vincristine
Ara-C
paclitaxel
MX
5
HL60/DNR
80 ( 21
32 ( 11
6.1 ( 2.1
>1000
>1000
>1000
1.3 ( 0.5
2.2 ( 0.1
0.88 ( 0.16
>1000
7.5 ( 0.6
11 ( 0.5
2.0 ( 0.5
0.77 ( 0.15
0.93 ( 0.15
1.32 ( 0.02
K562/HHT300
CCRF-CEM/VLB100
91 ( 15
266 ( 16
a Results are the mean of two independent experiments in triplicate in each experiment.
known as HA 14-1 (1) (Figure 1). 1 was selected for study
because it overcomes drug resistance in cancer cells that over-
express antiapoptotic Bcl-2 proteins and demonstrates synergism
with a variety of standard cancer therapies.15,16 However, 1 has a
short half-life and therefore is not an appropriate drug candi-
date.17 On the basis of the decomposition pathway of 1, we have
successfully developed a stable analogue, namely, sHA 14-1 (2)
(Figure 1).18
drug-resistant cancer cell lines, HL60/MX2 and CCRF-CEM/
CT, which are cross-resistant to standard cancer therapies. In this
report, we provide additional evidence toward the unique ability
of 5 to target drug resistant cancers and discuss an extended SAR
study of 5 to understand the role of various functional groups on
its cytotoxicity and selectivity toward drug resistant cancers.
One remarkable feature of 2 was that cancer cells genetically
overexpressing the antiapoptotic Bcl-2 family proteins reveal
no resistance to 2,18 despite the fact that such overexpression
conferred significant resistance against standard cancer therapies.
In addition, 2 functions quite differently from ABT-737 (3)
(Figure 1), a well-known inhibitor against Bcl-2 and Bcl-XL
proteins, in that 2 induces programmed cell death mainly through
the endoplasmic reticulum (ER) pathway by inducing ER stress
and ER Ca2+ release.19 2 also inhibits the ATPase activity of
sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA),
while its anticancer profile differs from that of thapsigargin (4)
(Figure 1) as well, a standard SERCA inhibitor.19 These data,
overall, suggest that 2 represents a novel scaffold that may be used
to develop therapies against drug-resistant cancers.
On the basis of the interesting anticancer profile of 2, we
performed a structureꢀactivity relationship (SAR) study that
led to the discovery of CXL017 (5) (Figure 1), which exhibits
cytotoxicity comparable to that of 3.20 Compound 5 demon-
strated low micromolar cytotoxicity over 11 different cancer
cell lines, including both solid and hematological malignancies.20
Of interest, 5 demonstrated selective cytotoxicity toward two
’ RESULTS AND DISCUSSION
Cytotoxicity of 5 in Multidrug Resistant Cancer Cells.
Previously, we have shown that multidrug resistant (MDR)
cancer cells like HL60/MX2 and CCRF-CEM/CT show no
cross-resistance to 5.20 Instead 5 demonstrates increased cyto-
toxicity toward such MDR cancer cells relative to the parental
counterparts. To evaluate the scope of 5 in targeting drug-
resistant cancers, we acquired several additional drug resistant
cancer cell lines, such as HL60/DNR, K562/HHT300, CCRF-
CEM/VLB100, selected for resistance toward daunorubicin
(DNR), homoharringtonine (HHT), and vinblastine (VLB),
respectively. We first evaluated these cell lines for their potential
cross-resistance to standard therapies. Our results (Table 1)
show that these drug-resistant cell lines reveal cross-resistance
to all standard therapies tested. Evaluation of compound 5 in
these cell lines again revealed that none of these resistant cell
lines show cross-resistance to 5 (Table 1) and that one cell line
(HL60/DNR) may be collaterally sensitive to 5. These results
further support the unique ability of 5 to target drug-resistant
malignancies.
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Table 2. CI Values of Vincristine and Paclitaxel with 5 in
MDR (HL60/DNR, K562/HHT300, and CCRF-CEM/
VLB100) Cancer Cell Linesa
Table 3. Cytotoxicity of 5 in μM across a panel of 60 cancer
cell lines from NCI
panel/cell lines
GI50 (μM)
panel/cell lines
melanoma
GI50 (μM)
combination index (CI)
leukemia
HL-60(TB)
K-562
0.35
0.39
0.45
0.81
0.80
1.9
UACC-62
M14
0.55
0.55
0.72
0.89
1.4
treatment
HL60/DNR K562/HHT300 CCRF-CEM/VLB100
vincristine + 5
paclitaxel + 5
0.61
0.77
0.56
0.53
0.55
0.53
SR
LOX IMVI
SK-MEL-5
SK-MEL-2
SK-MEL-28
UACC-257
ovarian cancer
OVCAR-3
NCI/ADR-RES
IGROV1
CCRF-CEM
MOLT-4
RPMI-8226
non-small-cell lung cancer
NCI-H460
NCI-H522
HOP-62
a The MDR cell lines were treated with single drugs vincristine,
paclitaxel, and 5 or in combination in a fixed ratio for 48 h. The fixed
ratios used for vincristine with 5 are 1:2.5, 1:5, and 1:1 and those for
paclitaxel with 5 are 1:8, 1:8, and 1:1 for HL60/DNR, K562/HHT300
and CCRF-CEM/VLB100, respectively. The CI (combination index)
was calculated by the combination index equation of Chou and Talalay.
CI < 1, CI = 1, and CI > 1 indicate synergism, additive effect, and
antagonism, respectively.
2.6
12
0.54
0.62
0.89
1.2
0.32
0.49
0.93
1.4
A549/ATCC
EKVX
1.3
SK-OV-3
OVCAR-8
OVCAR-5
OVCAR-4
renal cancer
A498
Synergism of 5 with Standard Therapies. 5 has been shown
to synergize with mitoxantrone in HL60/MX2 resistant cells.20
In this study, we tested the potential synergism of 5 in combina-
tion with vincristine or paclitaxel in the three new MDR cell lines.
The combination index (CI) of these drugs was calculated using
the Chou and Talalay method.21 Our results show that 5 syner-
gizes with these clinically used drugs with a CI of ∼0.5ꢀ0.8 in all
three MDR cell lines (Table 2), suggesting that 5 can be used
in combination with standard therapies to treat drug-resistant
cancer cells.
HOP-92
1.4
3.8
NCI-H23
NCI-H322M
NCI-H226
colon cancer
HT29
2.0
4.1
4.0
5.2
4.4
0.36
0.71
1.4
0.39
0.42
0.49
0.50
0.55
1.2
CAKI-1
HCT-116
SW-620
UO-31
ACHN
1.5
KM12
786ꢀ0
RXF 393
2.3
Cytotoxicity of 5 in the NCI-60 Cell Line Panel. The NCI-60
human tumor cell line anticancer drug screen was developed in
the late 1980s and has been used for a variety of purposes such as
identifying potential anticancer drug candidates, determining the
molecular target of a compound, gene, and chemosensitization
profiling.22 We used the NCI-60 cell line screen to evaluate the
activity profile of 5. Notably, 5 revealed decent cytotoxicity
across the entire NCI cell panel with a mean GI50 of 1.04 μM
(Table 3). Very interestingly, the natural MDR cell line HCT-15
and in vitro drug selected MDR cell line NCI/ADR-RES are both
sensitive to treatment by 5 with GI50 values of 0.55 and 0.49 μM,
respectively (Table 3). Further analysis of the NCI data suggests
that 5 was generally more effective against leukemia, colon cancer,
melanoma, and breast cancer cells but shows decreased activity
in renal, ovarian and non-small-cell lung cancer cells. These results
suggest that 5 may be useful for the treatment of a variety of
different cancers including naturally occurring MDR cancers.
COMPARE Analysis of 5. To explore the potential similarity of
5 to other compounds tested in the NCI-60 cell line screen, we
used the COMPARE algorithm provided by NCI to measure the
similarity of 5 (the seed compound) in responses to the 60 cell
lines of an entire database of compounds. It then ranks all the
compounds in the entire database in their order of similarity
toward the seed compound, in which a compound with a rank 1
signifies the highest similarity in the possible mechanism of
action to the seed compound. Using this approach, we compared
the mean GI50 profile (“fingerprint”) of 5 to the standard agents.
The standard agent database includes 171 compounds ranging
from cancer treatment new drug applications and investigational
new drug applications to compounds with a particularly high
interest at the NCI. The similarity pattern compared to that of
the seed is expressed using a Pearson’s correlation coefficient
(PCC). A PCC of >0.5 is generally considered significant based
on previous reports.23,24
HCT-15
2.5
COLO 205
HCC-2998
CNS cancer
SF-295
SN12C
2.8
2.5
TK-10
3.4
prostate cancer
PC-3
0.48
0.49
0.63
1.1
0.83
1.5
SF-539
DU-145
U251
breast cancer
HS 578T
MCF7
SF-268
0.32
0.45
0.67
0.77
1.5
SNB-75
1.3
SNB-19
1.9
MDA-MB-468
BT-549
melanoma
MDA-MB-435
MALME-3M
0.27
0.39
MDA-MB-231/ATCC
T-47D
3.7
Our COMPARE analysis results show that compound 5 is
unique in its mechanism of action with little correlation to
“standard” agents; specifically none of the 171 compounds
tested, which includes some clinically used agents, show a PCC of
>0.5 to 5 in the COMPARE analysis (Table 4). These data
suggest that 5 is a scaffold with a unique mechanism of action,
potentially accounting for its novel ability to selectively eliminate
drug-resistant cancers.
StructureꢀActivity Relationship (SAR) of 5. Rationale. Pre-
vious SAR studies of 5 mainly focused on its cytotoxicity. Here
we further explore the SAR of 5 to understand the importance of
various functional groups on 5 toward both its cytotoxicity and
selectivity. Since 5 has two methoxy groups on the 30 and 50
positions on the phenyl ring relative to 2, resulting in 10-fold
improvement in cytotoxicity, we designed molecules 6aꢀe
(Table 5) to investigate the importance of the number of
methoxy groups and their positions on the phenyl ring. Previous
SAR studies have also shown that the phenyl ring plays an
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ARTICLE
important role in the activity of 2; therefore, we evaluated
compounds 6fꢀk (Table 6) to determine the effect of the phenyl
substitution on selectivity toward MDR cells. Compounds 6lꢀp
(Table 7) were chosen to study the effect of electron donating
groups (EDGs) on the phenyl ring, while compounds 6q and 6r
were designed to determine the effect of electron withdrawing
groups (EWGs). Previous SAR study results also show that
substitution at the 40 position on the phenyl ring decreases
activity. Here we have expanded our SAR to include fused and
extended ring system analogues (6sꢀv) (Table 7) to further
understand the impact of sterics on the activity and selectivity
of 5. We also designed molecules (7aꢀf) (Table 8) with various
alkyl and alkynyl groups on the ester at the third and fourth
positions to evaluate the effect of modulating chain length and
flexibility on the ester functionality. To explore the importance
of ester functionality at the third and fourth position on the
chromene ring, analogues 8aꢀf (Table 9) were designed. Finally,
the role of the amino functionality at the second position of the
chromene ring was explored with the help of compounds 7g, 8g,
and 8h. These compounds were evaluated in HL60 and HL60/
MX2 (the drug-resistant cell line) to evaluate their cytotoxicity
and selectivity.
Chemistry. Compounds 6fꢀk and 8aꢀf were synthesized
previously with their synthesis and characterization reported
before.20 The general structures of the new series of 4H-
chromene derivatives, designed based on our lead compound
5, are depicted in Scheme 1 with compounds 6aꢀe,lꢀu synthe-
sized from their corresponding boronic acids with overall yields
of ∼70%. Compound 6v was synthesized via a nucleophilic
substitution reaction between 14 and 15 as outlined in Scheme 2
to give intermediate 16, followed by a Michael addition reaction
sequence to give 6v in 61% yield.25 Compounds 7aꢀf with
various alkyl or alkynyl groups on the ester functionality were
synthesized by treating 3,5-dimethoxyphenylcoumarin (17) with
ethyl cyanoacetate in the presence of various sodium alkoxides
generated in situ as outlined in Scheme 3 with overall yields
ranging from 45% to 60%. Compound 7g was synthesized
from 3,5-dimethoxyphenylsalicylaldehyde (18) using the meth-
od described in Scheme 4.26 Briefly, 18 was converted to the ester
Table 6. IC50 (μM) of Analogues 6fꢀk in HL60 and HL60/
MX2
Table 4. List of the Top Compounds with Growth Inhibitory
Patterns Similar to That of 5 (NSC No. S753690)a
rank PCC NSC no.
name
0.50 S35212 trimetrexate antimetabolite/antifolate
0.49 S163501 acivicin antimetabolite
mechanism
high concn
1
2
3
ꢀ6.0
ꢀ4.0
ꢀ3.6
IC50 ( SEMa
0.48 S126771 dichloroallyl antimetabolite
lawsone
selectivity HL60/
compd R5 R6 R7 R8
HL60
HL60/MX2 (HL60/MX2)
4
5
0.48 S153858 maytansine antimitotic agent
ꢀ3.6
ꢀ4.3
ꢀ5.6
ꢀ5.0
ꢀ4.0
ꢀ3.6
ꢀ6.0
6f
H
H
H
Ph
H
H
H
H
H
H
H
Ph
H
H
H
H
H
H
202 ( 10
99.1 ( 2.3
2.04
2.10
1.70
1.47
2.47
1.97
0.48 S332598 rhizoxin
antimitotic agent
antimitotic agent
antimitotic agent
6g
6h
6i
Br
n-Pr
H
74.4 ( 5.6 35.4 ( 0.7
79.2 ( 4.2 46.7 ( 1.9
56.8 ( 2.0 38.7 ( 1.2
84.5 ( 4.2 34.3 ( 5
14 0.40 S49842 vinblastine
16 0.39 S619003 vincristine
21 0.38 S19893 5-fluorouracil antimetabolite
6j
H
22 0.38 S740
methotrexate antimetabolite/antifolate
32 0.36 S125973 paclitaxel
antimitotic agent
6k
H
Ph 87.3 ( 6.5 44.4 ( 3.4
a Seed NSC: S753690 (5). EXP ID: AVGDATA. High concn: ꢀ4.0.
a Results are given as the mean of three independent experiments in
triplicate in each experiment.
Database: Standard Agents. Seed level: GI50. High concn: ꢀ4.0.
Table 5. IC50 (μM) of Analogues 2 and 6aꢀe in HL60 and HL60/MX2
IC50 ( SEMa
HL60/MX2
0
0
0
0
compd
R2
R3
R4
R5
HL60
selectivity HL60/(HL60/MX2)
5
H
OMe
H
H
OMe
H
10.7 ( 0.5
91.0 ( 0.3
38.7 ( 1.2
33.3 ( 0.8
66.6 ( 0.3
68.4 ( 1.3
54.6 ( 7.2
2.43 ( 0.18
23.2 ( 1.5
9.7 ( 0.5
4.41
3.93
4.02
1.19
4.41
4.28
3.77
2
H
H
H
H
H
H
6a
6b
6c
6d
6e
OMe
OMe
H
H
OMe
OMe
H
OMe
H
28.1 ( 1.0
15.1 ( 0.6
16.0 ( 0.3
14.5 ( 0.4
OMe
H
H
H
OMe
OMe
H
a Results are given as the mean of three independent experiments in triplicate in each experiment.
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Table 7. IC50 (μM) of Analogues 6lꢀv in HL60 and HL60/MX2
a Results are given as the mean of three independent experiments in triplicate in each experiment.
Table 8. IC50 (μM) of Analogues 7aꢀg in HL60 and HL60/MX2
IC50 ( SEMa
HL60/MX2
compd
R2
R3
R4
HL60
selectivity HL60/(HL60/MX2)
7a
7b
7c
7d
7e
7f
NH2
NH2
NH2
NH2
NH2
NH2
Me
n-Pr
n-Bu
allyl
C3H3
Et
n-Pr
7.6 ( 0.3
32 ( 1.7
4.7 ( 0.3
1.5 ( 0.1
4.7 ( 0.2
6.7 ( 0.5
63.6 ( 7.4
3.9 ( 0.3
1.97
2
n-Bu
allyl
14.1 ( 0.5
2.5 ( 0.1
1.62
2.38
2.54
1.87
6.66
C3H3
C3H3
0.64 ( 0.01
1.87 ( 0.06
3.55 ( 0.45
9.55 ( 0.90
cyclopropylmethyl
cyclopropylmethyl
7g
Et
Et
a Results are given as the mean of three independent experiments in triplicate in each experiment.
intermediate 19 using the Wittig reaction with 72% yield. Inter-
mediate 19 then underwent an oxa-Michael/Michael addition
sequence with ethyl buta-2,3-dieonate (20) to give compound 7g
in 82% yield.
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Table 9. IC50 (μM) of Analogues 8aꢀh in HL60 and HL60/MX2
a Results are given as the mean of three independent experiments in triplicate in each experiment.
Scheme 1a
further supported by the data from compounds 6cꢀe (Table 5).
Compounds 6c and 6d that have methoxy groups at 40 and 20
positions of the phenyl ring, respectively, showed decreased
activity in comparison to 6a that has a methoxy group at the 30
position. In addition, 6e with methoxy groups at 30 and 40
positions was less active than 5. Taken together, these data
suggest that the number and relative position of the methoxy
groups play an important role in the activity of 5.
We showed previously that removal of the phenyl group from
the sixth position of the chromene ring leads to a decrease in
activity in JURKAT cells.20 A similar trend was observed when
these analogues (6fꢀh) (Table 6) were tested in HL60 and
HL60/MX2 cells. In addition, moving the phenyl ring to posi-
tions 5, 7, and 8 (6iꢀk) on the chromene core also led to a
decrease in activity (Table 6), suggesting that the 6-phenyl is
probably optimal for activity.
a Reagents and condition: (i) 5-bromosalicylaldehyde, Pd(OAc)2, K2CO3,
DME/H2O (1:1), room temp; (ii) DMA, POCl3 60 °C, 4ꢀ8 h; (iii)
NaHCO3, 60 °C, 0.5 h; (iv) ethyl cyanoacetate, EtOH, NaOEt, room
temp.
When analogues with EDG (6lꢀp) (Table 7) were tested, we
found that small lipophilic groups like methyl (6l, 6m) led to an
improvement in activity while bulky lipophilic groups, such as
tert-butyl in 6p, led to a decrease in activity possibly due to
unfavorable steric interactions as observed for 6b. Interestingly,
replacing the lipophilic groups with hydrophilic groups like
hydroxyl (6n, 6o) also led to a decrease in activity, suggesting
that small lipophilic functional groups were optimal for activity.
We also tested the effect of EWG on the phenyl ring (6q and 6r)
and found that substituting electron withdrawing groups led to a
decrease in activity.
In Vitro Cytotoxicity in HL60 and HL60/MX2 Cells. The in vitro
cytotoxicity results of 2, 5, 6aꢀe (Table 5) revealed that the
number of methoxy groups on the phenyl ring played an
important role in the activity. For example, compound 2 with
no methoxy group shows a significant loss in activity in compar-
ison with analogues 6a (one methoxy at 30 position) and 5 (two
methoxy groups at 30 and 50 positions). Interestingly, further
increasing the number of methoxy groups (6b) led to decreased
activity in the HL60/MX2 cells. A possible reason for this
observation could be the change in sterics of the ring on addition
of a third methoxy group at the 40 position. This observation was
As observed in an earlier SAR study,20 steric bulkiness at the 40
position on the phenyl ring led to a decrease in activity. To
further explore the importance of the 40 position, we introduced
rigidity and flexibility in the scaffold with fused and extended ring
system analogues (6sꢀv) (Table 7). All of these analogues show
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Scheme 2a
a Reagents and condition: (i) NaH, DMF, room temp; (ii) ethyl cyanoacetate, EtOH, NaOEt, room temp.
Scheme 3a
a Reagents and condition: (i) ethyl cyanoacetate, R3OH, NaOR3, room temp.
Scheme 4a
a Reagents and condition: (i) Ph3PdCHCO2Et, toluene, 80 °C; (ii) H2CdCdCHCO2Et, K2CO3 (10 mol %), DMSO, 120 °C.
a decrease in activity in comparison to 5, further supporting that
increased sterics lead to decreased activity.
optimal for activity. Replacing the ester with CN or amide led to a
slight decrease in activity (Table 9), suggesting that the ester
functionality may be optimal for activity.
Previously, we showed that changes on the ester did not
significantly affect the activity in JURKAT cells.20 In this study,
we evaluated the ester functionality by designing analogues with
various alkyl and alkynyl substitutions at the ester position
(7aꢀf) (Table 8). Interestingly, replacing the ethyl ester with
an n-propyl (7a) slightly improves activity. However, a further
increase in chain length to the n-butyl ester (7b) led to a signi-
ficant decrease in activity. Interestingly, introducing unsaturation
in the propyl group led to a further improvement in activity (7c
and 7d). Analogue 7d with a propargyl group on the ester is the
most active compound with an IC50 of 1.53 μM in HL60 cells and
an IC50 of 0.64 μM in HL60/MX2 cells, about 4 to 7-fold more
active than 5.
Selectivity toward MDR Resistant HL60/MX2 Cells. The
selectivity of these analogues toward MDR resistant cell lines
was determined based on the ratio of their IC50 in HL60 cells
to that in HL60/MX2 cells. We used the Bonferroni multiple
comparison test of statistical analysis to classify the compounds
into three categories based on their selectivity ratio and statistical
analysis. Compounds with selectivity ratios between 0.5 and 2.0
had p > 0.05, indicating that they are likely to be nonselective.
Analogues with a selectivity ratio of 2ꢀ4 had p < 0.01 and were
considered to have medium selectivity. Analogues with a selec-
tivity ratio of >4 had p < 0.001 and were considered to have
significant selectivity.
Other positions on the chromene core, including the second,
third, and fourth positions, were also explored. The amino group
at the second position was evaluated by acetylation (8g and 8h)
(Table 9) or replacement by a methyl (7g) (Table 8), which
resulted in a loss in activity suggesting that the amino group was
On the basis of such criteria, 14 analogues demonstrate no
selectivity, 18 analogues demonstrate medium selectivity, while 7
analogues (5, 6a, 6c, 6d, 6n, 6o, and 7g) demonstrate significant
selectivity with 6n, 6o, and 7g being the most selective. Our
results show that removing a phenyl group (6fꢀh) from 5 led to
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Table 10. IC50 (μM) of 5 and Its Enantiomers in MDR Cell
action based on its low correlation with any known agents. With
these results, further SAR studies to make more potent and
selective analogues of 5 led to the discovery of compound 7d,
which is 4-fold more potent than 5. We were also able to improve
the selectivity of 5 with compounds 6m and 7g with a selectivity
of 7.1 and 6.7, respectively, toward HL60/MX2 MDR cells
relative to the parent HL60 cells. Finally, we have established
that the (ꢀ)-enantiomer for compound 5 is about 13 times
more active than the (+)-enantiomer; however, there is no dif-
ference in their selectivity toward MDR cancer cells. The signi-
ficant difference in cytotoxicity and lack of difference in selec-
tivity of the enantiomers suggest that 5 and its analogues have
distinct cellular targets responsible for cytotoxicity and selec-
tivity, which are currently under investigation. In conclusion, we
have demonstrated that compound 5 reveals a unique mechan-
ism of action to selectively kill MDR cancer cell lines, which
merits further investigation for its potential as a drug candidate
for the treatment of MDR cancers.
Linesa and Selectivity
cell line
5
(+)-enantiomer (ꢀ)-enantiomer
HL60, IC50
HL60/MX2, IC50
10.7 ( 0.5
2.4 ( 0.2
82.6 ( 1.4
21.8 ( 1.8
3.8
6.1 ( 0.1
1.9 ( 0.1
3.2
HL60/(HL60/MX2) 4.4
selectivity
a Results are given as the mean of two independent experiments in
triplicate in each experiment.
a decrease in selectivity (Table 6). However, removing the
methoxy groups (6a) or replacing them with other electron
donating groups such as a methyl (6m) does not change the
selectivity (Tables 5 and 7). In addition, replacing an EDG with
an EWG such as NO2 or CF3 does not cause significant change in
selectivity. These data overall suggest that electronic effects are
unlikely to play an essential role in the selectivity of 5 toward
drug-resistant cells. Interestingly, converting the methoxy to a
hydroxyl group, compounds 6n and 6o (Table 7), led to an
improvement in selectivity, suggesting that the hydrophilic
nature of a hydroxyl group might play a role in the improved
selectivity. Sterically hindered molecules like (6sꢀv) do not
show much change in selectivity in comparison to 5, indicating
that sterics do not play a major role in the selectivity of 5.
Converting the amino group at the second position to a methyl
group (7g) (Table 8) improves the selectivity, suggesting that
introducing lipophilic groups at the second position might lead
to more selective candidate.
The results of our current SAR studies also suggest that there is
no correlation between the selectivity and cytotoxicity of these
compounds in cancer cells. Nonetheless, both selectivity and
cytotoxicity of 5 are very sensitive to functional modifications.
For example, the methoxy groups on 5 play an important role in
its activity and selectivity (5 vs 6n). Similarly, replacement of the
amino group from 5 with a methyl (compound 7g) led to a slight
decrease in cytotoxicity but improvement in selectivity.
Chiral Separation and Cytotoxicity of Stereoisomers of 5.
As shown in Figure 1, compound 5 has one chiral center at
position 4 on the chromene core. Our study so far has only
evaluated the racemate of 5. We wished to determine whether the
optically pure isomers of compound 5 would present a different
in vitro activity profile. For this purpose, we used a chiral HPLC
column to separate the two enantiomers. The optical purity of
the enantiomers was determined using polarimetry. The optical
rotation for one enantiomer was +0.107 (c 1.0, MeOH) and that
for the other enantiomer was ꢀ0.102 (c 1.0, MeOH). The two
enantiomers were evaluated for their cytotoxicity and selectivity
in HL60 and HL60/MX2 cancer cells. The results show that
(ꢀ)-5 is about 13-fold more cytotoxic than the (+)-5 while there
is no significant change with respect to selectivity (Table 10).
’ EXPERIMENTAL SECTION
Chemistry. All commercial reagents and anhydrous solvents were
purchased from vendors and were used without further purification or
distillation unless otherwise stated. Analytical thin layer chromatography
was performed on Whatman silica gel 60 Å with fluorescent indicator
(Partisil K6F). Compounds were visualized by UV light and/or stained
with potassium permanganate solution followed by heating. Flash
column chromatography was performed on Whatman silica gel 60 Å
(230ꢀ400 mesh). NMR (1H, 13C) spectra were recorded on a Varian
300/400 MHz or a Bruker 400 MHz spectrometer and calibrated using
an internal reference. ESI mode mass spectra were recorded on a Bruker
BiotofII mass spectrometer. All compounds synthesized are racemic
mixtures and are more than 95% pure, analyzed using HPLC. Optical
rotation was measured using a Rudolph Research Autopol III polari-
meter at 589 nm Na D-line.
General Procedure for the Synthesis of Salicylaldehyde
(10aꢀu). 5-Bromosalicylaldehyde (1 g, 4.97 mmol), K2CO3 (2.061 g,
14.91 mmol), boronic acid (0.9954 g, 5.47 mmol), triphenylphosphine
(TPP) (1 mol %), and Pd(OAc)2 (1 mol %) were taken in DME/water
(1:1) (12 mL). The mixture was stirred at room temperature under an
atmosphere of nitrogen for 24 h. The reaction mixture was acidified
using HCl (1 N) on an ice bath, followed by extraction with ethyl acetate.
The extracts were combined, dried (MgSO4), and the solvent was
removed under vacuum. The crude solid was purified by flash chroma-
tography to isolate the desired salicylaldehyde.
4-Hydroxy-30-methoxy-[1,10-biphenyl]-3-carbaldehyde
(10a). Yield: 71%. 1H NMR (CDCl3): δ 11.01 (1H, s), 9.98 (1H, s),
7.76ꢀ7.78 (2H, m), 7.37 (1H, t, J = 4.0 Hz), 7.13 (1H, d, J = 7.6 Hz),
7.06ꢀ7.08 (2H, m), 6.90 (1H, dd, J = 2.0 Hz, 8.4 Hz), 3.87 (3H, s). 13C
NMR (75 MHz, CDCl3): δ 196.66, 161.08, 160.12, 140.83, 135.77,
133.17, 131.92, 130.03, 120.68, 119.08, 118.11, 112.60, 112.55, 55.35.
4-Hydroxy-40-methoxy-[1,10-biphenyl]-3-carbaldehyde
(10c). Yield: 76%. 1H NMR (CDCl3): δ 10.96 (1H, s), 9.97 (1H, s),
7.70ꢀ7.74 (2H, m), 7.47ꢀ7.49 (2H, m), 7.05 (1H, d, J = 8.4 Hz),
6.98ꢀ7.07 (2H, m), 3.86 (3H, s). 13C NMR (75 MHz, CDCl3): δ
196.70, 160.54, 159.20, 135.45, 133.07, 131.93, 131.34, 127.66, 120.70,
118.05, 114.41, 55.39.
4-Hydroxy-20-methoxy-[1,10-biphenyl]-3-carbaldehyde
(10d). Yield: 65%. 1H NMR (CDCl3): δ 11.02 (1H, s), 9.93 (1H, s),
7.70ꢀ7.73 (2H, m), 7.29ꢀ7.36 (2H, m), 7.01ꢀ7.06 (3H, m), 3.83 (3H, s).
13C NMR (75 MHz, CDCl3): δ 196.78, 160.63, 156.36, 138.43, 134.47,
130.38, 128.94, 128.67, 121.01, 120.34, 117.24, 111.24, 111.07, 55.55.
’ CONCLUSION
Given that the ability to selectively target MDR cancer cells is
one crucial factor for future cancer therapy development, we have
identified compound 5 as a potential lead. In this report, we
demonstrate that a variety of MDR cells show no cross-resistance
to 5. 5 synergizes with several standard anticancer agents in
MDR cells, including vincristine, paclitaxel, and mitoxantrone.
Additionally, we have shown that 5 is effective across the panel
of NCI-60 cell lines and seems to have a unique mechanism of
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4-Hydroxy-30,40-dimethoxy-[1,10-biphenyl]-3-carbaldehyde
(10e). Yield: 78%. 1H NMR (CDCl3): δ 10.97 (1H, s), 9.97 (1H, s),
7.70ꢀ7.73 (2H, m), 7.04ꢀ7.10 (3H, m), 6.95 (1H, d, J = 8.4 Hz), 3.96
(3H, s), 3.93 (3H, s). 13C NMR (75 MHz, CDCl3): δ 196.67, 160.64,
149.35, 148.72, 135.56, 133.26, 132.40, 131.46, 120.67, 118.96, 118.06,
111.62, 109.96, 56.02.
132.36, 132.36, 130.52, 125.62, 119.45, 119.01, 117.21, 117.04, 111.61,
110.30, 56.04.
6-(m-Tolyl)-2H-chromen-2-one (11l). Yield: 84%. 1H NMR
(CDCl3): δ 7.75 (2H, m), 7.66 (1H, d, J = 2.0 Hz), 7.34ꢀ7.40 (4H, m),
7.21 (1H, d, J = 6.4 Hz), 6.46 (1H, d, J = 9.4 Hz), 2.44 (3H, s). 13C
NMR (75 MHz, CDCl3): δ 160.74, 153.38, 143.50, 139.38, 138.70,
137.97, 130.79, 128.93, 128.54, 127.84, 126.04, 124.15, 119.00, 117.21,
117.01, 21.53.
4-Hydroxy-30-methyl-[1,10-biphenyl]-3-carbaldehyde (10l).
Yield: 67%. 1HNMR(CDCl3):δ10.99(1H, s), 9.98(1H, s), 7.76(2H, m),
7.35 (3H, m), 7.18 (1H, m), 7.07 (1H, d, J = 8.4 Hz), 2.43 (3H, s).
13C NMR (75 MHz, CDCl3): δ 196.69, 160.92, 139.32, 138.66, 135.79,
133.46, 131.86, 128.90, 128.15, 127.39, 123.70, 120.71, 118.06, 21.54.
4-Hydroxy-30,50-dimethyl-[1,10-biphenyl]-3-carbaldehyde
(10m). Yield: 53%. 1H NMR (CDCl3): δ 10.99 (1H, s), 9.99 (1H, s),
7.77 (2H, m), 7.18 (2H, s), 7.07 (1H, d, J = 8.4 Hz), 7.02 (1H, s), 2.40
(6H, s). 13C NMR (75 MHz, CDCl3): δ 196.72, 160.86, 139.31, 138.56,
135.83, 133.57, 131.85, 129.03, 124.52, 120.68, 117.98, 21.39.
4-Hydroxy-30,50-bis(trifluoromethyl)biphenyl-3-carbalde-
6-(3,5-Dimethylphenyl)-2H-chromen-2-one (11m). Yield:
79%. 1H NMR (CDCl3): δ 7.76 (1H, d, J = 9.4 Hz), 7.72 (1H, d, J =
2.0 Hz), 7.65 (1H, d, J = 2.4 Hz), 7.19 (2H, s), 6.46 (1H, d, J = 9.2 Hz),
2.40 (6H, s). 13C NMR (75 MHz, CDCl3): δ 160.79, 153.33, 143.53,
139.38, 138.61, 138.09, 130.82, 129.42, 126.02, 124.97, 118.96, 117.13,
116.96, 21.39.
6-(3,5-Bis(trifluoromethyl)phenyl)-2H-chromen-2-one (11q).
Yield: 65%. 1H NMR (CDCl3): δ 8.01 (2H, s), 7.89 (1H, s), 7.81ꢀ7.76
(2H, m), 7.73 (1H, d, J = 2.4 Hz), 7.49 (1H, d, J = 8.8 Hz), 6.53 (1H, d,
J = 9.6 Hz). 13C NMR (75 MHz, CDCl3): δ 160.08, 154.33, 142.86,
141.53, 134.71, 132.64, 132.30, 130.57, 127.14, 127.10, 126.45, 124.55,
121.47, 121.40, 119.42, 117.94, 117.82.
1
hyde (10q). Yield: 60%. H NMR (CDCl3): δ 11.13 (1H, s), 10.03
(1H, s), 7.98 (2H, s), 7.87 (1H, s), 7.82ꢀ7.79 (2H, m), 7.16 (1H, d, J =
8.4 Hz). 13C NMR (75 MHz, CDCl3): δ 196.28, 162.09, 141.46, 135.47,
132.59, 132.26, 132.14, 130.18, 126.64, 126.6, 124.6, 121.88, 121.05,
120.96, 120.86, 118.96.
1
6-(3-Nitrophenyl)-2H-chromen-2-one (11r). Yield: 72%. H
NMR (CDCl3): δ 8.46 (1H,s), 8.27ꢀ8.24 (1H, m), 7.93ꢀ7.91 (1H, m),
7.81ꢀ7.78 (2H,m), 7.737 (1H, d, J = 2 Hz), 7.66 (1H,t, J = 8 Hz), 7.48
(1H, d, J = 8.4 Hz), 6.53 (1H, d, J = 9.2 Hz). 13C NMR (75 MHz,
CDCl3): δ 160.23, 154.15, 142.99, 141.07, 135.21, 132.86, 130.55,
130.06, 126.33, 122.55, 121.88, 119.35, 117.82, 117.68.
6-(Benzo[d][1,3]dioxol-5-yl)-2H-chromen-2-one (11u).
Yield: 72%. 1H NMR (CDCl3): δ 7.72 (1H, d, J = 8.8 Hz), 7.66
(1H, dd, J = 2.4 Hz, 8.8 Hz), 7.58 (1H, d, J = 2.0 Hz), 7.37 (1H, d, J =
8.4 Hz), 7.03 (2H, m), 6.90 (1H, m), 6.47 (1H, d, J = 8.4 Hz), 6.02
(2H, s). 13C NMR (75 MHz, CDCl3): δ 160.72, 153.18, 148.38, 147.52,
143.44, 137.59, 133.70, 130.51, 125.69, 120.67, 119.01, 117.07, 108.77,
107.53, 101.35.
General Procedure for the Synthesis of Substituted Ethyl
4H-Chromene-3-carboxylate Compounds (6aꢀv, 7aꢀg).
Freshly cut sodium (0.096 mmol) was added to anhydrous ethanol
(2 mL), followed by the addition of ethyl cyanoacetate (0.192 mmol).
The reaction mixture was stirred at room temperature under an inert
atmosphere for 30 min, followed by the addition of a solution of the
corresponding coumarin (0.08 mmol) in anhydrous ethanol (1 mL).
The resulting reaction mixture was stirred at room temperature. Upon
consumption of the coumarin, the reaction mass was concentrated,
diluted with water (30 mL), and extracted using methylene chloride
(3 ꢁ 20 mL). The organics were combined, dried (MgSO4), and the
solvent was removed under vacuum to afford an oil. This crude oil was
subjected to column chromatography to afford the pure product.
Ethyl 2-Amino-4-(2-ethoxy-2-oxoethyl)-6-(3-methoxyphe-
nyl)-4H-chromene-3-carboxylate (6a). Yield: 74%. 1H NMR
(CDCl3): δ 7.46 (1H, d, J = 2.4 Hz), 7.40 (1H, dd, J = 2.0 Hz, 8.4
Hz), 7.32 (1H, t, J = 8.0 Hz), 7.11 (1H, d, J = 8.0 Hz), 7.06 (1H, t, J = 2.0
Hz), 7.00 (1H, d, J = 8.4 Hz), 6.87 (1H, dd, J = 2.0 Hz, 8.0 Hz), 6.34 (2H,
bs), 4.35 (1H, m), 4.23 (2H, q, J = 7.2 Hz), 4.02 (2H, q, J = 7.2 Hz), 3.85
(3H, s), 2.59ꢀ2.71 (2H, m), 1.33 (3H, t, J = 7.2 Hz), 1.13 (3H, t, J =
7.2 Hz). 13C NMR (75 MHz, CDCl3): δ 171.73, 169.04, 161.49, 159.96,
149.54, 141.81, 137.32, 129.78, 127.07, 126.42, 125.89, 119.37, 116.10,
112.60, 112.59, 60.20, 59.54, 55.28, 43.67, 31.38, 14.59, 14.07. MS (ESI,
positive) m/z 434.36. Calcd for C23H25NO6Na: 434.17.
4-Hydroxy-30-nitro-[1,10-biphenyl]-3-carbaldehyde (10r).
Yield: 51%. 1H NMR (CDCl3): δ 11.11 (1H, s), 10.02 (1H, s), 8.434ꢀ
8.425 (1H, m), 8.23ꢀ8.20 (1H, m), 7.90ꢀ7.88 (1H, m), 7.84ꢀ7.80
(2H, m), 7.64 (1H, m) 7.15 (1H, d, J = 8.4 Hz). 13C NMR (75 MHz,
CDCl3): δ 196.34, 162.16, 147.08, 140.64, 135.64, 132.35, 130.73,
127.32, 127.19, 124.38, 124.22, 120.84, 118.83.
5-(Benzo[d][1,3]dioxol-5-yl)-2-hydroxybenzaldehyde(10u).
1
Yield: 58%. H NMR (CDCl3): δ 10.96 (1H, s), 9.95 (1H, s), 6.67
(2H, m), 7.01 (3H, m), 6.88 (1H, d, J = 8.0 Hz), 6.01 (2H, s). 13C NMR
(75 MHz, CDCl3): δ 196.64, 160.70, 148.33, 147.17, 135.54, 133.73,
133.14, 131.53, 120.65, 120.12, 118.08, 108.73, 107.19, 101.28.
General Procedure for the Synthesis of Coumarin (11aꢀu).
To N,N-dimethylacetamide (1.98 mmol) stirred at 0 °C, phosphorus
oxychloride (1.98 mmol) was added slowly. The reaction mixture was
allowed to stir at 0 °C for 30 min followed by addition of the cor-
responding salicylaldehyde (0.99 mmol). The reaction mass was then
heated at 68ꢀ70 °C for 3 h. Following this, the reaction mass was cooled
to room temperature and saturated NaHCO3 solution (10 mL) was
added to it. The reaction mass was heated at 68ꢀ70 °C for another
30 min, cooled, and acidified (1 N HCl), followed by extraction with
methylene chloride. The extracts were combined, dried (anhydrous
MgSO4), and concentrated under reduced pressure to afford a residue,
which upon column chromatography afforded the desired coumarin.
6-(3-Methoxyphenyl)-2H-chromen-2-one (11a). Yield: 79%.
1H NMR (CDCl3): δ 7.75 (2H, m), 7.66 (1H, d, J = 2.4 Hz), 7.36ꢀ7.41
(2H, m), 7.14ꢀ7.17 (1H, m), 7.09 (1H, t, J = 2.0 Hz), 6.92ꢀ6.95 (1H,
m), 6.46 (1H, d, J = 9.2 Hz), 3.88 (3H, s). 13C NMR (75 MHz, CDCl3):
δ 160.68, 160.11, 153.51, 143.45, 140.89, 137.69, 130.78, 130.07, 126.11,
119.51, 119.01, 117.25, 117.08, 113.01, 112.98, 55.57.
6-(2-Methoxyphenyl)-2H-chromen-2-one (11d). Yield: 82%.
1H NMR (CDCl3): δ 7.74 (1H, d, J = 9.6 Hz), 7.70 (1H, dd, J = 2.4 Hz,
8.4 Hz), 7.63 (1H, d, J = 2.0 Hz), 7.34ꢀ7.38 (2H, m), 7.31 (1H, dd, J =
2.0 Hz, 7.6 Hz), 6.99ꢀ7.07 (2H, m), 6.44 (1H, d, J = 9.6 Hz), 3.83
(3H, s). 13C NMR (75 MHz, CDCl3): δ 160.97, 156.34, 153.07, 143.73,
135.02, 133.36, 130.69, 129.28, 128.73, 128.54, 121.02, 118.52, 116.61,
116.46, 111.27, 55.56.
6-(3,4-Dimethoxyphenyl)-2H-chromen-2-one (11e). Yield:
75%. 1H NMR (CDCl3): δ 7.76 (1H, d, J = 9.6 Hz), 7.71 (1H, dd, J =
2.0 Hz, 8.4 Hz), 7.61 (1H, d, J = 2.4 Hz), 7.38 (1H, d, J = 8.4 Hz), 7.13
(1H, dd, J = 2.0 Hz, 8.4 Hz), 7.07 (1H, d, J = 2.0 Hz), 6.96 (1H, d, J =
8.4 Hz), 6.46 (1H, d, J = 9.6 Hz), 3.97 (3H, s), 3.93 (3H, s). 13C NMR
(75 MHz, CDCl3): δ 160.73, 153.13, 149.38, 149.04, 143.45, 137.72,
Ethyl 2-Amino-4-(2-ethoxy-2-oxoethyl)-6-(4-methoxy-
phenyl)-4H-chromene-3-carboxylate (6c). Yield: 79%. 1H
NMR (CDCl3): δ 7.44ꢀ7.47 (2H, m), 7.42 (1H, d, J = 2.0 Hz), 7.35
(1H, dd, J = 2.4 Hz, 8.4 Hz), 6.99 (1H, d, J = 8.4 Hz), 6.95 (1H, d, J =
8.8 Hz), 6.32 (2H, bs), 4.32ꢀ4.35 (1H, m), 4.23 (2H, q, J = 7.2 Hz), 4.02
(2H, q, J = 7.2 Hz), 3.84 (3H, s), 2.58ꢀ2.70 (2H, m), 1.33 (3H, t, J =
7.2 Hz), 1.12 (3H, t, J = 7.2 Hz). 13C NMR (75 MHz, CDCl3): δ 171.76,
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169.06, 161.52, 159.05, 149.00, 137.12, 132.88, 127.85, 126.54, 125.92,
125.84, 116.05, 114.21, 60.19, 59.51, 55.34, 43.70, 31.38, 14.58, 14.07.
MS (ESI, positive) m/z 434.35. Calcd for C23H25NO6Na: 434.17.
Ethyl 2-Amino-4-(2-ethoxy-2-oxoethyl)-6-(2-methoxy-
Ethyl 2-Amino-6-(benzo[d][1,3]dioxol-5-yl)-4-(2-ethoxy-
2-oxoethyl)-4H-chromene-3-carboxylate (6u). Yield: 52%. H
1
NMR (CDCl3): δ 7.26ꢀ7.48 (2H, m), 6.93ꢀ7.01 (2H, m), 6.80ꢀ
6.89 (2H, m), 5.98 (2H, s), 4.06ꢀ4.18 (5H, m), 2.89ꢀ3.13 (2H, m),
1.14ꢀ1.22 (6H, m). 13C NMR (75 MHz, CDCl3): δ 171.71, 166.04,
165.17, 152.80, 148.09, 146.77, 134.81, 134.10, 127.62, 127.18, 124.82,
120.10, 117.00, 108.58, 107.29, 101.12, 63.00, 61.24, 41.79, 36.31, 34.99,
14.00, 13.79. MS (ESI, positive) m/z 448.42. Calcd for C23H23NO7Na:
448.15.
1
phenyl)-4H-chromene-3-carboxylate (6d). Yield: %. H NMR
(CDCl3): δ 7.40 (1H, d, J = 2.0 Hz), 7.36 (1H, dd, J = 2.4 Hz, 8.4 Hz),
7.26ꢀ7.32 (2H, m), 6.96ꢀ7.03 (3H, m), 6.35 (2H, bs), 4.32ꢀ4.35
(1H, m), 4.23 (2H, q, J = 7.2 Hz), 4.02 (2H, q, J = 7.2 Hz), 3.83 (3H, s),
2.58ꢀ2.69 (2H, m), 1.33 (3H, t, J = 7.2 Hz), 1.12 (3H, t, J = 7.2 Hz). 13C
NMR (75 MHz, CDCl3): δ 171.75, 169.13, 161.56, 156.37, 148.93,
134.71, 130.68, 129.76, 129.48, 128.90, 128.60, 125.08, 120.84, 115.32,
111.21, 60.13, 59.49, 55.53, 43.75, 31.34, 14.59, 14.04. MS (ESI,
positive) m/z 434.36. Calcd for C23H25NO6Na: 434.17.
Ethyl 2-Amino-6-(3,4-dimethoxyphenyl)-4-(2-ethoxy-2-
oxoethyl)-4H-chromene-3-carboxylate (6e). Yield: 71%. 1H
NMR (CDCl3): δ 7.42 (1H, d, J = 2.0 Hz), 7.36 (1H, dd, J = 2.0 Hz,
8.4 Hz), 7.07 (1H, dd, J = 2.0 Hz, 8.0 Hz), 7.04 (1H, d, J = 2.0 Hz), 7.00
(1H, d, J = 8.4 Hz), 6.92 (1H, d, J = 8.0 Hz), 6.34 (2H, bs), 4.35 (1H, m),
4.23 (2H, q, J = 7.2 Hz), 4.02 (2H, q, J = 7.2 Hz), 3.94 (3H, s), 3.92
(3H, s), 2.58ꢀ2.67 (2H, m), 1.33 (3H, t, J = 7.2 Hz), 1.13 (3H, t, J =
7.2 Hz). 13C NMR (75 MHz, CDCl3): δ 171.76, 169.05, 161.50, 149.15,
149.11, 148.54, 137.33, 133.36, 126.67, 126.06, 125.88, 119.12, 116.06,
111.48, 110.17, 60.18, 59.53, 55.98, 55.93, 43.72, 31.37, 14.58, 14.09. MS
(ESI, positive) m/z 464.68. Calcd for C24H27NO7Na: 464.18.
Ethyl 2-Amino-6-((3,5-dimethoxybenzyl)oxy)-4-(2-ethoxy-
2-oxoethyl)-4H-chromene-3-carboxylate (6v). Yield: 61%. H
1
NMR (CDCl3): δ 6.84ꢀ6.88 (2H, m), 6.78 (1H, dd, J = 3.2 Hz, 8.8 Hz),
6.56 (2H, d, J = 2.4 Hz), 6.40 (1H, t, J = 2.0 Hz), 6.29 (2H, bs), 4.93 (2H,
s), 4.19ꢀ4.26 (3H, m), 4.01 (2H, q, J = 7.2 Hz), 3.79 (6H, s), 2.54ꢀ2.65
(2H, m), 1.31 (3H, t, J = 7.2 Hz), 1.16 (3H, t, J = 7.2 Hz). 13C NMR (75
MHz, CDCl3): δ 171.76, 169.06, 161.73, 160.99, 155.22, 144.18, 139.28,
126.53, 116.53, 114.45, 113.78, 105.16, 99.90, 70.40, 60.18, 59.45, 55.35,
43.58, 31.54, 14.58, 14.10. MS (ESI, positive) m/z 494.27. Calcd for
C25H29NO8Na: 494.19. HPLC purity: 85%
Propyl 2-Amino-6-(3,5-dimethoxyphenyl)-4-(2-oxo-2-
propoxyethyl)-4H-chromene-3-carboxylate (7a). Yield: 58%.
1H NMR (400 MHz, CDCl3): δ 7.45 (d, J = 2.26 Hz, 1H), 7.40 (dd, J =
2.13, 8.41 Hz, 1H), 7.02 (d, J = 8.28 Hz, 1H), 6.67 (d, J = 2.26 Hz, 2H),
6.46 (t, J = 2.26 Hz, 1H), 6.34 (br s, 2H), 4.37 (dd, J = 4.02, 7.78 Hz, 1H),
4.15 (t, J = 6.65 Hz, 2H), 3.94 (dt, J = 2.38, 6.71 Hz, 2H), 3.85 (s, 6H),
2.58ꢀ2.75 (m, 2H), 1.75 (sxt, J = 7.08 Hz, 2H), 1.53 (qd, J = 7.19, 14.31
Hz, 2H), 1.03 (t, J = 7.40 Hz, 3H), 0.81 (t, J = 7.40 Hz, 3H). 13C NMR
(101 MHz, CDCl3): δ 171.74, 169.10, 161.43, 161.05, 149.56, 142.53,
137.44, 127.05, 126.45, 125.79, 116.08, 105.19, 99.21, 65.89, 65.25,
55.40, 43.67, 31.42, 29.69, 22.31, 21.84, 10.66, 10.28. MS (ESI, positive)
m/z calcd for C26H31NO7 (M + H), 470.22; found, 470.20.
Butyl 2-Amino-4-(2-butoxy-2-oxoethyl)-6-(3,5-dimethoxy-
phenyl)-4H-chromene-3-carboxylate (7b). Yield: 63%. 1H NMR
(400 MHz, CDCl3): δ 7.45 (d, J = 2.26 Hz, 1H), 7.40 (dd, J = 2.26,
8.28 Hz, 1H), 7.02 (d, J = 8.28 Hz, 1H), 6.68 (d, J = 2.26 Hz, 2H), 6.46
(t, J = 2.26 Hz, 1H), 6.34 (br s, 2H), 4.35 (dd, J = 4.02, 7.53 Hz, 1H), 4.19
(t, J = 6.15 Hz, 2H), 3.98 (t, J = 7.53 Hz, 2H), 3.85 (s, 6H), 2.56ꢀ2.74 (m,
2H), 1.65ꢀ1.76 (m, 2H), 1.41ꢀ1.55 (m, 4H), 1.24 (qd, J = 7.38, 15.00
Hz, 2H), 0.98 (t, J = 7.40 Hz, 3H), 0.84 (t, J = 7.40 Hz, 3H). 13C NMR
(101 MHz, CDCl3): δ 171.74, 169.10, 161.41, 161.04, 149.53, 142.50,
137.42, 127.05, 126.43, 125.76, 116.06, 105.19, 99.17, 64.18, 63.45, 55.38,
43.66, 31.39, 31.01, 30.53, 19.33, 19.03, 13.78, 13.62. MS (ESI, positive)
m/z calcd for C28H35NO7 (M + H), 498.25; found, 498.21.
Ethyl 2-Amino-4-(2-ethoxy-2-oxoethyl)-6-(m-tolyl)-4H-
chromene-3-carboxylate (6l). Yield: 69%. H NMR (CDCl3): δ
1
7.46 (1H, d, J = 2.4 Hz), 7.40 (1H, dd, J = 2.4 Hz, 8.4 Hz), 7.28ꢀ7.34
(3H, m), 7.15 (1H, d, J = 6.4 Hz), 7.01 (1H, d, J = 8.4 Hz), 6.33 (2H, bs),
4.35 (1H, m), 4.23 (2H, q, J = 6.8 Hz), 4.02 (2H, q, J = 7.2 Hz), 2.63
(2H, m), 2.41 (3H, m), 1.33 (3H, t, J = 6.8 Hz), 1.13 (3H, t, J = 7.2 Hz).
13C NMR (75 MHz, CDCl3): δ 171.74, 169.07, 161.50, 149.38, 140.27,
138.35, 137.60, 128.68, 127.91, 127.66, 127.01, 126.37, 125.84, 123.95,
116.05, 60.20, 59.53, 43.68, 31.38, 21.52, 14.58, 14.06. MS (ESI, posi-
tive) m/z 418.28. Calcd for C23H25NO5Na: 418.17. HPLC purity: 93%
Ethyl 2-Amino-6-(3,5-dimethylphenyl)-4-(2-ethoxy-2-
oxoethyl)-4H-chromene-3-carboxylate (6m). Yield: 61%. 1H
NMR (CDCl3): δ 7.45 (1H, d, J = 2.0 Hz), 7.39 (1H, dd, J = 2.4 Hz,
8.4 Hz), 7.14 (2H, d, J = 2.0 Hz), 7.00 (1H, d, J = 8.4 Hz), 6.97 (1H, s),
6.32 (2H, bs), 4.35 (1H, m), 4.23 (2H, q, J = 7.2 Hz), 4.02 (2H, q, J = 7.2
Hz), 2.66 (2H, m), 2.37 (6H, m), 1.33 (3H, t, J = 7.2 Hz), 1.13 (3H, t, J =
7.2 Hz). 13C NMR (75 MHz, CDCl3): δ 171.74, 169.08, 161.51, 149.32,
104.28, 138.27, 137.71, 128.80, 126.98, 126.36, 125.77, 124.79, 115.98,
60.20, 59.52, 43.68, 31.38, 24.74, 21.39, 14.57, 14.06. MS (ESI, positive)
m/z 432.38. Calcd for C24H27NO5Na: 432.19. HPLC purity: 92.2%
Ethyl 2-Amino-6-(3,5-bis(trifluoromethyl)phenyl)-4-(2-
ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (6q). Yield:
55%. 1H NMR (CDCl3): δ 7.95 (2H, s), 7.83 (1H, s), 7.51 (1H, d, J =
2.0 Hz), 7.45 (1H, dd, J = 2.4 Hz, 8.4 Hz), 7.11 (1H, d, J = 8.4 Hz), 6.4
(2H, bs), 4.40ꢀ4.37 (1H, m), 4.27 (2H, q, J = 7.2 Hz), 4.07 (2H, q, J =
7.2 Hz), 2.74ꢀ2.6 (2H, m), 1.35 (3H, t, J = 7.2 Hz), 1.17 (3H, t, J =
7.2 Hz). 13C NMR (75 MHz, CDCl3): δ 171.56, 168.87, 161.16, 150.58,
142.39, 134.36, 132.31, 131.98, 127.34, 126.72, 121.95, 120.79, 116.76,
60.34, 59.66, 43.56, 31.22, 14.54, 14.06. MS (ESI, positive) m/z 517.27.
Calcd for C24H21F6NO5.: 517.13.
Allyl 4-(2-(Allyloxy)-2-oxoethyl)-2-amino-6-(3,5-dimethoxy-
phenyl)-4H-chromene-3-carboxylate (7c). Yield: 47%. H NMR
1
(400 MHz, CDCl3): δ 7.46 (d, J = 2.01 Hz, 1H), 7.40 (dd, J = 2.13, 8.41
Hz, 1H), 7.02 (d, J = 8.53 Hz, 1H), 6.67 (d, J = 2.26 Hz, 2H), 6.46 (t, J =
2.13 Hz, 1H), 6.38 (br s, 2H), 5.95ꢀ6.08 (m, 1H), 5.73ꢀ5.85 (m, 1H),
5.39 (d, J = 17.32 Hz, 1H), 5.07ꢀ5.28 (m, 3H), 4.70 (d, J = 5.27 Hz,
2H), 4.48 (d, J = 5.77 Hz, 2H), 4.41 (dd, J = 4.27, 7.28 Hz, 1H), 3.85
(s, 6H), 2.62ꢀ2.79 (m, 2H). 13C NMR (101 MHz, CDCl3): δ 171.23,
168.54, 161.75, 161.05, 149.53, 142.44, 137.54, 133.00, 132.02, 127.06,
126.52, 125.66, 118.18, 117.18, 116.11, 105.21, 99.21, 65.01, 64.25,
55.39, 43.58, 31.35. MS (ESI, positive) m/z calcd for C26H27NO7
(M + H), 466.19; found, 466.21.
Prop-2-yn-1-yl 2-Amino-6-(3,5-dimethoxyphenyl)-4-(2-
oxo-2-(prop-2-yn-1-yloxy)ethyl)-4H-chromene-3-carboxy-
late (7d). Yield: 43%. 1H NMR (CDCl3): δ 7.46 (1H, d, J = 2.0 Hz),
7.40 (1H, dd, J = 2.0 Hz, 8.4 Hz), 7.02 (1H, d, J = 8.4 Hz), 6.66 (2H, d, J =
2.0 Hz), 6.44 (1H, t, J = 2.0 Hz), 6.39 (2H, br s), 4.78ꢀ4.79 (2H, m),
4.54ꢀ4.64 (2H, m), 4.39 (1H, dd, J = 4.8 Hz, 8.4 Hz), 3.84 (6H, s),
2.66ꢀ2.77 (2H, m), 2.47 (3H, t, J = 2.4 Hz), 2.31 (3H, t, J = 2.4 Hz). 13C
NMR (75 MHz, CDCl3): δ 170.64, 167.88, 162.20, 161.05, 149.45,
142.40, 137.78, 127.09, 126.70, 125.39, 116.19, 105.31, 99.24, 78.68,
Ethyl 2-Amino-4-(2-ethoxy-2-oxoethyl)-6-(3-nitrophenyl)-
4H-chromene-3-carboxylate (6r). Yield: 45%. 1H NMR (CDCl3):
δ 8.39ꢀ8.38 (1H, m), 8.19ꢀ8.16 (1H, m), 7.87ꢀ7.85 (1H, m), 7.6 (1H,
t, J = 8.0 Hz), 7.53 (1H, d, J = 2.4 Hz), 7.47 (1H, dd, J = 2.0 Hz, 8.4 Hz),
7.1 (1H, d, J = 8.4 Hz), 6.4 (2H, bs), 4.39ꢀ4.36 (1H, m), 4.27 (2H, q, J =
6.8 Hz), 4.07 (2H, q, J = 7.2 Hz), 2.73ꢀ2.61 (2H, m), 1.35 (3H, t, J = 7.2
Hz), 1.17 (3H, t, J = 6.8 Hz). 13C NMR (75 MHz, CDCl3): δ 171.65,
168.89, 161.24, 150.34, 148.72, 141.96, 134.84, 132.69, 129.75, 127.27,
126.54, 121.92, 121.61, 116.62, 60.31, 59.63, 43.56, 31.24, 14.56, 14.08.
MS (ESI, positive) m/z 426.4. Calcd for C25H29NO8Na: 426.14.
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Journal of Medicinal Chemistry
ARTICLE
75.86, 74.89, 74.28, 55.43, 51.81, 51.16, 43.33, 31.12. MS (ESI, positive)
m/z 484.46. Calcd for C26H23NO7Na: 484.15.
with 10% FBS. All cell lines were incubated at 37 °C with 5% CO2 in air
atmosphere.
Cell Viability Measurement. The in vitro cytotoxicity of these
small molecules was assayed by determining their ability to inhibit
the growth of the tumor cells. In brief, the tumor cells were plated in a
96-well plate (a density of 1 ꢁ 104 cells/well). The cells were treated
with a series of dilutions of the test compounds of varied concentrations
with 1% DMSO in the final cell medium (cells treated with medium
containing 1% DMSO served as a control). After a 48 h treatment, the
relative cell viability in each well was determined by using CellTiter-Blue
cell viability assay kit. The IC50 of each candidate was determined by
fitting the relative viability of the cells to the drug concentration by using
a doseꢀresponse model in the Prism program from GraphPad Software,
Inc. (San Diego, CA).
Synergism Assay. Synergistic interactions of 5 with vincristine
were examined by using median doseꢀeffect analysis as described by
Chou and Talalay.21 Briefly, tumor cells were treated with serial dilutions
of each agent individually and in combination simultaneously at a fixed
dose ratio for 48 h. The fixed ratios used for vincristine with 5 are 1:2.5,
1:5, and 1:1 and those for paclitaxel with 5 are 1:8, 1:8, and 1:1 for
HL60/DNR, K562/HHT300, and CCRF-CEM/VLB100, respectively.
The cytotoxic effects of the treatment were measured by evaluating the
cell viability using the cell viability assay and the long-term survival assay.
Fractional effect was calculated as fraction of cells killed by the individual
agent or the combination, in treated versus untreated cells. Median dose
effect analysis was performed using CompuSyn program from Combo-
Syn, Inc. (Paramus, NJ). The software computes combination index
(CI) values based on the following equation: CI = (D)1/(Dx)1 + (D)2/
(Dx)2 + (D)1(D)2/(Dx)1(Dx)2, where (D)1 and (D)2 are the doses of
drug 1 and drug 2 that have x effect when used in combination and
(Dx)1 and(Dx)2 are the doses of drugs 1 and 2 that have the same
x effect when used alone. The CI values indicate synergism (<1), addi-
tivity (1), or antagonism (>1). CIs of 0.1ꢀ0.3, 0.3ꢀ0.7, and 0.7ꢀ0.85
are considered to indicate strong synergism, synergism, and moderate
synergism, respectively.
Ethyl 2-Amino-6-(3,5-dimethoxyphenyl)-4-(2-oxo-2-(prop-
2-yn-1-yloxy)ethyl)-4H-chromene-3-carboxylate (7e). Yield:
22%. 1H NMR (CDCl3): δ 7.46 (1H, d, J = 2.4 Hz), 7.39 (1H, dd, J =
2.4 Hz, 8.4 Hz), 7.01 (1H, d, J = 8.8 Hz), 6.67 (2H, d, J = 2.0 Hz), 6.44
(1H, t, J = 2.0 Hz), 6.32 (2H, br s), 4.53ꢀ4.62 (2H, m), 4.38 (1H, q, J =
7.2 Hz), 4.23 (2H, dd, J = 4.8 Hz, 8.4 Hz), 3.84 (6H, s), 2.62ꢀ2.75 (2H,
m), 2.30 (3H, t, J = 2.4 Hz), 1.33 (3H, t, J = 7.2 Hz). 13C NMR (75 MHz,
CDCl3): δ 170.80, 168.92, 161.50, 161.03, 149.56, 142.49, 137.58,
127.09, 126.61, 125.57, 116.16, 105.32, 99.18, 77.58, 74.87, 59.60, 55.43,
51.73, 43.35, 31.29, 14.59. MS (ESI, positive) m/z 474.15. Calcd for
C25H25NO7Na: 474.16. HPLC purity: 88%
Cyclopropylmethyl 2-Amino-4-(2-(cyclopropylmethoxy)-
2-oxoethyl)-6-(3,5-dimethoxyphenyl)-4H-chromene-3-car-
boxylate (7f). Yield: 46%. 1H NMR (400 MHz, CDCl3): δ 7.48 (d, J =
2.26 Hz, 1H), 7.40 (dd, J = 2.26, 8.28 Hz, 1H), 7.02 (d, J = 8.53 Hz, 1H),
6.67 (d, J = 2.26 Hz, 2H), 6.45 (t, J = 2.26 Hz, 1H), 6.00ꢀ6.42 (br s, 2H),
4.40 (dd, J = 4.27, 7.28 Hz, 1H), 3.98ꢀ4.08 (m, 2H), 3.85 (s, 6H), 3.80
(dd, J = 1.25, 7.28 Hz, 2H), 2.65ꢀ2.79 (m, 2H), 1.27 (s, 2H), 0.56ꢀ0.62
(m, 2H), 0.42ꢀ0.48 (m, 2H), 0.31ꢀ0.37 (m, 2H), 0.13ꢀ0.18 (m, 2H).
13C NMR (101 MHz, CDCl3): δ 170.8, 168.1, 160.4, 160.0, 148.6, 141.5,
136.4, 126.1, 125.4, 124.8, 115.1, 104.2, 98.2, 76.3, 76.0, 75.7, 68.0, 67.1,
54.4, 42.5, 30.4, 28.7, 9.1, 8.7, 2.2, 2.1, 2.1, 2.1. MS (ESI, positive) m/z
calcd for C28H31NO7 (M + H), 494.22; found, 494.40. HPLC purity: 90%
Ethyl 6-(3,5-Dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-
2-methyl-4H-chromene-3-carboxylate (7g). Yield: 82%. 1H
NMR (CDCl3): δ 7.43 (1H, d, J = 2.0 Hz), 7.40 (1H, dd, J = 2.4 Hz,
8.4 Hz), 7.03 (1H, d, J = 8.4 Hz), 6.66 (2H, d, J = 2.4 Hz), 6.44 (1H, t, J =
2.0 Hz), 4.24 (1H, m), 4.05 (2H, q, J = 7.2 Hz), 4.03 (2H, q, J = 7.2 Hz),
3.83 (6H, s), 2.68 (1H, dd, J = 4.4 Hz, 14.8 Hz), 2.56 (1H, dd, J = 8.0 Hz,
15.2 Hz), 2.45 (3H, s), 1.34 (3H, t, J = 6.8 Hz), 2.45 (3H, s), 1.13 (3H, t,
J = 6.8 Hz). 13C NMR (75 MHz, CDCl3): δ 171.32, 166.90, 162.52,
161.07, 150.30, 142.57, 137.47, 126.87, 126.59, 124.49, 116.27, 105.14,
104.72, 99.18, 60.36, 60.35, 55.40, 43.59, 32.4.
Statistical Analysis. The in vitro cytotoxicity assay was performed
at least twice with triplicates in each experiment. Data are presented as
the mean ( SD, and comparisons were made using Student’s t test. The
synergism assay was performed as a single replicate with triplicates in
each experiment. We performed the Bonferroni multiple comparison
analysis for determining significant selectivity ratios in comparison to no
selectivity with a ratio of 1. A probability of 0.05 or less was considered
statistically significant.
6-((3,5-Dimethoxybenzyl)oxy)-2H-chromen-2-one (11v).
Yield: 56%. 1H NMR (CDCl3): δ 7.63 (1H, d, J = 8.4 Hz), 7.26 (1H, d,
J = 8.4 Hz), 7.16ꢀ7.19 (1H, m), 6.97 (1H, d, J = 2.4 Hz), 6.57 (1H, d,
J = 2.4 Hz), 6.40ꢀ6.43 (2H, m), 5.04 (2H, s), 3.80 (6H, s). 13C NMR
(75 MHz, CDCl3): δ 161.12, 160.92, 155.10, 148.63, 143.16, 138.69,
120.17, 119.19, 117.94, 117.11, 111.40, 105.16, 99.88, 70.63, 55.38.
(E)-Ethyl 3-(4-Hydroxy-30,50-dimethoxy-[1,10-biphenyl]-3-
yl)acrylate (19). Yield: 72%. 1H NMR (CDCl3): δ 8.06 (1H, d, J =
16 Hz), 7.67 (1H, d, J = 2.4 Hz), 7.45 (1H, dd, J = 2.4 Hz, 8.4 Hz), 6.91
(1H, d, J = 8.4 Hz), 6.73 (1H, d, J = 16 Hz), 6.67 (2H, d, J = 2 Hz), 6.50
(1H, s), 6.44 (1H, t, J = 2.0 Hz), 4.30 (2H, q, J = 6.8 Hz), 3.85 (6H, s),
1.36 (3H, t, J = 5.4 Hz). 13C NMR (75 MHz, CDCl3): δ 168.23, 161.11,
154.90, 142.44, 140.30, 134.01, 130.11, 127.84, 121.88, 119.04, 116.75,
105.05, 99.00, 60.74, 55.45, 14.33.
’ ASSOCIATED CONTENT
S
Supporting Information. Purity results of final com-
b
pounds and chiral HPLC spectra. This material is available free
of charge via the Internet at http://pubs.acs.org.
Determination of Purity and Chiral Separation. The purity
of the final compounds were determined using 85:15 (ACN/H2O) as the
mobile phase with a flow rate of 1.0 mL/min on a C18 column. The chiral
separation was done using a RegisPack chiral column (Regis Tech-
nologies). The enantiomers of 5 were separated using 70:30 (hexane/
IPA) with a flow rate of 1.5 mL/min. The retention time for the (+)-
enantiomer was 3.8 min, and that for the (ꢀ)-enantiomer was 5.0 min.
Cell Cultures. CCL-240 (HL-60) and CRL-2258 (HL-60/
mitoxantrone (MX) resistant) cells were purchased from ATCC. K562,
K562/HHT300 and HL60/DNR cell lines were developed by one of us
(R.T.). CCRF-CEM and CCRF-CEM-VLB100 were developed and
described by one of us (W.T.B.). The HL-60 cell line was grown in
IMDM Glutamax medium supplemented with 20% FBS. All other cell
lines were grown in RPMI 1640 purchased from ATCC supplemented
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 612-626-5675. Fax: 612-624-0139. E-mail: xingx009@
umn.edu.
Present Addresses
Bioorganic Chemistry Division, Life Technologies Corporation,
2130 Woodward Street, Austin, TX 78744-1832.
’ ACKNOWLEDGMENT
We thank National Institutes of Health National Cancer
Institute (Grant R01-CA114294 to C.X.); the Leukemia
5947
dx.doi.org/10.1021/jm200764t |J. Med. Chem. 2011, 54, 5937–5948

Journal of Medicinal Chemistry
ARTICLE
Research Fund, Masonic Cancer Center, University of Minnesota
(LRF seed grant, C.X.); and University of Minnesota (Ph.D.
Dissertation Fellowship to S.G.D.) for financial support. Drug-
resistant CCRF-CEM cell lines were developed and characterized
in work funded by NCI Grant CA40570 (to W.T.B.).
(17) Doshi, J. M.; Tian, D.; Xing, C. Ethyl-2-amino-6-bromo-4-
(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1), a
prototype small-molecule antagonist against antiapoptotic Bcl-2 proteins,
decomposes to generate reactive oxygen species that induce apoptosis.
Mol. Pharmaceutics 2007, 4, 919–928.
(18) Tian, D.; Das, S. G.; Doshi, J. M.; Peng, J.; Lin, J.; Xing, C. sHA
14-1, a stable and ROS-free antagonist against anti-apoptotic Bcl-2
proteins, bypasses drug resistances and synergizes cancer therapies in
human leukemia cell. Cancer Lett. 2008, 259, 198–208.
’ ABBREVIATIONS USED
MDR, multidrug resistance; AML, acute myeloid leukemia;
PCC, Pearson’s correlation coefficient; SAR, structureꢀactivity
relationship; EDG, electron donating group; EWG, electron
withdrawing group; Ara-C, cytarabine; MX, mitoxantrone; DNR,
daunorubicin; HHT, homoharringtonine; VLB, vinblastine; ER,
endoplasmic reticulum; SERCA, sarcoplasmic/endoplasmic re-
ticulum Ca2+-ATPase; CI, combination index; GI50, 50% growth
inhibitory concentration
(19) Hermanson, D.; Addo, S. N.; Bajer, A. A.; Marchant, J. S.; Das,
S. G.; Srinivasan, B.; Al-Mousa, F.; Michelangeli, F.; Thomas, D. D.;
Lebien, T. W.; Xing, C. Dual mechanisms of sHA 14-1 in inducing cell
death through endoplasmic reticulum and mitochondria. Mol. Pharma-
col. 2009, 76, 667–678.
(20) Das, S. G.; Doshi, J. M.; Tian, D.; Addo, S. N.; Srinivasan, B.;
Hermanson, D. L.; Xing, C. Structureꢀactivity relationship and molec-
ular mechanisms of ethyl 2-amino-4-(2-ethoxy-2-oxoethyl)-6-phenyl-
4H-chromene-3-carboxylate (sha 14-1) and its analogues. J. Med. Chem.
2009, 52, 5937–5949.
(21) Chou, T. C.; Talalay, P. Quantitative analysis of dose-effect
relationships: the combined effects of multiple drugs or enzyme inhi-
bitors. Adv. Enzyme Regul. 1984, 22, 27–55.
(22) Shoemaker, R. H. The NCI60 human tumour cell line antic-
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