Structure-activity studies of substituted quinoxalinones as multiple-drug-resistance antagonists

Article abstract of DOI:10.1021/jm000282d

A significant problem in the clinical treatment of cancer relates to the development of tumor resistance to many chemotherapeutic agents. Acquired drug resistance is often mediated through overexpression of membrane transport proteins that effectively efflux anticancer agents. Two of the best-studied transporters, P-glycoprotein (Pgp) and MRP1, have pharmacological properties that only partially overlap. In our search for improved drug-resistance antagonists, we have identified a family of substituted quinoxalines that selectively antagonizes Pgp over MRP1. Consequently, a focused library of congeners was designed and synthesized starting with a parent bromomethylquinoxalinone. This parent quinoxalinone was then condensed with a series of phenols to yield a family of substituted phenoxymethylquinoxalinones. These compounds were evaluated for their toxicity toward drug-sensitive MCF-7 breast carcinoma cells and for their abilities to antagonize Pgp and MRP1 in drug-resistant cell lines (NCI/ADR and MCF-7/VP, respectively). The results of this structure-activity study indicate that compounds with carbonyl substitutions of the phenoxy group (ester, amide, or ketone moieties) demonstrate excellent antagonism of Pgp while having relatively low toxicity toward drug-sensitive cells. Importantly, none of these compounds antagonized MRP1. Because of their transporter selectivity, we predict that substituted quinoxalinones may be more effective MDR modulators in vivo than are nonselective transporter antagonists.

Full text of DOI:10.1021/jm000282d

594
J . Med. Chem. 2001, 44, 594-601
Str u ctu r e-Activity Stu d ies of Su bstitu ted Qu in oxa lin on es a s
Mu ltip le-Dr u g-Resista n ce An ta gon ists
David S. Lawrence, J ean E. Copper, and Charles D. Smith*
Department of Pharmacology, Pennsylvania State University, Hershey, Pennsylvania 17033
Received J une 28, 2000
A significant problem in the clinical treatment of cancer relates to the development of tumor
resistance to many chemotherapeutic agents. Acquired drug resistance is often mediated
through overexpression of membrane transport proteins that effectively efflux anticancer agents.
Two of the best-studied transporters, P-glycoprotein (Pgp) and MRP1, have pharmacological
properties that only partially overlap. In our search for improved drug-resistance antagonists,
we have identified a family of substituted quinoxalines that selectively antagonizes Pgp over
MRP1. Consequently, a focused library of congeners was designed and synthesized starting
with a parent bromomethylquinoxalinone. This parent quinoxalinone was then condensed with
a series of phenols to yield a family of substituted phenoxymethylquinoxalinones. These
compounds were evaluated for their toxicity toward drug-sensitive MCF-7 breast carcinoma
cells and for their abilities to antagonize Pgp and MRP1 in drug-resistant cell lines (NCI/ADR
and MCF-7/VP, respectively). The results of this structure-activity study indicate that
compounds with carbonyl substitutions of the phenoxy group (ester, amide, or ketone moieties)
demonstrate excellent antagonism of Pgp while having relatively low toxicity toward drug-
sensitive cells. Importantly, none of these compounds antagonized MRP1. Because of their
transporter selectivity, we predict that substituted quinoxalinones may be more effective MDR
modulators in vivo than are nonselective transporter antagonists.
In tr od u ction
expressed by certain types of secretory cells, including
capillary endothelial cells in the brain and testis, and
at sites within the pancreas, kidney, and liver.1,10 By
comparison, MRP1 mRNA has been observed in virtu-
ally every type of tissue11 and is prominently expressed
in peripheral blood mononuclear cells.3,12-15 An ad-
ditional difference between Pgp and MRP1 relates to
their expression in human tumors. Extensive expression
analyses have demonstrated that Pgp is often increased
in tumor cells from patients undergoing chemotherapy,
indicating that this mechanism of MDR is clinically
important.16-19 In contrast, although MRP1 is expressed
in a high percentage of solid tumors and leukemias,20
its overexpression is not consistently found in tumors.
For example, MRP1 levels detected in normal and
malignant hematopoietic cells were equivalent,12,13 and
the MRP1 levels in lung tumors were found to be lower
than those in normal lung tissue.21 The MRP1 mRNA
levels in malignant melanoma,22 acute lymphocytic
leukemia,14 or chronic lymphocytic leukemia10 were not
altered by chemotherapy but did increase moderately
in acute myelogenous leukemia.13,14 Therefore, it seems
that overexpression of Pgp activity is clinically more
significant than elevation of MRP1 levels. A final
difference between Pgp and MRP1 relates to their drug-
resistance profiles which are only partially overlap-
ping.23,24 For example, the resistance factors for vin-
cristine, etoposide, and doxorubicin in MRP1-transfected
cells are greater than those for vinblastine and pacli-
taxel. Conversely, Pgp-overexpressing cells are ex-
tremely resistant to the latter two drugs. The capacity
of certain compounds to antagonize either Pgp or MRP1
has also been suggested.25-27
A significant problem in the clinical treatment of
cancer arises from the development of tumor resistance
to a wide variety of chemotherapeutic agents. The
overexpression of drug transport proteins is a major
mechanism for this multiple drug resistance (MDR).1,2
The drug transporter, P-glycoprotein (Pgp), is an ATP-
dependent drug efflux pump whose biochemistry and
pharmacology have been intensely studied. A series of
homologous proteins termed multidrug-resistance-
related proteins (MRPs) have been discovered more
recently, and these proteins share many pharmacologi-
cal characteristics with Pgp.3-5 Because of the impor-
tance of MDR in clinical oncology, an intensive search
for antagonists of these transporters has developed since
the demonstration that verapamil has this property.6
Consequently, a variety of compounds have since been
shown to reverse Pgp-mediated MDR in vitro,7 and
several of these agents have been evaluated in clinical
trials.8,9 Unfortunately, most of these agents suffer
clinically from their intrinsic toxicity or from undesired
effects on the pharmacokinetics of the accompanying
anticancer drugs.
It seems likely that these clinical results may stem
from incomplete attention to the pharmacologies of the
different transport proteins, making it worthwhile to
compare the properties of Pgp and MRP1. One signifi-
cant difference between Pgp and MRP1 relates to their
tissue distributions. Studies have shown that Pgp is
* Address for correspondence: Dept. of Pharmacology, H078, Penn
State College of Medicine, 500 University Dr., Hershey, PA 17033-
2390. Tel: (717) 531-1672. Fax: (717) 531-5013. E-mail: cdsmith@
psu.edu.
10.1021/jm000282d CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/22/2000

SAR of Substituted Quinoxalinones as MDR Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4 595
Sch em e 1. Synthesis of Substituted Quinoxalinones
Ta ble 1. Reversal of Pgp- and MRP1-Mediated Resistance by 2-Oxoquinoxalines
compd
R1
R2
benzo substituent
toxicitya
Pgpb
MRP1c
Pgp/MRP1
9
10
11
12
13
14
15
16
H
H
H
H
2-aminophenyl
2-acetamidophenyl
2-aminophenyl
2-amino-5-bromophenyl
2-methylamino-5-methoxyphenyl
2-methylamino-5-methoxyphenyl
2-methylaminophenyl
none
none
22
0
37
49
25
7
1.7
1.0
4.6
9.2
1.1
1.1
1.0
1.1
1.1
1.0
1.3
1.3
1.0
3.5
1.1
2.1
1.5
1.0
3.5
7.1
1.1
0.3
0.9
0.5
benz[g]
benz[g]
benz[g]
none
6-nitro
benz[g]
H
CH3
CH3
CH3
7
3
2-methylamino-5-methoxyphenyl
a
b
Toxicity is calculated as the percentage of MCF-7 cells killed by 10 µg/mL of the indicated compounds. The Pgp antagonism score
) percentage of NCI/ADR cells surviving in the absence of vinblastine/percentage of NCI/ADR cells surviving in the presence of vinblastine.
c The MRP1 antagonism score ) percentage of MCF-7/VP cells surviving in the absence of vincristine/percentage of MCF-7/VP cells surviving
in the presence of vincristine.
Because of these issues, we hypothesize that the
selectivity of individual MDR modulators toward Pgp
or MRP1 will be critical in determining their therapeutic
utility.28 Antagonists of these transport proteins en-
hance the intracellular accumulation of many anticancer
drugs, thereby increasing their toxicities. The MDR
modulators most extensively evaulated in clinical trials,
verapamil23-26 and the cyclosporin analogues,24,29,30
antagonize both Pgp and MRP1. Thus, the clinical
failure of these nonselective modulators may result from
increased systemic toxicity of the anticancer drug due
to inhibition of MRP1 in normal tissues. Superior results
may be obtained with modulators that are specifically
directed toward Pgp, and some newer agents including
XR9576,31 LY335979,32 and R10193333 have demon-
strated improved Pgp selectivity and pharmacological
properties. To extend the portfolio of transporter-selec-
tive antagonists, we now describe structure-activity
relationships (SARs) of substituted quinoxalinones that
reverse MDR mediated by Pgp but not MRP1.
tively, indicating selective antagonism of Pgp or MRP1.
Compound 12 was moderately cytotoxic toward MCF-7
cells, a property undesired for an MDR modulator. We
therefore set out to expand the diversity of compounds
of this type to explore the quinoxalinone scaffold for its
potential to provide selective Pgp and MRP1 antagonists
without concomitant toxicity toward nonresistant cells.
A series of substituted quinoxalinones, shown in Table
2, were synthesized by standard literature techniques
centered around the condensation of an o-phenylenedi-
amine with an R-keto acid (Scheme 1). These simple
structures were designed to assess some of the basic
requirements for selective MDR antagonists as well as
to determine parameters for their synthesis. Compound
1e was formed by N-benzylation of compound 1b, which
also produced an enol ether side product 4. While the
quinoxalinones in Table 2 were essentially nontoxic to
MCF-7 cells, they demonstrated only modest MDR
antagonism scores. For comparison purposes, the well-
established MDR antagonist verapmil demonstrated an
IC50 > 51 µM toward MCF-7 cells, and Pgp and MRP1
scores of 5.9 and 1.9, respectively. Importantly, antago-
nism by the substituted quinoxalinones was selective
for Pgp, whereas verapmil caused significant reversal
of both Pgp- and MRP1-mediated MDR. Benzyl substi-
tution in either position 1 or 3 resulted in enhanced Pgp
antagonism over methyl substitution or no substitution
(in position 1) and low toxicity to MCF-7 cells. Therefore,
substitution in the 3-position was further explored to
provide compounds with improved antagonism of Pgp.
In work reported by Corona et al.,34 phenoxy- and
phenoxymethylquinoxalines (without the 2-oxo moiety)
were synthesized and tested for inhibitory activity
Resu lts a n d Discu ssion
Screening 11 000 compounds from commercially avail-
able libraries identified several structural platforms
with good potential as MDR antagonists. One of these,
the 2-oxoquinoxaline or quinoxalinone heterocycle,
showed promise as a versatile scaffold with which to
study SARs. The initial screening was conducted at a
single final dose of 10 µg/mL for the test compounds,
equating to concentrations of approximately 10-50 µM.
Table 1 shows that compounds 12 and 14 selectively
potentiated the cytotoxicity of vinblastine toward NCI/
ADR cells or vincristine toward MCF-7/VP cells, respec-

596 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4
Lawrence et al.
Sch em e 2. Synthesis of Substituted Phenoxymethylquinoxalinones
Ta ble 2. Cytotoxicity and MDR Antagonism by Substituted
Quinoxalinones
Ta ble 3. Cytotoxicity and MDR Antagonism by
Phenoxymethylquinoxalinones
compd
1a
1b
1c
1d
1e
R1
R2
IC50 (µM)a Pgp MRP1 Pgp/MRP1
H
H
benzyl
CH3
CH3
>106
>156
>144
>100
>100
>51
2.8
1.0
1.1
2.9
3.1
5.9
1.0
1.1
0.7
1.1
0.8
1.9
2.8
0.9
1.7
2.7
3.7
3.1
IC50
Pgp/
CH3
CH3
benzyl CH3
compd
R1
R2
(µM) Pgp MRP1 MRP1
benzyl
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
>94
26
3.7
1.2
3.2
2.1
1.9
4.1
1.1
4.7
1.6
5.6
3.6
1.1
3.8
1.3
1.0
1.0
2.5
12.4
1.0
1.2
1.0
0.8
0.8
0.8
0.9
0.9
0.9
0.9
1.1
1.1
1.0
0.9
0.9
0.9
1.1
1.1
0.7
0.9
0.9
0.9
3.7
1.4
3.9
2.6
2.2
4.5
1.3
4.9
1.4
5.2
3.6
1.1
4.2
1.5
0.9
0.9
3.7
13.4
1.2
1.3
4-fluoro
4-cyano
2,3,4,5,6-pentafluoro >70
4-benzyloxy
3-tert-butyl
3,4-dichloro
4-benzoyl
3-methyl-4-nitro
4-CO2CH3-2-OCH3
2-C(O)NH-phenyl
3-dimethylamino
ethyl 4-carboxylate
3-(N-phenylamine)
2-benzyl
verapamil
>43a
a
IC50 ) concentration required to kill 50% of MCF-7 cells.
>40
19
against dihydrofolate reductase and thymidylate syn-
thase. This type of substitution is amenable to a parallel
synthesis strategy and allows the production of many
analogues in a facile manner and was therefore adapted
to a quinoxalinone scaffold. As indicated in Scheme 2,
the synthesis of substituted phenoxymethylquinoxali-
nones began with selective bromination of the 3-methyl
group of compound 1c (or 1e) to give the parent
bromomethylquinoxalinone 2 (or 2a ). The various phen-
oxymethylquinoxalinones were then formed in parallel
by condensation of the bromomethylquinoxalinone with
the appropriate phenolate anion. Because preliminary
experiments demonstrated that the quinoxalinone struc-
ture is sensitive to some basic conditions (e.g. cesium
carbonate in DMF at 70 °C), the reactions were per-
formed under phase-transfer-catalyzed conditions to
minimize exposure of the quinoxalinone heterocycle to
base. Furthermore, because N-alkylation would occur
with quinoxalinones unsubstituted in the 1-position, all
of the parent bromomethylquinoxalinones were substi-
tuted with either methyl or benzyl groups. The selec-
tions of solvent and temperature were done to ensure
convenience in a parallel synthesis approach. While the
unoptimized reaction yields were generally low, this
synthetic strategy allowed for sufficient quantities (>5
mg) of many different compounds to be made with
relative ease. Workups and purifications were done with
only slight variation among samples to maximize the
number of compounds that could be synthesized even
though reaction yield was thereby reduced. These reac-
tion conditions were found to be unsuitable for several
substituent groups, including -NH2, -CO2H, and in-
dole, which did not form condensation products due to
competing reactions. Additionally, methoxy-substituted
>37a
>67
>77
71
>32a
57
>74
25
8.1
>56
30
>54
20
40
3-hydroxypyridine
4-phenyl
benzyl 2-C(O)NH-phenyl
CH3
CH3
3s
3t
2-benzo
3-benzo
a
Accurate IC50 values could not be obtained because of low
solubilities of the compounds. The values indicate the maximum
doses tested.
phenols subjected to these reaction conditions formed
products that showed extra carbonyl stretches in the
infrared spectra, suggesting that a base-catalyzed rear-
rangement of the methoxyphenol had taken place.
Twenty substituted phenoxymethylquinoxalinones were
synthesized and evaluated. Lack of substitution of the
phenoxy moiety resulted in a nontoxic compound (3a )
with a moderate Pgp score (Table 3). Activity was
substantially improved in compounds with carbonyl
substitutions of the phenoxy group. Thus, compounds
3h , 3j, 3k , 3m , and 3r are substituted with ester, amide,
or ketone moieties and demonstrate good antagonism
of Pgp while having relatively low toxicity toward drug-
sensitive cells. Additional active compounds include
compounds 3c and 3f. While a cyano group (3c) can be
viewed as a bioisostere to the carbonyl group, the tert-
butyl group (3f) merely adds steric bulk and resulted
in a compound more toxic than the parental phenoxy-
methylquinoxalinone. Notably, the replacement of the

SAR of Substituted Quinoxalinones as MDR Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4 597
F igu r e 1. Dose-response curves for antagonism of Pgp and MRP1 by substituted phenoxymethylquinoxalinones. Pgp (A and B)
or MRP1 (C and D) antagonism scores were calculated at the indicated concentration of test compound as described in the
Experimental Section. No values beyond the IC50 of the compound toward MCF-7 cells are indicated. Compounds plotted in A
and C include 3a (9), 3b (4), 3c (2), 3d (1), 3e ([), 3f (b), 3g (0), 3h (3), 3i (]), 3j (O), verapamil (×). Compounds plotted in B
and D include 3k (9), 3l (4), 3m (2), 3n (1), 3o ([), 3p (b), 3q (0), 3r (3), 3s (]), 3t (O), verapamil (×).
N-methyl group of 3k with an N-benzyl group of 3r not
only decreased cytotoxicity but also improved Pgp
antagonism by more than 3-fold. Consequently, replace-
ment of N-methyl with larger substituents throughout
the entire sequence of substituted phenoxymethylquin-
oxalinones might yield larger antagonism scores. Of
central importance to the goal of developing transporter-
selective antagonists, none of the phenoxymethylquin-
oxalinones showed any antagonism toward MRP1,
indicating clear pharmacophore differences between Pgp
and MRP1.
The antagonism scores listed in Tables 2 and 3 were
calculated at concentrations of the test compound that
killed no more than a 25% of MCF-7 cells (in parallel
experiments). While this cutoff is somewhat arbitrary,
it simplifies the data significantly and makes a direct,
compound-to-compound comparison possible. A more
complete representation of the data relating to com-
pounds 3a -3t is given by Figure 1, where antagonism
scores up to the IC50 (against MCF-7 cells) are plotted
against the concentrations of test compounds. Results
obtained with verapamil, the current “benchmark” MDR
antagonist, demonstrate that this compound has little
selectivity for Pgp versus MRP1. The plots also show
that, while none of the substituted phenoxymethylquin-
oxalinones have higher peak Pgp scores than verapamil,
compound 3r is both more potent and more selective.
Screening libraries of structurally diverse compounds
resulted in the identification of several scaffolds on
which to build potential MDR antagonists. The phenoxy-
methylquinoxalinone heterocycles were chosen for their
amenability to synthesis and possession of several
substitution sites. A qualitative SAR analysis of 20
substituted phenoxymethylquinoxalinones showed that
the best combination of Pgp-selective antagonism and
low intrinsic toxicity was demonstrated by compounds
containing carbonyl substitutions of the phenoxy group,
with maximal activity demonstrated by compound 3r .
This quinoxalinone possesses an N-phenylamide on the
phenoxy moiety and a benzyl substitution on position
1. Further exploration of this platform to refine the
structural requirements for selective Pgp and MRP1
antagonism is currently underway. Cell lines are widely
used as model systems for the development of anticancer
drugs and are highly reasonable systems for developing
SARs but do not always adequately mimic clinical tumor
responses. Therefore, the ultimate therapeutic utility
of these agents will require evaluation through the
established clinical trial process.
Exp er im en ta l Section
Reagents and starting materials were generally obtained
from Aldrich Chemical (Milwaukee, MN) and used as received
unless otherwise noted. Solvents were either purchased as
anhydrous or ACS grade and stored over 4 Å molecular sieves.
Flash chromatography refers to the method of Still et al.35 and
generally used Selecto Scientific silica gel (32-63 µm). Thin-
layer chromatography was performed on either BakerFlex IB-F
or EM Science silica gel 60 F-254 plates (250-µm thick). Radial
chromatography was performed on a Chromatotron model
7924T (Harrison Research, Palo Alto, CA) using 1-, 2-, or 4-mm
Con clu sion s
We began this project with the hypothesis that
selective antagonism of Pgp versus MRP1 will be
important to effectively clinically potentiate the toxicity
of chemotherapeutic agents toward resistant tumors.

598 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4
Lawrence et al.
thick silica rotors. Melting points were determined in an open
capillary on a MelTemp II melting point apparatus and are
uncorrected. Infrared spectra were measured on a Nicolet
Avatar 360 FT-IR; values are expressed in wavenumbers
(cm-1). 1H and 13C NMR spectra were obtained on a Bruker
200AM spectrometer. Chemical shifts are reported in ppm (δ)
using tetramethylsilane (TMS) as reference and coupling
constants (J ) are reported in hertz. Mass spectra were obtained
from Mass Consortium (San Diego, CA). Elemental analysis
was performed by Midwest Microlab (Indianapolis, IN). All
chemical yields are unoptimized and generally represent the
result of a single experiment.
MCF-7 breast carcinoma cells and NCI/ADR cells, an MDR
line that expresses Pgp,36 were obtained from the Division of
Cancer Treatment of the National Cancer Institute. Drs.
Schneider and Cowan provided MCF-7/VP cells, which express
MRP1 but not Pgp.37 Libraries of compounds for screening
were purchased from MicroSource Discovery Systems, Inc.
(Gaylordsville, CT) and ChemBridge Corp. (San Diego, CA).
3-Ben zyl-1H -q u in oxa lin -2-on e (1a ). 1,2-Phenylenedi-
amine (1.00 g, 9.25 mmol) and phenylpyruvic acid (1.52 g, 9.25
mmol) were dissolved in EtOH (35 mL) and heated to reflux.
After ∼1.5 h, heat was removed and product spontaneously
precipitated. Crude product was collected and recrystallized
from EtOH to give 989 mg of off-white fibrous crystals (mp
178-179 °C, 4.19 mmol, 45.3%). FT-IR (KBr): 3425-3190
cm-1 (NH stretches), 3060-2975 (aromatic CH), 2900-2825
(aliphatic CH), 1660 (CdO), 1600 (CdN). 1H NMR (CDCl3): δ
12.39 (bs, 1H, NH), 7.835 (d, 1H, J ) 8.1 Hz, H8), 7.50-7.45
(m, 3H, H5/6/7), 7.35-7.17 (m, 5H, phenyl), 4.30 (s, 2H, benzyl
-CH2). Anal. (C15H12N2O‚H2O) C, H; N: calcd, 11.02; found,
13.28.
3-Met h yl-1H -q u in oxa lin -2-on e (1b ). 1,2-Phenylenedi-
amine (1.00 g, 9.25 mmol) and pyruvic acid (0.643 mL, 9.25
mmol) were dissolved in EtOH (35 mL) and heated to reflux.
After ∼1.5 h, heat was removed and product spontaneously
precipitated after cooling to 0 °C. Crude product was collected
and recrystallized from EtOH to give 1.001 g of yellow needles
(mp 241-243 °C dec, 6.25 mmol, 67.6%). FT-IR (KBr): 3450-
3100 cm-1 (NH stretches), 3000-2950 (aromatic CH), 2900-
2825 (aliphatic CH), 1660 (CdO), 1600 (CdN). 1H NMR
(CDCl3): δ 7.82-7.79 (m, 1H, H8), 7.51-7.47 (m, 1H, H5),
7.36-7.31 (m, 2H, H6/7), 2.63 (s, 3H, CH3). Anal. (C9H8N2O)
C, H, N.
1,3-Dim eth yl-1H-qu in oxa lin -2-on e (1c). N-Methyl-1,2-
phenylenediamine (6.68 g, 54.7 mmol) and pyruvic acid (3.8
mL, 54.7 mmol) were dissolved in EtOH (230 mL) and warmed
to ∼50 °C. At this point, the color changed dramatically from
a dark, cloudy brown to clear orange. Ethanol was evaporated
and the crude product was filtered through a silica bed with
CH2Cl2/EtOAc (1:1) to give 7.36 g of product as a yellow solid
(mp 75-77 °C, 42.3 mmol, 77.2%). FT-IR (KBr): 3075-2975
cm-1 (aromatic CH), 2950-2850 (aliphatic CH), 1650 (CdO),
1600 (CdN). 1H NMR (CDCl3): δ 7.80 (d, 1H, J ) 7.9 Hz, H8),
7.52 (t, 1H, J ) 7.8 Hz, H5), 7.33 (m, 2H, H6/7), 3.70 (s, 3H,
NCH3), 2.59 (s, 3H, CH3). Anal. (C10H10N2O) C, H, N.
3-Ben zyl-1-m eth yl-1H-qu in oxa lin -2-on e (1d ). N-Methyl-
1,2-phenylenediamine (1.00 g, 8.18 mmol) and phenylpyruvic
acid (1.34 g, 8.18 mmol) were dissolved in EtOH (31 mL) and
the reaction was refluxed for 1.5 h. Ethanol was evaporated
and the crude product was chromatographed through a Biotage
flash chromatography silica cartridge with 40% EtOAc/Hex (at
10 mL/min) to give 400 mg of product as a light yellow solid
(mp 72-74 °C, 1.60 mmol, 19.5%). FT-IR (KBr): 3075-3025
cm-1 (aromatic CH), 2950-2900 (aliphatic CH), 1650 (CdO),
1600 (CdN). 1H NMR (CDCl3): δ 7.83 (dd, 1H, J ) 1.2, 8.0
Hz, H8), 7.46 (d, 3H, J ) 7.3 Hz, H5/6/7), 7.30-7.19 (m, 5H,
phenyl), 4.25 (s, 2H, benzyl CH2), 3.55 (s, 3H, NCH3). Anal.
(C16H14N2O) H, N; C: calcd, 76.78; found, 76.10.
× 30 mL). Combined EtOAc extracts were washed with brine
(30 mL) and dried over MgSO4. Recrystallization of the crude
product from EtOH (0 °C) gave 384 mg of product as a light
yellow solid. Purification of the concentrated mother liquor by
radial chromatography (silica, 15% EtOAc/Hex) gave another
144 mg of product along with 110 mg (0.436 mmol, 13.7%) of
a by-product (2-ben zyloxy-3-m eth yl-1,2-d ih yd r oqu in oxa -
lin e, 4). Total yield of product was 528 mg (mp 90-91 °C, 2.11
mmol, 66.1%). FT-IR (KBr): 3033 cm-1 (aromatic CH), 2918
(aliphatic CH), 1649 (CdO), 1601 (CdN). 1H NMR (CDCl3): δ
7.82 (dd, 1H, J ) 1.3, 7.7 Hz, H8), 7.44-7.22 (m, 8H, H5/6/7
+
phenyl), 5.50 (s, 2H, NCH2), 2.67 (s, 3H, CH3). Anal. (C16H14N2O)
C, H, N. 4: FT-IR (KBr): 3115-3029 cm-1 (aromatic CH),
2955-2889 (aliphatic CH), 1609-1581 (CdC, CdN). 1H NMR
(CDCl3): δ 7.95 (d, 1H, J ) 6.5 Hz, H8), 7.93 (m, 1H, H5), 7.62-
7.38 (m, 7H, H6/7, phenyl), 5.57 (s, 2H, CH2O), 2.68 (s, 3H, CH3).
Anal. (C16H14N2O) C, H, N.
3-Br om om eth yl-1H-1-m eth ylqu in oxa lin -2-on e (2). Di-
methylquinoxalinone (1c; 133 mg, 0.763 mmol) was dissolved
in CCl4 (10 mL); N-bromosuccinimide (144 mg, 0.809 mmol)
and benzoyl peroxide (0.4 mg, 0.002 mmol) were added and
the mixture was heated to reflux. After 10.5 h, TLC (silica,
40% EtOAc/Hex) showed the reaction to be complete. Reaction
was cooled to room temperature and filtered through a plug
of silica with 40% EtOAc/Hex. Purification of the crude product
by radial chromatography (silica, 40% EtOAc/Hex) gave 71 mg
of pure product (mp 180-182 °C dec, 0.28 mmol, 49%). FT-IR
(KBr): 3045 cm-1 (aromatic CH), 2975 (aliphatic CH), 1645
1
(CdO), 1598 (CdN). H NMR (CDCl3): δ 7.87 (d, 1H, J ) 8.0
Hz, H8), 7.61 (t, 1H, J ) 7.3 Hz, H5), 7.41-7.33 (m, 2H, H6/7),
4.66 (s, 2H, CH2Br), 3.74 (s, 3H, NCH3). 13C NMR (CDCl3): δ
155.2 (2), 153.9 (3), 134.0 (10), 132.7 (9), 131.4 (6), 130.7 (7),
124.2 (5), 114.0 (8), 29.5 (CH3).
1-Ben zyl-3-b r om om et h yl-1H -q u in oxa lin -2-on e (2a ).
1-Benzyl-3-methyl-1H-quinoxalin-2-one (1e; 3.20 g, 12.8 mmol)
was dissolved in CCl4 (200 mL); N-bromosuccinimide (2.41 g,
13.5 mmol) and benzoyl peroxide (6.2 mg, 0.026 mmol) were
added and the mixture was heated to reflux. After 16.5 h, TLC
(silica, 25% EtOAc/Hex) showed the reaction to be complete.
Reaction was cooled to room temperature, CCl4 was removed
by evaporation, and the crude mixture was purified by flash
chromatography (silica, 25% EtOAc/Hex) to give 1.41 g of pure
product as a pale yellow solid (mp 131-133 °C dec, 4.28 mmol,
33.5%) along with 1.68 g (6.71 mmol, 52.4%) of unreacted
starting material. FT-IR (CH2Cl2): 3061 cm-1 (aromatic CH),
2979 (aliphatic CH), 1660 (CdO), 1603 (CdN). 1H NMR
(CDCl3): δ 7.88 (d, 1H, J ) 7.8 Hz, H8), 7.49-7.31 (m, 8H,
phenyl), 5.54 (s, 2H, benzyl CH2), 4.73 (s, 2H, CH2Br).
Gen er a l Con d en sa tion P r oced u r e for Su bstitu ted
1-Meth yl-3-p h en oxym eth yl-1H-qu in oxa lin -2-on es 3. Bro-
momethylquinoxalinone (2; 100 mg, 0.395 mmol) and a specific
phenol (0.395 mmol) were dissolved in CHCl3 (10 mL). A
solution of NaOH (23.7 mg, 0.593 mmol) and benzyltriethyl-
ammonium chloride (9 mg, 0.040 mmol) in H2O (10 mL) was
added and the mixture stirred rapidly while heating to 50 °C.
TLC (silica, 40% EtOAc/Hex or 10% EtOAc/CH2Cl2) was used
to monitor the reaction progress. Workup consisted of cooling
to room temperature, separating layers, extracting the aqueous
layer with fresh CHCl3 (2 × 10 mL), washing the combined
chloroform extracts with brine (10 mL), and drying over Na2-
SO4. Evaporation of the solvent, followed by filtration through
a plug of silica with 40% EtOAc/Hex, gave the crude product;
purification by radial chromatography (silica, 40% EtOAc/Hex)
gave the pure product.
1-Meth yl-3-p h en oxym eth yl-1H-qu in oxa lin -2-on e (3a ):
pale yellow film (41%). FT-IR (KBr): 3059 cm-1 (aromatic CH),
2933 (aliphatic CH), 1647 (CdO), 1603 (CdN). 1H NMR
(CDCl3): δ 7.95 (d, 1H, J ) 7.9 Hz, H8), 7.59 (m, 1H, H5), 7.39-
7.26 (m, 4H, H6/7 + phenolic Hb), 7.08 (d, 2H, J ) 7.8 Hz,
phenolic Ha), 6.97 (m, 1H, phenolic Hc), 5.38 (s, 2H, CH2O),
3.74 (s, 3H, NCH3). Anal. (C16H14N2O2‚0.5H2O) C, H, N.
3-(4-F lu or op h en oxym eth yl)-1-m eth yl-1H-qu in oxa lin -
2-on e (3b): clear film (14%). FT-IR (CCl4): 3063 cm-1 (aromatic
CH), 2922-2852 (aliphatic CH), 1651 (CdO), 1601 (CdN). 1H
1-Ben zyl-3-m eth yl-1H-qu in oxa lin -2-on e (1e). 3-Methyl-
quinoxalinone (1b; 511 mg, 3.19 mmol) was stirred with benzyl
chloride (0.367 mL, 3.19 mmol) and potassium carbonate (904
mg, 6.54 mmol) in DMF (10 mL) overnight. Reaction mixture
was poured into 30 mL of water and extracted with EtOAc (3

SAR of Substituted Quinoxalinones as MDR Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4 599
NMR (CDCl3): δ 7.97-7.93 (m, 1H, H8), 7.68-7.52 (m, 3H,
H5 + Hâ), 7.38-7.34 (m, 2H, H6/7), 7.01-6.97 (m, 2H, HR), 5.33
(s, 2H, CH2O), 3.75 (s, 3H, NCH3). Anal. (C16H13N2O2F‚2.5H2O)
C; H: calcd, 5.51; found, 4.03; N: calcd, 8.51; found, 9.71.
3-(4-Cya n op h en oxym eth yl)-1-m eth yl-1H-qu in oxa lin -2-
on e (3c): pale yellow solid (mp 133 °C dec, 17.4%). FT-IR
(KBr): 3068 cm-1 (aromatic CH), 2923 (aliphatic CH), 2222
(CtN), 1654 (CdO), 1606 (CdN). 1H NMR (CDCl3): δ 7.90
(d, 1H, J ) 7.1 Hz, H8), 7.59 (d, 3H, H5 + phenolic Hb), 7.39 (t,
2H, J ) 6.8 Hz, H6/7), 7.12 (d, 2H, J ) 8.8 Hz, phenolic Ha),
5.42 (s, 2H, CH2O), 3.76 (s, 3H, NCH3). Anal. (C17H13N3O2‚
H2O) C, H; N: calcd, 13.58; found, 12.99.
7.80-7.05 (m, 13H, aromatic protons), 5.59 (s, 2H, CH2O), 3.76
(s, 3H, NCH3). Anal. (C23H19N3O3‚1.5H2O) C, N; H: calcd, 5.38;
found, 4.68.
3-(3-Dim e t h yla m in op h e n oxym e t h yl)-1-m e t h yl-1H -
q u in oxa lin -2-on e (3l): orange-brown film (11%). FT-IR
(CDCl3): 3079 cm-1 (aromatic CH), 2924-2802 (aliphatic CH),
1
1657 (CdO), 1604 (CdN). H NMR (CDCl3): δ 7.95 (d, 1H, J
) 7.7 Hz, H8), 7.57 (d, 1H, J ) 7.2 Hz, H5), 7.40-7.33 (m, 2H,
H
6/7), 7.14 (m, 1H, H2′), 6.53 (m, 3H, H4′/5′/6′), 5.37 (s, 2H, CH2O),
3.74 (s, 3H, NCH3), 2.94 (s, 6H, N(CH3)2). Anal. (C18H19N3O2‚
1.5H2O) H; C: calcd, 64.27; found, 64.96; N: calcd, 12.49;
found, 10.08.
1-Meth yl-3-(2,3,4,5,6-p en ta flu or op h en oxym eth yl)-1H-
qu in oxa lin -2-on e (3d ): pale yellow solid (mp 128-130 °C,
27%). FT-IR (KBr): 3059 cm-1 (aromatic CH), 2933 (aliphatic
CH), 1647 (CdO), 1603 (CdN). 1H NMR (CDCl3): δ 7.88 (d,
1H, J ) 7.9 Hz, H8), 7.60 (m, 1H, H5), 7.38 (m, 2H, H6/7), 5.47
(s, 2H, CH2O), 3.73 (s, 3H, NCH3). Anal. (C16H9N2O2F5) C, H,
N; F: calcd, 26.66; found, 23.20.
3-(4-Ben zyloxyp h en oxym eth yl)-1-m eth yl-1H-qu in oxa -
lin -2-on e (3e): yellow film (9%). FT-IR (KBr): 3033 cm-1
(aromatic CH), 2926 (aliphatic CH), 1656 (CdO), 1603 (Cd
N). 1H NMR (CDCl3): δ 7.95 (d, 1H, J ) 3.9 Hz, H8), 7.63-
7.55 (m, 1H, H5), 7.42-7.22 (m, 7H, H6/7 + benzyl), 7.07-6.83
(m, 4H, H2′/3′/5′/6′), 5.32 (s, 2H, quinoxalines CH2O), 5.01 (s, 2H,
benzyl CH2O), 3.74 (s, 3H, NCH3). Anal. (C23H20N2O3‚2H2O)
C, N; H: calcd, 5.92; found, 5.18.
3-(3-ter t-Bu tylp h en oxym eth yl)-1-m eth yl-1H-qu in oxa -
lin -2-on e (3f): yellow film (6%). FT-IR (KBr): 3077 cm-1
(aromatic CH), 2963-2868 (aliphatic CH), 1658 (CdO), 1601
(CdN). 1H NMR (CDCl3): δ 8.32 (d, 1H, J ) 6.3 Hz, NH),
7.88-6.98 (m, 8H, aromatic protons), 6.88 (d, 1H, J ) 8.1 Hz,
CdCH-O), 5.59 (s, 1H, CH2O), 3.77 (s, 3H, NCH3), 1.26 (s,
9H, [CH3]3).
4-(4-Meth yl-3-oxo-3,4-dih ydr oqu in oxalin -2-ylm eth oxy)-
ben zoic a cid eth yl ester (3m ): light yellow solid (mp 152
°C dec, 39%). FT-IR (KBr): 3071 cm-1 (aromatic CH), 2983-
2910 (aliphatic CH), 1710 (ester CdO), 1656 (CdO), 1602 (Cd
N). 1H NMR (CDCl3): δ 8.02-7.90 (m, 2H, H3′/5′), 7.61-7.56
(m, 2H, H5/8), 7.41-7.33 (m, 2H, H6/7), 7.11-7.00 (m, 2H, H2′/6′),
5.46 (d, 2H, J ) 16 Hz, CH2O), 4.34 (q, 2H, J ) 7,1 Hz, ester
CH2), 3.75 (s, 3H, NCH3), 1.37 (t, 3H, J ) 7.1 Hz, ester CH3).
Anal. (C19H18N2O4‚1.75H2O) C, N; H: calcd, 5.86; found, 4.90.
1-Meth yl-3-(3-p h en yla m in op h en oxym eth yl)-1H-qu in -
oxa lin -2-on e (3n ): yellow film (17%). FT-IR (CDCl3): 3345
cm-1 (NH), 3050 (aromatic CH), 2923 (aliphatic CH), 1653 (Cd
1
O), 1595 (CdN). H NMR (CDCl3): δ 7.95 (d, 1H, J ) 7.7 Hz,
H8), 7.58 (t, 1H, J ) 7.7 Hz, H5), 7.41-7.33 (m, 2H, H6/7), 7.22-
6.62 (m, 9H, phenyl), 5.34 (s, 2H, CH2O), 3.73 (s, 3H, NCH3).
Anal. (C22H19N3O2‚3H2O) C; H: calcd, 6.12; found, 5.35; N:
calcd, 10.21; found, 9.24.
3-(2-Ben zylp h en oxym eth yl)-1-m eth yl-1H-qu in oxa lin -
2-on e (3o): yellow film (6%). FT-IR (CDCl3): 3026 cm-1
(aromatic CH), 2925 (aliphatic CH), 1655 (CdO), 1603 (Cd
N). 1H NMR (CDCl3): δ 8.07 (d, 1H, J ) 8.6 Hz, H8), 7.66-
7.32 (m, 5H, H5/6/7/3′/5′), 7.16 (bs, 5H, phenyl), 7.08-6.95 (m,
2H, H4′/6′), 5.22 (s, 2H, CH2O), 3.80 (s, 2H, benzyl CH2), 3.75
(s, 3H, NCH3).
3-(3,4-Dich lor op h en oxym eth yl)-1-m eth yl-1H-qu in oxa -
lin -2-on e (3g): orange solid (mp 105-107 °C, 66%). FT-IR
(KBr): 3084 cm-1 (aromatic CH), 2921 (aliphatic CH), 1658
1-Meth yl-3-(p yr id in -3-yloxym eth yl)-1H-qu in oxa lin -2-
on e (3p ): off-white film (3%). FT-IR (CDCl3): 3070 cm-1
(aromatic CH), 2919-2850 (aliphatic CH), 1651 (CdO), 1602
1
(CdO), 1608 (CdN). H NMR (CDCl3): δ 7.95 (d, 1H, J ) 7.9
Hz, H8), 7.63-7.59 (m, 1H, H5), 7.44-7.17 (m, 2H, H6/7), 6.98-
6.95 (m, 2H, phenolic H5/6), 6.72 (d, 1H, J ) 7.7 Hz, phenolic
H2), 5.34 (s, 2H, CH2O), 3.76 (s, 3H, NCH3). Anal. (C16H12N2O2-
Cl2‚H2O) N; C: calcd, 54.41; found, 54.87; H: calcd, 4.00; found,
3.48; Cl: calcd, 20.07; found, 23.44.
1
(CdN). H NMR (CDCl3): δ 7.88 (d, 1H, J ) 7.9 Hz, H8), 7.60
(m, 1H, H5), 7.42-7.19 (m, 6H, H6 + pyridyl), 5.45 (s, 2H,
CH2O), 3.75 (s, 3H, NCH3). Anal. (C15H13N3O2) C: calcd, 67.40;
found, 45.68; H: calcd, 4.90; found, 3.95; N: calcd, 15.72;
found, 7.48.
3-(4-Ben zoylp h en oxym eth yl)-1-m eth yl-1H-qu in oxa lin -
2-on e (3h ): cream-colored solid (mp 145-147 °C, 45%). FT-
IR (KBr): 3053 cm-1 (aromatic CH), 2903 (aliphatic CH), 1655
(CdO), 1600 (CdN). 1H NMR (CDCl3): δ 7.93 (dd, 1H, J )
1.1, 8.0 Hz, H8), 7.85-7.73 (m, 4H, H3′/5′/2′′/6′′), 7.62-7.42 (m,
4H, H5/3′′/4′′/5′′), 7.37 (d, 2H, J ) 8.0 Hz, H6/7), 7.13 (d, 2H, J )
8.9 Hz, H2′/6′) 5.46 (s, 2H, CH2O), 3.76 (s, 3H, NCH3). Anal.
(C23H18N2O3‚2H2O) C, N; H: calcd, 5.46; found, 4.73.
3-(Bip h en yl-4-yloxym et h yl)-1-m et h yl-1H -q u in oxa lin -
2-on e (3q): pale yellow film (27%). FT-IR (CDCl3): 3031 cm-1
(aromatic CH), 2934 (aliphatic CH), 1654 (CdO), 1604 (Cd
N). 1H NMR (CDCl3): δ 7.96 (d, 1H, J ) 6.0 Hz, H8), 7.81-
7.09 (m, 12H, phenyl), 5.44 (d, 2H, J ) 11.5 Hz, CH2O), 3.74
(s, 3H, NCH3). Anal. (C22H18N2O2‚3H2O) C, N; H: calcd, 6.10;
found, 4.65.
1-Meth yl-3-(3-m eth yl-4-n itr op h en oxym eth yl)-1H-qu i-
n oxa lin -2-on e (3i): off-white solid (mp 140-143 °C dec, 13%).
FT-IR (KBr): 3088 cm-1 (aromatic CH), 2930 (aliphatic CH),
1661 (CdO), 1607 (CdN), 1509 (assym. NO2), 1341 (symm.
2-(4-Ben zyl-3-oxo-3,4-dih ydr oqu in oxalin -2-ylm eth oxy)-
N-p h en ylb en za m id e (3r ): condensed as in 3k , but using
1-ben zyl-3-br om om eth yl-1H-qu in oxalin -2-on e (2a) as start-
ing material to give product as a yellow solid (mp 167-169
°C, 70%). FT-IR (KBr): 3446-3318 cm-1 (amide NH), 3064-
3060 (aromatic CH), 2924-2835 (aliphatic CH), 1655 (amide
1
NO2). H NMR (CDCl3): δ 8.08 (d, 1H, J ) 9.8 Hz, H5′), 7.89
(m, 1H, H8), 7.60 (m, 1H, H5), 7.43-7.35 (m, 2H, H6/7), 6.94
(bs, 2H, H2′/6′), 5.43 (s, 2H, CH2O), 3.76 (s, 3H, NCH3), 2.63 (s,
3H, CH3). Anal. (C17H15N3O4‚H2O) C, N; H: calcd, 4.99; found,
4.46.
1
CdO), 1599 (CdN). H NMR (CDCl3): δ 10.79 (bs, 1H, NH),
8.34 (dd, 1H, J ) 1.8, 7.9 Hz, H3′), 7.79 (d, 2H, J ) 7.6 Hz,
H
8/5′), 7.67 (d, 1H, J ) 7.0 Hz, phenyl), 7.52-7.14 (m, 14H,
phenyl), 5.66 (s, 2H, benzyl CH2), 5.54 (s, 2H, CH2O). Anal.
(C29H23N3O3) C, H, N.
3-Meth oxy-4-(4-m eth yl-3-oxo-3,4-d ih yd r oqu in oxa lin -2-
ylm eth oxy)ben zoic a cid m eth yl ester (3j): yellow film
(7.1%). FT-IR (KBr): 3087 cm-1 (aromatic CH), 2921-2850
(aliphatic CH), 1715 (ester CdO), 1653 (CdO), 1602 (CdN).
1H NMR (CDCl3): δ 7.92 (d, 1H, J ) 7.7 Hz, H8), 7.64-7.33
(m, 5H, H5/6/7 + H2’/6′), 6.99 (d, 1H, J ) 8.4 Hz, H5′), 5.49 (s,
2H, CH2O), 3.97 (s, 3H, CO2CH3), 3.89 (s, 3H, OCH3), 3.75 (s,
3H, NCH3). Anal. (C19H18N2O5‚2.5H2O) C, N; H: calcd, 5.80;
found, 5.08.
2-(4-Meth yl-3-oxo-3,4-dih ydr oqu in oxalin -2-ylm eth oxy)-
N-p h en ylben za m id e (3k ): yellow solid (mp 148 °C dec, 43%).
FT-IR (KBr): 3315 cm-1 (amide NH), 3040 (aromatic CH),
2916 (aliphatic CH), 1656 (amide CdO), 1643 (CdO), 1598 (Cd
N). 1H NMR (CDCl3): δ 8.31 (dd, 1H, J ) 1.8, 7.8 Hz, NH),
1-Meth yl-3-(n aph th alen -1-yloxym eth yl)-1H-qu in oxalin -
2-on e (3s): light yellow film (12%). FT-IR (CDCl3): 3053 cm-1
(aromatic CH), 2936 (aliphatic CH), 1653 (amide CdO), 1603
(CdN). 1H NMR (CDCl3): δ 8.49-8.44 (m, 1H, H8′), 7.94 (d,
1H, J ) 7.7 Hz, H8), 7.82-7.78 (m, 1H, H5), 7.64-7.32 (m, 7H,
phenyl), 6.98 (d, 1H, J ) 7.3 Hz, H2′), 5.53 (s, 2H, CH2O), 3.79
(s, 3H, NCH3). Anal. (C20H16N2O2‚1.25H2O) C, N; H: calcd,
5.50; found, 4.89.
1-Meth yl-3-(n aph th alen -2-yloxym eth yl)-1H-qu in oxalin -
2-on e (3t): light yellow film (17%). FT-IR (CDCl3): 3057 cm-1
(aromatic CH), 2936 (aliphatic CH), 1653 (amide CdO), 1602
(CdN). 1H NMR (CDCl3): δ 7.97-7.89 (m, 1H, H8), 7.79-7.33

600 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4
Lawrence et al.
(7) Ford, J . M.; Hait, W. N. Pharmacology of drugs that alter
multidrug resistance in cancer. Pharmacol. Rev. 1990, 42, 155-
199.
(8) Kaye, S. B. Multidrug resistance: clinical relevance in solid
tumors ans strategies for circumvention. Curr. Opin. Oncol.
1998, 10, S15-S19.
(9) Sikic, B. I. Modulation of multidrug resistance: a paradigm for
translational clinical research. Oncology 1999, 13, 183-187.
(10) Leveille-Webster, C. R.; Arias, I. M. The biology of the P-
glycoproteins. J . Membr. Biol. 1995, 143, 89-102.
(11) Zaman, G. J .; Versantvoort, C. H.; Smit, J . J .; Eijdems, E. W.;
de Haas, M.; Smith, A. J .; Broxterman, H. J .; Mulder, N. H.; de
Vries, E. G.; Baas, F.; et al. Analysis of the expression of MRP,
the gene for a new putative transmembrane drug transporter,
in human multidrug resistant lung cancer cell lines. Cancer Res.
1993, 53, 1747-1750.
(12) Abbaszadegan, M. R.; Futscher, B. W.; Klimecki, W. T.; List,
A.; Dalton, W. S. Analysis of multidrug resistance-associated
protein (MRP) messenger RNA in normal and malignant he-
matopoietic cells. Cancer Res. 1994, 54, 4676-4679.
(13) Schneider, E.; Cowan, K. H.; Bader, H.; Toomey, S.; Schwartz,
G. N.; Karp, J . E.; Burke, P. J .; Kaufmann, S. H. Increased
expression of the multidrug resistance-associated protein gene
in relapsed acute leukemia. Blood 1995, 85, 186-193.
(14) Hart, S. M.; Ganeshaguru, K.; Hoffbrand, A. V.; Prentice, H. G.;
Mehta, A. B. Expression of the multidrug resistance-associated
protein (MRP) in acute leukaemia. Leukemia 1994, 8, 2163-
2168.
(15) Burger, H.; Nooter, K.; Sonneveld, P.; Van Wingerden, K. E.;
Zaman, G. J .; Stoter, G. High expression of the multidrug
resistance-associated protein (MRP) in chronic and prolympho-
cytic leukaemia. Br. J . Haematol. 1994, 88, 348-356.
(16) Goldstein, L. J .; Galski, H.; Fojo, A.; Willingham, M.; Lai, S. L.;
Gazdar, A.; Pirker, R.; Green, A.; Crist, W.; Brodeur, G. M.; et
al. Expression of a multidrug resistance gene in human cancers.
J . Natl. Cancer Inst. 1989, 81, 116-124.
(m, 10H, phenyl), 5.49 (s, 2H, CH2O), 3.75 (s, 3H, NCH3). Anal.
(C20H16N2O2‚1.5H2O) C, N; H: calcd, 5.58; found, 4.83.
Assa y of Cytotoxicity a n d Rever sa l of MDR. To test for
reversal of Pgp-mediated MDR, NCI/ADR cells were placed
into 96-well tissue culture plates at approximately 15%
confluency and were allowed to attach and recover for 24 h.
The cells were then treated with varying concentrations (as
allowed by solubility) of the test compound in the presence of
0 or 50 nM vinblastine for 48 h according to previously
described procedures.38,39 After 48 h, cell survival was assayed
using the sulforhodamine B (SRB) binding assay.40 The
percentage of cells killed was calculated as the percentage
decrease in SRB binding as compared with control cultures
and was taken from the mean of the absorbance measurements
of three equally treated wells. Reversal of MDR is indicated if
the compound enhanced the toxicity of vinblastine toward the
NCI/ADR cells. The reversal index (Pgp Antagonism Score)
was calculated as the percentage of surviving NCI/ADR cells
in the absence of vinblastine/the percentage of surviving NCI/
ADR cells in the presence of vinblastine. Control cultures
included equivalent amounts of ethanol (as the solvent control),
which does not modulate the growth or drug sensitivity of
these cells at the doses used in these studies. Inhibition of Pgp
was manifested as the ability of the compound to potentiate
the cytotoxicity of vinblastine toward the NCI/ADR cells. To
assess the toxicity of the compounds toward drug-sensitive
cells, the effects of the test modulators on the growth of drug-
sensitive MCF-7 cells were determined by the same methods.
To test for reversal of MRP1-mediated MDR, MCF-7/VP cells
were placed into 96-well tissue culture plates at approximately
15% confluency and were allowed to attach and recover for 24
h. The cells were then treated with varying concentrations (as
allowed by solubility) of the test compound in the presence of
0 or 1 nM vincristine for 48 h as above. After 48 h, cell survival
was assayed using the SRB binding assay. Reversal of MDR
is indicated if the compound enhanced the toxicity of vincris-
tine toward the MCF-7/VP cells. The reversal index (MRP1
antagonism score) was calculated as the percentage of surviv-
ing MCF-7/VP cells in the absence of vincristine/the percentage
of surviving MCF-7/VP cells in the presence of vincristine.
Control cultures included equivalent amounts of ethanol (as
the solvent control), which does not modulate the growth or
drug sensitivity of these cells at the doses used in these studies.
Inhibition of MRP1 was manifested as the ability of the
compound to potentiate the cytotoxicity of vincristine toward
the MCF-7/VP cells.
(17) Fojo, A. T.; Ueda, K.; Slamon, D. J .; Poplack, D. G.; Gottesman,
M. M.; Pastan, I. Expression of a multidrug-resistance gene in
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Ack n ow led gm en t. This work was supported by
NIH Grant CA 64983 (C.D.S.) and NRSA Fellowship
F32 CA 74549 (D.S.L.) from the National Cancer
Institute.
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