Synthesis and chemical characterization of 2-methoxy-N10-substituted acridones needed to reverse vinblastine resistance in multidrug resistant (MDR) cancer cells

Article abstract of DOI:10.1016/S0968-0896(02)00068-8

In an attempt to find clinically useful modulators of multidrug resistance (MDR), a series of 19 N¹⁰-substituted-2-methoxyacridone analogues has been synthesized. 2-Methoxyacridone and its derivatives (1-19) were synthesized. Compound 1 was pre

Full text of DOI:10.1016/S0968-0896(02)00068-8

Bioorganic & Medicinal Chemistry 10 (2002) 2367–2380
Synthesis and Chemical Characterization of 2-Methoxy-
N -substituted Acridones Needed to Reverse Vinblastine
Resistance in MultidrugResistant (MDR) Cancer Cells
10
a,y
b
b,y
c
d
Gowdahalli Krishnegowda, Padma Thimmaiah, Ravi Hegde, Chhabil Dass,
b,
Peter J. Houghton and Kuntebommanahalli N. Thimmaiah *
aDepartment of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Road,
NW, Washington DC 20007-219, USA
b
Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, TN38105-2794, USA
cDepartment of Studies in Chemistry, University of Mysore, Mysore-570 006, India
dDepartment of Chemistry, University of Memphis, Memphis, TN38152, USA
Received 15 October 2001; accepted 26 December 2001
1
0
Abstract—In an attempt to find clinically useful modulators of multidrug resistance (MDR), a series of 19 N -substituted-2-
methoxyacridone analogues has been synthesized. 2-Methoxyacridone and its derivatives (1–19) were synthesized. Compound 1 was
prepared by the Ullmann condensation of o-chlorobenzoic acid and p-anisidine followed by cyclization using polyphosphoric acid.
This compound undergoes N-alkylation in the presence of phase transfer catalyst (PTC). Stirring of 2-methoxy acridone with
1
-bromo-3-chloropropane or 1-bromo-4-chlorobutane in a two-phase system consisting of organic phase (tetrahydrofuran) and 6 N
potassium hydroxide in the presence of tetrabutylammonium bromide leads to the formation of compounds 2 and 11 in good yield.
N-(o-Chloroalkyl) analogues were found to undergo iodide catalyzed nucleophilic substitution reaction with various secondary
amines. Products were characterized by UV, IR, H and C NMR, mass-spectral data and elemental analysis. The lipophilicity
1
13
expressed in log10 P and pK of compounds have been determined. All compounds were examined for their ability to increase the
a
R
uptake of vinblastine (VLB) in MDR KBCh -8-5 cells and the results showed that the compounds 7, 10, 12, and 15–19 at 100 mM
caused a 1.05- to 1.7-fold greater accumulation of vinblastine than did a similar concentration of the standard modulator, ver-
apamil (VRP). However, the effects on VLB uptake were specific because these derivatives had little effect in the parental drug
sensitive line KB-3-1. Steady state accumulation of VLB, a substrate for P-glycoprotein (P-gp) mediated efflux, was studied in the
R
MDR cell line KBCh -8-5 in the presence and absence of novel MDR modulators. Results of the efflux experiment showed that
VRP and each of the modulators (1–19) significantly inhibited the efflux of VLB, suggesting that they may be competitors for P-gp.
From among the compounds examined, 14 except 1, 2, 4, 8, and 11, exhibited greater efflux inhibiting activity than VRP. All the 19
3
compounds effectively compete with [ H] azidopine for binding to P-gp, pointed out this transport membrane protein as their likely
site of action. Cytotoxicity has been determined and the IC50 values lie in the range 8.00–18.50 mM for propyl and 4–15 mM for
butyl derivatives against KBCh -8-5 cells suggesting that the antiproliferative activity increases as chain length increases from 3 to
R
1
0
R
4
cells and found that the modulators enhanced the cytotoxicity of VLB by 5- to 35-fold. Modulators 12, 14–16, and 19 like VRP, were
carbons at N -position. Compounds at IC were evaluated for their efficacy to modulate the cytotoxicity of VLB in KBCh -8-5
10
R
able to completely reverse the 24-fold resistance of KBCh -8-5 cells to VLB. Examination of the relationship between lipophilicity and
antagonism of MDR showed a reasonable correlation suggesting that hydrophobicity is one of the determinants of potency for anti-
MDR activity of 2-methoxyacridones. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
in cross-resistance to many structurally unrelated
agents, has been and continues to be a major focus in
1
,2
Multidrug resistance (MDR) is the phenomenon by
which tumor cells exhibit an intrinsic resistance or
where acquired resistance to one cytotoxic agent results
experimental therapy of human cancer. The phenom-
enon is caused by the overexpression of a 170 kDa
transmembrane glycoprotein known as P-glycoprotein
3
(P-gp) encoded by the mdr1 gene in MDR cells. P-gp
has been shown to bind ATP and drug analogues,
4
,5
6,7
*
1
y
Corresponding author. Tel.: +1-901-495-2067; fax: +1-901-521-
668; e-mail: kn.thimmaiah@stjude.org
The first and second authors have made equal contributions to this work.
8
has ATPase activity and catalyzes ATP-dependent drug
efflux to effectively reduce intracellular accumulation in
0
968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00068-8

2368
G. Krishnegowda et al. / Bioorg. Med. Chem. 10 (2002) 2367–2380
Scheme 1.
resistant cells.3,9 Many diverse compounds that are cap-
able of modulating P-gp include calcium channel block-
among them one derivative, GF120918,26 was found to
be extremely potent in reversing the MDR. In two ani-
mal models of MDR, the P388/DOX leukemia and the
C26 colon carcinoma, a single iv dose of GF120918
restores the antiproliferative action of doxorubicin.
Further, in an ongoing program, the investigators’
group has already demonstrated a novel acridone deri-
vative [1,3-bis (9-oxoacridin-10-yl)propane], as a potent
and poorly reversible modulator of P-glycoprotein
1
0
11
12,13
ers,
antimalarials, and other lysosomotropic agents, ster-
calmodulin inhibitors,
4
antiarrythmics,
15
1
oids, antiestrogens,17 and cyclic peptide antibiotics.
1
6
18
They lower the IC50 values of a variety of drugs inclu-
ded in the MDR family and they increase intracellular
drug concentrations in resistant cells. The mechanisms
responsible for this reversal of resistance is believed to be
competition between the modulator and cytotoxic drug
2
7
mediated vinblastine transport. This interesting data
has inspired the authors to synthesize and describe the
properties of 19 2-methoxyacridones as potential mod-
19,20
for binding to the ATP-dependent efflux pump, P-gp.
The clinical utility of any modulator, however, depends
not only on its ability to reverse drug resistance at low
concentrations but also on whether it has a low toxicity
in vivo. The cardiac toxicity seen during the clinical
evaluation of verapamil as a chemosensitizing agent
R
ulators of MDR against KBCh -8-5 cells.
Chemistry
2
1,22
pointed out the need for less toxic modulators.
Two
other antiarrythmic drugs, quinidine and amiodarone,
have also entered clinical trials as chemosensitizing
2-Methoxyacridone (1) and its derivatives (2–19) were
prepared by the synthetic route as outlined in Scheme 1.
Compound 1 was prepared by the Ullmann condensa-
tion of o-chlorobenzoic acid and p-anisidine to form
2
3
agents and both drugs have produced a number of
2
4
adverse clinical side effects. While a number of phar-
macological agents have been shown to reverse MDR in
vitro, there remains a need to identify more potent, more
specific and less toxic chemosensitizers for clinical use.
0
4 -methoxydiphenylamine-2-carboxylic acid (C). The
reactants were heated preferably at reflux in the pre-
sence of copper powder and potassium carbonate in
0
2-carboxylic acid (C) was cyclized with sulfuric acid at
100 C on a water-bath to form 2-methoxyacridone (1)
isoamylalcohol medium. The 4 -methoxydiphenylamine-
The reversal of MDR can be obtained by treatment
with numerous classes of lipophilic positively, charged
drugs known as MDR inhibitors. Latest studies have
raised hopes that addition of MDR inhibitors to cyto-
toxics might improve chemotherapy success rate. The
design of potent MDR inhibitors devoid of other phar-
macological activities has thus become a desirable goal
to test the MDR reversal hypothesis in the clinic. In the
course of a chemical program aimed at identifying
ꢀ
(60%) and sulphonated 2-methoxyacridone (40%) as
evidenced by TLC. When cyclization was carried out
with polyphosphoric acid instead of sulfuric acid on a
ꢀ
water-bath at 100 C only single product of 2-methoxy-
acridone (1) with better yield (90%) was obtained.
The weakly basic nature of nitrogen atom of the acri-
done nucleus usually resists to undergo N-alkylation
with alkyl halides. However, it can be achieved in the
2
5
potent MDR inhibitors, Hyafil et al. have identified a
series of acridone carboxamide derivatives and from

G. Krishnegowda et al. / Bioorg. Med. Chem. 10 (2002) 2367–2380
2369
presence of basic condensing agents like sodium amide
or sodium hydride. The general procedure for preparing
N-alkyl derivative consists of condensation of acridone
with requisite alkyl halide in the presence of a strong
acid binding agent like sodium amide in anhydrous
aromatic solvents such as toluene or benzene. The reac-
tion of 2-methoxyacridone with mixed chloro-
bromoalkanes in the presence of sodium amide in
and unable to diffuse out of the cell. The magnitude of
the biological activity depends on pK of compounds
a
besides other factors. For the series of compounds
examined, the pK values (Table 1) ranging from 8.3 to
a
9.6 lie closer to physiological pH, which may suggest
that these compounds accumulate in MDR cells as free
bases rather than in protonated form. The lipophilicity
data varying from 1.3 to 2.3, expressed in log P for 19
1
0
1
0
anhydrous toluene under reflux conditions gave N -
chloroalkyl)-2-methoxyacridone, but the yield was low,
2-methoxyacridones are given in Table 1. Within the
series, all compounds are highly lipophilic at pH 7.4 and
it is expected that they will accumulate rapidly into cells.
Analysis of the relationship between lipid solubility
(log P) and the effectiveness of the modulators to
(
besides requiring drastic experimental conditions. How-
ever, this compound undergoes N-alkylation in the pre-
sence of PTC more easily compared to previously
described preparative procedure.
1
0
R
increase VLB accumulation in drug-resistant KBCh -8-5
cells showed a good correlation (R=0.78, data not
shown). The major outliyer in this analysis was that the
degree of lipophilicity of each drug would seem to be
important and it is one of the determinants of potency
for the P-gp modulating activity of 2-methoxyacridones.
Stirring of 2-methoxyacridone (1) at room temperature
with alkylating agent (Br-(CH ) -Cl or Br-(CH ) -Cl) in
2
3
2 4
a two-phase system consisting of an organic solvent
tetrahydrofuran) and 6 N aqueous potassium
hydroxide solution in the presence of tetra-
(
a
+
À
butylammonium bromide [(n-C H ) N Br ] leads to
4
9 4
10
Effect of 2-methoxy-N -substituted acridones on the
R
the formation of the compound 2 or 11 in good yield.
Here, ammonium salt transports hydroxide ion from
aqueous phase to organic phase where the actual reac-
tion takes place. These results are interpreted by depro-
accumulation of VLB in KBCh -8-5 cells
1
0
In order to explore the potential of 2-methoxy-N -sub-
stituted acridones to enhance the uptake of VLB, the
effect of 19 compounds at 100 mM was determined in
À
tonation of 1 by the OH , transferred by the catalyst
R
into the organic layer. The anion formed may be regar-
ded as phenolate stabilized anion, which subsequently
undergoes alkylation to form the aromatized system.
MDR KBCh -8-5 cells. As shown in Table 1, com-
pounds 1–19 at 100 mM concentration exhibited sig-
nificant VLB accumulation enhancing effect (7- to 20-
fold relative to control) compared to a standard mod-
ulator verapamil (VRP) (11.9-fold). Eleven compounds
1–6, 8, 9, 11, 13, and 14 were found to possess less VLB
enhancing effect (7- to 11.2-fold) compared to VRP
(11.9-fold) relative to control. Remaining eight com-
pounds (7, 10, 12, and 15–19) caused a 1.05- to 1.7-fold
greater accumulation of VLB than did a similar con-
centration of VRP. The enhancement of VLB uptake
was specific for MDR cell line since all the compounds
1
0
Iodide catalyzed nucleophilic substitution of the N -
1
0
propyl or N -butylchloride of 2-methoxyacridone (1)
with various secondary amines (N,N-diethylamine, N,N-
diethanolamine, pyrrolidine, piperidine, morpholine,
thiomorpholine, 1-methylpiperazine and (b-hydro-
xyethyl)piperazine) by refluxing for different times in the
presence of potassium carbonate in acetonitrile gave the
free bases 3–10 and 12–19.
(
1–19) had very little effect in sensitive KB-3-1cells
All the products were separated and purified by column
chromatography or recrystallization method and dried
under high vacuum for more than 12 h. The purified
(data not shown ). A similar effect was observed on the
effect of phenoxazines on the accumulation of VLB in
2
9
drug sensitive KB-3-1cells.
The VLB uptake enhan-
1
13
compounds were characterized by UV, IR, H and
NMR, mass spectral, and elemental analysis.
C
cing effects with respect to percentage of control are in
the range 842–1278% for propyl derivatives and 996–
1998% for butyl derivatives suggesting that the butyl
derivatives seem to rank the list of these compounds.
Comparative study of the VLB uptake data of the
modulators within the propyl derivatives revealed that
all the propyl compounds except 7 and 10, exhibited
almost the same uptake enhancing effect. Similar com-
parative study within the butyl derivatives on the uptake of
Pharmacological Results and Discussion
pK and lipophilicity effects
a
The effectiveness of any agent as an MDR modulator
will depend in part on its ability to accumulate in cells.
The 2-methoxyacridones are weak bases and able to exist
in both charged (protonated) and uncharged (unproto-
nated) forms. The unprotonated or neutral form of
compounds will be highly membrane permeable and
able to diffuse freely and rapidly across biological
membranes. In contrast, the protonated form would be
at least an order of magnitude less membrane permeable
and diffuse across membranes at a much reduced rate. In
addition, if the unprotonated form of the molecule dif-
fuses across the membrane and enters an acidic compart-
ment within the cell, it will rapidly become protonated
R
VLB into KBCh -8-5 cells revealed that the compounds
follow the order: 11<13ꢁ14<12<17ꢁ18<16<15<19.
Further, the effects of varying concentrations of mod-
ulators 5, 6, 7, 8, 9, 10, 12, 15, 19, or VRP on the uptake
R
of VLB in MDR cells were studied. The KBCh -8-5
3
cells were exposed for 2 h to 49.9 nM [ H] VLB in the
absence or presence of 10 mM, 25 mM, 50 mM, or 100
mM concentration of the above nine compounds or
VRP and the intracellular concentration of VLB in
2
8
6
picomoles/10 cells was calculated. The results revealed
that all the modulators at 10 mM exhibited the least
VLB enhancing effect whereas at 25, 50, and 100 mM,

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G. Krishnegowda et al. / Bioorg. Med. Chem. 10 (2002) 2367–2380
Table 1. Effect of 2-methoxy-N10-substituted acridones on accumulation of vinblastine in KBCh -8-5-cells
R
b
Compd
R
pK
a
log10Pa
2.11
Vinblastine uptake (% control) ÆSEM
1
-H
8.29
8.80
699Æ2.11
842Æ1.32
2
1.32
3
5.35
9
1.35
866Æ1.35
900Æ1.30
933Æ1.34
927Æ1.52
1278Æ1.70
900Æ1.30
996Æ1.42
1245Æ1.75
.16
4
5
6
7
8
9
4.69
.25
1.30
1.34
1.52
1.70
1.30
1.42
1.75
9
5.20
.29
9
5.34
.30
9
4.77
.20
9
4.95
.30
9
5.27
.15
9
10
5.24
.09
9
1
1
1
1
1
1
1
2
3
4
5
6
8.82
1.40
1.85
1.60
1.65
2.30
2.00
996Æ1.40
1301Æ1.85
1099Æ1.60
1116Æ1.65
1919Æ2.30
1673Æ2.00
5.43
9
.36
4.78
.30
9
5.24
.60
9
5.21
.60
9
4.93
.10
9
(
continued on next page)

G. Krishnegowda et al. / Bioorg. Med. Chem. 10 (2002) 2367–2380
2371
Table 1( continued)
log10Pa
1.90
Vinblastine uptake (% control) ÆSEM
b
Compd
R
pK
a
17
5.24
.25
1410Æ1.90
9
1
1
8
9
5.65
.21
1.90
1410Æ1.90
9
5.27
.38
2.30
ND
1998Æ2.30
1190Æ1.72
9
Verapamil
ND
ND, not determined.
a
Octanol/water partition coefficient.
ꢀ
ꢁ
b
Vinblastine uptake with modulator
Vinblastine uptake without modulator
 100: Compounds were tested at 100 mM. All values represent the mean of two separate experiments and each
experiment was done in triplicate.
the modulators exhibited a significant increase on the
accumulation of VLB suggesting that the uptake of
VLB into KBCh -8-5 cells is dependent on the con-
accumulation and inhibit significantly the efflux of VLB
R
from MDR KBCh -8-5 cells.
R
centration of modulator. The most effective modulator
is 19 allowing an almost 20-fold increase in accumula-
tion of VLB relative to control. The presence of an
The time-course of VLB efflux from MDR cells was also
3
done (Fig. 1). The cells were loaded with 49.9 nM [ H]
VLB for 2 h at room temperature and washed and the
retained radioactivity was measured at the indicated
times. When efflux was examined in the absence of mod-
ulator, less than 20% of the cellular VLB remained after 2
h. When a similar experiment was performed, but with 100
mM concentration of VRP or compound 19 present con-
tinuously, about 67% or 80% of the cell-associated VLB
remained at the end of a 2 h efflux period. This data sug-
gests that the 2-methoxyacridones like VRP, are able to
1
0
N -substitution seems to be necessary to optimize the
activity in 2-methoxyacridones. Comparison of the
derivatives for their ability to potentiate the uptake of
R
VLB in KBCh -8-5 cells revealed that they follow the
1
0
10
order N -butyl>N -propyl, although there are one or
two exceptions.
Effect of substituted acridones on the active outward
R
R
inhibit P-gp mediated VLB efflux from KBCh -8-5 cells.
transport of vinblastine from KBCh -8-5 cells
Decreased uptake of cytotoxics in many MDR cells is
attributed to increased efflux mediated by P-gp. To
determine whether the increase in VLB accumulation
upon coincubation with 2-methoxyacridones (1–19) was
Table 2. Effect of 2-methoxy-N10-substituted acridones on the cel-
R
lular retention of VLB in KBCh -8-5 cells after 2 h efflux
Compd
Cellular retention (%) of VLB after 2 h efflux
R
due to a slowing of P-gp mediated VLB efflux, KBCh -
3
1
2
3
4
5
6
7
8
9
1
1
12
13
1
1
1
1
18
1
56.00
63.00
77.00
62.00
68.50
78.40
77.15
57.30
72.40
71.00
63.00
86.20
79.60
79.80
81.00
82.00
84.00
84.00
80.00
67.00
19.50
8
-5 cells were loaded with 49.9 nM [ H] VLB for 2 h in
3
the absence of modulators and resuspended in [ H] VLB
free buffer with or without 100 mM modulators (1–19)
or VRP for an additional 2 h at room temperature. The
cell associated radiolabel remaining after a 2 h efflux
was determined and calculated as a percentage of the
VLB present after loading. The data on the fraction of
VLB remaining after 2 h are given in Table 2. Results of
the efflux experiment showed that VRP and each of the
modulators (1–19) significantly inhibited the efflux of
VLB, suggesting that they may be competitors for P-gp.
After incubation of the cells for 2 h in the absence of
modulator, more than 80% of VLB was lost from the
cells, whereas 56–86% of VLB in the presence of mod-
ulators (1–19) or 67% of VLB in the presence of VRP
was retained in the cells. From among 19 modulators,
0
1
4
5
6
7
9
Verapamil
Control
1
greater efflux inhibiting activity than VRP. Possibly all
4 compounds except 1, 2, 4, 8, and 11, exhibited
All values represent the mean of two separate experiments with a SD
of less than 10% of the mean; each experiment was done in triplicate.
3
the modulators (1–19) are able to both enhance [ H] VLB

2372
G. Krishnegowda et al. / Bioorg. Med. Chem. 10 (2002) 2367–2380
To investigate whether 2-methoxyacridones interact with
3
competing for the drug binding site on the protein. The
fact that all the compounds (1–19) have reduced the
photoaffinity labeling of azidopine appreciably, the
results predict that the modulators compete for azido-
pine for binding to P-gp. Careful evaluation of the data
of the modulators showed that butyl derivatives have
exhibited greater competition than that of propyl deri-
vatives suggesting that this is due to enhanced lipophi-
licity. From among the compounds tested, one propyl
derivative (10) and two butyl derivatives (15 and 19)
exhibited the maximum competition (85–88%) and the
remaining butyl derivatives except (11), quoted to be
around 76–80% competition. Comparison of the inhibi-
tion data has revealed that those modulators, which have
exhibited greater VLB accumulation effect and maximum
VLB efflux preventing ability from MDR cells, have
exhibited greater competition for azidopine labeling, sug-
gesting that the activity of 2-methoxyacridones (1–19)
may be mediated through P-gp-dependent mechanism.
P-gp by photolabeling this protein with [ H] azidopine
Cornwell et al.30 originally showed that P-gp of the
MDR KB cells is specifically labeled with VLB analo-
gues and that VRP blocked the specific labeling. A
dihydropyridine analogue has been found to specifically
interact with P-gp by a photoaffinity labeling assay. The
reversing of drug resistance by chemosensitizers seems
3
to be correlated with the inhibition of [ H] azidopine
3
photolabeling of P-gp. Photoaffinity labeling with [ H]
azidopine was not detected under our experimental
conditions when used membranes prepared from drug-
sensitive KB-3-1cells or from KBCh R-8-5 cells, which
have a low level of drug resistance (data not shown). A
highly VLB resistant and P-gp expressing KB cell sub-
3
line KB-V1was therefore used for [ H] azidopine com-
petition studies.
The VLB transport and cell survival data suggest that
the modulatory effect of 2-methoxyacridones is through
an interaction with P-gp. To confirm this suggestion the
competition between [ H] azidopine and 19 2-methoxy-
acridones (1–19) was determined. At 100 mM
1
0
Effect of 2-methoxy-N -substituted acridones on
inhibition of cellular proliferation
3
The cytotoxicity of 2-methoxyacridones was examined by
incubating the drug-sensitive (KB-3–1) cells continuously
for 7 days with a range of concentrations up to 100 mM.
Examination of the IC50 values has revealed that the
compounds are relatively nontoxic (data not given).
2
-methoxyacridones inhibited the labeling of P-gp by
3
3
[
after inhibition by acridones expressed in percentage of
H] azidopine. The binding of [ H] azidopine to P-gp
control (no competitor) is as follows: 1 by 65%, 2 by
0%, 3 by 60%, 4 by 55%, 5 by 45%, 6 by 40%, 7 by
0%, 8 by 54%, 9 by 50%, 10 by 10%, 11 by 65%, 12
7
2
The cytotoxicity of each of the 19 2-methoxyacridones
R
by 19%, 13 by 20%, 14 by 24%, 15 by 12%, 16 by 18%,
7 by 20%, 18 by 19%, 19 by 15%, and VRP by 60%.
Comparison of the data on the competition between
(1–19) was examined in MDR KBCh -8-5 cells. The
concentration of modulators (1–19) that reduced colony
1
formation by 10 and 50% (IC10 and IC ) were
50
3
[
concentration revealed that the ability of all the com-
H] azidopine and each acridone modulator at 100 mM
determined from concentration percent survival
curves and the results are given in Table 3. The IC10
and IC50 values for 19 modulators (1–19) lie, respec-
tively, in the range 0.5–5 and 4.0–25.0 mM for
KBCh -8–5 cells. Careful examination of IC values
for -N -chloropropyl (8.0-18.5 mM) and -N -chlor-
pounds, except compounds 1–3 and 11, to inhibit the
3
[
H] azidonpine labeling of P-gp, is greater than that of
R
the standard modulator VRP by 1.1- to 5-fold. If a
modulator inhibits labeling by the probe of interest,
then it is said that this modulator probably functions by
50
10
1
0
R
obutyl (4.0–9.3 mM) derivatives against KBCh -8-5
3
R
Figure 1. Effect of modulator 19 and verapamil on the outward transport of [ H]vinblastine from KBCh -8-5 cells. Efflux studies were done in the
presence of vehicle control, 0.1% DMSO (~), modulator 19 (&) or verapamil (^) as described in Experimental section.

G. Krishnegowda et al. / Bioorg. Med. Chem. 10 (2002) 2367–2380
2373
cells revealed that antiproliferative activity largely
increased as the chain length increased from 3 to 4
suggesting that the hydrophobicity plays an impor-
tant role on the biological activity. Increasing the
distance between the ring nucleus and amino group
increased the antiproliferative activity of these com-
pounds. Comparison of the cytotoxicity of the butyl
derivatives has shown that the cell killing potency fol-
lows the order: 19>16ꢁ15ꢁ13>14>12ꢁ18>17>11
between structure and antiproliferative activity from
these studies.
R
Sensitization of drug-resistant KBCh -8-5 cells by
1
0
2
-methoxy-N -substituted acridones
We have evaluated the ability of 19 compounds to mod-
ulate the cytotoxicity of VLB in MDR cells. Cells (KB-3-1
R
or KBCh -8-5) were exposed continuously to 0–100 nM
R
against KBCh -8–5 cells. The structural features
VLB for 7 days in the absence and presence of IC10 con-
centrations of 2-methoxyacridone modulators (1–19).
The concentration response curves were determined by
clonogenic assay, and the IC50 and fold-potentiation of
VLB cytotoxicity are summarized in Table 4. The IC
required to cause a maximum antiproliferative activity
include a hydrophobic acridone nucleus and a piper-
azinylamine with a para-CH group, joined by a four
3
alkyl bridge to the nucleus. The structural features
required within the series to cause a maximum anti-
5
0
R
values for VLB against KBCh -8-5 cells in the presence of
IC10 of modulator (1–19) lie in the range of 2–14 nM.
Examination of IC50 values of VLB in the presence of
propyl derivatives (4–12 nM) or butyl derivatives (2–6
nM) of acridone has revealed that the butyl derivatives
R
proliferative activity in KBCh -8–5 cells include
hydrophobic acridone ring nucleus with a-OCH sub-
3
stitution at C-2 and a side-chain tertiary cationic
amino group that is separated from the aromatic ring
by at least three to four carbons. However, it is not
possible to draw conclusions about the correlation
R
have sensitized the MDR KBCh -8-5 cells to a greater
extent presumably due to increased hydrophobicity.
Comparative study of the abilities of the modulators to
potentiate the cytotoxicity of VLB has revealed that the
modulator 19 demonstrated the greatest effect followed
by 16, 14, 12, 15, 18, and so on. Only five 2-methox-
yacridones (12, 14, 15, 16, and 19), like VRP, were able
Table 3. Cytotoxicity of 2-methoxy-N10-substituted acridones in
R
human KBCh -8-5 cell line
Compd
IC10 (mM)
IC50 (mM)
R
1
2
3
4
5
6
7
8
9
1.0
1.5
1.4
1.8
0.9
1.2
5.0
1.3
25.0
18.0
12.8
10.8
12.5
14.5
11.0
18.5
15.0
8.0
15.0
8.0
5.0
7.0
5.0
5.0
9.3
8.0
to completely reverse the 24-fold resistance of KBCh -
8-5 cells to VLB. The IC50 values for continuous expo-
sure to VLB was 3 nM in KB-3-1and 72 nM in
R
KBCh -8-5 cells in the absence of modulating agent.
The most effective modulators (19, 16, and 12) in
R
KBCh -8-5 cells were subsequently tested in KB-3-1
and all three were shown to cause a small sensitization
4.0
1.4
(2- to 3-fold) of the drug-sensitive line to VLB. How-
1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
9
ever, a similar degree of sensitization was also found
when the classical MDR modulator, VRP (3.5-fold) was
used. The influence of alkyl bridge length connecting the
acridone nucleus to the amino group was examined.
Increasing the distance between the ring nucleus and the
amino group from three to four carbons showed appre-
ciable difference in the antiproliferative and anti-MDR
effects of these compounds. Further, a careful analysis of
the relationship between hydrophobicity and antagonism
of MDR showed a reasonable correlation (Fig. 2). Thus,
the degree of lipophilicity is one of the determinants of
potency for anti-MDR activity of 2-methoxyacridones.
1.50
1.50
0.65
0.60
0.70
0.90
0.50
1.00
0.50
[30]
4.0
[55]
Verapamil
IC10 and IC50 are the concentrations (mM) required to produce 10%
and 50% reduction respectively, in clonogenic survival of KBCh -8–5
cells under the conditions described in the Experimental section.
R
Table 4. Effect of 2-methoxy-N10-substituted acridones on the potentiation of vinblastine cytotoxicity in drug resistant KBCh -8-5 cells
R
Compda
VLB ICb50 (nM)
FPc
Compda
VLB ICb50 (nM)
FPc
1
2
3
4
5
6
7
8
9
14.0
7.5
8.0
7.0
8.0
7.0
4.0
12.0
7.5
4.0
5.0
9.0
9.0
10.0
9.0
10.0
17.5
5.8
11
12
13
14
15
16
17
18
19
6.00
2.14
3.80
2.10
2.20
2.10
5.00
3.00
2.00
11.7
comp.d
18.4
comp.
comp.
comp.
14.0
23.33
comp.
9.0
17.5
10
a
Modulators used at the IC10 concentration (Table 3).
Concentration of modulators required for 50% reduction of cells compared to controls.
b
c
Fold-potentiation.
comp., Complete reversal of VLB resistance.
d

2374
G. Krishnegowda et al. / Bioorg. Med. Chem. 10 (2002) 2367–2380
Figure 2. Correlation between the log10 P of 2-methoxy-N10-substituted acridones (1–19) and antagonism of MDR in KBCh -8-5 cells.
R
In summary, we have synthesized a series of novel acri-
dones that showed significant anti-MDR activity at their
analyte molecules and no fragmentation provided the
sampling cone voltage <30 V. In order to reduce the
thermally induced fragmentation, the temperature of
R
IC10 values against KBCh -8-5 cells. Five compounds
(12, 14–16, and 19) were able to reverse completerly the
ꢀ
the ion source was adjusted to 60 C. The quadrupole
R
2
4-fold resistance of KBCh -8-5 cells to vinblastine.
was scanned in the mass range of 50 to 450 Da. In the
EI technique used an AutoSpec Q (VG Analytical,
Manchester, UK) hybrid tandem mass spectrometer of
E BE -qQ geometry (where E is an electric sector, B a
Based on the promissing results, the compounds are
considered to be potential lead molecules for the future
development of clinically useful chemosensitizers.
1
2
magnetic sector, q an rf-only quadrupole and Q a
quadrupole mass analyzer). Only the front end (i.e.,
E BE ) was used for this study. The data accumulation
1
2
Experimental
and manipulation were done under digital Vax Station
100-based Opus Software. Electron ionization-ionizing
3
General
energy, 70 eV; emission current, 100 mA; source tempera-
ture, 100 C; mass resolution 2000; accelerating potential,
ꢀ
Reactions were monitored by TLC. Column chromato-
graphy utilized silica gel Merck Grade 60 (230–400
8000 V, mass range 50–500 Da; spectral scan rate, 5 s/
decade; samples were introduced via direct insertion
probe, which was not heated; the only source of heat to
the sample was through contact with the ion source.
˚
mesh, 60 A). Melting points were recorded on a Tem-
pirol hot-stage with microscope and are uncorrected.
UV spectra were recorded in MeOH on a Shimadzu-
UV-1601 spectrophotometer; IR spectra were recorded
on a Perkin–Elmer Model 1320 spectrometer as KBr pel-
lets. Elemental analyses were performed at Central Drug
Research Institute, Lucknow, India. Found values are
within 0.4% of theoretical values unless otherwise noted.
Materials
All the chemicals and supplies were obtained from
standard commercial sources unless otherwise indicated.
DMEM media, Hank’s balanced salt and trypsin–
EDTA were purchased from Imperial (UK). Vinblastine
sulfate was purchased from Cetus Corporation (Emery-
ville, CA, USA). RPMI-1640 medium with glutamine
and without sodium bicarbonate and sodium pyruvate
were purchased from Gibco BRL (Grand Island, NY,
1H and 13C NMR spectra were recorded in DMSO
solution in a 5-mm tube on a Bruker drx 500 Fourier
transform spectrometer with tetramethylsilane as inter-
nal standard. Chemical shifts are expressed as d (ppm)
values. The spectrometer was internally locked to the
deuterium frequency of the solvent. To obtain mole-
cular weight information, acridone derivatives were
analyzed by electro spray ionization (ESI) and electron
ionization (EI) mass spectrometry. In ESI, the sample
solution is injected into a continuous stream of a solu-
tion that consists of 1:1 mixture aq 0.1% trifluoroacetic
acid and acetonitrile. In this study, a single quadrupole
mass spectrometer (platform II, micromass) was
employed to analyse acridones. ESI is a gentle mode of
ionization. It mainly produces protonated ions of the
3
USA). [ H] vinblastine (sp. act. 9.4 Ci/mmol) was pur-
chased from Amersham Pharmacia Biotech., UK, Ltd.
Verapamil hydrochloride, colchicine, dimethyl sulfoxide
were purchased from Sigma Chemical Co. (St. Louis,
MO, USA).
Synthesis
0
Preparation of 4 -methoxydiphenylamine-2-carboxylic
acid (C). Ullmann condensation. To a mixture of
o-chlorobenzoic acid (A) (12.5 g, 0.08 mol), p-anisidine

G. Krishnegowda et al. / Bioorg. Med. Chem. 10 (2002) 2367–2380
2375
(
B) (9.84 g, 0.08 mol) and copper powder (0.2 g) in 60
(8652) nm. IR 3446, 3010, 2945, 1638, 1605, 1506, 1475,
À1
1
mL of isoamylalcohol, dry potassium carbonate (12 g)
was slowly added and the contents were allowed to
reflux for 6 h on an oil bath at about 160 C. The iso-
1367, 1274, 1175, 1061, 812, 780, 761 cm . H NMR
(DMSO-d ) d 7.73–8.78 (m, 7H, Ar-H, H , H , H , and
6
1
3
4
ꢀ
H -H ), 3.74 (s, 3H, OCH ), 3.73 (m, 2H, H ), 3.71(m,
5 8 3 m
1
3
amylalcohol was removed by steam distillation and the
mixture poured into 1L of hot water and acidified with
concentrated hydrochloric acid. The bluish black pre-
cipitate which formed was filtered, washed with hot
water and collected. The crude acid was dissolved in aq
sodium hydroxide solution, boiled in the presence of
activated charcoal and filtered. On acidification of the
filtrate with concentrated hydrochloric acid, bluish
black precipitate of (C) was obtained which was washed
with hot water and recrystallized from aq methanol to
2H, H ), 2.66 (m, 2H, H ); C NMR (DMSO-d ) d
k
l
6
110.17 (C ), 157.91 (C ), 125.01 (C ), 108.79 (C ),
119.21 (C ), 137.02 (C ), 123.07 (C ), 126.21 (C ), 179.9
1
2
3
4
5
6
7
8
(C ), 138.0 (C
9
0
), 140.10 (C10
), 59.33 (OCH ), 46.54 (C ), 33.56 (C ), 46.89 (C ).
0
), 121.17 (C8
0
), 121.42
4
(C9
MS m/z (%) 301(M , 100), 238 (70), 224 (35), 210 (25),
0
3
k
l
m
+
Á
195 (25), 167 (30), 182 (10), 153 (10), 77 (5), 41 (25).
0
10-[3 -(N-Diethylamino)propyl]-2-methoxyacridone (3).
Compound 2 (1.11 g, 3.68 mmol) was dissolved in 100
mL of anhydrous acetonitrile and 1.57 g of KI, 2.54 g of
K CO and 1.17 g (16.02 mmol) of N,N-diethylamine
ꢀ
give greenish yellow needles (14 g, 72%, mp 187 C).
Á
+
MS (m/z) 243 (M ).
2
3
were added. The mixture was refluxed for 16 h, and the
contents were cooled, diluted with water and extracted
with chloroform. The chloroform layer was washed
thrice with water, dried over anhydrous sodium sulfate
and evaporated to give an oily product. The oily residue
was purified by column chromatography using the sol-
vent system chloroform–acetone (8+1mL) to give a
2
-Methoxyacridone (1). Twelve grams of C were taken
in a round-bottom flask to which was added 120 g of
polyphosphoric acid. Shaken well and heated on a water
bath at 100 C for about 3 h. Appearance of yellow
color indicated the completion of the reaction. Then, it
was poured into 1L of hot water and made alkaline by
liquor ammonia. The yellow precipitate which formed
was filtered, washed with hot water, and collected. The
sample of 2-methoxyacridone (1) was recrystallized
ꢀ
0
light-yellow oil of 10-[3 -(N-diethylamino)propyl]-2-
methoxyacridone (3). An acetone solution of the free
base was treated with ethereal hydrochloride to give the
hydrochloride salt, which was dried over high vacuum
from methanol:1N potassium hydroxide solution
ꢀ
ꢀ
[
60+40 mL] (10 g, 90%, mp 282–283 C). Further, the
to obtain pure solid (3) (0.81g, 40%, mp 9 1– 93 C). UV
purity was checked on TLC plate using the solvent sys-
tem chloroform:methanol [24+1mL] and the purified
product was characterized by spectral methods. UV
lmax (2) (MeOH) 211 (16,824), 250 (40,472), 269
lmax (2) (MeOH) 212 (8828), 252 (33,359), 271 (28,789),
397 (6562) nm. IR 3415, 2985, 2400, 1646, 1567, 1544,
1492, 1144, 1072, 820, 796 cm . H NMR (DMSO-d )
À1
1
6
d 7.34–8.38 (m, 7H, Ar-H, H , H , H , H -H ), 3.90 (s,
1
3
4
5
8
(
38,085), 394 (7477) nm. IR 3436, 2987, 2940, 1636,
3H, OCH ), 3.72 (m, 2H, H ), 3.46–3.79 (m, 6H, H ,
H , H ), 1.64 (m, 2H, H ), 1.60–1.67 (m, 6H, H and
b m l c
3 k a
1
8
533, 1605, 1476, 1502, 1398, 1372, 1238, 1166, 1036,
À1
1
13
29, 762, 705 cm . H NMR (DMSO-d ) d 7.21–8.23
Hd); C NMR (DMSO-d ) d 115.6 (C ), 154.0 (C ),
6
6
1
2
(
m, 7H, Ar-H, H , H , H , H -H ), 3.85 (s, 3H, OCH ),
125.06 (C ), 106.34 (C ), 117.55 (C ), 135.66 (C ), 122.73
1
3
4
5
8
3
3 4 5 6
13
1
1
1
1
5
1.73 (s, 1H, NH); C NMR (DMSO-d ) d 115.12 (C ),
(C ), 127.07 (C ), 177.51 (C ), 136.32 (C ), 140.81 (C10
7
0
0
),
0 0
), 121.53 (C9 ), 56.26 (OCH ), 49.58 (C ), 22.83
6
1
8
9
4
54.28 (C ), 124.56 (C ), 105.26 (C ), 117.68 (C ),
33.33 (C ), 121.33 (C ), 126.26 (C ), 176.46 (C ),
120.25 (C8
(C ), 43.93 (C ), 48.84 (C and C ), 9.54 (C and C ). MS
(m/z) 338 (M , 10), 337 (12), 86 (100), 72 (22), 36 (12).
2
3
4
5
3
k
6
7
8
9
l
m
a
b
c
d
+
Á
36.10 (C4
0
), 140.83 (C10
0
), 119.57 (C8
0
), 121.01 (C
+
0
),
5.68 (OCH ). MS m/z (%) 226 (M+H) , 100. Anal.
9
3
0
done (4). Amounts of 1.16 g (3.84 mmol) of 2, 1.64 g of
(
C H NO ) C, H, N.
1
10-[3 -[N-Bis(hydroxyethyl)amino]propyl]-2-methoxyacri-
4
11
2
KI, 2.65 g of K CO and 1.20 mL (1.28 g, 9.83 mmol) of
3
1
0
2
Synthesis of N -alkylated acridones via phase-transfer
catalysis
N,N-diethanolamine were refluxed for 24 h and fol-
lowed the rest of the procedure used for 3. The product
was purified by column chromatography to give yellow
oily product and it was converted into hydrochloride
0
4.44 mmol) of 2-methoxyacridone was dissolved in 20
1
(
0-(3 -Chloropropyl)-2-methoxyacridone (2). One gram
mL of tetrahydrofuran and 25 mL of 6 N potassium
hydroxide and 0.74 g of tetrabutylammonium bromide
salt, which was dried over high vacuum to get pure solid
(4) (0.93g, 45%, mp 192–193 C). UV l
ꢀ
212 (8177), 252 (32,838), 270 (30,211), 397 (6271) nm.
(2) (MeOH)
max
(
2.3 mmol) was added to it. The reaction mixture was
stirred at room temperature for 30 min. Added
-bromo-3-chloropropane (11.5 mmol) slowly into the
IR 3379, 2970, 2867, 2779, 1636, 1594, 1501, 1480, 1434,
À1
1
1
1274, 1258, 1227, 1129, 1196, 829, 767 cm . H NMR
(DMSO-d ) d 7.70–8.77 (m, 7H, Ar-H, H , H , H , H -
reaction mixture and stirred at room temperature for 24
h. Tetrahydrofuran was evaporated and the aq layer
extracted with chloroform. The chloroform layer was
washed with water and the organic layer dried over
anhydrous sodium sulfate and rotavaporated. The
crude product was purified by column chromatography
using the solvent system chloroform–acetone (8+1mL)
6
1
3
4
5
H ), 4.17 (s, 2H, H and H ), 3.93 (s, 3H, OCH ), 3.64–
3
8
e
f
3.88 (m, 8H, H , H , H , H ), 2.50–2.75 (m, 4H, H and
k
m
c
d
a
1
3
H ), 1.40–1.60 (m, 2H, H ); C NMR (DMSO-d ) d
b
l
6
114.04 (C ), 150.09 (C ), 121.16 (C ), 113.67 (C ),
116.00 (C ), 132.75 (C ), 118.85 (C ), 123.14 (C ), 173.58
1
2
3
4
5
6
7
8
(C ), 132.39 (C
9
0
), 136.88 (C10
0
), 116.29 (C8
0
), 117.34 (C9
0
),
4
0
to give yellow crystals of 10-(3 -chloropropyl)-2-meth-
ꢀ
56.44 (OCH ), 47.74 (C ), 18.47 (C ), 40.01(C ), 52.37 (C
3 k l m a
+
oxyacridone (2) (1.12 g, 51%, mp 130 C). UV l
(2)
and C ), 52.70 (C and C ). MS m/z (%) 371(M+H)
b
,
max
c
d
(
MeOH) 213 (13,262), 252 (44,645), 271 (36,241), 399
100), 226 (30), 146 (21). Anal. (C H N O ) C, H, N.
21 26 2 4

2376
G. Krishnegowda et al. / Bioorg. Med. Chem. 10 (2002) 2367–2380
0
The procedure used for 4 was repeated with 1.05 g
1
0 - (3 - N - Pyrrolidinopropyl) - 2 - methoxyacridone (5).
K CO and 1.25 g (12.12 mmol) of thiomorpholine. The
2 3
oily product was purified by column chromatography
and the yellow oily product was converted into hydro-
(
3.33 mmol) of 2, 1.48 g of KI, 2.4 g of K CO and 1g
2
3
ꢀ
(MeOH) 215 (9950), 252 (28,168), 271 (24,752), 399
(14.08 mmol, 1.2 mL) of pyrrolidine. The residue was
purified by column chromatography, and the oil was
chloride salt of 8 (0.85 g, 40%, mp 152 C). UV l
(2)
max
converted into hydrochloride salt of 5 (0.72 g, 41%, mp
1
(
(5630) nm. IR 3431, 2919, 2789, 1646, 1620, 1589, 1465,
À1
ꢀ
22,688), 272 (19,103), 400 (4587) nm. IR 3415, 2971,
1
56–158 C). UV l
(2) (MeOH) 214 (4050), 252
1424, 1253, 1139, 1072, 999, 891 cm
.
H NMR
max
(DMSO-d ) d 7.2–8.3 (m, 7H, Ar-H, H , H , H , H -
6
1
3
4
5
2
684, 1646, 1596, 1560, 1501, 1146, 1134, 1072, 876, 749
À1
H ), 3.85 (s, 3H, OCH ), 3.72 (m, 2H, H ), 2.50–2.54 (m,
8 3 k
1
cm . H NMR (DMSO-d ) d 7.3–8.4 (m, 7H, Ar-H,
H , H , H , H -H ), 3.89 (s, 3H, OCH ), 3.86 (m, 2H,
H ), 2.99–3.54 (m, 6H, H , H , H ), 1.86–2.35 (m, 6H,
H , H and H ); C NMR (DMSO-d ) d 115.74 (C ),
1
6H, H , H and H ), 2.49 (m, 4H, H and H ), 1.85 (m, 2H,
a b m c d
6
1
3
Hl). C NMR (DMSO-d ) d 117.68 (C ), 154.29 (C ),
1
3
4
5
8
3
6
1
2
124.57 (C ), 105.19 (C ), 119.58 (C ), 133.31 (C ), 121.3
k
a
b
m
3 4 5 6
1
3
(C ), 126.21 (C ), 176.46 (C ), 136.14 (C ), 140.85 (C10
7
0
0
),
0 0
), 121.01 (C9 ), 55.71(OCH ), 53.35 (C ), 24.05
l
c
d
6
1
8
9
4
54.16 (C ), 125.24 (C ), 106.43 (C ), 117.72 (C ), 135.7
0
119.95 (C8
(C ), 42.71(C ), 53.21(C and C ), 26.44 (C and C ). MS
m/z (%) 369 (M+H) , (5), 247 (100), 226 (90), 162 (58).
2
3
4
5
3
k
(
C ), 122.79 (C ), 127.19 (C ), 177.92 (C ), 136.51 (C
6
),
), 56.35 (OCH3),
7
8
9
4
l
m
a
b
c
d
+
1 ), 121.74 (C9
52.76 (C ), 24.80 (C ), 43.85 (C ), 55.52 (C and C ), 23.92
40.96 (C10
0
), 120.45 (C8
0
0
k
l
m
a
b
+
0
9). The procedure used for 4 was employed with 1g
(C and C ). MS m/z (%) 337 (M+H) , 100), 338 (22).
c
10-(3 -N-[Methylpiperazino)propyl]-2-methoxyacridone
(
(3.32 mmol) of 2, 1.41 g of KI, 2.29 g of K CO and
d
0
0-(3 -N-Piperidinopropyl)-2-methoxyacridone (6). The
1
2
3
procedure used for 4 was repeated with 1.08 g (3.58
mmol) of 2, 1.58 g of KI, 2.47 g of K CO and 1.25 g
(14.7 mmol, 1.45 mL) of piperidine. The crude product
was purified by column chromatography to give a yel-
1.10 g (11.0 mmol, 1.22 mL) of 1-methylpiperazine. The
oily residue was purified by column chromatography
and it was treated with ethereal hydrochloride to give
2
3
ꢀ
hydrochloride salt of 9 (0.82 g, 42%, mp 218 C). UV
low oily product which was converted into hydrochlo-
ride salt of 6 (0.88 g, 44%, mp 220 C). UV l
(
lmax (2) (MeOH) 212 (4701), 251 (29,003), 270 (27,569),
ꢀ
MeOH) 214 (8217), 252 (33,062), 271 (27,751), 399
(2)
396 (5816) nm. IR 3421, 2955, 2665, 2567, 1636, 1599,
1517, 1465, 1253, 1113, 1015, 968, 777 cm . H NMR
max
À1
1
(
1
6589) nm. IR 3405, 2944, 2692, 1625, 1599, 1568, 1512,
À1
(DMSO-d ) d 7.42–8.47 (m, 7H, Ar-H, H , H , H , H -
6
1
3
4
5
1
475, 1294, 1284, 1180, 1067, 761, 697 cm . H NMR
H ), 3.30–3.98 (m, 15H, H , H , H , H , H , H and
OCH ), 2.91(s, 3H, H ), 2.20 (m, 2H, H ); C NMR
3 e l
8 a b c d k m
1
3
(
H ), 3.84 (s, 3H, OCH ), 3.20–3.75 (m, 4H, H and H ),
DMSO-d ) d 7.6–8.75 (m, 7H, Ar-H, H , H , H , H -
6
1
3
4
5
(DMSO-d ) d 116.5 (C ), 154.68 (C ), 123.85 (C ),
8
3
k
m
6
1
2
3
2
.90–2.95 (m, 4H, H and H ), 1.82–2.23 (m, 8H, H ,
l
106.94 (C ), 118.44 (C ), 135.93 (C ), 123.11 (C ),
0
a
b
4 5 6 7
1
3
H , H and H ); C NMR (DMSO-d ) d 115.59 (C ),
1
127.53 (C ), 178.38 (C ), 137.15 (C
8
0
), 141.54 (C10
), 55.84 (OCH ), 44.16 (C ),
),
c
d
e
6
1
9
4
53.9 (C ), 124.9 (C ), 106.31 (C ), 117.53 (C ), 135.57
0
120.94 (C8
0
), 122.21 (C9
0
2
3
4
5
3
k
(
C ), 122.62 (C ), 127.02 (C ), 177.61 (C ), 136.27 (C
6
),
), 56.22 (OCH3),
23.09 (C ), 42.49 (C ), 50.09 (C and C ), 51.45 (C and
l m a b c
7
8
9
4
+
Á
C ), 27.58 (C ). MS m/z (%) 365 (M , 20), 266 (5), 252
d e
1
2
2
40.78 (C10
0
), 120.23 (C8
0
), 121.5 (C9
2.85 (C ), 22.32 (C ), 44.06 (C ), 54.66 (C , C and C ),
0
(12), 239 (20), 225 (10), 210 (12), 141 (10), 127 (35), 113
(90), 99 (100), 85 (80). Anal. (C H N O ) C, H, N.
l
e
m
a
b
k
+
Á
3.92 (C and C ). MS m/z (%) (M , 10), 349 (15), 124
c
d
22 27
3
2
(
20), 98 (100). Anal. (C H N O ) C, H, N.
22 26 2 2
0
0-(3 -N-[(ꢀ-Hydroxyethyl)piperazino]propyl)-2-methoxy-
acridone (10). Amounts of 1.09 g (3.61 mmol) of 2, 1.54
1
0
1
0-(3 -N-Morpholinopropyl)-2-methoxyacridone (7). The
experimental procedure used for 4 is applicable with
.05 g (3.48 mmol) of 2, 1.48 g of KI, 2.4 g of K CO
and 1.12 g (12.8 mmol) of morpholine. The oily product
was chromatographed on the silica gel to get pure pro-
duct 7 and it was treated with ethereal hydrochloride to
g of KI, 2.49 g of K CO and 2.03 g of (17.5 mmol, 2.15
2
3
1
mL) of (b-hydroxyethyl)piperazine were refluxed and
processed according to the procedure used for 3. The
free base was purified by column chromatography and
2
3
treated with ethereal hydrochloride to give hydrochlo-
ride salt of 10 (1.2 g, 43%, mp 228–230 C). UV l
ꢀ
(MeOH) 214 (15,600), 252 (46,800), 271 (39,266), 399
give hydrochloride salt of 7 (0.80 g, 42%, mp 212–
13 C). UV l
(2)
max
ꢀ
30,350), 270 (25,136), 398 (5486) nm. IR 3421, 2910,
2
(
(2) (MeOH) 209 (17,159), 252
max
(9400) nm. IR 3436, 2937, 2690, 1641, 1599, 1517, 1470,
À1
1
2
7
7
735, 1641, 1599, 1506, 1424, 1144, 1108, 1088, 946,
À1
1289, 1056, 1024, 824, 761 cm . H NMR (DMSO-d ) d
6
1
82, 709 cm . H NMR (DMSO-d ) d 7.73–8.78 (m,
7.63–8.8 (m, 7H, Ar-H, H , H , H , H -H ), 4.29 (t, 1H,
H ), 3.75 (s, 3H, OCH ), 3.00–3.92 (m, 4H, H and H )
g 3 k f
6
1
3
4
5
8
H, Ar-H, H , H , H , H -H ), 3.71–3.84 (m, 7H, H ,
1
3
4
5
8
c
H and OCH ), 3.46 (m, 4H, H and H ), 2.68 (t, 4H, H
d
2.36–3.00 (m, 12H, H , H , H , H , H and H ), 1.54 (m,
a b c d e m
3
k
m
a
1
3
13
and H ), 1.40–1.51 (m, 2H, H ); C NMR (DMSO-d ) d
2H, Hl); C NMR (DMSO-d ) d 115.7 (C ), 153.99 (C ),
b
l
6
6
1
2
1
15.7 (C ), 154.08 (C ), 125.14 (C ), 106.39 (C ), 117.66
124.34 (C ), 106.08 (C ), 117.93 (C ), 134.07 (C ), 122.24
1
2
3
4
3 4 5 6
(
1
C ), 135.7 (C ), 122.77 (C ), 127.12 (C ), 177.78 (C ),
6
(C ), 126.72 (C ), 176.07 (C ), 136.34 (C ), 141 (C10
7
0
0
),
0 0
), 121.08 (C9 ), d 55.78 (OCH ), 53.36 (C ),
5
7
8
9
8
9
4
36.43 (C4
0
), 140.89 (C10
0
), 120.34 (C8
0
), 121.63 (C9
0
), 56.32
120.71 (C8
23.74 (C ), 43.13 (C ), 49.79 (C and C ), 51.35 (C and
3
k
(
C ), 65.05 (C and C ). MS m/z (%) 353 (M+H) , 1 00.
OCH ), 54.98 (C ), 22.57 (C ), 43.83 (C ), 53.08 (C and
3 k l m a
l
m
a
b
c
+
+
C ), 55.45 (C ), 57.97 (C ). MS m/z (%) 396 (M+H) , 100.
d e f
b
c
d
0
0
1
0-(3 -N-Thiomorpholinopropyl)-2-methoxyacridone (8).
10-(4 -Chlorobutyl)-2-methoxyacridone (11). Compound
11 (1.24 g, 53%), in the pure form was prepared by fol-
lowing the procedure used for 2, with 1g (4.44 mmol) of
The experimental steps used for 4 were repeated by
taking 1.13 g (3.74 mmol) of 2, 1.59 g of KI, 2.58 g of

G. Krishnegowda et al. / Bioorg. Med. Chem. 10 (2002) 2367–2380
2377
2
-methoxyacridone and 1-bromo-4-chlorobutane (11.5
mmol). UV lmax (2) (MeOH) 213 (16,165), 253
48,667), 271 (41,275), 399 (9813) nm. IR 3436, 3012,
(7354) nm. IR 3431, 2955, 2701, 1636, 1599, 1553, 1512,
À1
1
1475, 1289, 1186, 1051, 849, 813, 767 cm . H NMR
(DMSO-d ) d 7–7.85 (m, 7H, Ar-H, H , H , H , H -H ),
(
6
1
3
4
5
8
2
8
968, 1645, 1636, 1599, 1478, 1369, 1276, 1179, 1067,
À1
3.59 (s, 3H, OCH ), 3.80 (m, 2H, H ), 3.51(m, 2H, H ),
3 k n
1
16, 784, 776 cm . H NMR (DMSO-d ) d 7.22–8.35
2.89–2.99 (m, 4H, H and H ) 1.87–2.02 (m, 4H, H and
a b c
6
1
3
(
3
m, 7H, Ar-H, H , H , H , H -H ), 3.85 (s, 3H, OCH ),
.70–3.75 (m, 2H, H ), 3.62 (m, 2H, H ), 2.01(m, 2H,
H ), 1.44–1.61 (m, 4H, H and H ); C NMR (DMSO-
d l m
1
3
4
5
8
3
d ) d 116.34 (C ), 154.58 (C ), 125.66 (C ), 106.79 (C ),
n
k
6
1
2
3
4
1
3
H ), 1.89 (m, 2H, H ); C NMR (DMSO-d ) d 115.86
118.25 (C ), 135.82 (C ), 122.99 (C ), 127.45 (C ),
0
l
m
6
5
6
7
8
(
1
C ), 153.9 (C ), 122.32 (C ), 106.12 (C ), 115.86 (C ),
33.85 (C ), 120.96 (C ), 125.91 (C ), 175.76 (C ), 135.66
178.27 (C ), 137.02 (C
9
0
), 141.41 (C10
), 120.86 (C8
0
),
1
2
3
4
5
4
122.12 (C9
(C ), 46.59 (C ), 55.57 (C and C ), 24.07 (C and C ).
0
), 56.77 (OCH ), 55.74 (C ), 25.13 (C ), 23.83
6
7
8
9
3 k l
(
C4
OCH ), 45.03 (C ), 45.19 (C ), 24.39 (C ), 25.65 (C ). MS
), 140.42 (C10 ), 118.05 (C8 ), 120.65 (C9 ), 55.38
0 0 0 0
m
n
a
+
b
c
d
(
m/z (%) 316 (M+H) , (10), 315 (100), 224 (30), 91 (40).
MS m/z (%) 351(M+H) , 100.
3
k
n
l
m
+
0
1
0-(4 -N-Piperidinobutyl)-2-methoxyacridone (15). The
0
1
0-[4 -(N-Diethylamino)butyl] - 2 - methoxyacridone (12).
method employed for 4 was used with 1.08 g (3.42
mmol) of 11, 1.41 g of KI, 2.36 g of K CO and 1.4 g
The procedure used for 3 was followed with 1.2 g (3.8
mmol) of 11, 1.57 g of KI, 2.62 g of K CO and 1.3 g
(17.8 mmol) of N,N-diethylamine. The oily residue was
purified by column chromatography and the pure base
was converted into hydrochloride salt of 12 (1.26 g,
2
3
(16.44 mmol) of piperidine. The crude product was
purified by column chromatography and it was con-
verted into hydrochloride salt of 15 (1.2 g, 53%, mp
2
3
ꢀ
119–121 C). UV l
(2) (MeOH) 214 (8348), 253
max
ꢀ
52 (42,527), 271(35,583), 399 (84 61 ) nm. IR 3440,
5
2
2
1
7
6%, mp 166 C). UV l
(2) (MeOH) 213 (15,361),
(36,071), 271 (30,401), 399 (7455) nm. IR 3415, 2987,
2704, 1646, 1631, 1625, 1501, 1279, 1181, 1025, 839, 755
cm . H NMR (DMSO-d ) d 6.86–7.75 (m, 7H, Ar-H,
max
À1
1
951, 2684, 1625, 1599, 1553, 1501, 1480, 1284, 1191,
À1
6
1
030, 849, 839, 756 cm . H NMR (DMSO-d ) d 6.86–
H , H , H , H -H ), 3.53 (s, 3H, OCH ), 3.68 (t, 2H,
H ), 3.30 (t, 2H, H ), 2.70–2.95 (m, 4H, H and H ),
k n a b
6
1
3
4
5
8
3
.78 (m, 7H, Ar-H, H , H , H , H -H ), 3.71(m, 2H,
1
3
4
5
8
1
3
H ), 3.55 (s, 3H, OCH ), 2.87–3.08 (m, 6H, H , H , H ),
k
1.31–1.77 (m, 10H, H , H , H , H and H ); C NMR
l m c d e
3
a
b
n
1
.38–1.62 (m, 4H, H and H ), 1.15 (m, 6H, H and H );
l
(DMSO-d ) d 112.27 (C ), 150.35 (C ), 121.6 (C ),
m
c
d
6
1
2
3
1
3
C NMR (DMSO-d ) d 114.5 (C ), 152.52 (C ), 123.82
102.44 (C ), 114.09 (C ), 131.88 (C ), 119.04 (C ),
0
6
1
2
4 5 6 7
(
C ), 106.31 (C ), 116.3 (C ), 135.56 (C ), 121.25 (C ),
123.45 (C ), 174 (C ), 132.78 (C
9
0
), 137.22 (C10
), 53.68 (OCH ), 18.77 (C ),
),
3
4
5
6
7
8
4
1
1
2
8
25.62 (C ), 176.13 (C ), 134.97 (C
8
0
), 139.41 (C10
0
),
), 54.86 (OCH ), 50.86 (C ),
116.65 (C8
0
), 119.84 (C9
0
9
4
3
l
18.81 (C8
0
), 119.99 (C9
3.35 (C ), 20.25 (C ), 45.11 (C ), 47.17 (C and C ),
.10 (C and C ). MS m/z (%) 353 (M+H) , 100. Anal.
c
0
18.11 (C ), 21.15 (C ), 42.92 (C ), 50.83 (C and C ),
e m n a b
3
k
52.68 (C ), 20.46 (C and C ). MS m/z (%) 365
k c d
l
m
n
a
b
+
+
(M+H) , 100).
d
(
C H N O ) C, H, N.
22 28 2 2
0
1
0-(4 -N-Morpholinobutyl)-2-methoxyacridone (16). The
0
10-[4 -[N-Bis (hydroxyethyl)amino]butyl]-2-methoxyacri-
done (13). Amounts of 1.01 g (3.20 mmol) of 11, 1 .32 g of
procedure used for 4 was repeated with 1.19 g (3.77
mmol) of 11, 1.55 g of KI, 2.59 g of K CO and 1.05 g
2
3
KI, 2.21g K CO and 1.2 mL (9.83 mmol) of bishydroxy-
2
(1.06 mL, 12.14 mmol) of morpholine. The product was
purified by column chromatography and converted into
hydrochloride salt of 16 (1.1 g, 53%, mp 206 C). UV
3
ethylamine were refluxed and processed according to the
procedure used for 4. The crude product was chroma-
tographed on silica gel to get pure free base and it was
ꢀ
lmax (2) (MeOH) 214 (18,622), 253 (37,097), 272
(30,932), 399 (7521) nm. IR 3436, 2963, 2649, 1645,
1636, 1506, 1487, 1274, 1210, 1183, 1065, 942, 839, 727
converted into hydrochloride salt of 13 (1g, 5 1% , mp
1
ꢀ
37,050), 372 (31,320), 399 (7443) nm. IR 3410, 3074,
52 C). UV l
(2) (MeOH) 214 (11,432), 253
max
À1
1
(
cm . H NMR (DMSO-d ) d 6.89–7.81(m, 7H, Ar-H,
6
2
1
8
883, 1625, 1599, 1558, 1501, 1470, 1279, 1263, 1186,
À1
H , H , H , H -H ), 4.00 (m, 2H, H ), 3.70 (m, 4H, H
and H ), 3.56 (s, 3H, OCH ), 3.38 (m, 2H, H ), 3.05 (m,
d 3 n
4H, H and H ), 1.41–1.65 (m, 4H, H and H ); C NMR
a b l m
1
3
4
5
8
k
c
1
077, 854, 823, 767 cm . H NMR (DMSO-d ) d 7.29–
6
1
3
.32 (m, 7H, Ar-H, H , H , H , H -H ), 4.17 (s, 2H, H
1
3
4
5
8
e
and H ), 3.83 (s, 3H, OCH ), 3.73 (m, 6H, H , H and H ),
d
(DMSO-d ) d 114.47 (C ), 152.5 (C ), 123.85 (C ), 104.53
f
3
k
c
6
1
2
3
3
4
1
.61(m, 2H, H ), 2.47 (m, 4H, H and H ), 1.78–1.91 (m,
b
(C ), 116.27 (C ), 134.13 (C ), 121.28 (C ), 125.59 (C ),
0
n
a
4
5
6
7
8
1
3
H, H and H ); C NMR (DMSO-d ) d 114.47 (C ),
175.8 (C ), 134.91 (C ), 139.33 (C10
0
), 118.65 (C8 ), 119.84
), d56.27 (OCH ), 54.90 (C ), 23.22 (C ), 20.02 (C ),
0
l
m
6
1
9
4
52.54 (C ), 123.78 (C ), 104.66 (C ), 116.28 (C ), 134.1
(C9
45.27 (C ), 51.58 (C and C ), 63.68 (C and C ). MS m/z
0
2
3
4
5
3
k
l
m
(
C ), 121.26 (C ), 125.66 (C ), 176.07 (C ), 134.97 (C ),
0
6 7 8 9 4
n
a
b
c
d
+
1
5
39.42 (C10
3.11 (C ), 23.34 (C ), 19.97 (C ), 45.23 (C ), 54.75 (C
a
0
), 118.83 (C8
0
), 120.01 (C9
0
), 54.92 (OCH3),
(%) 367 (M+H) , 1 00. Anal. (C H N O ).
22 26 2 3
k
l
m
n
+
0
and C ), 55.23 (C and C ). MS m/z (%) 385 (M+H) ,
c
10-(4 -N-Thiomorpholinobutyl)-2-methoxyacridone (17).
Amounts of 1.11 g (3.52 mmol) of 11, 1.45 g of KI,
b
d
100.
2
.42 g K CO and 1.3 g (12.6 mmol) of thiomorpholine
2 3
0
Compound 14 as its hydrochloride salt (0.96 g, 47%,
1
0 - (4 - N - Pyrrolidinobutyl) - 2 - methoxyacridone (14).
were refluxed and processed according to the procedure
used for 4. The crude product was purified by column
chromatography and the light-yellow oily product was
ꢀ
with 1g (3. 19 mmol) of 11, 1.31 g KI, 2.18 g K CO
mp 132 C) was obtained by following the procedure of
4
converted into hydrochloride salt of 17 (1g, 49%, mp
163 C). UV l
2
3
ꢀ
271(30,689), 399 (7564) nm. IR 3426, 2965, 2872, 16 36,
and 1.26 g (17.74 mmol) of pyrrolidine. UV lmax (2)
(2) (MeOH) 214 (13,189), 255 (35,412),
max
(
MeOH) 214 (13,822), 252 (34,903), 271 (29,725), 399

2378
G. Krishnegowda et al. / Bioorg. Med. Chem. 10 (2002) 2367–2380
1
8
609, 1599, 1512, 1465, 1361, 1289, 1175, 1030, 945,
À1
hydrochloric acid or sodium hydroxide using a combi-
nation glass electrode to measure pH. The relative lipo-
philicity was assessed for each of the derivatives
1
36, 761cm . H NMR (DMSO-d ) d 7.2–8.3 (m, 7H,
6
Ar-H, H , H , H , H –H ), 3.83 (s, 3H, OCH ), 3.76
1
3
4
5
8
3
3
2
(
(
m, 2H, H ), 2.52–2.56 (m, 6H, H , H and H ), 2.43
n
m, 4H, H and H ), 1.85–2.00 (m, 4H, H and H );
m
using an adaptation of the method of Zamora et al.
k
a
b
1
3
C
This method involves measuring the partitioning of
the compound between 1-octanol and 0.1 M PBS
buffer (pH 7.4). HPLC-grade 1-octanol was pre-
saturated with PBS, and aqueous phase PBS was satu-
rated with 1-octanol before use. The derivatives were
c
d
l
NMR (DMSO-d ) d 114.74 (C ), 151.69 (C ), 122.98
6
1
2
(
C ), 106.11 (C ), 116.27 (C ), 134.27 (C ), 121.7 (C ),
3 4 5 6 7
1
1
2
2
25.87 (C ), 177.12 (C ), 134.89 (C ), 139.33 (C10
18.62 (C8
3.22 (C ), 20.21(C m), 42.77 (C ), 53.22 (C and C ),
0
0
),
0 0
), 119.74 (C9 ), 55.73 (OCH ), 53.30 (C ),
8
9
4
3
k
each dissolved in 1-octanol/buffer phase at final
À4
l
n
a
+
b
6.38 (C and C ). MS m/z (%) 383 (M+H) , 10), 224
concentration of 10
M, and an equal volume of
c
d
(
100).
buffer/1-octanol was added. The tubes were then
inverted continuously for 15 min; control experiments
confirmed that equilibrium was reached within this
time. The final concentration of compound in both
aqueous and octanol phases was measured by UV
spectrophotometry. The partition coefficient, P, was
determined by dividing the concentration of the deriva-
tive in 1-octanol by the concentration in the aqueous
phase.
0
0-(4 -N-(Methylpiperazino)butyl)-2-methoxyacridone (18).
1
The procedure used for 3 was followed with 1.09 g (3.45
mmol) of 11, 1.43 g of KI, 2.38 g of K CO and 1.56 g
2
3
(
15.6 mmol) of 1-methylpiperazine. The crude product
was purified by column chromatography and the oily
product was treated with ethereal hydrochloride to get
hydrochloride salt of 18 (1.4 g, 57%, mp 223–224 C).
ꢀ
UV lmax (2) (MeOH) 209 (27,923), 253 (32,140), 271
(
25,814), 399 (5846) nm. IR 3436, 2965, 2761, 1634,
537, 1501, 1478, 1287, 1174, 1031, 847, 767 cm . H
Cell lines and cell culture. Human epidermoid carci-
noma KB-3-1cells and a colchicine-selected MDR
variant, KBCh -8-5 cells cross-resistant to vincris-
À1
1
1
R
NMR (DMSO-d ) d 7.18–8.08 (m, 7H, Ar-H, H , H ,
6
1
3
H , H -H ), 3.73 (s, 3H, OCH ), 3.25–3.56 (m, 12H, H ,
H , H , H , H and H ), 2.94 (s, 3H, H ), 1.70–1.79 (m,
tine (45-fold) and VLB (ꢂ10-fold) and KB-V1 cells
were grown in monolayer culture in antibiotic-free
Dulbecco’s modified Eagle medium (DMEM) con-
taining 10% fetal bovine serum and l-glutamine in a
4
5
8
3
a
b
c
d
k
n
e
1
3
4
1
1
1
5
H, H and H ,); C NMR (DMSO-d ) d 116.5 (C ),
l
m
6
1
54.68 (C ), 125.77 (C ), 106.94 (C ), 118.44 (C ),
35.93 (C ), 123.11 (C ), 127.53 (C ), 178.38 (C ),
2
3
4
5
humidified atmosphere of 5% CO in air. The
6
7
8
9
2
R
37.15 (C4
0
), 141.54 (C10
0
), 120.94 (C8
0
), 122.21 (C9
0
),
7.85 (OCH ), 56.90 (C ), 25.13 (C ), 22.05 (C ), 46.53
resistance of KBCh -8-5 and KB-V1cells was
maintained by culturing them respectively with 10
ng/mL of colchicine and 1ng/mL of VLB.
3
k
l
m
(
MS m/z (%) 380 (M+H) , 100).
C ), 50.11 (C and C ), 51.69 (C and C ) 44.45 (C ).
n a b c d e
+
6
Accumulation studies. Cells (2Â10 cells) were plated
0
cridone (19). Amounts of 1.2 g (3.80 mmol) of 11, 1.57 g
ꢀ
1
0-[4 -N-(ꢀ-Hydroxyethyl)piperazino)butyl]-2-methoxya-
and incubated overnight at 37 C to attach to plastic.
Medium was aspirated and cells were washed with
2Â2 mL PT buffer (120 mM NaCl, 20 mM Tris-base,
3 mM K HPO , 10 mM glucose, 0.5 mM MgCl and 1
of KI, 2.62 g of K CO and 2.2 g (2.1mL, 16 .9 mmol)
2
3
of (b-hydroxyethyl)piperazine were refluxed and pro-
cessed according to the experimental conditions for 4.
The free base was purified by column chromatography,
2
4
2
mM CaCl , pH 7.4). Monolayers were incubated at
2
room temperature for 10 min in PT buffer prior to
aspiration and adding 1mL of serum-free RPMI-
1640HEPES buffer (10.4 g RPMI-1640 medium in 1
L of 25 mM HEPES, pH 7.4) containing 49.9 nM
a pure yellow solid of 19 was obtained (1.3 g, 43%, mp
ꢀ
1
36 C). UV l
(2) (MeOH) 213 (21,410), 252
max
(
2
51,534), 272 (43,360), 399 (10,569) nm. IR 3379, 2953,
676, 1642, 1594, 1506, 1491, 1274, 1174, 1067, 863, 767
3
10
[ H] VLB with or without VRP or 2-methoxy-N -
substituted acridones (1–19) at 1 00mM dissolved in
DMSO (final culture concentration <0.1% DMSO).
After 2 h of incubation at room temperature, med-
ium was rapidly aspirated to terminate the drug
accumulation and monolayers were washed four times
À1
1
cm . H NMR (DMSO-d ) d 7.7–8.77 (m, 7H, Ar-H,
6
H , H , H , H –H ), 4.77 (t, 1H, H ), 3.73 (s, 3H,
OCH ), 3.92 (m, 2H, H ), 3.71(m, 2H, H ), 2.49–3.00
1
3
4
5
8
g
3
f
k
(
m, 12H, H , H , H , H , H and H ), 2.08–2.25 (m, 4H,
a b c d e n
1
3
H and H ); C NMR (DMSO-d ) d 116.14 (C ), 154.2
l
m
6
1
.
with ice-cold PBS (g/L:NaCl 8.0; Na HPO 12H O
2 4 2
(
C ), 124.41 (C ), 106.35 (C ), 118.32 (C ), 134.1 (C ),
2
3
4
5
6
1
1
5
22.65 (C ), 127.01 (C ), 176.12 (C ), 136.6 (C
7
0
),
), 58.89 (OCH3),
2.9; KCl 0.2; KH PO 0.2) buffer and drained. To
2 4
8
9
4
41.29 (C10
0
), 121.12 (C8
0
), 121.12 (C9
0
each dish 1mL of trypsin-EDTA (0.05% trypsin,
0.53 mM EDTA) was added. After 1min, mono-
layers were triturated to give a uniform suspension of
cells and radioactivity in 0.75 mL was determined
by scintillation counting. Cell number per dish was
determined using hemocytometer and amount of
intracellular VLB was determined (Table 1).
5.73 (C ), 24.40 (C ), 22.80 (C ), 45.39 (C ), 53.10 (C
a
and C ), 53.73 (C and C ), 56.67 (C ), 60.79 (C ). MS
b
m/z (%) 410 (M+H) , 100. Anal. (C H N O ) C, H,
2
k
l
m
n
c
d
e
f
+
4
31
3
3
N.
Biological activity
6
Determination of pK and partition coefficient. The pK s
a
Measurement of VLB efflux. Cells (2Â10 /dish) were
a
ꢀ
were determined according to a previously published
3
plated and incubated overnight at 37 C in a CO incu-
2
bator to attach to plastic. Medium was removed and
monolayer cells were washed once with 3 mL of PT
1
À3
M
method. Briefly, 10 mL of an approximately 10
solution of each compound was titrated with 1M

G. Krishnegowda et al. / Bioorg. Med. Chem. 10 (2002) 2367–2380
2379
buffer and incubated for 10 min in another 2 mL of the
R
twice with distilled water and dried overnight. The IC10
and IC50 values (Table 3) were determined from con-
centration percent cell survival curves and were defined
as the concentrations required for 10 and 50% reduc-
tion in colonies compared to controls.
same buffer. KBCh -8-5 cells were incubated with 1mL
of serum-free RPMI-1640HEPES buffer, pH 7.4 con-
3
taining 49.9 nM [ H] VLB for 2 h at room temperature.
Drug solutions were aspirated and 1mL of the same
buffer without or with modulators (1–19) or VRP at 100
mM were added and incubated for 2 h more at room
temperature. The medium was aspirated from each dish
and the cells were washed four times in ice-cold PBS
and drained. Cells were harvested and radioactivity per
dish was calculated as described above and the results
are given in Table 2.
1
0
Effect of 2-methoxy-N -substituted acridones on in vitro
cytotoxicity of VLB. To determine the effects of the
R
modulators on the cytotoxicity of VLB, KBCh -8–5
cells were treated with serial dilutions of VLB (0.0–100
nM) in the absence or presence of IC10 concentration of
ꢀ
modulators. After incubation for 7 days at 37 C, colo-
nies were enumerated as described above. IC50 values
were determined as previously described and the fold-
potentiation (Table 4) was calculated from dividing the
IC50 for VLB in the absence of modulator by the IC50 in
the presence of modulating agent.
3
Competition for [ H] azidopine labelingof P- gl ycopro-
tein. Preparation of plasma membrane fractions and pho-
toaffinity labeling. Competition assay for photolabeling
of P-gp used membranes from KB-V1cells, which have
higher P-gp levels than do KBCh -8-5 cells. Crude
membranes were prepared from the MDR variant, KB-
R
3
3
V1, essentially as described previously.
9
Briefly >10 KB-V1cells were harvested, washed sev-
eral times in 25 mM HEPES, 130 mM NaCl, 5 mM
KCl, 1mM MgCl , 10 mM glucose, 5 mM NaH PO ,
Acknowledgements
2
2
4
7
pH 7.4, finally resuspended at 10 cells/mL in 20 mM
HEPES, 150 mM NaCl, 2 mM MgCl , 1mM EDTA, 1
This work was supported by the Department of Atomic
Energy (DAE), Government of India (to K.N.T.).
G.K.G. is thankful to PES College of Science, Mandya,
for their cooperation and encouragement and to the
University of Mysore, Mysore, for providing the
research facilities.
2
mM phenylmethylsulfonyfluoride, pH 7.4 and lysed by
nitrogen cavitation; membrane pellets were suspended
ꢀ
in 100 mM Tris, pH 7.4 and frozen at À70 C until use.
For photoaffinity labeling, membrane protein (200 mg)
in buffer containing 250 mM sucrose and 10 mM
ꢀ
3
.
Tris HCl, pH 7.4 at 25 C, was mixed with 100 nM [ H]
azidopine (50 Ci/mmol; Amersham) in the absence or
presence of 100 mM modulators (1–19), in a total
volume of 150 mL. After incubating for 20 min in the
dark, the mixture was then irradiated with a germicidal
UV-light (30 W), commonly used in laminar flow
hoods, for 20 min at a distance of 10 cm.
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