Targeting efflux pumps-In vitro investigations with acridone derivatives and identification of a lead molecule for MDR modulation

Article abstract of DOI:10.1016/j.bmc.2010.05.003

To search multi drug resistance modulators, acridones carrying hydroxyl amine substituent at N-10 and COOH/Cl at C-4 were investigated for their interactions with the three components of efflux pump viz. P-gp, ATP, and Mg²⁺. Experimental and th

Full text of DOI:10.1016/j.bmc.2010.05.003

Bioorganic & Medicinal Chemistry 18 (2010) 4212–4223
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Targeting efflux pumps—In vitro investigations with acridone derivatives
and identification of a lead molecule for MDR modulation
a
b
b,
Palwinder Singh a, , Jatinder Kaur , Bhawna Yadav , Sneha Sudha Komath
*
*
a Department of Chemistry, Guru Nanak Dev University, Amritsar 143 005, India
b School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India
a r t i c l e i n f o
a b s t r a c t
Article history:
To search multi drug resistance modulators, acridones carrying hydroxyl amine substituent at N-10 and
COOH/Cl at C-4 were investigated for their interactions with the three components of efflux pump viz.
P-gp, ATP, and Mg2+. Experimental and theoretical results indicated that the compounds with COOH
group at C-4 interact with P-gp and Mg2+ while other set of compounds with Cl at C-4 interact with
ATP and Mg2+. Spot assay and R6G influx/efflux assay of compound 3 using Candida albicans showed
decrease in the fungal growth and efflux of R6G, respectively, in presence of compound 3 suggesting
the suitability of this compound for MDR modulation.
Received 11 March 2010
Revised 30 April 2010
Accepted 1 May 2010
Available online 7 May 2010
Keywords:
Multi drug resistance
P-Glycoprotein/cdr1p/cdr2p
ATP
Ó 2010 Elsevier Ltd. All rights reserved.
Mg2+
Acridone derivatives
Candida albicans
1. Introduction
been investigated for their P-gp and cdr1p/cdr2p drug resistance
modulating properties.8–10 Since drug transportation is an energy
The practice of chemotherapy for various diseases like cancer,
AIDS, malaria, bacterial and fungal infections etc. suffers from the
development of resistance for the prescribed drugs. This resistance
to multiple drugs (MDR)1,2 occurs due to the natural process of
activation of ATP-binding cassette transporter proteins3,4 when a
foreign particle enters the cell. Among various ABC transporter
proteins, P-glycoprotein (P-gp) having two or more binding sites
and a flip–flop mechanism of action, results in the transportation
of a number of substances (including lipophilic drugs) out of the
mammalian cell and causes decrease in intracellular drug concen-
tration.5 Likewise cdr1p and cdr2p6,7 functional homologues of
P-gp play vital roles in the efflux of antifungal drugs out of the cells
of Candida albicans which might be the cause of antifungal drug
resistance. Hence occurrence of MDR becomes an important issue
in current medicinal chemistry due to which a combination of
drugs (the modulator and the chemotherapeutic agent) are recom-
mended for the treatment of a disease. The modulator, through its
interactions with P-gp/cdr1p/cdr2p avoids the transportation of
the chemotherapeutic agent out of the cell; thereby an optimum
concentration of the chemotherapeutic agent could be maintained
inside the cells. In this context a number of chemical entities have
driven process getting energy from ATP hydrolysis, the inhibition
of ATP hydrolysis could be an alternative approach for blocking
the P-gp activity. ATP hydrolysis may get blocked either through
interactions with ATP or by Mg2+ chelation.11–13 Therefore, the
drug transporting activity of P-gp could be blocked through the
interruption of components of efflux pump like P-gp, ATP, and
Mg2+. In continuation with our earlier work14 and the importance
of acridone derivatives as anti-tumor,15–17 anti-protozoan,18–20
anti-viral,21 and multi drug resistance (MDR) modulating15,22,23
agents; compounds 3–8 and 11–16 were investigated for their
interactions with P-gp, ATP, and Mg2+. Based upon these results,
some of the compounds were subjected to R6G influx/efflux assay
on C. albicans. Compound 6 was identified as a lead for anti-candi-
diasis therapy24 while compound 3 exhibits significant inhibition of
R6G efflux from the cells ofC. albicans and seems to be the inhibitor
of cdr1p and cdr2p.
2. Results and discussion
2.1. Chemistry
Ullmann type arylation25–27 of anthranilic acid with o-chloro-
benzoic acid gave 1a which after cyclization provided the acridone
skeleton (1) bearing –COOH group at C-4 position. Treatment of
acridone 1 with NaH in DMSO followed by stirring with epichloro-
* Corresponding authors. Tel.: +91 183 2258802-09x3495; fax: +91 183 2258819.
E-mail address: palwinder_singh_2000@yahoo.com (P. Singh).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.003

P. Singh et al. / Bioorg. Med. Chem. 18 (2010) 4212–4223
4213
O
O
K2CO3, CuO
conc. H2SO4
heat
OH
NH
COOH
Cl
+
Isoamyl alcohol,
reflux
N
H
H2N
COOH
COOH
1
HOOC
NaH, DMSO
epichlorohydrin,
60-70oC
1a
O
O
9
1
8
5
3-8: NR1R2
2
3
NHR1R2
7
10
N
3
4
N
N
N
N
6
MWI, 5-7min
N
4
11
Hd
He
COOH
COOH
12
HO
O
Hc
13
Ha
R2R1N
3-8
Hb
O
5
2
6
N
7
N(C2H5)2
N(i-pr)2
8
Scheme 1.
hydrin provided N-substituted acridone 2. Irradiation of an
equimolar mixture of acridone 2 and pyrrolidine in microwave
oven resulted in the formation of compound 3. Similarly, the reac-
tions of acridone 2 with other amines under microwaves28,29 pro-
vided compounds 4–8 in 5–7 min (Scheme 1).
Since the acridone derivatives with COOH group at C-4 position
showed better potency24 in comparison to the compounds without
substituent at C-4,14 further to look into the role of substituent at
C-4 position, Cl was introduced through the same sequence of
reactions as depicted in Scheme 1, using o-chloroaniline in place
of anthranilic acid and compounds 11–16 (Scheme 2) were
prepared.
2.2. Biology
The interactions of compounds 3–8 and 11–16 with P-gp were
studied using ‘Drug-P-glycoprotein Interaction’ assay kit30 which
contains the P-gp vesicles prepared from highly resistant MDR
cells, the DC-3F/ADX line. The interactions of compounds with
P-gp were assessed in terms of modulation of basal activity
(MgATP hydrolysis activity in the absence of drug) of P-gp mea-
sured by spectrophotometric method by continuous monitoring
of ADP formation in the vesicle suspension medium. The interac-
tions of test compound with P-gp (in the ATP binding site)/ATP/
Mg2+ slows down the ATPase activity of P-gp which results in the
O
O
K2CO3, CuO
COOH
conc. H2SO4
heat
OH
+
Isoamyl alcohol,
reflux
NH
N
H2N
H
Cl
Cl
9
Cl
Cl
NaH, DMSO
epichlorohydrin,
60-70oC
O
O
N
11-16 : NR1R2
NHR1R2
11
N
N
N
N
MWI, 5-7min
N
Hd
He
Cl
Cl
HO
O
12
13
14
Hc
Ha
R2R1N
11-16
Hb
O
10
N
15 N(C2H5)2
16
N(i-pr)2
Scheme 2.

4214
P. Singh et al. / Bioorg. Med. Chem. 18 (2010) 4212–4223
Table 1
Relative activity and percentage increase of basal activity of P-gp by compounds 3–8
Compound
Relative activity of compd (% increase of basal
activity of P-gp)
5
lM
0.5
l
M
0.05 lM
3
4
5
6
7
8
2.00 (100)
2.44 (144)
2.66 (166)
2.47 (147)
2.12 (112)
2.05 (105)
2.80
1.85 (85)
1.66 (66)
2.00 (100)
1.73 (73)
1.70 (70)
1.60 (60)
2.01
1.60 (60)
1.57 (57)
1.63 (63)
1.64 (64)
1.65 (65)
1.53 (53)
1.11
Verapamil*
Vinblastine*
Progesterone*
1.21
1.42
1.37
1.11
0.98
1.00
*
Relative activity values of these compounds were taken from the booklet avail-
able with the ‘Drug–P-gp interaction’ assay kit.
Figure 1. Absorption spectra of compound 14 (50 lM) on titration with 0–3 equiv
of ATP in HEPES buffer. Inset: Benesi–Hildebrand plot (stoichiometry 1:1).
slow conversion of phosphoenolpyruvate to pyruvate and slow for-
mation of lactate. This will decrease the conversion of NADH to
NAD+ and hence higher absorbance at 340 nm. Compounds were
2.3. Interactions of compounds 3–8 and 11–16 with ATP and
Mg2+
tested for their interactions with P-gp at 0.05
lM, 0.5 lM, and
5
lM concentrations in triplicate. Verapamil, vinblastine and pro-
gesterone, as provided in the assay kit, were taken for comparison.
The compounds under present investigations differ from each
other in the nature of the cyclic tertiary base at the end of the
N-10 substituent and presence of COOH/Cl group at C-4 of acridone
ring. Compounds 3–8 exhibited more than 50% increase in the ba-
Looking into the second approach for modulating the drug
transportation activity of P-gp via blocking of ATP hydrolysis, the
interactions of acridones 3–8 and 11–16 with ATP and Mg2+ were
investigated spectrophotometrically. UV–vis spectra of compounds
3–8 and 11–16 at 50 lM concentration in HEPES Buffer (pH 7.2)
exhibited absorption bands in the region 230–240 nm,
250–260 nm, and 385–420 nm. Addition of 0–3 equiv of ATP to
50 lM solution of compound 14 shows that the absorption max-
ima at 255 nm decreases continuously and also shifts to 247 nm
with a hypsochromic shift of 8 nm (Fig. 1) but further addition of
ATP leads to no decrease in absorbance.
Similar trend in absorption spectra has been observed for com-
pounds 11, 12, 13, 15, and 16. The association constants of com-
pounds 11–16 with ATP indicate their appreciable interactions
with ATP (Table 2). Compound 14 with piperidino piperidine at
the end of N-10 substituent exhibits significantly large association
constant than the other compounds. However, no change in the
absorption spectra of compounds 3–8 was observed on addition
of ATP which may be due to the repulsive interactions between
the carboxyl group and the phosphate group (However further
investigations are needed to prove it). The Benesi–Hildebrand plot
(inset of Fig. 1) and Job plot (Supplementary Fig. S1) show 1:1 stoi-
chiometry of compound–ATP complexes. There was no change in
the UV–vis spectra of compounds 11–16 on addition of adenosine,
AMP and ADP indicating the selectivity of these compounds for
ATP among its analogues (Fig. 2). As per the literature survey, this
sal activity of P-gp at 0.05 lM concentration (as per the kit manu-
facturer’s guidelines, a 30% or more increase in the basal activity of
P-gp implies the interactions of the compound with P-gp) indicat-
ing appreciable interactions with P-gp (Table 1). Compounds 5 and
6 with, respectively, morpholine and piperidino piperidine at the
end of N-10 substituent showed better interactions with P-gp in
comparison to other compounds. Looking at the various physico-
chemical parameters of these molecules, a variation in their parti-
tion coefficient (Log P) and total polar surface area 0 (TPSA) was
observed. Compound 5 with Log P 0.6 and TPSA 92 ÅA2 and com-
0
pound 6 with Log P 2.08 and TPSA 86 ÅA2 show a difference in these
two parameters from that of compounds 3, 4, 7, and 8 which have
0
Log P 1.16, 1.66, 1.51, 2.10, respectively, and TPSA 82.7 ÅA2 indicat-
ing that a combination of these two parameters might be respon-
sible for the better interactions of compounds 5 and 6 with P-gp.
All the compounds with COOH group present on the acridone
moiety (3–8) showed comparative or better interactions with
P-gp than those exhibited by the reference compounds verapamil,
progesterone and vinblastine. Surprisingly, none of the compounds
11–16 (with a Cl at C-4 of acridone) modulated the basal activity of
P-gp pointing towards no interactions of these compounds with
P-gp. In comparison to the interactions of acridones with P-gp
reported earlier14 (acridones lacking COOH group), compounds
3–8 exhibited better interactions with this protein. This trend of
interactions of the compounds with P-gp seems to be in parallel
with their Log P and TPSA values (Supplementary Table S1). Among
the three categories of compounds (no substituent at C-4, Cl at C-4
and COOH at C-4) compounds 3–8 have highest TPSA values and
smallest (of the three categories) Log P, compounds 11–16 have
highest Log P values and smaller TSPA (than compounds 3–8)
while compounds with unsubstituted C-414 have same TPSA values
as for compounds 11–16 but their Log P values are in between the
Log P values of compounds 3–8 and 11–16. Therefore, it seems that
more the hydrophilicity of the compounds, better the interactions
with P-gp. Therefore, a polar group like COOH is preferred on the
phenyl component of acridone. This also supports the previous
observation (for compounds 5 and 6) that the Log P and TPSA
might be the controlling parameters for the interactions of these
compounds with P-gp.
Table 2
Association constants of compounds 3–8 and 11–16 with ATP and Mg2+ in HEPES
buffer (Mꢀ1
)
2+
Compound
Ka
(105)
Ka
(105)
)
(ATP)
(Mg
3
4
5
6
7
—
—
—
—
—
—
6.57
5
4.08
11
1.50
19.7
3.82
1.18
7.74
2.00
0.95
1.48
17.1
18.6
0.39
2.20
8
11
12
13
14
15
16
7.9
1.42
—: not interacting with ATP.

P. Singh et al. / Bioorg. Med. Chem. 18 (2010) 4212–4223
4215
Figure 2. Absorbance change of compound 11–16 (red pyramid) at wavelength 255 nm, in the presence of ATP (green pyramid), adenosine (silver pyramid), AMP (purple
pyramid), ADP (blue pyramid) indicating selectivity for ATP.
is the first report where acridone derivatives selectively bind to
ATP.31
show that Mg2+ is interacting with the carbonyl oxygen of acridone
and various parts of the N-10 substituent and there is no interaction
between the C-4 substituent of acridone and metal ion.
Since the role of Mg2+ in ATP hydrolysis is well established, cap-
turing of this metal ion could slow down ATP hydrolysis and hence
interruption in the supply of energy during drug effluxing. New de-
signed acridones were investigated for their interactions with Mg2+
with the help of UV–vis spectral studies. Solutions of compounds
3–8 and 11–16 were prepared at 10ꢀ4 M concentrations in HEPES
buffer (10ꢀ2 M) at pH 7.2 and titrated with Mg2+ solution
(0–0.5 ꢁ 10ꢀ4 M). All compounds exhibited regular increase in
absorbance in the region 270–310 nm, 390–415 nm and a decrease
in absorbance in the region 320–340 nm with isosbestic points at
311 nm and 360 nm (Figs. 3 and 4). The association constants
(Table 2) indicate significant bindings of compounds 3–8 and
11–16 with Mg2+. Here also the Benesi–Hildebrand plot (inset of
Figs. 3 and 4) and Job plots (Supplementary Fig. S2) indicate the
1:1 stoichiometry of compound–Mg2+ complexes.
2.4. Influx–efflux assay using C. albicans
Further to explore the drug influx/efflux properties of these
molecules, cell based assays using C. albicans were carried out.
Compounds 4, 5, and 6 have already been studied on C. albicans
where compound 6 was causing enhanced levels of both influx
and efflux of R6G due to the weakening of the cell wall of C. albi-
cans.24 For compounds 3 and 11–16, first the MIC80 values were
determined. Compound 3 had a MIC80 value of 781 lg/ml while
for the other compounds the values were much higher (MIC80 for
compound 15 was 1.5 mg/ml, for rest, it was >3.0 mg/ml). So fur-
ther experiments were focused on compound 3. To look into the
inhibition of Candida growth in presence of compound 3, spot as-
says were performed. Surprisingly, there was no significant inhibi-
tion of Candida growth with either 0.9 mg/ml or 1 mg/ml
concentration of compound 3 although some reduction in colony
size is clearly visible at higher dilutions (Fig. 5). Such differences
in growth of C. albicans between solid and liquid media are well
documented.32 Due to problems of solubility in aqueous media,
higher concentrations of the drug could not be tested. Next the ac-
tion of compound 3 was tested in combination with cell wall per-
turbing agents Congo red and calcofluor white (Figs. 6 and 7,
Hence the present investigations have identified two sets of
compounds, one which shows higher bindings with P-gp (com-
pounds 3–8) and second showing better interactionswith ATP (com-
pounds 11–16) while both the sets of compounds show appreciable
bindings with Mg2+. The observation that both the sets of com-
pounds (3–8 and 11–16) interact with Mg2+ led us to investigate
the probable sites on the compound where Mg2+ binding occurs.
The energy minimized structures of compounds 6 (Supplementary
Fig. S3) and 14 (Supplementary Fig. S4) in presence of Mg2+ clearly
Figure 3. Absorption spectra of compound 4 in the presence of increasing concentration of Mg2+ (0–0.5 equiv). Arrows denote the change in absorbance in the presence of
Mg2+. Inset: Benesi–Hildebrand plot (stoichiometry 1:1).

4216
P. Singh et al. / Bioorg. Med. Chem. 18 (2010) 4212–4223
Figure 4. Absorption spectra of compound 14 in the presence of increasing concentration of Mg2+ (0–0.5 equiv). Arrows denote the change in absorbance in the presence of
Mg2+. Inset: Benesi–Hildebrand plot (stoichiometry 1:1).
Figure 5. Plate assay in the presence of compound 3. Details are given under
methods. Spot numbers along with cell dilutions in parenthesis.
Figure 6. Plate assay in the presence of a combination of compound 3 and Congo
red. Details are given under methods. Spot numbers along with cell dilutions in
parenthesis.
respectively). No significant effect was seen in either of these com-
binations, clearly indicating that compound 3 did not influence the
integrity of the cell wall of the organism. Since it appeared from
the MIC80 values that compound 3 was toxic to Candida cells, we
next looked to see if it would in any way influence the drug efflux
pumps in the organism. For this, spot assays were carried in the pres-
ence of a combination of compound 3 and rhodamine 6G (R6G),
which is also a substrate for both cdr1p and cdr2p in C. albicans. It
was found that a combination of compound 3 and R6G in the growth
medium inhibited the cell growth (Fig. 8). These results could sug-
gest that either compound 3 is facilitating the entry of R6G inside
the cells, or it is somehow negatively affecting the efflux of R6G from
the cells.
R6G influx inside the cells (Fig. 9). However, it may be pointed
out that the total amount of R6G in the cells is roughly the same
at the end of 3 h, indicating that the toxicity of the combination
of compound 3 with R6G cannot be due to enhanced levels of the
toxin entering the cell. In the absence of glucose the efflux of
R6G was the same for all sets and was far less than in the presence
of glucose, indicating that (i) the presence of compound 3 did not
significantly perturb the cell wall of the organism and lead to high-
er levels of passive efflux from the cells and (ii) the efflux of R6G
was primarily due to an active process. The efflux data in the pres-
ence of glucose suggests that in the presence of compound 3 the
level of R6G efflux from the cells is less than the control set. On
comparison with the solvent control, it was observed that the ef-
flux in the presence of the compound was inhibited more relative
to the solvent control up to a period of 60 min (Fig. 10). It may be
pointed out, however, that at higher time points, the solvent itself
appeared to influence the efflux of R6G from the cells, indicating
that the cells were probably being affected by the organic solvent
present in the assay buffer.
To investigate this further, R6G influx–efflux assays were done
in the presence of compound 3, to see if there was any change in
the influx or efflux levels of R6G in the presence of this compound.
Influx and efflux of R6G was monitored in the Candida cells in the
absence and the presence (800 lg/ml) of compound 3. A solvent
control set was also assayed to account for the action of the solvent
on this process. Within limits of experimental error, the influx data
suggests that, the presence of compound 3 increases the rate of

P. Singh et al. / Bioorg. Med. Chem. 18 (2010) 4212–4223
4217
Control
Solvent control
Compound 3 (0.8 mg/ml)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
15
30
45
60
90
120 150 180
Time (minutes)
Figure 9. R6G influx in CAI4 cells in the presence of compound 3.
A
higher
O.D.527 nm indicates higher levels of R6G in the buffer and hence a lower influx
inside the cells.
Figure 7. Plate assay in the presence of
a combination of compound 3 and
calcofluor white.
450
Control (no glucose)
Solvent control (no glucose)
Compound 3 (no glucose)
400
Control (with glucose)
Solvent control (with glucose)
350
Compound 3 (with glucose)
300
250
200
150
100
50
0
0
15
30
40
60
TIME (minutes)
Figure 10. R6G efflux from CAI4 cells in the presence of compound 3. A higher
intensity level indicates higher levels of R6G in buffer and hence a higher value of
R6G influx from the cells.
binding site cavity. Thus, docking occurs between the flexible
ligand parts of the compound and enzyme. The ligand orientation
was determined by a shape scoring function based on Ascore and
the final positions were ranked by lowest interaction energy
values.
Figure 8. Plate assay in the presence of
rhodamine 6G.
a combination of compound 3 and
2.5. Docking studies
Docking of compounds 3–8 in the ATP binding site of P-gp indi-
cate appreciable interactions between these compounds and the
amino acids of P-gp. Compound 3 on docking in the ATP binding
site of P-gp exhibited H-bond interaction from its carbonyl oxygen
with Y1393, OH group with D1353 and COOH group with D1353
and Y1352 residues (Fig. 11). Docking of compound 4 in the ATP
binding site of P-gp also indicated H-bond interactions between
the carbonyl oxygen of acridone moiety, OH group of hydroxyl
amine chain with Y1393 and D1353 residues. Compound 5 on
docking in the ATP binding site showed interactions between car-
bonyl oxygen and OH of carboxyl group with Y1352, Y1393, and
D468, respectively. Docking of compound 6 in the ATP binding site
of P-gp showed interactions through nitrogen of piperidino piper-
idine moiety, OH of hydroxyl amine chain and OH of carboxyl
group with Y2393, Y1393, V479, and E473, respectively. The
H-bond interaction between the nitrogen of piperidino piperidine
moiety of compound 6 and Y2393 (which is not observed with
other amines) indicates that a large group (which could extend
Since a difference in the behavior of compounds 3–8 and 11–16
for interactions with P-gp was observed in ‘Drug–P-glycoprotein
Interaction Assay’, it was planned to perform the docking of these
two categories of compounds in the ATP and drug binding sites of
P-gp. Compounds 3–8 and 11–16 were built using the builder tool-
kit of the software package ArgusLab 4.0.133 and energy minimized
using semi-empirical quantum mechanical method PM3. The crys-
tal coordinates of the P-glycoprotein (pdb ID 1MV5, pdb ID 3G60)5
were downloaded from protein data bank. For the docking of com-
0
pounds 3–8 and 11–16, a volume of 15 ÅA3 was taken around ADP in
case of 1MV5 crystal coordinates representing the ATP binding site
0
and 15 ÅA3 around hexapeptide in case of 3G60 crystal coordinates
representing drug binding site. Mg2+ is also visible in the ATP bind-
ing site of P-gp. For the docking process, the energy minimized
molecule was pasted on the protein. The docking program imple-
ments an efficient grid based docking algorithm which approxi-
mates an exhaustive search within the free volume of the

4218
P. Singh et al. / Bioorg. Med. Chem. 18 (2010) 4212–4223
towards Y2393) with H-donor/acceptor sites is desirable at the end
of N-10 substituent. Compounds 7 and 8 also exhibit similar inter-
actions in the ATP binding site of P-gp.
Docking of compounds 3–8 in the drug binding site of P-gp indi-
cate that these compounds show less interactions with the amino
acids here in comparison to their interactions in the ATP binding
site (Table 3), supporting the experimental observation (P-gp–drug
interaction assay). However, compounds 11–16 did not enter
either the ATP binding site or drug binding site during the docking
of these compounds in the respective crystal coordinates of the
protein. Therefore, the docking studies support the experimental
observations of appreciable interactions of compounds 3–8 and
no interactions of compounds 11–16 with P-gp and also indicate
the preference of compounds 3–8 for the ATP binding site over
the drug binding site of P-gp.
3. Conclusions
1. ‘Drug–P-gp interaction’ experiment showed that compounds
3–8 exhibit significant interactions with P-gp while compounds
11–16 did not interact with P-gp which is also supported by
docking studies. Log P and TPSA values of these compounds
Figure 11. Compound 3 docked in the ATP binding site of P-gp. Hs0 are omitted for
clarity.
Table 3
0
Comparison of interactions of compounds in the ATP binding site and the drug binding site of P-gp. H-bond distances are given in AÅ
Compound
Interactions in ATP binding site
Interactions in drug binding site
0
Y1393
O
2.66 A
O
N
Y1352
O
OH
2.87 A
S975
0
N
2.66 A
3
0
HO
O
OH
0
N
2.26 A
OH
0
N
D1353
2.60A
D1353
0
O
N
2.51 A
Y1393
O
N
O
OH
4
O
OH
0
OH
N
2.62 A
OH
D1353
0
N
2.89 A
D1353
No H-bond Interactions
O
O
D468
0
N
N
2.41 A
O
OH
O
OH
0
2.90 A
0
5
OH
OH
2.90 A
0
N
N
Y1393
2.34 A
Y1352
O
O
No H-bond Interactions

P. Singh et al. / Bioorg. Med. Chem. 18 (2010) 4212–4223
4219
Table 3 (continued)
Compound
Interactions in ATP binding site
Interactions in drug binding site
O
O
0
E473
2.72 A
N
N
O
OH
O
OH
0
2.96 A
6
OH
OH
0
N
N
2.99 A
V479
Y1393
0
0
N
N
2.40 A
2.67A
Y2393
Y303
O
N
O
0
N
2.17 A
O
O
D1353
O
OH
H
7
0
2.99A
OH
OH
N
N
0
D1353
2.64 A
D1353
No H-bond Interactions
0
O
2.63A
Y1393
O
N
N
O
OH
0
8
2.46 A
OH
O
O
D1353
N
H
OH
N
0
No H-bond Interactions
2.15 A
Y1352
O
O
N
N
Cl
Cl
11
OH
OH
N
N
No H-bond Interactions
No H-bond Interactions
O
O
N
N
Cl
Cl
12
OH
N
OH
N
No H-bond Interactions
No H-bond Interactions
(continued on next page)

4220
P. Singh et al. / Bioorg. Med. Chem. 18 (2010) 4212–4223
Table 3 (continued)
Compound
Interactions in ATP binding site
Interactions in drug binding site
O
O
N
N
Cl
Cl
13
OH
OH
N
N
O
O
No H-bond Interactions
No H-bond Interactions
O
O
N
N
Cl
Cl
OH
N
OH
N
14
N
N
No H-bond Interactions
No H-bond Interactions
O
O
N
N
Cl
Cl
15
OH
N
OH
N
No H-bond Interactions
No H-bond Interactions
O
O
N
N
Cl
Cl
16
OH
N
OH
N
No H-bond Interactions
No H-bond Interactions
indicate that the hydrophilicity might be the controlling param-
eter for their interactions with P-gp since more hydrophilic
compounds (3–8) exhibit better interactions with P-gp.
2. On the basis of UV–vis spectral studies, irrespective of the nat-
ure of C-4 substituent, appreciable interactions were observed
between compounds 3–8, 11–16 with Mg2+ indicating that
Mg2+ might be interacting at sites other than the C-4 substitu-
ent (probably OH and N present at N-10 substituent, as shown
by the energy minimized structures of compound–Mg2+
complex). Only compounds 11–16 interact with ATP and the
non-interactions of compounds 3–8 with ATP seems to be
due to the repulsion between carboxyl group and phosphate
group.
3. Influx–efflux assay on C. albicans indicate that the presence of
compound 3 decreases the efflux of R6G from the Candida cells
which might be occurring through the inhibition of cdr1p/cdr2p
transporting proteins. Compound 3 is suitable candidate for fur-
ther investigations for MDR modulating properties.
4. Experimental
4.1. General note
Melting points were determined in capillaries and are uncor-
rected. 1H and 13C NMR spectra were recorded on JEOL 300 MHz
and 75 MHz NMR spectrometer, respectively, using CDCl3 as solvent.

P. Singh et al. / Bioorg. Med. Chem. 18 (2010) 4212–4223
4221
Chemical shifts are given in ppm with TMS as an internal reference.
J values are given in Hertz. IR and UV spectral data were recorded on
FTIR 8400S Shimadzu and BioTek PowerWave XS instruments,
respectively. The reactions corresponding to epoxy ring opening
with amines were performed in domestic microwave oven (INALSA
model 1MW17EG) with microwave power 700 W and operating fre-
quency 2450 MHz. Reactions were monitored by thin layer chroma-
tography (TLC) on glass plates coated with Silica Gel GF-254. Column
chromatography was performed with 100–200 mesh silica. In 13C
NMR spectral data, +ve, ꢀve terms correspond to CH3, CH, and CH2
signals, respectively, in DEPT-135 NMR spectra.
isolate pure product. Spectral data for compounds 3–6 has already
been reported.24
4.4.1. 10-(3-Diethylamino-2-hydroxy-propyl)-9-oxo-9,10-dihydro-
acridine-4-carboxylic acid (7)
Compound 7 (0.08 g, 0.23 mmol) was synthesized using com-
pound 2 (0.1 g, 0.34 mmol) according to the synthetic procedure
C as a yellow solid in a yield of 67%, mp 115 °C; IR (KBr, cmꢀ1):
1610 (C@O), 1680 (C@O), 3250 (OH), 3360 (OH); UV (etha-
nol + HEPES buffer) kmax
(e) 232 (9880), 256 (25,530), 412
(6040); 1H NMR (300 MHz, CDCl3) 1.25–1.40 (m, 6H, 2 ꢁ CH3),
3.20–3.53 (m, 6H, 13-H + 2 ꢁ NCH2), 4.67–4.92 (m, 3H,
12-H + 11-H), 7.26–7.40 (m, 2H, ArH), 7.42–7.46 (m, 3H, ArH),
8.35–8.64 (m, 2H, ArH), 12.09 (br s, 1H, COOH); 13C NMR (nor-
mal/DEPT-135) d 12.02 (+ve, CH3), 47.75 (ꢀve, CH2), 50.20 (ꢀve,
CH2), 57.64 (ꢀve, CH2), 66.36 (+ve, C-12), 115.80 (absent, ArC),
121.48 (+ve, ArCH), 122.55 (+ve, ArCH), 127.64 (+ve, ArCH),
128.42 (+ve, ArCH), 133.90 (+ve, ArCH), 167.12 (C@O), 178.52
(C@O), MS (FAB) 369 (M++1). Anal. Calcd for C21H24N2O4: C,
68.46; H, 6.57; N, 7.60. Found: C, 68.48; H, 6.55; N, 7.65.
4.2. General procedure for the syntheses of compounds 1 and 9
(procedure A)22
A mixture of 2-chlorobenzoic acid (1.56 g, 10 mmol), anthranilic
acid/o-chloroaniline (10 mmol), powdered CuO (25 mg) and K2CO3
(1.5 g, ꢂ11 mmol) in isoamyl alcohol (10 ml) was heated at 160 °C
for 10 h. After cooling, the alcohol was evaporated under vacuum
and the residue was dissolved in hot water (120 ml) and acidified
with 10 N HCl. The precipitates were filtered and washed with
hot water. The solid product was dissolved in ethyl acetate, the
solution was dried over anhydrous Na2SO4 and evaporated to dry-
ness. The crude product was purified by column chromatography
over silica gel using a mixture of ethyl acetate and hexane (1:1)
as the eluent. This product was taken in concd H2SO4 (just to dis-
solve the compound) and heated on water bath for 1.5 h. Reaction
mixture was added to hot water and the resulting precipitates
were filtered to get acridone 1 and 9.
4.4.2. 10-(3-Diisopropylamino-2-hydroxy-propyl)-9-oxo-9,10-
dihydro-acridine-4-carboxylic acid (8)
Compound 8 (0.08 g, 0.21 mmol) was synthesized using com-
pound 2 (0.1 g, 0.34 mmol) according to the synthetic procedure
C as a light yellow solid in a yield of 62%, mp 120 °C; IR (KBr,
cmꢀ1): 1605 (C@O), 1685 (C@O), 3220 (OH); 3380 (OH); UV
(ethanol + HEPES buffer) kmax (e) 232 (10,050), 253 (26,780), 412
(6430); 1H NMR (300 MHz, CDCl3) d 1.56 (d, J = 6.6 Hz, 12H,
4 ꢁ CH3), 3.13–3.18 (m, 1H, NCH), 3.71–3.73 (m, 3H, 13-H +
NCH), 4.46–4.55 (m, 3H, 11-H + 12-H), 7.21–7.36 (m, 3H, ArH),
7.68–7.69 (m, 2H, ArH), 8.35–8.73 (m, 2H, ArH), 11.51 (br s, 1H,
COOH); 13C NMR (normal/DEPT-135) d 19.35 (+ve, CH3), 21.44
(+ve, CH3), 45.05 (ꢀve, CH2), 47.59 (ꢀve, CH2), 49.41 (+ve, CH),
50.42 (+ve, CH), 66.26 (+ve, C-12), 115.36 (absent, ArC), 121.74
(+ve, ArCH), 122.43 (+ve, ArCH), 127.85 (+ve, ArCH), 133.78 (+ve,
ArCH), 134.00 (+ve, ArCH), 167.35 (C@O), 178.08 (C@O); MS
(FAB) 397 (M++1). Anal. Calcd for C23H28N2O4: C, 69.67; H, 7.12;
N, 7.07. Found: C, 69.65; H, 7.16; N, 7.09.
4.3. General procedure for the syntheses of compounds 2 and 10
(procedure B)
Sodium hydride (3.0 mmol) was washed with dry hexane and
taken in 15 ml of dimethyl sulphoxide. To this solution, acridone
1/9 (2.5 mmol) and epichlorohydrin (3.0 mmol) were added and
the reaction mixture was stirred for 17–18 h at 70–80 °C (TLC
monitoring). The reaction mixture was extracted with ethyl
acetate. The organic phase was dried over anhydrous Na2SO4. The
solvent was distilled off and the residue was column chromato-
graphed using ethyl acetate and hexane (7:1) as eluents to isolate
pure compound 224 and 10.
4.4.3. 4-Chloro-10-(2-hydroxy-3-(pyrrolidin-1-yl)propyl)acridin-
9(10H)-one (11)
Compound 11 (0.08 g, 0.24 mmol) was synthesized using com-
pound 10 (0.1 g, 0.35 mmol) according to the synthetic procedure
C as a yellow solid in a yield of 68%, mp 105 °C; IR (KBr, cmꢀ1):
4.3.1. 4-Chloro-10-((oxiran-2-yl)methyl)acridin-9(10H)-one (10)
Compound 10 (0.84 g, 2.95 mmol) was synthesized using com-
pound 9 (1 g, 4.36 mmol) according to the synthetic procedure B as
a yellow solid in a yield of 68%, mp 130 °C; IR (KBr, cmꢀ1): 1635
1595 (C@O), 3355 (OH); UV (ethanol + HEPES buffer) kmax (e) 235
(10,320), 256 (22,600), 413 (5150); 1H NMR (300 MHz, CDCl3) d
1.79–1.84 (m, 4H, 2 ꢁ CH2pyroll), 2.58–2.66 (m, 3H, 1H of 13-
H + 2H of NCH2), 2.75 (dd, 2H, 2J = 6.7 Hz, 3J = 2.2 Hz, NCH2), 2.78
(dd, 1H, 2J = 11.8 Hz, 3J = 9.7 Hz, 1H of 13-H), 4.29–4.32 (m, 1H,
12-H), 4.45 (dd, 1H, 2J = 16.2 Hz, 3J = 4.2 Hz, 11-H), 4.57 (dd, 1H,
2J = 16.0 Hz, 3J = 7.05 Hz, 11-H), 7.29 (dd, 2H, 2J = 8.1 Hz,
3J = 4.2 Hz, ArH), 7.71 (dd, 3H, 2J = 4.2 Hz, 3J = 0.9 Hz, ArH), 8.53 (t,
1H, J = 1.2 Hz, ArH), 8.56 (t, 1H, J = 1.2 Hz, ArH); 13C NMR (nor-
mal/DEPT-135) d 23.82 (ꢀve, CH2), 50.09 (ꢀve, CH2), 54.00 (ꢀve,
CH2), 59.76 (ꢀve, CH2), 67.77 (+ve, C-12), 115.33 (+ve, ArCH),
121.38 (+ve, ArCH), 122.37 (absent, ArC), 122.75 (+ve, ArCH),
127.73 (+ve, ArCH), 133.69 (+ve, ArCH), 177.88 (C@O); MS (FAB)
356, 358 (3:1) (M+). Anal. Calcd for C20H21ClN2O2: C, 67.32; H,
5.93; N, 7.85. Found: C, 67.34; H, 5.99; N, 7.88.
(C@O); UV (ethanol + HEPES buffer) kmax (e) 231 (8870), 246
(21,560), 404 (11,090); 1H NMR (300 MHz, CDCl3) d 2.68 (dd, 1H,
2J = 2.7 Hz, 3J = 2.4 Hz, Hb), 2.93 (dd, 1H, 2J = 3.39 Hz, 3J = 4.8 Hz,
Ha), 3.48–3.50 (m, 1H, Hc), 4.39 (dd, 1H, 2J = 16.9 Hz, 3J = 4.6 Hz,
He), 4.85 (dd, 1H, 2J = 16.9 Hz, 3J = 1.9 Hz, Hd), 7.26–7.32 (m, 2H,
ArH), 7.56–7.74 (m, 3H, ArH), 8.53 (dd, 2H, 2J = 8.1 Hz, 3J = 1.5 Hz,
ArH); 13C (Normal/DEPT-135) d 45.03 (ꢀve, CH2), 47.55 (ꢀve,
CH2), 50.23 (+ve, CH), 115.10 (+ve, ArCH), 122.69 (+ve, ArCH),
122.42 (absent, ArC), 127.78 (+ve, ArCH), 133.95 (+ve, ArCH),
142.51 (+ve, ArCH), 178.05 (C@O), MS (FAB) 285, 287 (3:1) (M+).
Anal. Calcd for C17H13NO4: C, 67.26; H, 4.23; N, 4.90. Found: C,
67.24; H, 4.25; N, 4.87.
4.4. General procedure for the syntheses of compounds 3–8 and
11–16 (procedure C)
4.4.4. 4-Chloro-10-(2-hydroxy-3-(piperidin-1-yl)propyl)acridin-
9(10H)-one (12)
A mixture of acridone 2 (1 mmol) and appropriate amine
(1 mmol) was irradiated in microwave oven for 5–7 min. On com-
pletion of the reaction (TLC), it was washed with diethyl ether to
Compound 12 (0.08 g, 0.22 mmol) was synthesized using com-
pound 10 (0.1 g, 0.35 mmol) according to the synthetic procedure
C as a yellow solid in a yield of 63%, mp 110 °C; IR (KBr, cmꢀ1):

4222
P. Singh et al. / Bioorg. Med. Chem. 18 (2010) 4212–4223
1585 (C@O), 3354 (OH); UV (ethanol + HEPES buffer) kmax
(
e
) 233
57.35 (ꢀve, CH2), 66.45 (+ve, C-12), 115.70 (+ve, ArCH), 121.48
(+ve, ArCH), 122.36 (absent, ArC), 127.54 (+ve, ArCH), 128.04
(+ve, ArCH), 133.90 (+ve, ArCH), 178.52 (C@O), MS (FAB) 358,
360 (3:1) (M+). Anal. Calcd for C20H23ClN2O2: C, 66.94; H, 6.46; N,
7.81. Found: C, 66.98; H, 6.47; N, 7.86.
(10,810), 252 (24,850), 404 (7110); 1H NMR (300 MHz, CDCl3) d
1.63 (br s, 6H, 3 ꢁ CH2), 2.51–2.62 (m, 6H, 3 ꢁ NCH2), 4.30–4.40
(m, 1H, 12-H), 4.44 (dd, 1H, 2J = 16.2 Hz, 3J = 3.6 Hz, 11-H), 4.55
(dd, 1H, 2J = 16.0 Hz, 3J = 7.0 Hz, 11-H), 7.23–7.29 (m, 2H, ArH),
7.70–7.73 (m, 3H, ArH), 8.49 (t, 1H, J = 1.2 Hz, ArH), 8.52 (t, 1H,
J = 1.0 Hz, ArH); 13C NMR (normal/DEPT-135) d 25.86 (ꢀve, CH2),
50.24 (ꢀve, CH2), 54.74 (ꢀve, CH2), 62.36 (ꢀve, CH2), 65.98 (+ve,
C-12), 115.41 (+ve, ArCH), 117.30 (+ve, ArCH), 119.52 (+ve, ArCH),
121.34 (absent, ArC), 127.66 (+ve, ArCH), 133.66 (+ve, ArCH), 178.53
(C@O), MS (FAB) 370, 372 (3:1) (M+). Anal. Calcd for C21H23ClN2O2:
C, 68.01; H, 6.25; N, 7.55. Found: C, 68.06; H, 6.28; N, 7.57.
4.4.8. 4-Chloro-10-(3-(diisopropylamino)-2-hydroxypropyl)acridin-
9(10H)-one (16)
Compound 16 (0.09 g, 0.24 mmol) was synthesized using com-
pound 10 (0.1 g, 0.35 mmol) according to the synthetic procedure
C as a yellow solid in a yield of 67%, mp 100 °C; IR (KBr, cmꢀ1):
1592 (C@O), 3342 (OH); UV (ethanol + HEPES buffer) kmax (e) 237
(11,270), 254 (20,870), 401 (6550); 1H NMR (300 MHz, CDCl3): d
1.07 (d, J = 6.8 Hz, 6H, 2 ꢁ CH3), 1.10 (d, J = 6.6 Hz, 6H, 2 ꢁ CH3),
2.63 (dd, 1H, 2J = 13.6 Hz, 3J = 9.4 Hz, 13-H), 2.79 (dd, 1H,
2J = 13.8 Hz, 3J = 4.0 Hz, 13-H), 3.07–3.16 (m, 2H, NCH), 4.19–4.26
(m, 1H, 12-H), 4.42 (dd, 1H, 2J = 16.0 Hz, 3J = 3.4 Hz, 11-H), 4.54
(dd, 1H, 2J = 16.0 Hz, 3J = 7.3 Hz, 11-H), 7.22–7.33 (m, 2H, ArH),
7.69–7.75 (m, 3H, ArH), 8.52 (t, 1H, J = 1.0 Hz, ArH), 8.59 (t, 1H,
J = 0.9 Hz, ArH); 13C NMR (normal/DEPT-135): d 19.89 (+ve CH3),
23.06 (+ve, CH3), 45.06 (ꢀve, CH2), 47.58 (ꢀve, CH2), 50.33 (+ve,
CH), 50.79 (+ve, CH), 66.63 (+ve, C-12), 115.12 (+ve, ArCH),
115.48 (+ve, ArCH), 118.18 (+ve, ArCH), 120.32 (+ve, ArCH),
121.33 (absent, ArC), 122.39 (+ve, ArCH), 178.09 (C@O); MS (FAB)
386, 388 (3:1) (M+). Anal. Calcd for C22H27ClN2O2: C, 68.29; H,
7.03; N, 7.24. Found: C, 68.27; H, 7.06; N, 7.28.
4.4.5. 4-Chloro-10-(2-hydroxy-3-morpholinopropyl)acridin-
9(10H)-one (13)
Compound 13 (0.08 g, 0.22 mmol) was synthesized using com-
pound 10 (0.1 g, 0.35 mmol) according to the synthetic procedure
C as a yellow solid in a yield of 63%, mp 120 °C; IR (KBr, cmꢀ1):
1592 (C@O), 3343 (OH); UV (ethanol + HEPES buffer) kmax (e) 235
(16,670), 252 (27,860), 413 (8040); 1H NMR (300 MHz, CDCl3) d
2.53–2.64 (m, 2H, 13-H), 2.66–2.71 (m, 4H, 2 ꢁ NCH2), 3.75 (t,
4H, J = 4.5 Hz, 2 ꢁ OCH2), 4.33–4.42 (m, 1H, 12-H), 4.48 (dd, 1H,
2J = 16.2 Hz, 3J = 3.6 Hz, 11-H), 4.58 (dd, 1H, 2J = 16.0 Hz,
3J = 7.2 Hz, 11-H), 7.19–7.26 (m, 2H, ArH), 7.65–7.70 (m, 3H,
ArH), 8.42 (t, 1H, J = 0.9 Hz, ArH), 8.45 (t, 1H, J = 1.2 Hz, ArH); 13C
NMR (normal/DEPT-135) d 50.16 (ꢀve, CH2), 53.85 (ꢀve, CH2),
62.33 (ꢀve, CH2), 66.10 (ꢀve, CH2), 66.92 (+ve, C-12), 115.37
(+ve, ArCH), 119.21 (+ve, ArCH), 120.23 (+ve, ArCH), 121.43 (ab-
sent, ArC), 127.70 (+ve, ArCH), 133.71 (+ve, ArCH), 177.65 (C@O),
MS (FAB) 372, 374 (3:1) (M+). Anal. Calcd for C20H21ClN2O3: C,
64.43; H, 5.68; N, 7.51. Found: C, 64.45; H, 5.71; N, 7.55.
4.4.9. P-gp interaction studies
Stock solutions of compounds 3–8 and 11–16 were prepared at
10ꢀ2 M concentration and diluted to three concentrations 0.5
5 lM, and 50 l
l
M,
M. Further dilutions take place during the assay
and the final concentrations become 0.05 M, 0.5 M, and 5 M.
Each well of 96-well plate was dispensed with 80 L of enzymatic
buffer, 20 L of PK/LDH solution, 10 L of PEP solution and 10 L of
NADH solution. Additionally, 60 L of enzymatic buffer was added
to the total activity well; 30 L of non-specific ATPase inhibitor
solution and 30 L enzymatic buffer was added to basal activity
well; 30 L non-specific ATPase inhibitor solution, 10 L each of
verapamil, progesterone, vinblastine, and 20 L enzymatic buffer
was added to the reference wells and 30 L non-specific ATPase
inhibitor solution, 30 L enzymatic buffer to non-specific activity
well. Blank well contains 200 L of enzymatic buffer. The plate
was incubated for 30 min at 37 °C. 10 L of enzymatic buffer was
added to non-specific activity wells and 10 L of membrane vesi-
cles were added to all other wells except blank well. Plate was
incubated for 5 min at 37 °C, dispensed 20 L of compound at each
concentration and again incubated for 5 min at 37 °C. Finally, 10
l
l
l
4.4.6. 4-Chloro-10-(2-hydroxy-3-(4-(piperidin-1-yl)piperidin-1-
yl)propyl)acridin-9(10H)-one (14)
Compound 14 (0.10 g, 0.23 mmol) was synthesized using com-
pound 10 (0.1 g, 0.35 mmol) according to the synthetic procedure
C as a yellow solid in a yield of 65%, mp 115 °C; IR (KBr, cmꢀ1):
l
l
l
l
l
l
l
1590 (C@O), 3345 (OH); UV (ethanol + HEPES buffer) kmax
(e
) 236
l
l
(11,720), 254 (29,680), 407 (7520); 1H NMR (300 MHz, CDCl3) d
1.45–1.60 (m, 2H, CH2), 1.74–1.75 (m, 8H, 4 ꢁ CH2), 1.98–2.25
(m, 1H, CH), 2.28–2.48 (m, 2H, NCH2), 2.50–2.58 (m, 6H, 3 ꢁ NCH2),
2.95–2.98 (m, 2H, 13-H), 4.27–4.32 (m, 1H, 12-H), 4.42 (dd, 1H,
2J = 16.2 Hz, 3J = 3.3 Hz, 11-H), 4.55 (dd, 1H, 2J = 15.9 Hz,
3J = 7.2 Hz, 11-H), 7.24–7.29 (m, 1H, ArH), 7.69–7.71 (m, 4H,
ArH), 8.50 (t, 1H, J = 1.0 Hz, ArH), 8.53 (t, 1H, J = 0.9 Hz, ArH); 13C
NMR (normal/DEPT-135) d 24.83 (ꢀve, CH2), 26.42 (ꢀve, CH2),
50.28 (ꢀve, CH2), 52.66 (ꢀve, CH2), 55.30 (ꢀve, CH2), 61.49 (ꢀve,
CH2), 62.15 (+ve, CH), 66.50 (+ve, C-12), 115.56 (+ve, ArCH),
119.25 (+ve, ArCH), 121.11 (+ve, ArCH), 121.23 (+ve, ArCH),
127.69 (absent, ArC), 133.62 (+ve, ArCH), 177.00 (C@O); MS (FAB)
453, 455 (3:1) (M+). Anal. Calcd for C26H32ClN3O2: C, 68.78; H,
7.10; N, 9.26. Found: C, 68.81; H, 7.16; N, 9.29.
l
l
l
l
l
l
l
lL
of MgATP was added to each well except blank well and plate was
incubated for 20 min at 37 °C. Plate was read at 340 nm followed
by incubation and again reading after 20 min.
4.4.10. Interactions of compounds 11–16 with ATP
Stock solutions of compounds 11–16 were prepared at 10ꢀ3 M
concentration by dissolving the compound in 2–3 drops of ethanol
and making final volume up to 10 ml in HEPES Buffer (10ꢀ2 M, pH
4.4.7. 4-Chloro-10-(3-(diethylamino)-2-hydroxypropyl)acridin-
9(10H)-one (15)
Compound 15 (0.08 g, 0.22 mmol) was synthesized using com-
pound 10 (0.1 g, 0.35 mmol) according to the synthetic procedure
C as a yellow solid in a yield of 64%, mp 120 °C; IR (KBr, cmꢀ1):
7.2). Thissolution is further diluted to 50 lM concentration in HEPES
buffer. Stock solution (10ꢀ2 M) of ATP was prepared by dissolving
NaATP in HEPES Buffer. Taking the ligand concentration constant,
upon addition of increasing concentration of ATP there is decrease
in absorbance up to the addition of 3 equiv of ATP solution. The bind-
ing constants of compounds 11–16 with ATP were calculated using
Benesi–Hildebrand equation34 (Supplementary data).
1595 (C@O), 3345 (OH); UV (ethanol + HEPES buffer) kmax (e) 234
(10,560), 253 (22,770), 405 (6600); 1H NMR (300 MHz, CDCl3) d
1.06 (t, 6H, J = 7.3 Hz, 2 ꢁ CH3), 2.54–2.74 (m, 6H, 13-H + 2 ꢁ
NCH2), 4.20–4.28 (m, 1H, 12-H), 4.42 (dd, 1H, 2J = 15.9 Hz,
3J = 3.3 Hz, 11-H), 4.54 (dd, 1H, 2J = 16.05 Hz, 3J = 7.35 Hz, 11-H),
7.25–7.27 (m, 2H, ArH), 7.70–7.72 (m, 3H, ArH), 8.48 (t, 1H,
J = 1.0 Hz, ArH), 8.51 (t, 1H, J = 0.3 Hz, ArH); 13C NMR (normal/
DEPT-135) d 11.82 (+ve, CH3), 47.56 (ꢀve, CH2), 50.20 (ꢀve, CH2),
4.4.11. Interactions of compounds 3–8 and 11–16 with Mg2+
Stock solutions of compounds 3–8 and 11–16 (10ꢀ3 M) were
prepared by dissolving the compounds 3–8 and 11–16 in 2–3 drops

P. Singh et al. / Bioorg. Med. Chem. 18 (2010) 4212–4223
4223
of ethanol and making final volume up to 10 ml in HEPES buffer (10
ꢀ2 M, pH 7.2). The solutions of compounds were further diluted to
10ꢀ4 M concentration in HEPES buffer. Upon titration with increas-
ing concentration of Mg2+ by taking ligand concentration constant,
into two sets each. PBS was added to one of the sets while 2% glu-
cose in PBS was added to the other set, to obtain a 2% cell suspen-
sion. The cell suspensions were then kept back at rocker and
aliquots were taken out at different time points, up to 3 h. The ali-
quots were spun at 2000 rpm for 2 min and the supernatant was
used for monitoring the amount of drug effluxed out from the cells.
The fluorescence intensity was monitored on a Cary-Varian spec-
trofluorimeter using excitation wavelength of 529 nm and emis-
sion wavelength of 553 nm (5 nm excitation and emission slit
widths).
regular increase, decrease was observed up to 0.5 equiv of Mg2+
.
The binding constants of the compounds 3–8 and 11–16 were cal-
culated using Benesi–Hildebrand equation.
4.4.12. MIC80 assays
MIC80 values for the compounds were determined using broth
dilution method. Briefly, the Candida CAI4 strain was streaked on
a YEPD plate and incubated at 30 °C for 24 h. In a 96-well flat
bottom microtitre plate, the compounds were serially diluted
twofold in YEPD medium upto the 10th column. The 11th and
12th columns were our control lanes to monitor for contamination
during the experiment and as growth control to determine MIC80
values, respectively. Similarly, a volume of solvent corresponding
to the volume of the compound used for dilution was also diluted
to see the effect of the solvent on the growth of the cells. The cells
were picked from the YEPD plate, and suspended in 0.9% saline to
obtain an O.D.600 nm equal to 0.1. This cell suspension was further
Acknowledgments
The financial assistance from DST, New Delhi is gratefully
acknowledged. J.K. thanks UGC, New Delhi for fellowship. CDRI,
Lucknow is acknowledged for recording mass spectra.
Supplementary data
Supplementary data (Job plots, Hildebrand equation, 1H NMR
spectra, physico-chemical parameters, docking complex and en-
ergy minimized compound–Mg2+ complexes) associated with this
article can be found, in the online version, at doi:10.1016/
j.bmc.2010.05.003.
diluted 100-fold in YEPD medium and 100 lL of this cell suspen-
sion was added to all the wells, except 11th column. The plates
were incubated at 30 °C for 48 h and O.D.600 nm was monitored
using ELISA plate reader. MIC80 values were calculated by deter-
mining the concentration of the compound which resulted in an
inhibition of cell growth by 80% as compared to the cell growth
in the absence of the compound.
References and notes
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4.4.14. Rhodamine 6G (R6G) influx/efflux assay (screening for
MDR modulation)
R6G influx and efflux in the CAI4 cells was monitored in the
presence and the absence of compound 3. 10 ml of CAI4 primary
culture was grown in YEPD medium at 30 °C for 24 h. Secondary
culture was grown in 600 ml YEPD with 2% inoculum from primary
culture, and grown at 30 °C for around 5 h. The cells were then pel-
leted at 4000 rpm and washed twice with PBS. The cell pellet was
divided into six sets, 2% cell suspension of each was made in PBS
with 2 mM 2-deoxyglucose and then kept on a rocker for 2 h. After
2 h 800
375 L of solvent (methanol/DMSO 1:1) was added to the solvent
control sets. 10 M/ml of R6G was added to each set. 700 L ali-
lg/ml of compound 3 was added to two of the sets while
l
l
l
quots were taken from each set at this time point (considered as
0 min), and then kept back at rocker. Aliquots were taken out at
different time points, spun at 2000 rpm for 2 min and the superna-
tant was transferred into a fresh Eppendorf. The amount of R6G in
the medium was monitored by measuring O.D.527 nm. After 3 h of
incubation with R6G, the cells were again pelleted at 2000 rpm
and washed twice with PBS. The cell pellets were further divided