Design, synthesis and evaluations of acridone derivatives using Candida albicans-Search for MDR modulators led to the identification of an anti-candidiasis agent

Article abstract of DOI:10.1016/j.bmc.2009.04.026

In order to search for MDR modulators, rationally designed acridone derivatives were investigated for their effect on influx or efflux of Rhodamine6G (R6G) in CAI4 cells. Results of these investigations indicate that in presence of compound 12, inhibition of growth of CAI4 cells and also an increased influx/efflux of R6G in CAI4 cells have been observed. This seems to be occurring due to the cell wall rupturing of Candida albicans. Compound 12 may be a suitable candidate for candidiasis therapy.

Full text of DOI:10.1016/j.bmc.2009.04.026

Bioorganic & Medicinal Chemistry 17 (2009) 3973–3979
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluations of acridone derivatives using
Candida albicans—Search for MDR modulators led to the
identification of an anti-candidiasis agent
a,*
a
b
b,*
Palwinder Singh , Jatinder Kaur , Bhawna Yadav , Sneha Sudha Komath
a
Department of Chemistry, Guru Nanak Dev University, Amritsar 143005, India
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 February 2009
Revised 4 April 2009
Accepted 6 April 2009
Available online 18 April 2009
In order to search for MDR modulators, rationally designed acridone derivatives were investigated for
their effect on influx or efflux of Rhodamine6G (R6G) in CAI4 cells. Results of these investigations indicate
that in presence of compound 12, inhibition of growth of CAI4 cells and also an increased influx/efflux of
R6G in CAI4 cells have been observed. This seems to be occurring due to the cell wall rupturing of Candida
albicans. Compound 12 may be a suitable candidate for candidiasis therapy.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Multi drug resistance
p-Glycoprotein
Acridone derivatives
Candida albicans
R6G influx/efflux
Cell wall rupturing
5–17
anti-protozoan,18–20 anti-viral21 drugs
1
. Introduction
of many anti-tumour,1
and MDR modulators
2
2,23
) substituted with a hydroxyl amine
Multi drug resistance (MDR),1–3 emanating due to the decrease
(pharmacophore of MDR modulators ) and investigated for their
24
in the intracellular drug concentration, is a great hurdle in the
successful practice of chemotherapy of various diseases like cancer,
drug influx/efflux behaviour over C. albicans.
4
,5
AIDS and even malaria. The role of p-glycoprotein (p-gp),
a
2
2
. Results
plasma membrane protein and a member of ABC family of proteins,
is well established in causing MDR due to the effluxing of the drug
molecules out of the cell. Similar to the role of p-gp, cdrp1 and
.1. Chemistry
6
cdrp2 are responsible for drug efflux in Candida albicans and hence
The synthesis of the target molecules have been achieved from
provide a hurdle in the anti-candidasis therapy. Accordingly, a
number of chemical entities have been investigated for their
the commercially available, cheaper materials. The Ullmann type
2
5–27
arylation
acridone skeleton of the molecule after the treatment of 1a and
b with acid. Treatment of acridone 1 with NaH in DMSO followed
of aniline with o-chlorobenzoic acid provided the
7
–13
14
p-gp
and cdrp1, cdrp2 modulating activities.
Since p-gp/cdrp1, cdrp2 interact with drugs (while effluxing) as
well as MDR modulators, combination of structural features of the
drug and modulator in a single molecule may enhance the interac-
tions of new molecule with these transporting proteins and hence
modulate their drug effluxing behaviour, better than the parent
modulator. Therefore, in order to find a potential candidate for
modulating the drug effluxing activities of transporting proteins,
in the present investigations, we have designed acridones (part
1
by stirring with epichlorohydrin gave N-substituted acridone 2.
NMR and mass spectral data confirm the formation of this com-
pound. Irradiating an equimolar mixture of acridone 2 and piperi-
dine in microwave oven for 5 min results in the formation of
compound 4. Likewise the reactions of acridone 2 with other
amines provided compounds 3, 5 and 6 in 5–7 min (Scheme 1). A
similar sequence of reactions of o-chlorobenzoic acid with anthra-
nilic acid provided compounds 9–12 (Scheme 2). Therefore, epoxy
ring opening with secondary amines under microwave irradia-
*
Corresponding authors. Tel.: +91 183 2258802 3495; fax: +91 183 2258819
P.S.).
E-mail address: palwinder_singh_2000@yahoo.com (P. Singh).
2
8,29
tions
constitutes a versatile approach to the synthesis of target
(
compounds.
0
968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.026

3
974
P. Singh et al. / Bioorg. Med. Chem. 17 (2009) 3973–3979
O
8
00
COOH
O
K CO
2
3
OH
+
c. H SO
MIC80
2
4
Cl
H N
CuO
700
600
500
400
300
200
2
N
H
N
H
1a
1
O
NaH, DMSO
3
-6:
NR R
Cl
1
2
O
1
2
9
8
O
3
4
N
N
7
NHR R
1 2
1
0
6
3
N
4
5
MWI
-7 min
1
1
N
Hd
He
Hc
12 OH
5
5
6
N
N
O
O
1
3
Ha
NR R
1
2
N
Hb
3-6
2
100
Scheme 1. Synthesis of acridone derivatives.
0
5
6
9
10
11
12
2
.2. Biology
Biological investigations of these molecules were performed on
COMPOUND
Figure 1. MIC80 for compounds 5, 6, 9, 10, 11 and 12. (Note: For compounds 5, 6
and 12 the duplicate values are identical and hence no error bar is visible.)
3
0
the strain CAI4, which is the derivative of wild type C. albicans
SC5314. Strain CAI4 is similar to wild type except for one mutation
at URA3 locus, that is, it is not able to grow on uridine deficient
medium.
also exhibit appreciable interactions with p-gp (evaluated using
3
1
‘Drug-p-glycoprotein interaction’ assay kit; compound 12 exhibits
3% increase in the basal activity of p-gp at 0.05 M concentration).
In comparison to the reported acridone derivatives
3
l
2
2,23
2
.2.1. MIC80 assays
compound
To test the sensitivity of the test compounds to C. albicans, MIC80
Minimum Inhibitory Concentration-80) assays, that is, the concen-
12 carries piperidino-piperidine moiety as a part of hydroxyl amine
substituent present at N-1 and a COOH group on the benzene ring of
acridone. Compounds 3 and 4 exhibit MIC80 values in the range of
mg/ml (not shown in Fig. 1).
(
tration of the toxin at which 80% of the cells are killed, were per-
formed. For this purpose, 96 well (8 row ꢀ 12 column) flat bottom
transparent cell culture plates were used. Serial dilution of the drugs
was done in a 96-well ELISA plate. Candida cells which were already
grown in rich medium (YEPD) for 24 h, at 30 °C were resuspended in
a 0.9% normal saline solution to give an optical density of 0.1 at
2.2.2. Spot assays
Spot assays were performed in small Petriplates, requiring
7.5 ml of YEPD-Agar medium per plate. Concentrations of com-
pounds 10, 11 and 12 were taken in the plates in the range of their
MIC. The solvent control plates were made corresponding to each
compound by taking the same volume of solvent, as the volume
of compound used in the corresponding plates. One control plate
was also made with YEPD alone. The plates were incubated over-
night at 30 °C to check for any contamination. The primary culture
was grown by taking CAI4 streak in 10 ml YEPD medium and grow-
ing for 24 h at 30 °C. Secondary culture was grown in 10 ml of
YEPD with 4% inoculums from primary culture, and grown at
30 °C for around 5 h. The cells were counted in 200-fold diluted
secondary culture using haemocytometer. Spots (labelled 1, 2, 3,
6
00 nm (OD600). The cells were then diluted 100 times in YEPD med-
ia. 100 l of diluted cells was added to each well except A11, which
l
was our media control to monitor contamination during the study.
A12 did not contain any of the test compounds and was our control
for comparison of growth. The plate was incubated at 30 °C for 48 h
and OD600 was monitored using a microplate reader. Based on OD600
we obtained the concentration of the compound which gave only
0% growth as compared to the control in the last well. This concen-
,
2
tration of the test compound corresponds to the MIC80 value (Fig. 1).
TheMIC80 assays wereperformedtwicefor eachcompoundand then
the MIC80 value was estimated. Compounds 10, 11 and 12 for which
theMIC80 wasestimatedto be 405
respectively, were selected for spot assays. These compounds (3–12)
l
g/ml, 132
l
g/ml, and 234
l
g/ml,
4, 5, 6) of 5 ll cell suspension were spotted on the plates corre-
sponding to 10 , 10 , 10 , 10 , 10 , 10 cells. The plates were
5
4
3
2
1
0
O
O
COOH
2
K CO3
OH
+
2
c. H SO4
H N
2
CuO
Cl
N
N
H
HOOC
COOH
H
7
COOH
Cl
1b
O
NaH, DMSO
O
N
9
-12
NR R2
1
O
9
1
N
N
1
NHR R2
COOH
OH
MWI
N
0
Hd
He
Hc
COOH
5
-7 min
O
NR R2
H
a
1
1
N
N
O
1
Hb
9
-12
1
2
N
8
Scheme 2. Synthesis of acridone derivatives.

P. Singh et al. / Bioorg. Med. Chem. 17 (2009) 3973–3979
3975
7
incubated at 30 °C for around 48 h and then checked for growth.
No significant difference in growth was observed in the plates for
compounds 10 and 11 (Fig. 2A and B) with their corresponding sol-
vent control plates despite the fact that they had shown inhibition
of Candida growth in liquid medium (as seen from MIC80). Such dif-
ferences between solid and liquid medium growth of Candida cells
have been previously reported as well.32 However, there was no
growth in plate of compound 12 (Fig. 2C) but visible growth in
its solvent control plate, suggesting that compound 12 is inhibiting
Candida growth in solid phase as well.
CAI4 cells were grown overnight in YEPD medium at 30 °C. 10
cells were spread on a 90 mm diameter YEPD-agar plate and the
plate was left to dry for about 10 min. Sterile filter paper discs of
6 mm diameter were placed on this plate with the help of sterile
forceps. Compound 12 (200
lg) dissolved in 20 ll of methanol
was spotted on one of the paper discs. As a solvent control 20
ll
of methanol alone was spotted on one paper disc. Two known anti-
fungal agents, fluconazole and miconazole, were also spotted on
similar size paper discs at a known inhibitory amount of 6 lg per
disc. The plate was incubated at 30 °C and the inhibition zone
diameters were measured after 24 h. As seen in Figure 3, com-
pound 12 clearly inhibits the Candida growth, though at a concen-
tration higher than the other antifungals. A derivative of this
compound could probably have improved antifungal activity.
Since compound 12 had an MIC80 value of 234
lg/ml on CAI4
strain of C. albicans, we used ꢁ300
l
g/ml of this compound in YEPD
plates and found that the compound greatly inhibited growth of
CAI4 in our spot assays (Fig. 2C). As it was clearly seen in Figure
2C, the inhibition in growth was a result of the compound and
not due to the solvent background.
2.2.4. Rhodamine 6G (R6G) influx/efflux assay (screening for
MDR modulation)
2
.2.3. Disc diffusion assay
Disc diffusion assay was performed to confirm the effect of
Since compound 12 was the best inhibitor of growth of CAI4
cells in both solid and liquid medium, we carried out influx/efflux
assays for this compound to understand what might be its effect on
compound 12 on Candida growth inhibition in solid medium.
A Compound 10 (0.5 mg/ml)
1
2
3
4
5
6
Solvent control (0.3 ml)
1
2
3
4
5
6
Positive control (YEPD)
5
4
3
2
1
0
1
(10 )
2(10 )
3(10 )
4(10 )
5(10 )
6(10 ) cells
B Compound 11 (0.2 mg/ml)
1
2
3
4
5
6
Solvent control (0.390 ml)
1
2
3
4
5
6
Positive control (YEPD)
5
4
3
2
1
0
1
(10 )
2(10 )
3(10 )
4(10 )
5(10 )
6(10 ) cells
C Compound 12 (0.313 mg/ml)
1
2
3
4
5
5
6
6
Methanol solvent control (0.470 ml)
1
2
3
4
Positive control (YEPD)
5
4
3
2
1
0
1
(10 )
2(10 )
3(10 )
4(10 )
5(10 )
6(10 ) cells
Figure 2. Plate assays on compounds: (A) 10, (B) 11, (C) 12 compared with solvent and medium controls. (Note: The white specks observed in plate for compound 12 are due
to precipitation of the drug upon long incubation and not fungal cell colonies.)

3
976
P. Singh et al. / Bioorg. Med. Chem. 17 (2009) 3973–3979
Compound 12 Solvent control Miconazole
Fluconazole
(6 µg)
(
200 µg)
(20 µl)
(6 µg)
Inhibition zone
diameter (mm)
10
7
12
15
Figure 3. Disc diffusion assay.
the drug influx/efflux ability of the cells. CAI4 primary culture
10 ml) 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 sterile water. The cell
pellet was divided into four sets, 2% cell suspension of each was
made in PBS with 2 mM 2-deoxyglucose and then kept on a rocker
the supernatant was used for monitoring the amount of drug
effluxed out from the cells in the absence and presence of
(
3
3
glucose. The fluorescence intensity was monitored on a Cary-
Varian spectrofluorimeter in these cases using excitation
wavelength of 529 nm and emission wavelength of 553 nm
(5 nm excitation and emission slit widths). Higher fluorescence
intensity corresponded to higher efflux of R6G from the cells.
Influx of R6G in cells takes place by a passive diffusion, while
efflux is either passive or ATP dependent (mediated by MDR pumps).
As can be seen fromFigure 4, compound 12 had a significant effect on
Candida cells, increasing the influx of R6G into the cells in its pres-
ence. Likewise, the efflux of R6G in the absence of glucose (passive
or ATP independent efflux) too appears to be higher. The relative
ratio of glucose stimulated or ATP driven efflux of R6G to the efflux
in the absence of glucose is the same in the control versus compound
12 treated cells. Hence, it seems that the higher influx and efflux of
R6G in the compound 12 treated cells is because of the effect of com-
pound 12 on the Candida cell wall, which leads to more influx and
more efflux passively, and the higher efflux is not because of the
function of the MDR pumps. It would therefore appear that com-
pound 12 does not directly affect the multi drug pumps inC. albicans,
although it appears to alter the cell wall integrity.
for 2 h. After 2 h 230
the sets while 10 M/ml of R6G was added to each set. Aliquots
700 l) were taken from each set at this time point (considered
lg/ml of compound 12 was added to two of
l
(
l
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 super-
natant was transferred into a fresh microcentrifuge tube. The
amount of R6G remaining in the medium was monitored by mea-
suring Abs527 nm. The data thus obtained corresponds to influx of
3
3
R6G. A higher Abs527 nm value corresponds to lesser amount of
the drug having entered the cell.
After 60 min incubation with R6G, the cells were again pelleted
at 2000 rpm and washed twice with water. The cell pellets were
further divided into two sets each. To each paired set of cells, after
making 2% cell suspensions made in PBS, 2% glucose was added to
one while PBS alone was added to the other. The cell suspensions
were then kept back at rocker and aliquots taken out at different
time points. The aliquots were spun at 2000 rpm for 2 min and
In order to verify this, we studied the effect of compound 12 in
combination with cell wall toxins CFW and CR that are known to
R6G INFLUX
R6G EFFLUX
CONTROL NO GLUCOSE
0.24
0.18
0.12
0.06
0.00
Compd 12
Control
200
CONTROL WITH GLUCOSE
COMPOUND 12 NO GLUCOSE
COMPOUND 12 WITH GLUCOSE
150
100
50
0
0
15
30
45
60
0
15
30
45
Time (minutes)
Time (minutes)
Figure 4. R6G influx/efflux in CAI4 cells in presence of compound 12.

P. Singh et al. / Bioorg. Med. Chem. 17 (2009) 3973–3979
3977
1
2
3
4
5
6
YEPD
Compd 12 (160 µg/ml)
MeOH (200 µl)
MeOH (200 µl) +
CR (300 µg/ml)
Compd 12 (160µg/ml) +CR
(
300 µg/ml) + MeOH
MeOH (200 µl) +
CFW (300 µg/ml)
Compd 12 (160µg/ml) +
CFW (300 µg/ml) + MeOH
Cells
1 (105)
2 (104) 3 (103)
4 (102)
5 (101)
6 (100)
Figure 5. Effect of compound 12 on CAI4 cells in combination with CFW and CR.
inhibit chitin polymerization and thereby affect the cell wall.34 For
this purpose, a concentration of compound 12 (160 g/ml) which
internal reference. J values are given in hertz. (+) Signs in DEPT
spectra correspond to signals due to CH
, CH groups while (ꢂ)
signs denote signals of CH groups. Chromatography was per-
formed with silica 100–200 mesh and reactions were monitored
by thin layer chromatography (TLC) with silica plates coated with
silica gel HF-254.
l
3
was not toxic to the cells was chosen (Fig. 5). As can be seen from
Figure 5 the presence of compound 12 alters the toxicity of CFW as
well as CR on CAI4. A combination of compound 12 and CFW/CR is
far more effective in inhibiting the Candida growth than any of
these acting alone, suggesting a possible synergistic mechanism,
although further detailed analysis will need to be done in order
to understand the mechanism of this action.
2
4.2. General procedure for the syntheses of 1 and 7(procedure A)
Thus it appears that compound 12 affects Candida cell wall and
is therefore toxic for this pathogenic fungus. It may be pointed out
that one of the major features of several anti-fungals currently
being used against candidiasis involves targeting and weakening
of the Candida cell wall.35 Since the cell wall is a distinct element
in fungal cells that does not exist in mammalian cells, such selec-
tive targeting is a particularly important strategy for therapy
against C. albicans.
A mixture of 2-chlorobenzoic acid (1.56 g, 10 mmol), aniline/
anthranillic acid (10 mmol), powdered CuO (25 mg) and K CO
2 3
(1.5 g, ꢁ11 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 concd HCl. The precipitates were washed with hot water, dis-
solved in ethyl acetate and the solution was dried over anhydrous
Na
umn chromatography over silica gel. This product was taken in con-
cd H SO (just to dissolve the compound) and heated on water bath
2 4
SO and then evaporated. The crude product was purified by col-
2
4
3
. Conclusions
for 1.5 h. Reaction mixture was added to hot water and the resulting
precipitates were filtered to get acridone 1/7.
In conclusion, during our attempt to find out suitable MDR
modulators from rationally designed acridone derivatives, we
found that compound 12 weakens the cell wall of C. albicans. Fur-
ther refinement of this molecule may develop it into an effective
agent for candidiasis therapy.
4
.3. General procedure for the syntheses of compounds 2 and 8
(
procedure B)
Sodium hydride (3.0 mmol) was washed with dry hexane and
taken in 15 ml of dimethyl sulfoxide. To this solution, acridone 1/
7 (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 ace-
4
4
. Experimental
.1. General note
2 4
tate. The organic phase was dried over anhydrous Na SO . The sol-
Melting points were determined in capillaries and are uncor-
vent was distilled off and the residue was column
chromatographed using ethyl acetate and hexane as eluents to iso-
late pure compound. Spectral data for compound 2 has already
1
13
rected. H and
00 MHz and 75 MHz NMR spectrometer, respectively, using CDCl
as solvent. Chemical shifts are given in ppm with TMS as an
C NMR spectra were run on JEOL JNM AL
3
3
3
1
been reported.

3
978
P. Singh et al. / Bioorg. Med. Chem. 17 (2009) 3973–3979
ꢂ1
4
.3.1. 10-Oxiranylmethyl-9-oxo-9,10-dihydro-acridine-4-
110 °C; IR (KBr, cm ): 1610 (C@O), 1680 (C@O), 3200 (OH), 3280
1
carboxylic acid (8)
Compound 8 was synthesized using compound 7 according to
the synthetic procedure B as a yellow solid in a yield of 65%. Mp
(OH); H NMR (300 MHz, CDCl
2.68–2.75 (m, 2H, C13
), 3.73–3.77 (m, 4H, 2 ꢀ OCH
1H, C12H), 4.36–4.42 (dd, 1H, J = 12.0 Hz, J = 6.0 Hz, C11H), 4.51–
3
) d 2.48–2.57 (m, 4H, 2 ꢀ CH
2
),
H
2
2
), 4.19 (m,
2
3
ꢂ1
1
2
3
1
05 °C; IR (KBr, cm ): 1605 (C@O), 1680 (C@O), 3400 (OH);
H
4.56 (dd, 1H, J = 12.0 Hz, J = 3.0 Hz, C11H), 7.23–7.41 (m, 3H,
2
NMR (300 MHz, CDCl
3
)
d
2.78–2.80 (dd, 1H, J = 4.8 Hz,
ArH), 7.65–7.71 (m, 1H, ArH), 8.43–8.47 (m, 2H, ArH), 8.71–8.74
3
2
3
2
3
13
J = 2.7 Hz, H
b
), 2.95–2.97 (dd, 1H, J = 4.8 Hz, J = 3.9 Hz, H
a
),
(dd, J = 8.1 Hz, 1H, J = 1.5 Hz, ArH), 11.63 (b, 1H, COOH); C (nor-
mal/DEPT-135) d 53.62 (ꢂve, CH ), 60.65 (ꢂve, CH ), 64.77 (+ve,
), 117.49 (+ve, ArCH), 119.83
2
3
3
H
.39–3.42 (m, 1H, H
c
), 4.25–4.31 (dd, 1H, J = 12.3 Hz, J = 6.3 Hz,
2
2
2
3
e
), 4.72–4.77 (dd, 1H, J = 12.3 Hz, J = 3.0 Hz, H
d
), 7.23–7.39 (m,
CH), 66.83 (ꢂve, CH
2
), 67.15 (ꢂve, CH
2
3
(
H, ArH), 7.65–7.70 (m, 1H, ArH), 8.41–8.71 (m, 2H, ArH), 8.74
(+ve, ArCH), 122.37 (+ve, ArCH), 126.92 (+ve, ArCH), 133.94 (+ve,
d, 1H, J = 1.8 Hz, ArH), 11.59 (b, 1H, COOH); 13C (normal/DEPT-
ArCH), 136.65 (+ve, ArCH), 167.58 (C@O), 177.84 (C@O); MS (FAB)
+
1
1
1
1
35) d 42.61 (ꢂve, CH
2
), 44.64 (ꢂve, CH
2
), 49.26 (+ve, CH),
m/z 383 (M +1). Anal. Calcd for C21
H
22
N
2
O
5
: C, 65.96; H, 5.80; N,
13.02 (+ve, ArCH), 119.88 (+ve, ArCH), 122.39 (+ve, ArCH),
26.95 (+ve, ArCH), 136.75 (+ve, ArCH), 139.87 (+ve, ArCH),
7.33. Found: C, 65.98; H, 5.84; N, 7.37.
+
0
67.37 (C@O), 178.15 (C@O), MS (FAB) 296 (M +1). Anal. Calcd
4.4.4. 10-(3-[1,4]Bipiperidinyl-1 -yl-2-hydroxypropyl)-9-oxo-
9,10-dihydroacridine-4-carboxylic acid (12)
4
for C17H13NO : C, 69.15; H, 4.44; N, 4.74. Found: C, 69.18; H,
4
.48; N, 4.79.
Compound 12 was synthesized using compound 8 according to
the synthetic procedure C as a light yellow solid in a yield of 78%.
ꢂ1
4
1
.4. General procedure for the syntheses of compounds 3–6, 9–
2 (procedure C)
Mp 100 °C; IR (KBr, cm ): 1604 (C@O), 1670 (C@O), 3240 (OH),
1
3355 (OH); H NMR (300 MHz, CDCl
3
) d 1.43–1.45 (m, 2H, CH
), 1.83–1.87 (m, 2H, CH ), 1.98–2.06
(m, 1H, CH), 2.23–2.38 (m, 2H, CH
), 2.45–2.52 (m, 6H, 3 ꢀ CH
2.89 (m, 1H, C13H), 3.07 (m, 1H, C13H), 4.11–4.13 (m, 1H, C12H),
2
),
1
.53–1.65 (m, 6H, 3 ꢀ CH
2
2
A mixture of acridone 2/8 (1 mmol) and appropriate amine
2
2
)
(
1 mmol) was irradiated in microwave oven for 5–7 min. On com-
2
3
pletion of the reaction (TLC), it was washed with diethyl ether to
isolate pure product. Spectral data for compounds 3–6 has already
been reported.
4.32–4.38 (dd, 1H, J = 11.4 Hz, J = 6.0 Hz, C11H), 4.48–4.53 (dd,
1H, J = 9.0 Hz, J = 3.0 Hz, C11H), 7.22–7.40 (m, 3H, ArH), 7.65–
2
3
3
1
7.70 (m, 1H, ArH), 8.41–8.46 (m, 2H, ArH), 8.70–8.73 (d, 1H,
1
3
J = 8.1 Hz, ArH), 11.63 (b, 1H, COOH); C NMR (normal/DEPT-
135) d 24.50 (ꢂve, CH ), 25.86 (ꢂve, CH ), 27.82 (ꢂve, CH ),
50.28 (ꢂve, CH ), 51.84 (ꢂve, CH ), 55.36 (ꢂve, CH ), 59.75 (ꢂve,
CH ), 62.39 (+ve, CH), 65.03 (+ve, CH), 117.47 (+ve, ArCH), 119.91
4
.4.1. 10-(2-Hydroxy-3-pyrrolidin-1-yl-propyl)-9-oxo-9,10-
2
2
2
dihydroacridine-4-carboxylic acid (9)
2
2
2
Compound 9 was synthesized using compound 8 according to
2
the synthetic procedure C as a yellow solid in a yield of 63%. Mp
(+ve, ArCH), 122.36 (+ve, ArCH), 126.84 (+ve, ArCH), 133.88 (+ve,
ꢂ1
6
5 °C; IR (KBr, cm ): 1620 (C@O), 1680 (C@O), 3210 (OH), 3320
ArCH), 136.81 (+ve, ArCH), 167.00 (C@O), 178.00 (C@O); MS
1
+
(
OH); H NMR (300 MHz, CDCl
3
) d 1.60–1.69 (m, 4H, 2 ꢀ CH
2
),
(FAB) m/z 464 (M +1). Anal. Calcd for C27
H
33
N
3
O
4
: C, 69.95; H,
2
.35–2.41 (m, 2H, NCH
2
), 3.74–3.78 (m, 4H, 2 ꢀ NCH ), 4.15–4.23
2
7.18; N, 9.06. Found: C, 69.98; H, 7.120; N, 9.10.
2
3
(
m, 1H, C12H), 4.36–4.41 (dd, 1H, J = 12.0 Hz, J = 6.0 Hz, C11H),
2
3
4
3
8
.51–4.56 (dd, 1H, J = 12.0 Hz, J = 3.0 Hz, C11H), 7.21–7.39 (m,
Acknowledgements
H, ArH), 7.65–7.70 (m, 1H, ArH), 8.42–8.45 (m, 2H, ArH), 8.69–
2
3
.73 (dd, 1H, J = 7.8 Hz, J = 1.8 Hz, ArH), 11.61 (b, 1H, COOH);
1
3
DST, New Delhi is gratefully acknowledged for financial assis-
tance. J.K. thanks UGC for fellowship. CDRI, Lucknow is acknowl-
edged for recording mass spectra and elemental analysis.
C (normal/DEPT-135) d 25.32 (ꢂve, CH
2
), 53.22 (ꢂve, CH
2
),
6
4.76 (+ve, CH), 66.86 (ꢂve, CH ), 67.13 (ꢂve, CH
2
2
), 117.49 (+ve,
ArCH), 119.84 (+ve, ArCH), 122.37 (+ve, ArCH), 126.93 (+ve, ArCH),
33.94 (+ve, ArCH), 136.66 (+ve, ArCH), 167.15 (C@O), 177.15
1
+
References and notes
(
22 2 4
C@O); MS (FAB) m/z 367 (M +1). Anal. Calcd for C21H N O : C,
6
8.84; H, 6.05; N, 7.65. Found: C, 68.89; H, 6.10; N, 7.69.
1
2
3
4
5
.
.
.
.
.
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4
.4.2. 10-(2-Hydroxy-3-piperidin-1-yl-propyl)-9-oxo-9,10-
dihydroacridine-4-carboxylic acid (10)
Compound 10 was synthesized using compound 8 according to
6
.
the synthetic procedure C as a yellow solid in a yield of 74%. Mp
ꢂ1
1
3
1
15 °C; IR (KBr, cm ): 1610 (C@O), 1680 (C@O), 3240 (OH),
1
320 (OH); H NMR (300 MHz, CDCl
.95–2.08 (m, 4H, 2 ꢀ CH ), 2.96 (m, 2H, CH
), 3.83–3.98 (m, 2H, CH ), 4.48–4.82 (m, 3H, C11H
2
3
) d 1.25–1.50 (m, 2H, CH
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2
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2
2
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2
2
9
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7
1
2
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1
1
1
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3.14 (ꢂve, CH
2
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2
), 59.96 (ꢂve, CH ), 64.06 (ꢂve,
2
CH
2
), 66.50 (+ve, CH), 117.68 (+ve, ArCH), 120.41 (+ve, ArCH),
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1
1
3
7
22.95 (+ve, ArCH), 126.46 (+ve, ArCH), 133.89 (+ve, ArCH),
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+
81 (M +1). Anal. Calcd for C22
H
24
N
2
4
O : C, 69.46; H, 6.36; N,
1
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2
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4
.4.3. 10-(2-Hydroxy-3-morpholin-4-yl-propyl)-9-oxo-9,10-
1
1
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