Structure-activity relationship studies of acridones as potential antipsoriatic agents. 2. Synthesis and antiproliferative activity of 10-substituted hydroxy-10H-acridin-9-ones against human keratinocyte growth

Article abstract of DOI:10.1016/j.ejmech.2010.08.059

A series of 10-substituted hydroxy-10H-acridin-9-ones were synthesized and studied as potential antipsoriatic agents. The antiproliferative activity of the novel derivatives, which can be considered as aza-analogues of the antipsoriatic drug anthralin, wa

Full text of DOI:10.1016/j.ejmech.2010.08.059

European Journal of Medicinal Chemistry 45 (2010) 5345e5352
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structureeactivity relationship studies of acridones as potential antipsoriatic
agents. 2. Synthesis and antiproliferative activity of 10-substituted
hydroxy-10H-acridin-9-ones against human keratinocyte growth
*
Aleksandar Putic, Lambert Stecher, Helge Prinz, Klaus Müller
Institute of Pharmaceutical and Medicinal Chemistry, Westphalian Wilhelms-University, Hittorfstraße 58e62, D-48149 Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 10-substituted hydroxy-10H-acridin-9-ones were synthesized and studied as potential anti-
psoriatic agents. The antiproliferative activity of the novel derivatives, which can be considered as aza-
analogues of the antipsoriatic drug anthralin, was determined using the human keratinocyte cell line
HaCaT. Structureeactivity relationships with respect to the nature of the N-substituent at the acridone
scaffold were delineated. Release of lactate dehydrogenase (LDH) was used to exclude non-specific
cytotoxic effects. As compared to anthralin, N-substitution of the acridone scaffold in the target
compounds provided agents devoid of radical producing properties, which was documented by their
ineffectiveness to interact with the free radical 2,2-diphenyl-1-picrylhydrazyl. This was in excellent
agreement with the data obtained from the LDH assay in which the novel compounds did not induce
membrane damage. Benzyl substitution at the 10-position yielded keratinocyte growth inhibitory
activity in the low micromolar range. The most potent inhibitor of keratinocyte hyperproliferation was
compound 8a having an N-methyl group and a 1,3-dihydroxy arrangement at the acridone scaffold, with
an IC50 value comparable to that of anthralin.
Received 26 April 2010
Received in revised form
25 August 2010
Accepted 26 August 2010
Available online 20 September 2010
Keywords:
Acridone derivatives
Antiproliferative activity
Antipsoriatic
HaCaT cells
Radical
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
with their capability to generate oxygen radicals [15,16], and the
methylene moietyat C-10 has been recognized as a keysite of radical
The acridone alkaloids constitute a small group of naturally
occurring compounds produced exclusively by higher plants
belonging to the order Rutales [1]. Because of their demonstrated
antiproliferative activity, interest continues unabated in the
synthesis of related synthetic analogues of the tetracyclic acridone
alkaloid acronycine (1, Chart 1) [2e6] and 10H-acridin-9-ones pos-
sessing antitumor or other interesting biological activities [7e12].
Acridones can be considered as 10-aza-analogues of the anthrone
class of topical agents against psoriasis, an inflammatory and scaling
skin disease, which is mainly characterized by hyperproliferation of
keratinocytes [13]. Owing to the proinflammatory and staining
effects of antipsoriatic anthrones such as anthralin (dithranol, 1,8-
dihydroxy-10H-anthracen-9-one, 2), this drug is best used in
hospital or day treatment centers [14], and there is need for
analogues with improved therapeutic properties. Several studies
have suggested that the side effects of the anthrones are associated
formation [17,18]. Indeed, appropriate chemical modification of the
critical C-10 of the anthrone pharmacophore provided agents with
diminished oxygen radical formation [19e22].
In this direction, our interest has focused on the synthesis of 10-
aza-analogues of anthrones in order to identify improved anti-
psoriatic agents that act via the keratinocyte as the primary target
cell [23]. The antiproliferative activity of anthralin is intimately
effected by the nature and position of the substituents at the
anthrone nucleus. Both hydroxy groups peri to the keto group are
required for high activity [24]. In our recent study [23] we have
shown that structureeactivity relationships (SAR) for N-unsub-
stituted acridone derivatives did not follow those of the anti-
psoriatic anthrones. Surprisingly, the aza-analogue of anthralin,
1,8-dihydroxy-10H-acridin-9-one (3), was only marginally active.
However, acridone
4 comprising a 1,3-dihydroxy-substitution
pattern was the most active analogue, with keratinocyte growth
inhibitory potency comparable to anthralin. In this communication,
we expand on this previous report and discuss SAR for modification
of the substituent at the acridone nitrogen atom.
We have tested the novel 10-substituted acridones as inhibitors
of keratinocyte hyperproliferation, which is one of the hallmarks of
Abbreviations: DPPH, 2,2-diphenyl-1-picrylhydrazyl; LDH, lactate dehydro-
genase; RT, room temperature; SAR, structureeactivity relationships.
* Corresponding author. Tel.: þ49 251 833 3324; fax: þ49 251 833 2144.
E-mail address: kmuller@uni-muenster.de (K. Müller).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.059

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A. Putic et al. / European Journal of Medicinal Chemistry 45 (2010) 5345e5352
performed in the presence of sodium hydride as described by Lewis
[25]. However, in both cases only the acetoacetyl-substituted
acridones 11 and 12, respectively, were obtained (Scheme 4).
3. Biological assay methods
In the present study, we used HaCaT keratinocytes [26] as
a model for the highly proliferative epidermis in psoriasis. This
rapidly dividing, non-transformed human cell line is a useful tool in
identifying new topical antipsoriatic agents, and it was described as
an extremely sensitive target for the antiproliferative action of the
antipsoriatic drug anthralin [27]. Accordingly, we evaluated the
effects of the novel acridones on the growth of cultured HaCaTcells,
which was determined directly by counting the dispersed cells
under a phase-contrast microscope after 48 h of treatment.
Keratinocytes were also tested for their susceptibility to the
action of the most potent members of this 10-substituted acridone
series on plasma-membrane integrity, to confirm that inhibition of
keratinocyte growth was not a result of membrane damage. This
was assessed by the activity of LDH released into the culture
medium [19,27].
Chart 1. Structures of acronycine (1), anthralin (2), aza-analogous anthralin (3), and
acridone derivative 4.
psoriasis. We also evaluated their capability to interact with a stable
free radical by use of 2,2-diphenyl-1-picrylhydrazyl (DPPH).
Furthermore, release of lactate dehydrogenase (LDH) was
measured to exclude cytotoxic effects mediated by membrane
damage.
Furthermore, free radical production by anthralin and the
resulting dimerization and polymerization of the molecule is
associated with the instability of the drug as well as with undesired
side effects such as staining and irritation of the skin [16]. In order
to compare the capability of the acridones to produce radicals by
loss of a hydrogen atom with that of anthralin, we determined the
extent of the N-substituted acridones to scavenge radical inter-
mediates by measuring spectrophotometrically the decrease of
DPPH concentration [28] at 520 nm. When DPPH reacts with an
electron-donating test compound (i.e., a hydrogen atom donor), it
is reduced to the corresponding hydrazine, whereas the primary
species derived from the test compound is a free radical.
2. Chemistry
All target compounds were easily prepared from N-unsub-
stituted acridones. Thus, 1-hydroxyacridone (5) was N-alkylated
with an appropriate alkyl halogenide and potassium carbonate
according to Scheme 1. The preparation of 6a and 6d was accom-
panied by side products, O-alkylated 6aa and 6dd, respectively.
Using benzyl bromide as an alkylating agent, also 2-benzylated
6ddd was obtained by aromatic electrophilic substitution. The
structure was confirmed by NOE experiments. On irradiation at the
frequency of the NCH2 protons (5.74 ppm), a positive NOE was
observed for the signals at 6.96 (H-4) and 7.64 ppm (H-5), whereas
on irradiation at the frequency of the ArCH2 protons (3.94 ppm),
a positive NOE was observed for the signal at 7.51 (H-3) and also
14.91 ppm (OH) was detectable.
4. Results and discussion
4.1. Inhibition of keratinocyte growth
In the case of 1,3-dihydroxyacridone (7), also the non-hydrogen-
bonded hydroxy group in 3-position was alkylated to give acri-
dones 8aae8ee, which were then smoothly converted into the final
1,3-dihydroxyacridones 8ae8e by ether cleavage with 48% hydro-
bromic acid (Scheme 2). Acridones 8aa, 8bb and 8dd were also
isolated and purified. Acridones 9be9e were prepared by reaction
of unsubstituted acridone (9a) with the approriate alkyl halogen-
ides (Scheme 3). Finally, acetylation of 9a or 1-hydroxylated 5 was
In the antiproliferative study, each acridone derivative was
tested for inhibitory action against HaCaT keratinocytes, as
demonstrated by reduction in cell number over time as compared
to control plates. The concentrations required to inhibit 50% of
keratinocyte growth are shown in Table 1.
From the present work, 1,3-dihydroxy-acridone 8a with an
N-methyl substituent was identified as a highly potent inhibitor of
keratinocyte proliferation, with an IC50 value (0.6
mM) in the
Scheme 1. Reagents: (a) alkyl halogenide, K2CO3, acetone, RT or reflux; (b) 48% HBr, reflux.

A. Putic et al. / European Journal of Medicinal Chemistry 45 (2010) 5345e5352
5347
Scheme 4. Reagents: (a) NaH, THF, AcCl, RT, N2.
There were four sets of compounds where the effect of
N-methylation of the acridone scaffold could be evaluated, by
comparing the corresponding NH and N-methyl derivatives. In two
cases (i.e., 7, 8a and 9a, 9b), the N-methyl analogues were the more
potent inhibitors of keratinocyte growth. In particular compound
8a showed a 33-fold improvement over N-unsubstituted 7. By
contrast, the most potent inhibitor of our previous report, NH
analogue 4, completely lost its activity upon methylation (i.e., 10).
One pair of compounds (5, 6a) could not be compared, as both were
inactive.
Scheme 2. Reagents: (a) alkyl halogenide, K2CO3, acetone, RT or reflux; (b) 48% HBr,
reflux.
submicromolar range and comparable to that of standard anthralin.
The naturally occurring alkaloid acronycine, which is also an
N-methylated acridone and has shown activity in a large number of
experimental tumor models [29], also displayed activity in the
keratinocyte assay, with an IC50 value in the low micromolar range.
By contrast, unsubstituted acridone (9a) was not appreciably active,
whereas the corresponding N-methylated 9b displayed moderate
activity. However, larger N-substituents as compared to 9b, as in
9ce9e, were detrimental for activity. In terms of SAR information,
N-methylated acridone 9b without hydroxy-substituent was
substantially less potent than that hydroxylated at the 1,3-positions
(8a), while compound 6a with only one hydroxy-substituent at the
1-position was virtually inactive. As compared to the N-unsub-
stituted analogue 7, potency of 8a was dramatically improved by
the 10-methyl substituent affording the most potent compound of
all N-substituted acridones in this series. However, alkylation of the
3-hydroxy group (8aa, 8bb, 8dd) or only slight enlargements of the
aliphatic 10-substituent as in N-ethylated 8b or in N-propylated 8c
resulted in total loss of activity. In contrast to simple aliphatic
substitution at the 10-position, compounds with an aliphatic chain
terminated with a phenyl ring, in particular a benzyl substituent,
were substantially more potent than their N-unsubstituted
analogues. In the 1-hydroxy-substituted series (6ae6l), the ben-
zylated analogues 6d and 6e as well as the methoxy-substituted
analogues 6h and 6i, with an additional spacer group in the linker
between the acridone scaffold and the phenyl ring, all displayed
IC50 values in the low micromolar range. On the other hand, further
elongation of the linker chain as in 6k and 6l with three and four
spacer groups, respectively, produced inactive compounds. In
the 1,3-dihydroxy series (8ae8e), N-benzylated 8d shared
a comparable potency with the N-benzylated 6d and 6e. Of interest,
also N-acylation of acridones 5 and 9a gave active analogues (i.e., 11
and 12), with analogue 12 being active in the low micromolar
range.
4.2. Membrane damage
The release of LDH is commonly used as an indicator of plasma-
membrane damage. In this assay, LDH release by the standard
anthralin, which was used as a positive control, significantly
exceeded that of the vehicle control. On the other hand, the kera-
tinocyte growth inhibitory activity of the naturally occurring
acronycine and the most potent acridone derivatives was due to
antiproliferative rather than non-specific cytotoxic effects, as LDH
release was unchanged as compared to controls at 2-
mM concen-
tration of the test compounds (Table 1). The only exception was 6e,
which caused a slight increase in LDH activity as compared to
vehicle controls. This may be related to its aromatic nitro group,
even though direct radical production was excluded by the DPPH
assay. However, enzymatic reduction of nitro aromatic compounds
gives rise to nitro radical anions that may undergo redox cycling
with oxygen to generate oxygen radicals [30]. Resultant oxidative
stress can produce membrane damage.
4.3. Interaction with the stable free radical DPPH
Although scarcely described in the literature and only after
photoirradiation and laser flash excitation of acridone, N-radical
formation by hydrogen transfer to pyridine has been observed
[31,32]. However, as N-substitution would prevent hydrogen
transfer to a stable free radical such as DPPH, it is not surprising that
with EC50 values greater than 500 mM, none of the 10-substituted
acridones showed any appreciable reactivity towards DPPH. These
results were in excellent agreement with our data obtained from
the LDH assay in which these novel compounds were devoid of
membrane-damaging properties. As a matter of fact, this is bene-
ficial as compared to anthralin, which was very effective in
donating an electron to the stable free radical DPPH and, as a result,
also caused desintegrity of keratinocyte membranes. The fact that
the ability of the 10-substituted acridones to arrest keratinocyte
growth was not mediated by non-specific redox properties or
cytotoxic effects on the plasma-membrane, as observed for the
antipsoriatic agent anthralin [15], opens new possibilities in the
search for antipsoriatic agents.
Scheme 3. Reagents: (a) alkyl halogenide, K2CO3, acetone, reflux.

5348
A. Putic et al. / European Journal of Medicinal Chemistry 45 (2010) 5345e5352
Table 1
Antiproliferative activity against HaCaT keratinocytes, lactate dehydrogenase release, and free radical interacting capability of 10-substituted 10H-acridin-9-ones.
O
R1
O
1
8
5
7
6
9
2
3
10
N
R2
N
4
H
R3
cpd
Substitution pattern
AAa IC50
(
m
M)
LDHb (mU/mL)
70.4
DPPHd EC50
(mM)
1
Acronycine
Anthralin
1,8-(OH)2
1,3-(OH)28eMe
3.2
0.7
39.5
0.8
>50
>50
>50
>50
>50
2.5
7.5
13.0
1.8
41.5
20.1
6.3
>500
16.7
>500
>500
2
122.8c
3f
e
4f
71.5
5f,g
6a
6aa
6b
6c
R1 ¼ OH, R2 ¼ H, R3 ¼ H
R1 ¼ OH, R2 ¼ H, R3 ¼ Me
R1 ¼ OMe, R2 ¼ H, R3 ¼ Me
R1 ¼ OH, R2 ¼ H, R3 ¼ Et
R1 ¼ OH, R2 ¼ H, R3 ¼ CH3(CH2)2
R1 ¼ OH, R2 ¼ H, R3 ¼ PhCH2
R1 ¼ OCH2Ph, R2 ¼ H, R3 ¼ PhCH2
1-OH-2,10-(PhCH2)2
>500
e
e
e
e
e
e
e
e
e
6d
6dd
6ddd
6e
74.9
>500
72.1
>500
e
e
R1 ¼ OH, R2 ¼ H, R3 ¼ 4-NO2ePhCH2
R1 ¼ OH, R2 ¼ H, R3 ¼ Ph(CH2)2
R1 ¼ OH, R2 ¼ H, R3 ¼ 4-OHePh(CH2)2
R1 ¼ OH, R2 ¼ H, R3 ¼ 4-OMeePh(CH2)2
86.7c
>500
e
e
6f
e
6g
6h
6i
6j
6k
6l
>500
>500
74.6
R1 ¼ OH, R2 ¼ H, R3 ¼ 3,4-(OMe)2ePh(CH2)2
R1 ¼ OH, R2 ¼ H, R3 ¼ 3,4,5-(OMe)3ePh(CH2)2
R1 ¼ OH, R2 ¼ H, R3 ¼ Ph(CH2)3
R1 ¼ OH, R2 ¼ H, R3 ¼ Ph(CH2)4
R1 ¼ OH, R2 ¼ OH, R3 ¼ H
7.0
>500
e
e
e
e
e
e
45.5
>50
>50
19.8
0.6
>50
>50
>50
>50
3.7
>50
40.5
>50
38.9
>50
46.3
>50
>50
27.8
3.3
e
e
7f,h
8a
8aa
8b
8bb
8c
8d
8dd
8e
9a
9bi
9ci
9dk
9e
>500
R1 ¼ OH, R2 ¼ OH, R3 ¼ Me
74.3
>500
R1 ¼ OH, R2 ¼ OMe, R3 ¼ Me
R1 ¼ OH, R2 ¼ OH, R3 ¼ Et
e
e
e
e
e
e
e
e
R1 ¼ OH, R2 ¼ OEt, R3 ¼ Et
R1 ¼ OH, R2 ¼ OH, R3 ¼ CH3(CH2)2
R1 ¼ OH, R2 ¼ OH, R3 ¼ PhCH2
R1 ¼ OH, R2 ¼ OPhCH2, R3 ¼ PhCH2
R1 ¼ OH, R2 ¼ OH, R3 ¼ Ph(CH2)3
R1 ¼ R2 ¼ R3 ¼ H
76.8
>500
e
e
e
e
e
e
e
e
e
e
e
e
e
e
R1 ¼ R2 ¼ H, R3 ¼ Me
R1 ¼ R2 ¼ H, R3 ¼ PhCH2
R1 ¼ R2 ¼ H, R3 ¼ 4-OMeePhCH2
R1 ¼ R2 ¼ H, R3 ¼ 3,4-(OMe)2ePhCH2
1,3-(OH)2-8,10-Me2
>500
>500
e
10
11
12
R1 ¼ R2 ¼ H, R3 ¼ COCH2COMe
e
R1 ¼ OH, R2 ¼ H, R3 ¼ COCH2COMe
74.0
>500
a
Antiproliferative activity against keratinocytes. IC50, concentration of test compound required for 50% inhibition of cell growth (HaCaT). Inhibition of cell growth was
significantly different with respect to that of the control, N ¼ 3, P < 0.05.
b
Activity of LDH (mU) release in HaCaT cells after treatment with 2
m
M test compound (N ¼ 3, SD < 10%).
c
Values are significantly different with respect to vehicle control (0.2% DMSO in the culture medium, 70.6 mU/mL), P < 0.05. Brij 35 (polyoxyethyleneglycol dodecyl ether)/
ultrasound was the positive control (251 mU/mL).
d
DPPH radical interacting capability. EC50, effective concentration of test compound (N ¼ 3, SD < 10%) required for 50% decrease in DPPH absorbance at 520 nm. NDGA
(IC50 ¼ 4.2
m
M) and
a
-tocopherol (IC50 ¼ 8.2
mM) were used as positive controls.
e
Not determined.
Ref. [23].
Ref. [36].
Ref. [33].
Ref. [34].
f
g
h
i
k
Ref. [8].
5. Conclusions
IC50 value of 0.6 mM, potency of the anthrone derivative anthralin was
retained. As already seen with the most potent analogue of our
previous series, acridone 4, which is an isomer of 8a, this N-methyl-
ated acridone was not capable of interacting with DPPH and thus
producing radicals that give rise to a non-specific cytotoxic action,
which would preclude a possible use in dermatology. This was in
excellent agreement with the fact that in contrast to anthralin, the
most potent acridones of this series did not disrupt membrane
integrity. Also of interest, acylation of the acridone nitrogen atom
resulted in active compounds such as 12, whose potency may further
be improved with an appropriate substitution pattern. In conclusion,
The aim of this study was to elucidate the SAR of 10-substituted
acridone analogues related to the antipsoriatic agent anthralin, to
further delineate the number and position of hydroxy and other
groups at the acridone scaffold that contribute to the antiproliferative
activity. AsalreadyobservedinourpreviousseriesofN-unsubstituted
acridones [23], a 1,3-dihydroxy-substitution pattern was most bene-
ficial for potency. One member of this series, acridone 8a, with an
N-methyl substituent at the 1,3-dihydroxy-substituted skeleton, was
detected as a highly potent inhibitor of keratinocyte growth. With an

A. Putic et al. / European Journal of Medicinal Chemistry 45 (2010) 5345e5352
5349
among our series of acridone analogues we were able to identify
potent inhibitors of human keratinocyte growth at sufficiently low
concentrations to warrant investigation as antipsoriatic agents in
more sophisticated systems.
6.1.1.4. 10-Benzyl-1-benzyloxy-10H-acridin-9-one (6dd). The title
compound was obtained as a side product of 6d. Purification by
chromatography (SiO2; CH2Cl2) gave gold-colored crystals; 5%
yield; mp 179 ꢁC; 1H NMR (DMSO-d6)
d
8.32 (d, J ¼ 6.7 Hz, 1H, H-8),
7.73e7.13 (m, 14H, Ar), 7.05 (d, J ¼ 8.7 Hz, 1H, H-4), 6.91 (d,
J ¼ 8.1 Hz, 1H, H-2), 5.68 (s, 2H, OCH2), 5.26 (s, 2H, NCH2); FTIR
1632 cmꢂ1 (CO); MS m/z ¼ 391 (100, Mþ). Anal. C27H21NO2 (C, H, N).
6. Experimental section
6.1. Chemistry
6.1.1.5. 2,10-Dibenzyl-1-hydroxy-10H-acridin-9-one
(6ddd). The
title compound was obtained as a side product of 6d, when the
mixture was refluxed for 50 h. Purification by chromatography
(SiO2; CH2Cl2) gave yellow crystals; 13% yield; mp 191 ꢁC; 1H NMR
Melting points were determined with a Kofler melting point
apparatus and are uncorrected. 1H NMR and 13C NMR spectra were
recorded with a Varian Gemini 200 (200 and 50.29 MHz, respec-
tively) spectrometer, using tetramethylsilane as an internal stan-
dard. Fourier-transform IR spectra (KBr) were recorded on a Bio-Rad
laboratories type FTS 135 spectrometer. Mass spectra were obtained
in the EI mode using a MAT GCQ Finnigan instrument. Thin layer
chromatography (TLC) was conducted on Merck 60 F254 precoated
silica gel plates. Chromatography refers to column chromatography,
which was performed on Merck silica gel (70e230 mesh). Yields
have not been optimized. Elemental analyses were determined by
the Microanalysis Laboratory at the University of Münster using
a Vario EL III elemental analyzer. Elemental analyses were within
ꢀ0.4% of calculated values, except where stated otherwise.
(DMSO-d6)
d
14.91 (s, 1H, OH), 8.39 (dd, J ¼ 8.1, 1.7 Hz, 1H, H-8),
7.83e7.10 (m, 14H, Ar), 6.96 (d, J ¼ 8.9 Hz, 1H, H-4), 5.74 (s, 2H,
NCH2Ar), 3.94 (s, 2H, ArCH2Ar); FTIR 1622 cmꢂ1 (CO); MS m/z ¼ 391
(100, Mþ). Anal. C27H21NO2 (C, H, N).
6.1.1.6. 1-Hydroxy-10-(4-nitrobenzyl)-10H-acridin-9-one (6e). The
title compound was obtained from 5 and 4-nitrobenzyl bromide
using the same procedure as for 6b, but the mixture was refluxed
for 9 h. Purification by chromatography (SiO2; CH2Cl2) gave yellow
crystals; 46% yield; mp 282 ꢁC; 1H NMR (DMSO-d6)
d 14.59 (s, 1H,
OH), 8.40 (dd, J ¼ 1.4, 8.1 Hz,1H, H-8), 8.18 (d, J ¼ 8.5 Hz, 2H, H-30, H-
50), 7.82 (t, J ¼ 7.3 Hz, 1H, H-6), 7.66e7.39 (m, 3H, H-3, H-5, H-7),
7.41 (d, J ¼ 8.5 Hz, 2H, H-20, H-60), 6.96 (d, J ¼ 8.7 Hz, 1H, H-4), 6.68
(d, J ¼ 8.0 Hz, 1H, H-2), 5.94 (s, 2H, CH2); FTIR 1638 cmꢂ1 (CO); MS
m/z ¼ 346 (100, Mþ). Anal. C20H14N2O4 (C, H, N).
Acridones 6a, 6aa [25], 8a, 8aa [33], 9b, 9c [34] 9d [8], and 10
[35] were prepared according to literature methods. Acridone 9a
was commercially available (SigmaeAldrich).
6.1.1. General procedure for alkylation of hydroxyacridones
6.1.1.7. 1-Hydroxy-10-(2-phenylethyl)-10H-acridin-9-one (6f). The
title compound was obtained from 5 and 2-bromoethylbenzene
using the same procedure as for 6b, but the mixture was refluxed
for 24 h. Purification by chromatography (SiO2; CH2Cl2/MeOH, 98/
2) gave yellow crystals; 12% yield; mp 162 ꢁC; 1H NMR (DMSO-d6)
6.1.1.1. 10-Ethyl-1-hydroxy-10H-acridin-9-one (6b). A mixture of
1-hydroxyacridone [36] (5, 0.20 g, 0.95 mmol), dry, finely mortared
K2CO3 (0.41 g, 3 mmol) and iodoethane (3.11 g, 19.95 mmol) in dry
acetone (30 mL) was stirred under reflux for 5 h. Then the mixture
was filtered by suction, the residue dissolved in CH2Cl2 and filtered.
The combined filtrate was evaporated and the residue was purified
by chromatography (SiO2; CH2Cl2/MeOH, 99/1) to provide yellow
d
14.59 (s, 1H, OH), 8.36 (d, J ¼ 7.9 Hz, 1H, H-8), 7.89e7.20 (m,
10H, Ar), 6.66 (d, J ¼ 7.8 Hz, 1H, H-2), 4.66 (t, J ¼ 8.0 Hz, 2H,
NCH2CH2), 3.10 (t, J ¼ 8.1 Hz, 2H, NCH2CH2); FTIR 1630 cmꢂ1 (CO);
MS m/z ¼ 315 (91, Mþ), 224 (100). Anal. C21H17NO2 (C, H, N).
crystals; 40% yield; mp 161 ꢁC; 1H NMR (DMSO-d6)
d 14.59 (s, 1H,
OH), 8.34 (d, J ¼ 7.7 Hz, 1H, H-8), 7.90e7.34 (m, 4H, H-3, H-5, H-6,
H-7), 7.19 (d, J ¼ 8.7 Hz, 1H, H-4), 6.64 (d, J ¼ 8.1 Hz, 1H, H-2), 4.53
(q, J ¼ 7.2 Hz, 2H, CH2CH3), 1.38 (t, J ¼ 7.1 Hz, 3H, CH2CH3); 13C NMR
6.1.1.8. 1-Hydroxy-10-[2-(4-hydroxyphenyl)ethyl]-10H-acridin-9-
one (6g). The title compound was obtained from 6h using the same
procedure as for 8b. Purification by chromatography (SiO2; CH2Cl2)
gave yellow crystals; 43% yield; mp 230 ꢁC; 1H NMR (DMSO-d6)
(DMSO-d6)
d 181.3, 162.8, 142.0, 141.1, 120.0, 109.2, 136.6, 135.2,
126.0, 121.7, 115.8, 106.7, 104.4, 41.1, 34.7; FTIR 1630 cmꢂ1 (CO); MS
m/z ¼ 239 (100, Mþ). Anal. C15H13NO2 (C, H, N).
d
14.59 (s, 1H), 9.28 (s, br, 1H), 8.35 (d, J ¼ 7.7 Hz, 1H), 7.89e6.64 (m,
10H), 4.58 (t, J ¼ 8.0 Hz, 2H), 2.98 (t, J ¼ 8.0 Hz, 2H); FTIR 3329,
1637 cmꢂ1 (CO); MS m/z ¼ 331 (48, Mþ), 212 (100). Anal. C21H17NO3
(C, H, N).
6.1.1.2. 1-Hydroxy-10-propyl-10H-acridin-9-one
(6c). The
title
compound was obtained from 5 and 1-iodopropane using the same
procedure as for 6b, but the mixture was refluxed for 24 h. Purifi-
cation by chromatography (SiO2; CH2Cl2) gave golden yellow
6.1.1.9. 1-Hydroxy-10-[2-(4-methoxyphenyl)ethyl]-10H-acridin-9-
one (6h). The title compound was obtained from 5 and 1-(2-bro-
moethyl)-4-methoxybenzene [37] using the same procedure as for
6b, but the mixture was refluxed for 24 h. Purification by chro-
matography (SiO2; twice: hexane, then CH2Cl2) gave yellow crys-
crystals; 63% yield; mp 92 ꢁC; 1H NMR (DMSO-d6)
d 14.59 (s, 1H),
8.32 (d, J ¼ 7.6 Hz, 1H), 7.87e7.32 (m, 4H), 7.15 (d, J ¼ 8.9 Hz, 1H),
6.62 (d, J ¼ 8.0 Hz, 1H), 4.37 (t, J ¼ 8.1 Hz, 2H), 1.83e1.72 (m, 2H),
1.05 (t, J ¼ 7.3 Hz, 3H); FTIR 1634 cmꢂ1 (CO); MS m/z ¼ 253 (100,
Mþ). Anal. C16H15NO2 (C, H, N).
tals; 24% yield; mp 126 ꢁC; 1H NMR (DMSO-d6)
d 14.59 (s, 1H), 8.35
(d, J ¼ 7.8 Hz, 1H), 7.88 (d, J ¼ 3.1 Hz, 1H), 7.71 (t, J ¼ 8.3 Hz, 1H),
7.39e7.33 (m, 2H), 7.35 (d, J ¼ 8.6 Hz, 2H), 7.21 (d, J ¼ 8.5 Hz, 1H),
6.88 (d, J ¼ 8.4 Hz, 2H), 6.66 (d, J ¼ 8.0 Hz, 1H), 4.60 (t, J ¼ 8.1 Hz,
6.1.1.3. 10-Benzyl-1-hydroxy-10H-acridin-9-one
(6d). The
title
compound was obtained from 5 and benzyl bromide using the
same procedure as for 6b, but the mixture was refluxed for 24 h.
Purification by chromatography (SiO2; CH2Cl2) gave gold-colored
2H), 3.72 (s, 3H), 3.03 (t, J ¼ 7.9 Hz, 2H); 13C NMR (DMSO-d6)
d 181.3,
162.8, 158.1, 142.1, 141.2, 136.6, 135.2, 130.0 (2C), 129.5, 125.9, 121.8,
120.0, 115.9, 113.9 (2C), 109.0, 106.9, 104.6, 55.0, 47.4, 31.6; FTIR
1633 cmꢂ1 (CO); MS m/z ¼ 345 (20, Mþ), 134 (100). Anal. C22H19NO3
(C, H, N).
crystals; 77% yield; mp 176 ꢁC; 1H NMR (DMSO-d6)
d 14.50 (s, 1H,
OH), 8.30 (d, J ¼ 7.3 Hz, 1H, H-8), 7.89e7.12 (m, 9H, Ar), 6.95 (d,
J ¼ 8.8 Hz, 1H, H-4), 6.65 (d, J ¼ 8.0 Hz, 1H, H-2), 5.79 (s, 2H, CH2);
13C NMR (DMSO-d6)
d 181.5, 162.7, 142.9, 142.0, 136.5, 135.8, 135.1,
128.7 (2C), 127.2, 125.8, 125.7 (2C), 122.0, 120.1, 116.1, 109.1, 107.1,
104.9, 49.6; FTIR 1630 cmꢂ1 (CO); MS m/z ¼ 301 (70, Mþ), 91 (100).
Anal. C20H15NO2 (C, H, N).
6.1.1.10. 1-Hydroxy-10-[2-(3,4-dimethoxyphenyl)ethyl]-10H-acridin-
9-one (6i). The title compound was obtained from 5 and 1-(2-bro-
moethyl)-3,4-dimethoxybenzene [38] using the same procedure as

5350
A. Putic et al. / European Journal of Medicinal Chemistry 45 (2010) 5345e5352
for 6b, but the mixture was refluxed for 24 h. Purification by chro-
matography (SiO2; twice: CH2Cl2, then CH2Cl2/MeOH, 98/2) gave
yellow-orange crystals; 28% yield; mp 125 ꢁC; 1H NMR (DMSO-d6)
bromide [40] using the same procedure as for 6b, but the mixture was
refluxed for 24 h. Purification by chromatography (SiO2; CH2Cl2) gave
orange crystals; 20% yield; mp 215e216 ꢁC;1H NMR (CDCl3)
d 8.60 (dd,
d
14.59 (s, 1H), 8.35 (d, J ¼ 7.7 Hz, 1H), 7.89e6.64 (m, 9H), 4.64 (t,
J ¼ 8.0 Hz, J ¼ 1.8 Hz, 2H, H-1, H-8), 7.76e7.54 (m, 2H, H-3, H-6),
7.46e7.16 (m, 4H, H-2, H-4, H-5, H-7), 7.02e6.74 (m, 2H, H-20, H-50),
6.46e6.28 (m,1H, H-60), 5.63 (s, 2H, NCH2), 4.07 (s, 3H, OCH3), 3.94 (s,
3H, OCH3); FTIR 3345, 1634 cmꢂ1 (CO); MS m/z ¼ 345 (38, Mþ), 151
(100). Anal. C22H19NO3 (C, H, N).
J ¼ 7.8 Hz, 2H), 3.74 (s, 3H), 3.70 (s, 3H), 3.02 (t, J ¼ 7.9 Hz, 2H); FTIR
1635 cmꢂ1 (CO); MS m/z ¼ 375 (17, Mþ), 164 (100). Anal. C23H21NO4
(C, H, N).
6.1.1.11. 1-Hydroxy-10-[2-(3,4,5-trimethoxyphenyl)ethyl]-10H-acri-
din-9-one (6j). The title compound was obtained from 5 and 1-(2-
bromoethyl)-3,4,5-trimethoxybenzene [38] using the same proce-
dure as for 6b, but the mixture was refluxed for 24 h. Purification by
chromatography (SiO2; twice: CH2Cl2, then CH2Cl2/MeOH, 98/2)
gave yellow-orange crystals; 5% yield; mp 156 ꢁC; 1H NMR (DMSO-
6.1.2. General procedure for the cleavage of ethers
6.1.2.1. 10-Ethyl-1,3-dihydroxy-10H-acridin-9-one (8b). A solution
of 8bb (0.62 g, 2.20 mmol) in 48% HBr (30 mL) was refluxed for 3 h.
Then the solution was allowed to cool to RT. The precipitating
crystals were filtered by suction, washed with water (2 ꢃ 100 mL),
dissolved in CH2Cl2 (100 mL), and the aqueous phase was extracted
with CH2Cl2 (2 ꢃ 100 mL). The combined organic phase was dried
over Na2SO4, evaporated, and the residue purified by chromatog-
raphy (SiO2; CH2Cl2/MeOH, 98/2) to provide yellow-green crystals;
d6)
d
14.60 (s, 1H), 8.35 (d, J ¼ 7.9 Hz, 1H), 7.88e6.61 (m, 8H), 4.71 (t,
J ¼ 7.5 Hz, 2H), 3.72 (s, 6H), 3.58 (s, 3H), 3.02 (t, J ¼ 7.6 Hz, 2H); FTIR
1630 cmꢂ1 (CO); MS m/z ¼ 405 (19, Mþ), 194 (100). Anal. C24H23NO5
(C, H, N).
94% yield; mp 293 ꢁC; 1H NMR (DMSO-d6)
d 14.90 (s, 1H, 1-OH),
6.1.1.12. 1-Hydroxy-10-(3-phenylpropyl)-10H-acridin-9-one
10.65 (s, 1H, 3-OH), 8.28 (d, J ¼ 7.4 Hz, 1H, H-8), 7.85e7.19 (m, 3H,
H-5, H-6, H-7), 6.43 (d, J ¼ 1.7 Hz, 1H, H-4), 6.11 (d, J ¼ 1.7 Hz, 1H,
H-2), 4.37 (q, J ¼ 6.9 Hz, 2H, CH2CH3), 1.36 (t, J ¼ 7.0 Hz, 3H,
(6k). The title compound was obtained from 5 and 3-bromopro-
pylbenzene using the same procedure as for 6b, but the mixture
was refluxed for 24 h. Purification by chromatography (SiO2;
CH2Cl2) gave yellow crystals; 55% yield; mp 120 ꢁC; 1H NMR
CH2CH3); 13C NMR (DMSO-d6)
d 179.9, 165.0, 164.9, 143.6, 140.9,
120.0, 103.7, 134.4, 125.8, 121.3, 115.3, 95.9, 90.8, 41.0, 11.9; FTIR
(DMSO-d6)
d
14.56 (s, 1H, OH), 8.31 (dd, J ¼ 1.7, 8.1 Hz, 1H, H-8),
1655 cmꢂ1 (CO); MS m/z ¼ 255 (100, Mþ). Anal. C15H13NO3 (C, H, N).
7.82e7.20 (m, 9H, Ar), 6.99 (d, J ¼ 8.6 Hz, 1H, H-4), 6.60 (d,
J ¼ 7.9 Hz, 1H, H-2), 4.39 (t, J ¼ 8.0 Hz, 2H, NCH2CH2CH2), 2.84 (t,
J ¼ 7.7 Hz, 2H, NCH2CH2CH2), 2.08e2.01 (m, 2H, NCH2CH2CH2); FTIR
1620 cmꢂ1 (CO); MS m/z ¼ 329 (69, Mþ), 224 (100). Anal.
C22H19NO2 (C, H, N).
6.1.2.2. 1,3-Dihydroxy-10-propyl-10H-acridin-9-one (8c). The title
compound was obtained from 7 [33] and 1-iodopropane using the
same procedure as for 6b, but the mixture was refluxed for 24 h.
The crude product 8cc was then processed as described for 8b.
Purification by chromatography (SiO2; CH2Cl2/MeOH, 98/2) gave
6.1.1.13. 1-Hydroxy-10-(4-phenylbutyl)-10H-acridin-9-one (6l). The
title compound was obtained from 5 and 4-bromobutylbenzene
[39] using the same procedure as for 6b, but the mixture was
refluxed for 24 h. Purification by chromatography (SiO2; hexane/
CH2Cl2, 85/15) gave yellow crystals; 90% yield; mp 108 ꢁC; 1H NMR
yellow crystals; 29% yield; mp 213 ꢁC; 1H NMR (DMSO-d6)
d 14.89
(s, 1H), 10.65 (s, 1H), 8.25 (d, J ¼ 8.2 Hz, 1H), 7.83e7.71 (m, 2H), 7.30
(t, J ¼ 7.0 Hz,1H), 6.38 (d, J ¼ 1.7 Hz,1H), 6.09 (d, J ¼ 1.6 Hz, 1H), 4.20
(t, J ¼ 7.8 Hz, 2H), 1.81e1.69 (m, 2H), 1.05 (t, J ¼ 7.3 Hz, 3H); FTIR
1639 cmꢂ1 (CO); MS m/z ¼ 269 (82, Mþ), 227 (100). Anal. C16H15NO3
(C, H, N).
(DMSO-d6)
d
14.58 (s, 1H), 8.31 (dd, J ¼ 1.5, 8.0 Hz, 1H), 7.83e7.15
(m, 9H), 7.07 (d, J ¼ 8.7 Hz, 1H), 6.61 (d, J ¼ 7.9 Hz, 1H), 4.42 (t,
J ¼ 8.3 Hz, 2H), 2.66 (t, J ¼ 6.8 Hz, 2H), 1.80e1.76 (m, 4H); FTIR
1628 cmꢂ1 (CO); MS m/z ¼ 343 (100, Mþ). Anal. C23H21NO2 (C, H, N).
6.1.2.3. 10-Benzyl-1,3-dihydroxy-10H-acridin-9-one (8d). The title
compound was obtained from 8dd as described for 8b, but the
mixture was refluxed for 9 h. Purification by chromatography (SiO2;
CH2Cl2) gave yellow crystals; 13% yield; mp 233 ꢁC; 1H NMR
6.1.1.14. 3-Ethoxy-10-ethyl-1-hydroxy-10H-acridin-9-one
(8bb). The title compound was obtained from 1,3-dihydrox-
yacridone (7) [33] and iodoethane using the same procedure as for
6b, but the mixture was refluxed for 24 h. Purification by chro-
matography (SiO2; CH2Cl2/MeOH, 98/2) gave yellow crystals; 68%
(DMSO-d6)
d
14.50 (s, 1H), 10.69 (s, br, 1H), 8.14 (dd, J ¼ 8.1, 1.2 Hz,
1H), 7.82e7.07 (m, 9H), 6.19 (s, 1H), 4.21 (s, 2H); FTIR 3402,
1650 cmꢂ1 (CO); MS m/z ¼ 317 (100, Mþ). Anal. C20H15NO2 (C, H, N).
yield; mp 170e172 ꢁC; 1H NMR (DMSO-d6)
d
14.92 (s, 1H, OH), 8.26
6.1.2.4. 1,3-Dihydroxy-10-(3-phenylpropyl)-10H-acridin-9-one
(8e). The title compound was obtained from 7 [33] and 3-bromo-
propylbenzene using the same procedure as for 6b, but the mixture
was refluxed for 48 h. The crude product 8ee was then processed as
described for 8b. Purification by chromatography (SiO2; CH2Cl2/
MeOH, 99/1) gave yellow crystals; 22% yield; mp 204 ꢁC; 1H NMR
(d, J ¼ 8.2 Hz, 1H, H-8), 7.81e7.78 (m, 2H, H-5, H-6), 7.31 (dd, J ¼ 8.0,
5.8, 2.1 Hz, 1H, H-7), 6.47 (d, J ¼ 2.1 Hz, 1H, H-4), 6.22 (d, J ¼ 1.9 Hz,
1H, H-2), 4.42 (q, J ¼ 7.0 Hz, 2H, OCH2CH3), 4.15 (q, J ¼ 6.9 Hz, 2H,
NCH2CH3), 1.39e1.30 (m, 6H, OCH2CH3, NCH2CH3); FTIR 1639 cmꢂ1
(CO); MS m/z ¼ 283 (100, Mþ). Anal. C17H17NO3 (C, H, N).
(DMSO-d6)
d
14.89 (s, 1H), 10.69 (s, 1H), 8.26 (dd, J ¼ 8.0, 1.3 Hz, 1H),
6.1.1.15. 10-Benzyl-3-benzyloxy-1-hydroxy-10H-acridin-9-one
(8dd). The title compound was obtained from 7 and benzyl
bromide using the same procedure as for 6b, but the mixture was
stirred at RT for 24 h. Purification by chromatography (SiO2;
hexane/CH2Cl2, 1/1) and recrystallization from CH2Cl2/MeOH (1/1)
gave pale-yellow crystals; 20% yield; mp 224e225 ꢁC; 1H NMR
7.75 (t, J ¼ 7.2 Hz,1H), 7.60 (d, J ¼ 8.7 Hz,1H), 7.34e7.17 (m, 6H), 6.36
(d, J ¼ 1.3 Hz,1H), 6.10 (d, J ¼ 1.4 Hz,1H), 4.27 (t, J ¼ 7.2 Hz, 2H), 2.84 (t,
J ¼ 7.6 Hz, 2H), 2.07e2.03 (m, 2H); 13C NMR (DMSO-d6)
d 179.3,165.0
(2C), 143.9, 141.1, 141.0, 134.4, 128.4 (2C), 128.2 (2C), 126.0, 125.7,
121.4, 119.9, 115.2, 103.7, 95.9, 90.6, 45.6, 31.9, 27.9; FTIR 1638 cmꢂ1
(CO); MS m/z ¼ 345 (99, Mþ), 241 (100). Anal. C22H19NO3 (C, H, N).
(DMSO-d6)
d
14.66 (s, 1H), 8.17 (d, J ¼ 8.1 Hz, 1H), 7.86e7.10 (m,
14H), 6.48 (s, 1H), 5.21 (s, 2H), 4.30 (s, 2H); FTIR 3345, 1635 cmꢂ1
6.1.3. 1-(9-Oxo-9H-acridin-10-yl)butane-1,3-dione (11)
(CO); MS m/z ¼ 407 (75, Mþ), 316 (100). Anal. C27H21NO3 (C, H, N).
To a solution of acridone (9a, 1.0 g, 5.1 mmol) in dime-
thylformamide (70 mL) was added NaH (60%, 0.19 g) under N2. The
mixture was stirred at RT for 1 h. Then acetyl chloride (2 mL,
28.0 mmol) was added. Three further successive additions of NaH
6.1.1.16. 10-(3,4-Dimethoxybenzyl)-10H-acridin-9-one (9e). The title
compound wasobtained from acridone (9a)and3,4-dimethoxybenzyl

A. Putic et al. / European Journal of Medicinal Chemistry 45 (2010) 5345e5352
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poured into ice-water (120 mL) and the residue filtered by suction.
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6.2.1. Keratinocyte culture and determination of cell growth
HaCaT cells [26] were cultivated and the cell proliferation assay
was performed as described [41]. After 48 h of incubation, cell
growth was determined by enumerating the dispersed cells by
phase-contrast microscopy. Inhibition was calculated by the
comparison of the mean values of the test compound (N ¼ 3) with
the control (N ¼ 6e8) activity: (1 - test compound/control) ꢃ 100.
Inhibition was statistically significant compared to that of the
control (Student’s t-test; P < 0.05). IC50 values were obtained by
nonlinear regression.
6.2.2. Lactate dehydrogenase release
The assay was performed as described [19,27]. HaCaT cells were
incubated with the test compounds (2 m
M) for 4 h at 37 ꢁC. Extra-
cellular LDH activity was measured using the UV method with
pyruvate and NADH and is expressed in mU/ml. Appropriate
controls with the vehicle were performed (P < 0.05; N ¼ 3,
SD < 10%). Brij 35 (polyoxyethyleneglycol dodecyl ether)/ultra-
sound was the positive control.
6.2.3. Determination of DPPH radical interacting capability
The assay was performed as described [23]. The capability to
interact with DPPH radical (%) was calculated by the comparison of
the mean values of the test compound (N ¼ 3) with those of the
control (N ¼ 8): (1ꢂabsorbance of test compound/absorbance of
control) ꢃ 100. Results were expressed as EC50, the effective
concentration of the test compound required for 50% decrease in
DPPH absorbance at 520 nm. Values were obtained by nonlinear
regression.
Appendix. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2010.08.059.
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