Structure-activity relationship studies of acridones as potentialantipsoriatic agents.1.Synthesis and antiproliferative activity of simple N-unsubstituted 10H-acridin-9-ones against human keratinocyte growth

Article abstract of DOI:10.1016/j.ejmech.2010.04.013

A series of N-unsubstituted hydroxy-10H-acridin-9-ones were synthesized and evaluated for inhibitory action against HaCaT keratinocyte growth,in order to explore their potential as antipsoriatic agents. For structureeactivity relationship studies,the numb

Full text of DOI:10.1016/j.ejmech.2010.04.013

European Journal of Medicinal Chemistry 45 (2010) 3299e3310
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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. 1. Synthesis and antiproliferative activity of simple N-unsubstituted
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 of Münster, 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 N-unsubstituted hydroxy-10H-acridin-9-ones were synthesized and evaluated for inhibitory
action against HaCaT keratinocyte growth, in order to explore their potential as antipsoriatic agents. For
structureeactivity relationship studies, the number and position of the hydroxyl groups were modified,
the oxygen functions substituted or replaced, or additional functional groups were introduced into the
acridone scaffold. 1,8-Dihydroxy-10H-acridin-9-one (4), which is an aza-analogue of the antipsoriatic
anthralin, was only marginally active. However, 1,3-dihydroxy-substituted 5ee was the most potent
acridone within this series and inhibited keratinocyte growth with an IC50 value comparable to that of
anthralin. In contrast to anthralin, nearly all members of the acridone series were devoid of radical
generating properties, which were determined by their capability to interact with the free radical 2,2-
diphenyl-1-picrylhydrazyl. Structures with a phenolic hydroxyl or an aromatic amine arranged ortho or
para to the acridone NH group were exceptions. Also in contrast to anthralin, membrane-damaging
effects as documented by the release of lactate dehydrogenase into the culture medium were not
observed for acridones.
Received 12 March 2010
Received in revised form
8 April 2010
Accepted 13 April 2010
Available online 18 April 2010
Keywords:
Acridone
Antiproliferative activity
Antipsoriatic
HaCaT cells
Radical
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
pharmacophore, which led to various series of 10-substituted 1,8-
dihydroxy-10H-anthracen-9-ones [9e12].
Psoriasis is a common, immune-mediated inflammatory and
scaling skin disease, mainly characterized by excessive growth of
keratinocytes [1]. Despite the importance of systemic treatment for
severe and life-threatening forms of psoriasis, most patients can be
treated with topical antipsoriatic agents such as vitamin D3
analogues, anthralin (1, dithranol, Chart 1), coal tar and retinoids
[2,3]. Among the classical treatments, the anthrone derivative
anthralin is best used in hospital or day treatment centers, because
it causes substantial skin irritation and staining of skin, clothing
and furniture [4]. The methylene moiety at C-10 of the anthrone
nucleus has been recognized as a key site of anthralin-derived
oxygen radicals [5,6] and anthralin metabolite radicals [7,8], which
play a fundamental role in the induction of these undesired effects.
Therefore, our strategy to overcome this problem was to modify the
molecule by partially blocking the critical 10-position of the
In this regard, our interest has focused on acridones, which can
be considered as 10-aza-analogues of the anthrone class of anti-
psoriatic agents. The acridone alkaloids constitute a small group of
naturally occurring compounds found exclusively in plants
belonging to the order Rutales [13], and interest continues
unabated in the synthesis of related synthetic analogues of the
tetracyclic acridone alkaloid acronycine (2) [14] or acridones
showing large numbers of oxygenated groups in particular patterns
such as glyfoline (3) [15]. In view of the hyperproliferative nature of
keratinocytes in psoriatic epidermis, compounds derived from the
acridone scaffold may be of special importance because of their
demonstrated antiproliferative activity [14e19] as well as other
interesting properties associated with cell growth [20e22]. Prom-
inent amongst some acridone alkaloids isolated from two Boronia
species (Rutaceae) [23,24] and also a Samadera species (Simar-
oubaceae) [25] found in Australia is an atypically substituted,
symmetrical acridone alkaloid, 1,8-dihydroxy-10H-acridin-9-one
(4), matching exactly the substitution pattern of anthralin.
Although this alkaloid is a relatively simple compound, its struc-
ture, which was determined originally by spectroscopic analysis
[23], has not yet been accessible by total synthesis.
Abbreviations: DPPA, diphenylphosphoryl azide; DPPH, 2,2-diphenyl-1-pic-
rylhydrazyl; LDH, lactate dehydrogenase; RT, room temperature; TPP, 5,10,15,20-
tetraphenyl-21H,23H-porphine.
* 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.2010.04.013

3300
A. Putic et al. / European Journal of Medicinal Chemistry 45 (2010) 3299e3310
substituted acridones with 48% hydrobromic acid (e.g., 5ff), 36% HCl
(e.g., 5y), boron tribromide (e.g., 5hh), or aluminum chloride (e.g.,
5nn). Finally, Fries reaction of 1-O-acylated acridones 5az and 5zz,
which were prepared by acylation of 5a, led exclusively to the ortho-
substituted 5xx and 5yy, respectively (Scheme 4).
In the procedures depicted in Schemes 1 and 2, the final cycli-
zation is compromised by the fact that two possible routes are
available, and depending on the substitution pattern, sometimes
a mixture of isomers is obtained. For a regiospecific route to the
aza-analogous anthralin, 1,8-dihydroxy-10H-acridin-9-one (4), it
was necessary to use starting material that incorporates from the
outset the requisite substitution pattern (Scheme 5). The benzo-
phenone precursor 9 was synthesized in high yields by techniques
analogous to those described by Franck [31], which have also been
employed for the synthesis of (ꢀ)-balanol [32]. Accordingly,
[4 þ 2]-cyloaddition of singlet molecular oxygen (1O2) in a tetra-
phenylporphine photosensitized oxidation of tetramethoxyan-
thracene 7 [33] provided the 9,10-endoperoxide 8 which upon acid
cleavage yielded the desired benzophenone 9. This conversion can
be rationalized in a straightforward manner as a process involving
regioselective ketal cleavage at C-9 followed by a BaeyereVilliger
type rearrangement of an intermediate peroxide [32]. Methylation
of phenolic 9 with dimethyl sulfate to 10, hydrolysis to the
carboxylic acid 11, and Curtius reaction [34] using diphenylphos-
phoryl azide (DPPA) afforded the carbamate 12, which was directly
cyclized to the acridone 5 by sodium hydride in dimethyl sulfoxide
according to a literature method [35]. Ether cleavage with hydro-
bromic acid provided the desired aza-anthralin (4).
Chart 1. Structures of anthralin (1), acronycine (2), glyfoline (3), and 1,8-dihydroxy-
10H-acridin-9-one (4).
We set out to determine the significant structural features
responsible for inhibitory action against keratinocyte growth
among a series of simple acridone analogues related to anthralin,
particularly by modifying the number and position of the hydroxyl
groups, substitution of the oxygen functions or introduction of
additional functional groups into the acridone scaffold. As in earlier
studies with anthrones, we evaluated the capability of the acri-
dones to interact with a stable free radical by use of 2,2-diphenyl-1-
picrylhydrazyl (DPPH), and we also examined the potential of the
compounds to exert membrane-damaging effects.
3. Biological assay methods
2. Chemistry
As antiproliferative action in cell cultures may be critical in the
management of the proliferative component of psoriasis, the
sensitivity of HaCaT keratinocytes to each N-unsubstituted acri-
done was determined directly by counting the dispersed cells
under a phase-contrast microscope after 48 h of treatment. HaCaT
is a rapidly dividing immortalized human keratinocyte line [36],
which mimics the hyperproliferative epidermis found in psoriasis,
one of the pathological features of the disease.
Secondly, the generation of free radicals by anthralin and the
resulting dimerization and polymerization is associated with the
instability of the drug as well as with undesired side effects such as
staining and irritation of the skin [6]. Therefore, we have used the
stable free radical DPPH (2,2-diphenyl-1-picrylhydrazyl) [37] as
a simple method to compare the capability of the acridones to
produce radicals by loss of a hydrogen atom with that of anthralin.
Scheme 1 presents the synthesis of monohydroxy- and 1,3-
dihydroxy-substituted acridones 5a, 5i, 5t, 5x, 5aae5ee which was
accomplished by the classical condensation of
a substituted
anthranilate with resorcinol or phloroglucinol [26], respectively, in
1-heptanol in the presence of 4-toluenesulfonic acid [27,28].
Methoxy-substituted acridones 5ee5h, 5j, 5m, 5p, 5r, 5w, 5z
(Scheme 2), 5gg, 5ii, 5oo, 5ss (Scheme 3) were obtained by the
conventional approach [29]. This involves a polyphosphoric acid-
catalyzed ring closure of a requisite phenylanthranilic acid (e.g., 6w)
prepared by Ullmann reaction (Scheme 2). In an alternative
approach (Scheme 3), phenylanthranilic acids (6gg, 6ii, 6oo, 6ss)
were obtained in a modified Ullmannreactionof diphenyliodonium-
2-carboxylate with substituted anilines [30]. Hydroxy-substituted
acridones were obtained by ether cleavage of appropriate methoxy-
Scheme 1. Reagents: (a) 4-toluenesulfonic acid, 1-heptanol, reflux.

A. Putic et al. / European Journal of Medicinal Chemistry 45 (2010) 3299e3310
3301
Scheme 2. Reagents: (a) Cu(OAc)2, dimethylformamide, 90 ꢁC (b) PPA, 90 ꢁC; (c) 48% HBr, reflux; (d) 36% HCl, reflux.
As would be the case with most other free radicals, DPPH does not
dimerize by virtue of the delocalization of the unpaired electron
over the whole molecule. This also gives rise to a deep-violet color,
characterized by absorbance 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.
assessed by the activity of LDH (lactate dehydrogenase) released
into the culture medium, which is commonly used as an indicator
of plasma membrane damage [9,38].
4. Results and discussion
4.1. Inhibition of keratinocyte growth
Thirdly, keratinocytes were also tested for their susceptibility for
the action of the most potent inhibitors of keratinocyte growth of
the acridone class on plasma membrane integrity. This was
It can be seen from Table 1 that the bioisosteric replacement of
the 10-methylene group of the antipsoriatic anthrone anthralin (1)

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A. Putic et al. / European Journal of Medicinal Chemistry 45 (2010) 3299e3310
In general, monohydroxy- (5ae5d), monomethoxy- (5ee5h),
dimethoxy- (5, 5j, 5p, 5r), and trimethoxy-substitution (5w, 5z)
resulted in almost total loss of activity. However, introduction of an
additional hydroxy-substituent into position 3 (5i) or 4 (5l) of
1-monohydroxy 5a gave moderate inhibitors of keratinocyte
growth, with the 1,6-dihydroxyacridone (5o) exhibiting activity
even in the lower micromolar range (IC50 of 4.5 mM). Improvement
over 5a could also be observed for the 2-acetylated and 2-ben-
zoylated analogues 5xx and 5yy, respectively. In particular, 5yy was
markedly active and retained the antiproliferative activity of
anthralin, although the potency was somewhat reduced.
The series 5ffe5uu explored the consequences of varying
4-substitution on monohydroxy 5a and its methyl ether 5e. An
active keratinocyte growth inhibitor was obtained by introduction
of a para-hydroxyl group (5l), but this caused chemical instability.
As compared to 5l, changing the para-hydroxyl group to lipophilic
or electron-withdrawing (5ff, 5hh, 5jj, 5nn) and also electron-
donating substituents, as in 5ll, was disadvantageous for anti-
proliferative potency. By contrast, substitution of the para-position
of 5a with a carboxamide (5pp) or methyl ester (5rr) group
improved potency, while the analogous free carboxylic acid 5tt was
inactive. In each case of the 1-hydroxy-4-substituted analogues, the
corresponding O-methylated derivatives were less active or inac-
tive, indicating that the presence of the intramolecular hydrogen
bond in 1-hydroxy-10H-acridin-9-ones played an important role
for their keratinocyte inhibitory action. This is in good agreement
with the results obtained for glyfoline analogues against human
leukemic cells [15].
Finally, we explored additional substituents on 1,3-dihydroxy-
acridone (5i). Introduction of a 5-hydroxyl group in 5s improved
potency, whereas its 3-O-methylated analogue (5t) was less potent.
This was in line with the observation that 3-O-alkylation of 5i with
methyl, benzyl or phenylethyl groups in 5k, 5vv and 5ww,
respectively, rather impaired antiproliferative action. Most
successfully, addition of a peri-hydroxyl group provided 1,3,8-
Scheme 3. Reagents: (a) Cu, K2CO3, 1-heptanol, reflux; (b) PPA, 90 ꢁC; (c) 48% HBr,
reflux; (d) BBr3, CH2Cl2, ꢀ78 ꢁC, N2; (e) AlCl3, NaCl, 160 ꢁC.
with an NH group to produce acridone 4 resulted in a dramatic loss
of antiproliferative potency (IC50 of 0.7 mM for 1 versus 39.5 mM for
4). Nevertheless, based upon the IC50 values of some acridone
derivatives presented in Table 1 one can quite reasonably suppose
that an antipsoriatic potential resides within the acridone scaffold.
Our screening of simple acridone analogues for keratinocyte
growth inhibitory action revealed that, given the appropriate
substitution pattern, representatives with IC50 values in the lower
micromolar range were obtained. As already seen in the anthrone
series of antipsoriatic agents [39], the nature of the substituents,
their number and their position at the tricyclic skeleton were
critical for the antiproliferative potency of acridones. Thus, the
modification of these features led to the establishment of the
following structureeactivity relationships.
trihydroxy-substituted 5v with an IC50 value of 2.0
mM, suggesting
the requirement of a 3-hydroxyl group in the aza-analogous
anthralin 4 for potent keratinocyte inhibition. Moreover, replace-
ment of the 8-hydroxyl with a methyl group in 5ee further
improved potency. With an IC50 value in the submicromolar range
the potency of 5ee to arrest the excessive growth of keratinocytes
was comparable to that of the antipsoriatic anthralin. The impor-
tance of the position of the methyl substituent on the 1,3-dihy-
droxyacridone skeleton was investigated by comparing analogues
5cce5ee. Moving the methyl substituent to the 5- or 6-position
produced inactive compounds 5cc and 5dd, respectively. Accord-
ingly, even minor changes in the arrangement of a promising
substitution pattern such as 5ee resulted in a dramatic loss of
activity.
4.2. Interaction with the stable free radical DPPH
Table 1 shows that in terms of its ineffectiveness to reduce the
free radical DPPH, the aza-analogous anthralin (4) is devoid of any
appreciable radical interacting activity. This is in sharp contrast to
anthralin (1) itself, which proved to be very effective in donating an
electron to the stable free radical DPPH. As a consequence, acridone
4 was also more stable than the corresponding anthrone 1 and did
not dimerize or polymerize to dark brown material. Obviously,
hydrogen atom abstraction from acridones by free radicals such as
DPPH did not easily occur, as most acridones of this study were not
reactive with DPPH. In particular, the most potent inhibitors of
keratinocyte growth such as 5v and 5ee, bearing hydroxyl groups in
meta-position of the acridone nitrogen atom (i.e., position 1, 3 or 8
at the acridone skeleton), did not exhibit radical generating
Scheme 4. Reagents: (a) NaH, THF, AcCl or BzCl, RT, N2; (b) AlCl3, 160 ꢁC.

A. Putic et al. / European Journal of Medicinal Chemistry 45 (2010) 3299e3310
3303
Scheme 5. Reagents: (a) Et2O, TPP, O2, h
DMSO, RT; (g) 48% HBr, reflux.
n
, 5 ꢁC; (b) H2SO4, acetone, RT; (c) Me2SO4, K2CO3, acetone, reflux; (d) 10% NaOH, EtOH, reflux; (e) NEt3, tert-BuOH, DPPA, N2, reflux; (f) NaH,
properties. This is not surprising, as radical production from acri-
dones has only been described scarcely in the literature. Only after
photoirradiation and laser flash excitation, N-radical formation by
hydrogen transfer to pyridine has been observed [40,41].
However, in some cases interaction of acridone derivatives with
DPPH did occur. Given a suitable arrangement of their substituents,
acridones were capable of interacting with the DPPH radical as
5. Conclusions
We screened a series of N-unsubstituted hydroxy-10H-acridin-
9-ones for their activities against the proliferation of human kera-
tinocytes using HaCaT cells. Structureeactivity relationships for
acridone derivatives did not follow those observed in the anthrone
class of antipsoriatics [39]. 1,8-Dihydroxy-substitution as seen in
the anthrone series was not a prerequisite of antiproliferative
potency in the HaCaT keratinocyte line, as the aza-analogous
representative, 1,8-dihydroxy-10H-acridin-9-one (4), was only
marginally active as compared to the antipsoriatic anthralin.
Rather, acridones comprising a 1,3-dihydroxy-substitution pattern
were among the most active analogues. Compound 5ee, with an
8-methyl substituent at the 1,3-dihydroxy-substituted skeleton,
was the most potent acridone within this series and displayed
keratinocyte growth inhibitory action with an IC50 value in the
submicromolar concentration range, which was comparable to that
of the antipsoriatic anthralin. However, only minor changes in the
position of the methyl substituent resulted in loss of activity.
In most cases, acridones were devoid of radical generating
properties. The only groups that had a substantial impact on
interaction with the free radical DPPH were a phenolic hydroxyl or
an aromatic amine arranged ortho or para (2-, 4-, 5-, or 7-position)
to the acridone NH group. Also of note is the fact that in contrast to
anthralin, the most potent acridones did not disrupt membrane
integrity. The results of this work suggest that the acridone scaffold
is a quite reasonable structure for the development of antipsoriatic
agents.
effectively as the antioxidants NDGA or a-tocopherol. In fact, our
results document the importance of the orientation of hydroxyl or
amino groups on the acridone scaffold for the capability of the
compounds to generate radicals, with the hydroxy- or amino-
substitution ortho (5d, 5l, 5s, 5ll, 5mm) or para (5b, 5q) to the NH
group of acridone being required for effective interacting with
DPPH. This observation is in good agreement with a recent report
on moderate free radical scavenging activity of some naturally
occurring alkaloids consisting of
a 1,3,5-trihydroxy-acridone
moiety [42]. Furthermore, our findings can be rationalized as
a result of the resonance stabilization afforded to the resulting
acridone N-radical (e.g., 5b, Scheme 6). The resulting primary
structure 13a would be stabilized by the so-called captodative
effect [43]. In such a scenario, pairs of substituents of opposite
polarity, an electron-acceptor group (9-carbonyl) together with an
electron-donor group (2-OH) act in synergy on the stabilization of
the captodative radical (e.g., 13ae13e) and contribute a stronger
stabilization to this radical than pairs of identical substituents. In
addition, a second hydrogen abstraction from the radical would
give rise to a para-quinone-imine derivative 14.
4.3. Membrane damage
6. Experimental section
As a result of its potential to generate radicals, LDH release by the
standard anthralin significantly exceeded that of the vehicle
control. On the other hand, LDH release of the most potent acri-
dones (Table 1) was generally unchanged as compared to controls,
documenting that their antiproliferative activity was not related to
nonspecific cytotoxic effects. The only exception was the 2-benzoyl-
substituted acridone 5yy. This compound showed enhanced LDH
activity, but somewhat less pronounced than anthralin.
6.1. Chemistry
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

Table 1
Antiproliferative activity against HaCaT keratinocytes, lactate dehydrogenase release, free radical interacting capability, and method of preparation of N-unsubstituted
10H-acridin-9-ones.
Compounds
Substitution pattern
AAa IC50
(
m
M)
LDHb (mU/mL)
DPPHd EC50
(
mM)
Methode
[60]
1
4
5
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
anthralin
1,8-(OH)2
1,8-(OMe)2
1-OH
2-OH
3-OH
4-OH
1-OMe
2-OMe
3-OMe
4-OMe
1,3-(OH)2
1,3-(OMe)2
1-OH-3-OMe
1,4-(OH)2
1,4-(OMe)2
1-OH-4-OMe
1,6-(OH)2
0.7
39.5
>50
>50
>50
>50
>50
>50
>50
>50
>50
19.8
>50
46.3
10.0
>50
17.5
4.5
122.8c
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
61.2
ND
ND
77.7
ND
ND
ND
69.9
ND
ND
69.3
ND
ND
ND
ND
ND
ND
ND
ND
71.5
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
68.1
66.5
58.8
63.7
ND
ND
ND
ND
ND
88.9c
ND
ND
16.7
>60
ND
>60
2.7
A
f
F
A
A
A
G
G
G
G
F
G
B
A
G
B
A
G
A
G
A
F
ND
6.8
>60
ND
ND
>60
>60
ND
ND
8.8
>60
>60
>60
ND
1,6-(OMe)2
1,7-(OH)2
>50
48.8
>50
8.4
21.3
34.3
2.0
3.9
1,7-(OMe)2
1,3,5-(OH)3
1,3-(OH)2-5-OMe
1-OH-3,5-(OMe)2
1,3,8-(OH)3
1,3,8-(OMe)3
1,3-(OH)2-8-OMe
1,8-(OH)2-4-OMe
1,4,8-(OMe)3
1-OH-3-Me
1-Me-3-OH
1,3-(OH)2-5-Me
1,3-(OH)2-6-Me
1,3-(OH)2-8-Me
1-OH-4-Me
1-OMe-4-Me
1-OH-4-Cl
1-OMe-4-Cl
1-OH-4-NO2
1-OMe-4-NO2
1-OH-4-NH2
1-OMe-4-NH2
1-OH-4-CN
1-OMe-4-CN
1-OH-4-CONH2
1-OMe-4-CONH2
1-OH-4-CO2Me
1-OMe-4-CO2Me
1-OH-4-CO2H
1-OMe-4-CO2H
1-OH-3-OBn
1-OH-3-O(CH2)2Ph
1-OH-2-COMe
1-OH-2-COPh
1-OAc
ND
15.0
ND
ND
>60
ND
E
A
G
F
B
G
F
F
F
F
F
5w
5x
5y
45.3
28.3
>50
>50
>50
27.3
>50
>50
0.8
25.7
46.7
26.3
43.8
47.3
>50
>50
>50
27.9
>50
4.7
ND
>60
>60
ND
ND
ND
5z
5aa
5bb
5cc
5dd
5ee
5ff
5gg
5hh
5ii
ND
>60
>60
> 60
>60
>60
>60
>60
7.6
A
G
C
G
5jj
5kk
5ll
[56]
f
H
H
D
G
D
G
C
5mm
5nn
5oo
5pp
5qq
5rr
5ss
5tt
5uu
5vv
5ww
5xx
5yy
5az
5zz
7.7
>60
>60
>60
>60
> 60
>60
>60
>60
ND
8.8
5.2
10.8
49.2
>50
47.8
21.0
17.1
1.6
G
D
f
E
E
I
ND
>60
>60
ND
I
21.5
12.4
[57]
f
1-OCOPh
>60
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 < 0.01.
b
Activity of LDH (mU) release in HaCaT cells after treatment with 2
m
M test compound (N ¼ 3, SD < 10%).
Values are significantly different with respect to vehicle control. Brij 35 (polyoxyethyleneglycol dodecyl ether)/ultrasound was the positive control (251 mU/mLc).
c
ND ¼ not determined.
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 M) were used as positive controls. ND ¼ not determined.
m
e
Method of preparation according to the Experimental section.
See Experimental section.
f

A. Putic et al. / European Journal of Medicinal Chemistry 45 (2010) 3299e3310
3305
Scheme 6. Hydrogen abstraction from acridone 5b and resonance stabilization of radical 13ae13e by an electron-withdrawing 9-carbonyl and an electron-donating 2-hydroxyl
group.
a Bio-Rad laboratories type FTS 135 spectrometer. Mass spectra
were obtained in the EI mode using a MAT GCQ Finnigan instru-
ment. 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) with CH2Cl2 as eluant, unless otherwise stated. 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 ꢂ0.4% of
calculated values, except where stated otherwise.
6.85e6.74 (m, 3H), 6.46 (d, J ¼ 7.7 Hz,1H); FTIR 1649 cmꢀ1 (CO); MS
m/z ¼ 227 (100, Mþ).Anal. C13H9NO3 (C, H, N).
6.1.1.3. 1-Hydroxy-4-methyl-10H-acridin-9-one
compound was obtained from 5gg by method A to provide a yellow
powder; 55% yield; mp 285e286 ꢁC; 1H NMR (DMSO-d6)
13.97
(5ff). The
title
d
(s, 1H), 10.86 (s, 1H, br), 8.21 (d, J ¼ 8.3 Hz, 1H), 7.95 (d, J ¼ 8.4 Hz,
1H), 7.78 (t, J ¼ 8.3 Hz, 1H), 7.45 (d, J ¼ 8.1 Hz, 1H), 7.32 (t, J ¼ 8.2 Hz,
1H), 6.48 (d, J ¼ 8.0 Hz, 1H), 2.45 (s, 3H); 13C NMR (DMSO-d6)
d
181.9, 159.9, 141.0, 139.7, 118.9, 113.1, 108.5, 136.5, 134.0, 124.8,
121.7, 118.0, 105.4, 17.0; FTIR 1643 cmꢀ1 (CO); MS m/z ¼ 225 (100,
6.1.1. General procedure for the cleavage of methyl ethers
Mþ). Anal. C14H11NO2 (C, H, N).
6.1.1.1. Method A: 1,8-dihydroxy-10H-acridin-9-one (4). A solution
of 5 (0.56 g, 2.20 mmol) in 48% HBr (30 mL) was refluxed for 10 h.
The solution was allowed to cool to RT. Then the precipitating
crystals were filtered by suction and dissolved in MeOH (30 mL). A
solution of 10% KH2PO4 (20 mL) was added to induce crystalliza-
tion of the product. The precipitate was filtered, washed with
water, and then purified by chromatography (SiO2; CH2Cl2/MeOH,
99/1) to provide bright yellow crystals; 82% yield; mp 298 ꢁC (lit.
Acridones 5b [20], 5c [44], 5d [45], 5l [46], 5q [47], 5s [48], and
5v [49] were prepared from the corresponding methyl ethers (5f,
5g, 5h, 5m, 5r, 5t, and 5w, respectively) by method A.
6.1.1.4. Method B: 1,8-dihydroxy-4-methoxy-10H-acridin-9-one
(5y). A mixture of 5z (0.57 g, 2.0 mmol), 1-octanol (16 mL) and 36%
HCl (7 mL) was heated to reflux for 4 h. The mixture was allowed to
cool to RT and then neutralized with NaOH (5 mol/L) on an ice-bath.
The crude product was isolated by suction, washed with n-hexane
(3 ꢃ 100 mL), dissolved in CH2Cl2 (100 mL), and dried over Na2SO4.
The solution was concentrated and purified by column chroma-
tography (SiO2; CH2Cl2/MeOH, 98/2) to provide yellow plates; 33%
[24] 252 ꢁC); 1H NMR (DMSO-d6)
d 12.65 (s, 2H), 12.30 (s, 1H,), 7.63
(t, J ¼ 8.2 Hz, 2H), 6.96 (d, J ¼ 8.1 Hz, 2H), 6.57 (d, J ¼ 8.0 Hz, 2H);
FTIR 1654 cmꢀ1 (CO); MS m/z ¼ 227 (100, Mþ).Anal. C13H9NO3
(C, H, N).
yield; mp 98 ꢁC; 1H NMR (DMSO-d6)
11.77 (s, 1H, br), 7.90e7.08 (m, 3H), 6.86e6.16 (m, 2H), 3.96 (s, 3H);
d 12.74 (s, 1H), 12.00 (s, 1H),
6.1.1.2. 1,6-Dihydroxy-10H-acridin-9-one (5o). The title compound
was obtained from 5p by method A to provide bronze needles; 66%
13C NMR (DMSO-d6)
d 184.5, 160.5, 153.2, 131.8, 141.7, 139.1, 107.8,
yield; mp 275 ꢁC; 1H NMR (DMSO-d6)
d
14.20 (s, 1H), 11.77 (s, 1H,
107.5, 136.1, 116.6, 107.4, 106.4, 104.3, 56.6; FTIR 1650 cmꢀ1
br), 10.70 (s, 1H), 8.04 (d, J ¼ 8.6 Hz, 1H), 7.50 (t, J ¼ 8.2 Hz, 1H),
(CO/HO); MS m/z 257 (50, Mþ), 242 (100). Anal. (C14H11NO4) C, H, N.

3306
A. Putic et al. / European Journal of Medicinal Chemistry 45 (2010) 3299e3310
Acridones 5k [20] and 5n [50] were prepared from 5i and 5m,
respectively, by method B.
6.1.2. General procedure for alkylation of 3-hydroxyacridones
6.1.2.1. Method E:3-(benzyloxy)-1-hydroxy-10H-acridin-9-one
(5vv). A mixture of 5i (0.23 g, 1.0 mmol), dry K2CO3 (3 mmol) and
benzyl chloride in dry acetone (30 mL) was stirred 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) to provide yellow
6.1.1.5. Method C: 4-chloro-1-hydroxy-10H-acridin-9-one (5hh). To
a solution of 5ii (0.26 g, 1.0 mmol) in dry CH2Cl2 (20 mL) was added
a solution of BBr3 in CH2Cl2 (1 mol/L, 3 mL) at ꢀ78 ꢁC under N2. The
solution was stirred for 4 h, then it was allowed to warm to RT and
stirred for an additional 18 h. Water (50 mL) was added, the solu-
tion was stirred for 2 h at RT, and the aqueous phase was extracted
with CH2Cl2 (2 ꢃ 50 mL). The combined CH2Cl2 phase was washed
with water (2 ꢃ 75 mL), dried over Na2SO4, and the solvent was
evaporated. The residue was purified by column chromatography
(SiO2; CH2Cl2/MeOH, 99/1) to provide yellow plates; 52% yield; mp
crystals; 13% yield; mp 293 ꢁC; 1H NMR (DMSO-d6)
d 14.24 (s, 1H),
8.16 (d, J ¼ 7.9 Hz,1H), 7.70 (t, J ¼ 7.5 Hz,1H), 7.51e7.24 (m, 8H), 6.44
(d, J ¼ 1.9 Hz, 1H), 6.24 (d, J ¼ 1.8 Hz, 1H), 5.22 (s, 2H); FTIR
1656 cmꢀ1 (CO); MS m/z ¼ 317 (100, Mþ). Anal. C20H15NO3 (C, H, N).
6.1.2.2. 1-Hydroxy-3-phenethoxy-10H-acridin-9-one
(5ww). The
262 ꢁC; 1H NMR (DMSO-d6)
d
14.17 (s, 1H), 11.29 (s, 1H, br), 8.22 (dd,
title compound was obtained from 5i and 2-bromoethylbenzene by
method E, but the mixture was refluxed for 48 h. Purification
afforded yellow crystals; 87% yield; mp 242 ꢁC; 1H NMR (DMSO-d6)
J1 ¼ 8.2 Hz, J2 ¼ 1.2 Hz, 1H), 8.08 (d, J ¼ 8.4 Hz, 1H), 7.92e7.70 (m,
2H), 7.37 (t, J ¼ 8.1 Hz, 1H), 6.59 (d, J ¼ 8.7 Hz, 1H); 13C NMR (DMSO-
d6)
d
181.7, 161.1, 140.9, 137.6, 119.1, 109.1, 107.7, 135.6, 134.7, 124.9,
d
14.21 (s, 1H), 11.86 (s, 1H, br), 8.15 (d, J ¼ 8.0 Hz, 1H), 7.71 (t,
122.5, 118.4, 106.7; FTIR 1641 cmꢀ1 (CO.HO); MS m/z 245 (100,
J ¼ 7.6 Hz, 1H), 7.46 (d, J ¼ 8.3 Hz, 1H), 7.33e7.21 (m, 6H), 6.36
(d, J ¼ 1.4 Hz,1H), 6.12 (d, J ¼ 1.4 Hz,1H), 4.27 (t, J ¼ 6.8 Hz, 2H), 3.06
(t, J ¼ 6.7 Hz, 2H); FTIR 1655 cmꢀ1 (CO); MS m/z ¼ 331 (19, Mþ), 227
(100). Anal. C21H17NO3 (C, H, N).
Mþ). Anal. (C13H8ClNO2) C, H, N.
6.1.1.6. Methyl 1-hydroxy-9-oxo-9,10-dihydroacridine-4-carboxylate
(5rr). The title compound was obtained from 5ss by method C to
provide light-yellow crystals; 31% yield; mp 186e187 ꢁC; 1H NMR
Acridone 5u [48] was prepared from 5t and iodopropane by
method E.
(DMSO-d6)
d
15.05 (s, 1H), 12.15 (s, 1H, br), 8.29 (d, J ¼ 8.2 Hz,
1H), 8.20 (d, J ¼ 8.7 Hz, 1H), 7.78e7.55 (m, 1H), 7.46e7.10 (m, 2H),
6.52 (d, J ¼ 9.0 Hz, 1H), 3.88 (s, 3H); FTIR 1688 (CO2Me),
1633 cmꢀ1 (CO); MS m/z ¼ 269 (68, Mþ), 237 (100). Anal.
C15H11NO4 (C, H, N).
6.1.3. 1,8-Dimethoxy-10H-acridin-9-one (5)
To a solution of carbamate 12 (2.84 g, 7.3 mmol) in DMSO
(55.0 mL) was added 60% NaH (1.0 g) with stirring. The reaction
mixture was stirred for 24 h at RT, then it was poured into water
(100 mL) and extracted with EtOAc (3 ꢃ 50 mL), and the organic
phase was dried over Na2SO4. The solvent was evaporated, and the
residue was purified by column chromatography (SiO2; CH2Cl2/
MeOH, 99/1) to provide orange crystals; 13% yield; mp 239 ꢁC; 1H
6.1.1.7. Method D: 1-hydroxy-9-oxo-9,10-dihydroacridine-4-carbon-
itrile (5nn). A mixture of 5oo (0.50 g, 2.0 mmol), anhydrous AlCl3
(1.33 g, 10.0 mmol), and NaCl (2.7 g, 46.2 mmol) was triturated and
then heated to 160 ꢁC for 30 min. Then it was allowed to cool to RT,
and the solidified mixture was stirred with HCl (1 mol/L, 100 mL)
for 20 min. The precipitate was isolated by suction, washed with
water (100 mL), and then heated to reflux in a mixture of EtOH
(50 mL) and NaOH (2 mol/L, 100 mL) for 2 h. The hot mixture was
filtered, the filtrate was acidified with HCl (2 mol/L), and the crude
product was crystallized on an ice-bath. The precipitate was iso-
lated by suction, washed with water (200 mL) and dried with
toluene (DeaneStark). The product was isolated by suction and
purified by column chromatography (SiO2; CH2Cl2/MeOH, 98/2) to
provide yellow crystals; 34% yield; mp 279 ꢁC; 1H NMR (DMSO-d6)
NMR (DMSO-d6)
d 11.11 (s, 1H, br), 7.98e6.59 (m, 6H), 3.79 (s, 6H);
FTIR 1638 cmꢀ1 (CO); MS m/z ¼ 255 (100, Mþ). Anal. C15H13NO3
(C, H, N).
6.1.4. General procedure for the condensation of anthranilates with
phloroglucinol
6.1.4.1. Method
F:
1,3-dihydroxy-8-methoxy-10H-acridin-9-one
(5x). Methyl 2-amino-6-methoxybenzoate (3.62 g, 20.0 mmol),
phloroglucinol (2.52 g, 20.0 mmol) and 4-toluenesulfonic acid
(0.2 g, 1.2 mmol) in 1-heptanol (40 mL) were refluxed for 4 h. The
mixture was allowed to cool to RT, and petroleum ether (150 mL)
was added with stirring. The precipitate was filtered, washed with
petroleum ether (3 ꢃ 100 mL) and CH2Cl2 (2 ꢃ 50 mL), and then
purified by chromatography (SiO2; CH2Cl2/MeOH, 94/6) to provide
d
15.18 (s, 1H), 11.93 (s, 1H, br), 8.60e7.62 (m, 4H), 7.38 (t,
J ¼ 7.0 Hz, 1H), 6.63 (d, J ¼ 8.3 Hz, 1H); 13C NMR (DMSO-d6)
d 181.5,
167.1, 117.0, 87.4, 143.0, 141.1, 119.4, 108.0, 141.8, 134.9, 124.8, 123.1,
118.7, 107.5; FTIR 2221 (CN), 1637 cmꢀ1 (CO.HO); MS m/z 236
(100, Mþ). Anal. (C14H8N2O2) C, H, N.
yellow crystals; 77% yield; mp 326 ꢁC; 1H NMR (DMSO-d6)
d 14.81
(s, 1H), 11.51 (s, 1H), 10.37 (s, 1H, br), 7.54 (t, J ¼ 8.3 Hz, 1H), 6.94 (d,
J ¼ 8.3 Hz, 1H), 6.66 (d, J ¼ 8.0 Hz, 1H), 6.17 (d, J ¼ 2.1 Hz, 1H), 5.90
(d, J ¼ 2.0 Hz, 1H), 3.83 (s, 3H); FTIR 1653 cmꢀ1 (CO); MS m/z ¼ 257
(100, Mþ). Anal. C14H11NO4 (C, H, N).
Analogously, acridones 5i [28], 5t [48], 5cce5ee [51] were
prepared by method F. Also, compound 5a [50] was prepared from
methyl 2-aminobenzoate and resorcinol, compounds 5aa [52] and
5bb [52] were prepared from 2-aminobenzoate and
methylresorcinol.
6.1.1.8. 1-Hydroxy-9-oxo-9,10-dihydroacridine-4-carboxamide (5pp).
The title compound was obtained from 5qq (0.30 g, 1.1 mmol) by
method D to provide yellow needles; 46% yield; mp 304 ꢁC; 1H NMR
(DMSO-d6, hydroxylimine tautomer)
d 14.98 (s, 1H), 13.70 (s, 1H),
8.54e8.05 (m, 3H), 7.95e7.51 (m, 3H), 7.38 (t, J ¼ 7.3 Hz, 1H), 6.62 (d,
J ¼ 8.6 Hz, 1H); FTIR 1684 (CONH2), 1634 cmꢀ1 (CO); MS m/z ¼ 254
(90, Mþ), 237 (100). Anal. C14H10N2O3 (C, H, N).
6.1.1.9. 1-Hydroxy-9-oxo-9,10-dihydroacridine-4-carboxylicacid(5tt).
The title compound was obtained from 5uu (2.0 g, 7.1 mmol) by
method D to provide a yellow powder; 50% yield; mp 275 ꢁC
6.1.5. General procedure for the cyclization of phenylanthranilic
acids to acridones
6.1.5.1. Method G: 1,3,8-trimethoxy-10H-acridin-9-one (5w). 6-
Methoxy-2-(3,5-dimethoxyphenylamino)benzoic acid (6w, 5.76 g,
19.0 mmol) and polyphosphoric acid (PPA, 15.0 g) were heated at
90 ꢁC for 6 h with stirring. The mixture was cooled to 50 ꢁC, triturated
with ice (100 g), and buffered to pH 5 with NaOH (2 mol/L). The
mixture was stirred at RT for 5 h, and the precipitate was filtered by
(decomp.); 1H NMR (DMSO-d6)
d 15.29 (s, 1H, br), 14.57 (s, 1H), 8.32
(d, J ¼ 8.3 Hz, 1H), 8.21 (d, J ¼ 8.0 Hz, 1H), 7.76 (t, J ¼ 7.3 Hz, 1H), 7.58
(d, J ¼ 8.3 Hz,1H), 7.30 (t, J ¼ 7.4 Hz, 1H), 6.50 (d, J ¼ 8.3 Hz, 1H); FTIR
1631, 1604 cmꢀ1; MS m/z ¼ 255 (62, Mþ), 237 (100). Anal. C14H9NO4
(C, H, N).

A. Putic et al. / European Journal of Medicinal Chemistry 45 (2010) 3299e3310
3307
suction. The crude product was dissolved in CH2Cl2 (200 mL), and the
filtratewasextractedwithCH2Cl2 (3ꢃ100mL).Thecombinedorganic
phase was dried over Na2SO4, concentrated, and the residue was
purified by column chromatography (SiO2; CH2Cl2/MeOH, 98/2) to
provide beige crystals; 37% yield; mp 299 ꢁC; 1H NMR (DMSO-d6)
(300 mL) was refluxed for 48 h. The solution was allowed to cool to
RT, water was added (100 mL), the precipitate isolated by suction
and washed with water (200 mL). The product was dissolved in
CH2Cl2, dried over Na2SO4, concentrated in vacuo, and purified by
column chromatography (SiO2; CH2Cl2/MeOH, 97/3) to provide
a dark-yellow powder; 61% yield; mp 253e254 ꢁC; 1H NMR (DMSO-
d
11.02 (s,1H, br), 7.43 (d, J ¼ 8.2Hz,1H), 6.85(d, J ¼ 8.3 Hz,1H), 6.59(d,
J ¼ 8.1 Hz, 1H), 6.35 (d, J ¼ 2.3 Hz,1H), 6.18 (d, J ¼ 2.2 Hz, 1H), 3.82 (s,
3H), 3.77(s, 3H),3.76(s, 3H);FTIR1638cmꢀ1 (CO);MSm/z¼ 285(100,
Mþ). Anal. C16H15NO4 (C, H, N).
d6)
d
11.83 (s, 1H, br), 8.67 (d, J ¼ 9.6 Hz, 1H), 8.40 (dd, J1 ¼ 8.0 Hz,
J2 ¼ 0.8 Hz, 1H,), 7.82e7.62 (m, 1H), 7.48e7.30 (m, 2H,), 6.75
(d, J ¼ 9.6 Hz, 1H), 4.17 (s, 3H); 13C NMR (CDCl3)
d 176.8, 168.3, 139.6,
138.2, 123.8, 112.1, 110.0, 134.0, 133.5, 127.3, 123.9, 117.4, 102.6, 57.2;
FTIR 1643 cmꢀ1 (CO); MS m/z ¼ 270 (100, Mþ). Anal. C14H10N2O4
(C, H, N).
6.1.5.2. 1,4,8-Trimethoxy-10H-acridin-9-one
compound was obtained from 6z by method G to provide yellow
crystals; 46% yield; mp 255 ꢁC; 1H NMR (DMSO-d6)
10.45 (s, 1H),
(5z). The
title
d
7.58e7.26 (m, 2H), 7.12 (d, J ¼ 8.7 Hz, 1H), 6.62 (d, J ¼ 7.8 Hz, 1H),
6.1.7. Method H: general procedure for the reduction of
nitroacridones
6.53 (d, J ¼ 8.9 Hz, 1H), 3.92 (s, 3H), 3.79 (s, 3H), 3.74 (s, 3H); 13C
NMR (DMSO-d6)
d
175.8,159.7,153.2,132.5,142.5,140.7,113.4,112.9,
6.1.7.1. 4-Amino-1-hydroxy-10H-acridin-9-one (5ll). To a solution
of 1-hydroxy-4-nitro-10H-acridin-9-one [56] (5jj, 5.12 g,
20.0 mmol) in EtOH (75 mL) was added a solution of Na2S2O4
(13.95 g, 80 mmol) in water (75 mL) with stirring. The solution was
refluxed for 4 h and the solvent evaporated in vacuo. The residue
was isolated by suction, washed with water (200 mL), and dried
with toluene (DeaneStark). Then it was filtered by suction, washed
with petroleum ether (150 mL), and purified by column chroma-
tography (SiO2; CH2Cl2/MeOH, 98/2) under light protection to
provide a dark-red powder; 69% yield; mp 246e247 ꢁC (decomp.);
132.6, 112.4, 109.4, 102.7, 101.6, 56.3, 55.9, 55.5; FTIR 1627 cmꢀ1
(CO); MS m/z ¼ 285 (53, Mþ), 270 (100). Anal. C16H15NO4 (C, H, N).
6.1.5.3. 1-Methoxy-4-methyl-10H-acridin-9-one (5gg). The title
compound was obtained from 6gg by method G. The crude product
was refluxed in toluene (DeaneStark). Then it was filtered by
suction, washed with petroleum ether (3 ꢃ 100 mL), and purified by
column chromatography (SiO2; CH2Cl2/MeOH, 97/3) to provide
lemon yellow plates; 80% yield; mp 274 ꢁC; 1H NMR (DMSO-d6)
d
10.12 (s, 1H, br), 8.11 (d, J ¼ 8.0 Hz, 1H), 7.84 (d, J ¼ 8.3 Hz, 1H), 7.64
1H NMR (DMSO-d6)
d
13.18 (s, 1H), 10.92 (s, 1H), 8.20 (d, J ¼ 8.1 Hz,
(t, J ¼ 7.6 Hz, 1H), 7.43 (d, J ¼ 8.0 Hz, 1H), 7.19 (t, J ¼ 7.6 Hz, 1H), 6.62
(d, J ¼ 8.2 Hz, 1H), 3.80 (s, 3H), 2.45 (s, 3H); FTIR 1628 cmꢀ1 (CO);
MS m/z ¼ 239 (82, Mþ), 210 (100). Anal. C15H13NO2 (C, H, N).
1H), 7.96e7.50 (m, 2H), 7.44e7.16 (m, 1H), 7.05 (d, J ¼ 8.3 Hz,
1H), 6.40 (d, J ¼ 8.3 Hz, 1H), 4.90 (s, 2H, br); 13C NMR (DMSO-d6)
d
181.8, 152.9, 140.7, 130.3, 126.4, 118.8, 108.8, 133.9, 125.0, 121.4,
120.7, 117.6, 105.1; FTIR 1644 cmꢀ1 (CO); MS m/z ¼ 226 (100, Mþ).
6.1.5.4. 4-Chloro-1-methoxy-10H-acridin-9-one
compound was obtained from 6ii by method G to provide pale
yellow crystals; 59% yield; mp 259 ꢁC; 1H NMR (DMSO-d6)
10.55
(5ii). The
title
Anal. C13H10N2O2 (C, H, N).
d
6.1.7.2. 4-Amino-1-methoxy-10H-acridin-9-one (5mm). The title
compound was obtained from 5kk by method H to provide a dark-
red powder; 73% yield; mp 234e235 ꢁC (decomp.); 1H NMR
(s, 1H, br), 8.11 (dd, J1 ¼ 8.2 Hz, J2 ¼ 1.4 Hz, 1H), 7.96 (d, J ¼ 8.2 Hz,
1H), 7.76 (d, J ¼ 8.7 Hz, 1H), 7.67 (t, J ¼ 8.2 Hz, 1H), 7.25 (t, J ¼ 8.2 Hz,
1H), 6.74 (d, J ¼ 9.0 Hz, 1H), 3.73 (s, 3H); FTIR 1623 cmꢀ1 (CO); MS
m/z ¼ 259 (79, Mþ), 230 (100). Anal. C14H10ClNO2 (C, H, N).
(DMSO-d6)
d
10.21 (s, 1H, br), 8.10 (d, J ¼ 8.1 Hz, 1H), 7.79e7.51 (m,
2H), 7.30e7.10 (m, 1H), 6.98 (d, J ¼ 8.5 Hz, 1H), 6.53 (d, J ¼ 8.5 Hz,
1H), 4.95 (s, 2H, br), 3.72 (s, 3H); FTIR 1625 cmꢀ1 (CO); MS m/
z ¼ 240 (64, Mþ), 225 (100). Anal. C14H12N2O2 (C, H, N).
6.1.5.5. 1-Methoxy-9-oxo-9,10-dihydroacridine-4-carbonitrile (5oo).
The title compound was obtained from 6oo by method G to provide
pale yellow needles; 87% yield; mp 250e251 ꢁC; 1H NMR (DMSO-d6)
6.1.8. 1-Methoxy-9-oxo-9,10-dihydroacridine-4-carboxylic acid
(5uu)
d
11.10 (s, 1H), 8.22e8.01 (m, 2H), 7.92 (d, J ¼ 8.3 Hz, 1H), 7.68 (t,
J ¼ 7.7 Hz, 1H), 7.27 (t, J ¼ 7.4 Hz, 1H), 6.86 (d, J ¼ 8.7 Hz, 1H), 3.94 (s,
3H); FTIR 2217 (CN), 1627 cmꢀ1 (CO); MS m/z ¼ 250 (84, Mþ), 221
(100). Anal. C15H10N2O2 (C, H, N).
A solution of 5ss (2.00 g, 7.1 mmol) in EtOH (60 mL) and NaOH
(2 mol/L, 120 mL) was refluxed for 2 h. The solution was acidified
with HCl (2 mol/L), and the product was crystallized on an ice-bath,
isolated by suction, washed with water (200 mL), and dried with
toluene (DeaneStark). Then it was filtered by suction, washed with
petroleum ether (100 mL), and recrystallized from MeOH to
provide light-yellow crystals; 89% yield; mp 264 ꢁC (lit. [30]
6.1.5.6. 1-Methoxy-9-oxo-9,10-dihydroacridine-4-carboxamide(5qq).
The title compound was obtained from 6oo by method G, but the
mixture was stirred for 1.5 h. The crude product was refluxed in
toluene (DeaneStark). Then it was filtered by suction, washed with
petroleum ether (3 ꢃ 100 mL), and purified by column chromatog-
raphy (SiO2; CH2Cl2/MeOH, 95/5) to provide light-yellow crystals;
64% yield; mp 298 ꢁC; 1H NMR (DMSO-d6, hydroxylimine tautomer)
266e268 ꢁC); 1H NMR (DMSO-d6)
d 12.34 (s, 1H), 8.33 (d,
J ¼ 9.0 Hz, 1H), 8.12 (d, J ¼ 7.8 Hz, 1H), 7.87e7.44 (m, 2H), 7.26 (t,
J ¼ 7.1 Hz, 1H), 6.83 (d, J ¼ 9.0 Hz, 1H), 3.95 (s, 3H); FTIR 1678
(COOH), 1619 cmꢀ1 (CO); MS m/z ¼ 269 (98, Mþ), 223 (100). Anal.
C15H11NO4 (C, H, N).
d
13.41 (s, 1H), 8.34 (s, 1H, br), 8.25 (d, J ¼ 8.8 Hz, 1H), 8.12 (d,
J ¼ 8.0 Hz,1H), 7.86e7.58 (m, H-6, 2H), 7.50 (d, J ¼ 8.0 Hz,1H), 7.23 (t,
J ¼ 7.0 Hz, 1H), 6.78 (d, J ¼ 8.8 Hz, 1H), 3.93 (s, 3H); FTIR 1680 cmꢀ1
(CONH2); MS m/z ¼ 268 (99, Mþ), 251 (100). Anal. C15H12N2O3 (C, H,
N).
6.1.9. Method I: general procedure for the Fries reaction of
1-acyloxyacridones
6.1.9.1. 2-Acetyl-1-hydroxy-10H-acridin-9-one (5xx). A mixture of
9-oxo-9,10-dihydroacridin-1-yl acetate [57] (5az, 0.25 g, 1.0 mmol)
and anhydrous AlCl3 (0.73 g, 5.5 mmol) was triturated and then
heated to 160 ꢁC for 2 h. The mixture was allowed to cool to RT, and
the solidified product was stirred with HCl (0.2 mol/L, 50 mL) on an
ice-bath for 20 min. The precipitate was washed with water
(100 mL) and dried with toluene (DeaneStark). The product was
isolated by suction, washed with petroleum ether (75 mL),
Analogously, methoxy-substituted acridones 5e [29], 5f [53], 5g
[29], 5h [29], 5j [20], 5m [54], 5p [55] and 5r [47] and 5ss [30] were
prepared by method G.
6.1.6. 1-Methoxy-4-nitro-10H-acridin-9-one (5kk)
A solution of 1-chloro-4-nitro-10H-acridin-9-one [56] (2.0 g,
7.3 mmol) and NaOMe (1.18 g, 21.9 mmol) in absolute MeOH

3308
A. Putic et al. / European Journal of Medicinal Chemistry 45 (2010) 3299e3310
recrystallized from MeOH and purified by column chromatography
6.1.11.3. Method K: 2-(5-methoxy-2-methylphenylamino)benzoic
acid (6gg). To a solution of diphenyliodonium-2-carboxylate [58]
(3.40 g, 10.5 mmol) and 5-methoxy-2-methylaniline (1.37 g,
10.0 mmol) in dry dimethylformamide (20 mL) was added anhy-
drous Cu(OAc)2 (0.10 g), and the mixture was heated at 90 ꢁC for
15 h. Then it was poured into HCl (0.2 mol/L, 300 mL) on an ice-
bath, and the precipitate was extracted with CH2Cl2 (3 ꢃ 100 mL).
The combined extracts were washed consecutively with HCl
(0.2 mol/L, 2 ꢃ 75 mL) and water (3 ꢃ 100 mL), and then extracted
with NaOH (0.2 mol/L, 3 ꢃ 100 mL). The combined extracts were
acidified with 35% HCl (pH 4e5), and the precipitate was extracted
with CH2Cl2 (3 ꢃ 100 mL). The combined extracts were washed
with water (3 ꢃ 100 mL), dried over Na2SO4, concentrated, and the
residue was purified by column chromatography (SiO2; CH2Cl2/
MeOH, 97/3) to provide a white powder; 67% yield; mp 159 ꢁC; 1H
(SiO2; CH2Cl2/MeOH, 98/2) to provide yellow-orange crystals; 53%
yield; mp 272 ꢁC; 1H NMR (DMSO-d6)
d 15.89 (s, 1H), 12.44 (s, 1H,
br), 8.20 (d, J ¼ 7.8 Hz, 1H), 8.00 (d, J ¼ 9.0 Hz, 1H), 7.82 (t, J ¼ 8.0 Hz,
1H), 7.57 (d, J ¼ 8.0 Hz,1H), 7.37 (t, J ¼ 7.8 Hz,1H), 6.91 (d, J ¼ 9.0 Hz,
1H), 2.57 (s, 3H); 13C NMR (DMSO-d6)
d 195.0, 182.1, 165.0, 144.8,
140.4, 119.4, 115.2, 107.7, 135.8, 134.9, 125.2, 123.0, 117.8, 106.7, 31.9;
FTIR 1639, 1620 cmꢀ1 (CO); MS m/z ¼ 253 (46, Mþ), 238 (100). Anal.
C15H11NO3 (C, H, N).
6.1.9.2. 2-Benzoyl-1-hydroxy-10H-acridin-9-one (5yy). The title
compound was obtained from (5zz) by method I to provide orange
crystals; 42% yield; mp 285 ꢁC; 1H NMR (DMSO-d6)
d 15.04 (s, 1H),
12.44 (s, 1H, br), 8.22 (d, J ¼ 7.6 Hz, 1H), 7.85 (t,1H), 7.78 (d, 1H), 7.76
(d, 2H), 7.63 (d, 1H), 7.61 (t, 1H), 7.50 (t, 2H), 7.39 (t, 1H), 7.05 (d,
J ¼ 8.8 Hz, 1H); FTIR 1654, 1635 cmꢀ1 (CO); MS m/z ¼ 315 (100, Mþ).
Anal. C20H13NO3 (C, H, N).
NMR (DMSO-d6)
d
9.50 (s, 1H, br), 7.88 (d, J ¼ 7.7 Hz, 1H), 7.36
(t, J ¼ 8.0 Hz,1H), 7.18 (d, J ¼ 8.5 Hz,1H), 6.94 (d, J ¼ 8.1 Hz,1H), 6.86
(d, J ¼ 2.1 Hz, 1H), 6.73 (t, J ¼ 7.7 Hz, 1H), 6.64 (dd, J1 ¼ 8.2 Hz,
J2 ¼ 2.2 Hz, 1H), 3.70 (s, 3H), 2.10 (s, 3H); 13C NMR (DMSO-d6)
6.1.10. 9-Oxo-9,10-dihydroacridin-1-yl benzoate (5zz)
A mixture of 5a (0.1 g, 1.0 mmol) and 60% sodium hydride
(0.1 g) in THF (25 mL) was stirred under N2 for 30 min. Then
a solution of benzoyl chloride (0.12 g, 0.84 mmol) in THF (5 mL)
was added and stirring was continued for 1.5 h. The mixture was
poured into ice-water (20 mL), extracted with CH2Cl2, dried over
Na2SO4, concentrated in vacuo, and purified by column chroma-
tography (SiO2; CH2Cl2/MeOH, 98/2) to provide light-yellow crys-
d 170.0,158.1,147.5, 139.5,122.8,112.1,134.1,131.7,131.5,116.9,113.6,
109.5,108.0, 55.0, 16.7; FTIR 1654 cmꢀ1 (COOH); MS m/z ¼ 257 (100,
Mþ). Anal. C15H15NO3 (C, H, N).
6.1.11.4. 2-(2-Chloro-5-methoxyphenylamino)benzoic acid (6ii). The
title compound was prepared from 2-chloro-5-methoxyaniline by
method
K
to provide
a
light-grey powder; 51% yield; mp
9.80 (s, 1H), 7.93 (d, J ¼ 7.8 Hz,
tals; 35% yield; mp 262 ꢁC; 1H NMR (DMSO-d6)
d
11.85 (s, 1H, br),
152e153 ꢁC; 1H NMR (DMSO-d6)
d
8.36e8.10 (m, 2H), 8.04 (d, J ¼ 8.2 Hz, 1H), 7.93e7.39 (m, 7H), 7.20
(t, J ¼ 7.6 Hz, 1H), 6.98 (d, J ¼ 7.6 Hz, 1H); FTIR 1738 (OCOPh),
1636 cmꢀ1 (CO); MS m/z ¼ 315 (30, Mþ), 105 (100). Anal.
C20H13NO3 (C, H, N).
1H), 7.56e7.34 (m, 2H), 7.28 (d, J ¼ 8.3 Hz, 1H), 7.05 (d, J ¼ 2.7 Hz,
1H), 6.87 (t, J ¼ 7.8 Hz, 1H), 6.66 (dd, J1 ¼ 8.9 Hz, J2 ¼ 2.8 Hz, 1H),
3.73 (s, 3H); FTIR 1657 cmꢀ1 (COOH); MS m/z ¼ 277 (63, Mþ), 224
(100). Anal. C14H12ClNO3 (C, H, N).
6.1.11. General procedures for the synthesis of phenylanthranilic
acids
6.1.11.5. 2-(2-Cyano-5-methoxyphenylamino)benzoic acid (6oo).
The title compound was prepared from 2-amino-4-methoxy-
benzonitrile by method K to provide a light-grey powder; 45%
6.1.11.1. Method J: 6-methoxy-2-(3,5-dimethoxyphenylamino)benzoic
acid (6w). To a solution of 2-chloro-6-methoxybenzoic acid (3.64 g,
19.5 mmol) and 3,5-dimethoxyaniline (3.98 g, 26.0 mmol) in
1-heptanol (20 mL) was added powdered copper (0.50 g). Then
anhydrous K2CO3 was added in portions with gentle heating and
stirring. The mixture was refluxed for 7 h, and then treated with
ice-water (300 mL), alkalinized with NaOH (0.1 mol/L), and filtered.
The solution was washed with Et2O (3 ꢃ 50 mL) and acidified with
18% HCl on an ice-bath with stirring. The precipitating crude
product was filtered by suction, dried in a drying apparatus in
vacuo and purified by column chromatography (SiO2; CH2Cl2/
MeOH, 98/2) to provide white crystals; 13% yield; mp 152 ꢁC; 1H
yield; mp 198e199 ꢁC; 1H NMR (DMSO-d6)
d 10.10 (s, 1H), 7.96
(d, J ¼ 6.8 Hz,1H), 7.70 (d, J ¼ 8.8 Hz,1H), 7.50 (t, J ¼ 7.6 Hz, 1H), 7.39
(d, J ¼ 8.0 Hz, 1H), 7.12e6.88 (m, 2H), 6.73 (dd, J1 ¼ 8.8 Hz,
J2 ¼ 2.2 Hz,1H), 3.80 (s, 3H); FTIR 2214 (CN),1665 cmꢀ1 (COOH); MS
m/z ¼ 268 (100, Mþ). Anal. C15H12N2O3 (C, H, N).
6.1.12. 1,8,9,10-Tetramethoxy-9,10-dihydro-9,10-
epidioxyanthracene (8)
A solution of 1,8,9,10-tetramethoxyanthracene [33] (7, 2.0 g,
6.70 mmol) and 5,10,15,20-tetraphenyl-21H,23H-porphine (TPP,
20 mg) in absolute Et2O (300 mL) was irradiated with halogen
lamps (Osram Halostar, 200 W) under continuous oxygen bubbling
in a water-cooled immersion well (Haake cryostat KT 33.0) for
90 min at 5 ꢁC. A solution of 40% KNO2 in water was used as a cutoff
filter (350 nm). The solvent was removed, the precipitate filtered
and washed with Et2O (3 ꢃ 10 mL) to provide colorless crystals; 80%
NMR (acetone-d6)
d
9.75 (s, 1H br), 7.35 (t, J ¼ 8.4 Hz, 1H), 7.02 (d,
J ¼ 8.4 Hz, 1H), 6.59 (d, J ¼ 8.2 Hz, 1H), 6.31 m, 3H), 4.06 (s, 3H), 3.78
(s, 6H); FTIR 1697 cmꢀ1 (COOH); MS m/z ¼ 303 (100, Mþ). Anal.
C16H17NO5 (C, H, N).
6.1.11.2. 2-(2,5-Dimethoxyphenylamino)-6-methoxybenzoic
acid
yield; mp 230 ꢁC; 1H NMR (CDCl3)
d 7.26e7.07 (m, 4H), 6.81 (dd,
(6z). The title compound was obtained from 2-chloro-6-
methoxybenzoic acid and 2,5-dimethoxyaniline by method J. The
solution of the crude product was extracted with CH2Cl2
(3 ꢃ 150 mL), the combined extracts were dried over Na2SO4, and
the residue was purified by column chromatography (SiO2; CH2Cl2/
MeOH, 97/3) to provide a light-yellow powder; 26% yield; mp
J ¼ 7.0, 2.0 Hz, 2H), 3.90 (s, 3H), 3.83 (s, 3H). The crude product was
used in the subsequent reaction.
6.1.13. Methyl 2-(2-hydroxy-6-methoxybenzoyl)-
3-methoxybenzoate (9)
To a solution of endoperoxide 8 (0.33 g, 1.0 mmol) in dry acetone
(300 mL) was added a solution of 96% H2SO4 (0.2 mL) in dry acetone
(10 mL). The solution was stirred for 3 h at RT, then diluted with
water, extracted with CH2Cl2 (3 ꢃ 100 mL), and the organic phase
was washed with water and dried over Na2SO4. The solvent was
evaporated, and the residue was purified by column chromatog-
raphy (SiO2; CH2Cl2/MeOH, 98/2) to provide light-green crystals;
106 ꢁC; 1H NMR (DMSO-d6)
d
7.78 (s, 1H, br), 7.29 (t, J ¼ 8.3 Hz, 1H),
7.06e6.84 (m, H-3, 2H), 6.76 (d, J ¼ 2.8 Hz, 1H), 6.61 (d, J ¼ 8.3 Hz,
1H), 6.44 (dd, J1 ¼ 8.8 Hz, J2 ¼ 2.8 Hz, 1H), 3.78 (s, 3H), 3.76 (s, 3H),
3.65 (s, 3H); 13C NMR (DMSO-d6)
d 168.2, 158.3, 153.4, 131.9, 143.3,
142.3, 111.6, 131.6, 112.2, 109.2, 104.8, 103.7, 103.3, 56.2, 55.8, 55.2;
FTIR 1692 cmꢀ1 (COOH); MS m/z ¼ 303 (100, Mþ). Anal. C16H17NO5
(C, H, N).
72% yield; mp 184 ꢁC; 1H NMR (CDCl3)
d 12.87 (s, 1H), 7.62 (d,

A. Putic et al. / European Journal of Medicinal Chemistry 45 (2010) 3299e3310
3309
J ¼ 8.0 Hz, 1H), 7.35 (t, J ¼ 8.0 Hz, 1H), 7.31 (t, J ¼ 8.0 Hz, 1H), 7.09 (d,
J ¼ 8.0 Hz, 1H), 6.62 (d, J ¼ 8.0 Hz, 1H), 6.22 (d, J ¼ 8.0 Hz, 1H), 3.71
(s, 3H), 3.68 (s, 3H), 3.30 (s, 3H); FTIR 1700 (CO2Me), 1620 cmꢀ1
(CO); MS m/z ¼ 316 (31, Mþ), 253 (100). Anal. C17H16O6 (C, H).
measured. Appropriate vehicle and DPPH controls as well as posi-
tive controls with NDGA (nordihydroguaiaretic acid) and
a-tocopherol were performed. Data were analyzed with the soft-
ware Softmax Pro. The capability to interact with DPPH radical (%)
was calculated by the comparison of the mean values of the test
6.1.14. Methyl 2-(2,6-dimethoxybenzoyl)-3-methoxybenzoate (10)
To a suspension of 9 (0.15 g, 4.74 mmol) and anhydrous K2CO3
(4.5 g, 3.26 mmol) in dry acetone (150 mL) was added dimethyl
sulfate (1.99 g, 15.78 mmol), and the mixture was heated to reflux
for 16 h. The reaction mixture was then filtered by suction, the
filtrate was cooled on an ice-bath, diluted with water, and extracted
with CH2Cl2 (3 ꢃ 50 mL). The organic phase was concentrated, and
the residue was purified by column chromatography (SiO2; CH2Cl2/
acetone, 99/1) to provide colorless prisms; 73% yield; mp 150 ꢁC; 1H
compound (N
¼
3) with those of the control (N
¼
8):
(1 ꢀ absorbance 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.
6.2.3. Lactate dehydrogenase release
The assay was performed as described [9,38]. HaCaT cells were
incubated with the test compounds (2 m
M) for 4 h at 37 ꢁC. Extra-
NMR (CDCl3)
d
7.43 (dd, J ¼ 8.0, 2.0 Hz, 1H), 7.31 (t, J ¼ 8.0 Hz, 1H),
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 < 0.01; N ¼ 3,
SD < 10%). Brij 35 (polyoxyethyleneglycol dodecyl ether)/ultra-
sound was the positive control.
7.28 (t, J ¼ 8.0 Hz, 1H), 7.01 (dd, J ¼ 8.0, 2.0 Hz, 1H), 6.52 (d,
J ¼ 8.0 Hz, 2H), 3.68 (s, 3H), 3.66 (s, 3H), 3.63 (s, 3H); FTIR 1700
(CO2Me), 1650 cmꢀ1 (CO); MS m/z ¼ 330 (48, Mþ), 165 (100). Anal.
C18H18O6 (C, H).
6.1.15. 3-Methoxy-2-(2,6-dimethoxybenzoyl)benzoic acid (11)
A solution of 10 (3.30 g, 10.0 mmol) in EtOH (16 mL) and 10%
NaOH (8 mL) was refluxed for 6 h. The solution was allowed to cool
to RT, then acidified with HCl (0.1 mol/L), extracted with CH2Cl2
(3 ꢃ 50 mL), dried over Na2SO4 and concentrated in vacuo. The
product was crystallized by addition of small amounts of petroleum
ether. The precipitate was isolated by suction and washed with
petroleum ether to provide white crystals; 90% yield; mp 176 ꢁC; 1H
Appendix. Supplementary material
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.ejmech.2010.04.013.
References
NMR (CDCl3)
d 7.44e6.61 (m, 6H), 3.59 (s, 3H), 3.54 (s, 6H); FTIR
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