Synthesis and?biological evaluation of?modified acridines: the?effect of?N- and?O- substituent in?the?nitrogenated ring on?antitumor activity

Article abstract of DOI:10.1016/j.ejmech.2005.11.006

A series of new acridines has been prepared by cyclodehydration of N-(2,3-dihydro-1,4-benzodioxin-6-yl)anthranilic acid in acidic media following classical procedures. All these compounds have in common a dioxygenated ring fused to the acridine. The tetra

Full text of DOI:10.1016/j.ejmech.2005.11.006

European Journal of Medicinal Chemistry 41 (2006) 340–352
http://france.elsevier.com/direct/ejmech
Original article
Synthesis and biological evaluation of modified acridines:
the effect of N- and O- substituent
in the nitrogenated ring on antitumor activity
Isabel Sánchez a, Rosa Reches a, Daniel Henry Caignard b, Pierre Renard b, Maria Dolors Pujol a,*
a
Laboratori de Química Farmacèutica (Unitat Associada al CSIC), Facultat de Farmàcia,
Universitat de Barcelona, Av. Diagonal 643, E-08028 Barcelona, Spain
b
Les Laboratoires Servier, 1, rue Carle-Hébert, 92415 Courbevoie cedex, France
Received 20 September 2005; received in revised form 17 November 2005; accepted 30 November 2005
Available online 18 January 2006
Abstract
A series of new acridines has been prepared by cyclodehydration of N-(2,3-dihydro-1,4-benzodioxin-6-yl)anthranilic acid in acidic media
following classical procedures. All these compounds have in common a dioxygenated ring fused to the acridine. The tetracyclic system possesses
a linear or angular structure formed by intramolecular cyclisation. The last ring and the substituent of the system modify, in an interesting way,
the antitumor activity of acridines. Several of the studied compounds displayed significant cytotoxic activity (inhibition of L1210 and HT-29 cell
proliferation). The most cytotoxic compound 13a, shows more activity than m-AMSA in inhibiting L1210 and HT-29 cell proliferation and this
compound has been selected as a development candidate for further evaluation. The activity results also indicate that the new 11-O-substituted
compounds are of considerable interest with high levels of cytotoxic activity. The angular or non-linear dioxinoacridine 10 was equiactive with
the linear structure 7. Pentacyclic analogues (14 and 15) were more cytotoxic than the tetracyclic compounds (up to twofold).
© 2006 Elsevier SAS. All rights reserved.
Keywords: Acridines; Antitumor agents; Cytotoxicity; Polycyclic systems
1. Introduction
be clinically useful (Fig. 1). Amsacrine 2 is the best-known
compound of 9-anilinoacridines series. It was one of the first
DNA-intercalating agents to be considered as a topoisomerase
II inhibitor. The significant clinical use of several of these com-
pounds is limited by problems such as side effects, drug resis-
tance and poor bioavailability, which have encouraged further
modifications to these compounds. At present, almost all the
reported antitumor agents in the acridine series have been de-
rived from pattern compounds and they have incorporated
changes in the substituents or heterocyclic system modifica-
tions.
Acridine derivatives are one of the more studied chemother-
apeutic compounds, widely used as antimalarial [1], antiproto-
zoal [2], antibacterial [3] and antitumor agents [4].
Antitumor cytotoxic agents with DNA-intercalative proper-
ties have been studied. These compounds are characterized by
the presence of a planar chromophore, generally a tri- or tetra-
cyclic ring system and one or two flexible substituent groups.
The acridine derivatives known as DNA-intercalators, such as
nitracrine 1 [5], m-AMSA 2 [6], DACA 3 [7] and others DNA-
intercalative agents as proflavine 4 [8] and ellipticine 5 [9] ex-
hibit cytotoxic activity and some of them have been found to
The intercalation process is the strongest type of reversible
binding to the double helical DNA in compounds with suffi-
* Corresponding author.
E-mail address: mdpujol@ub.edu (M.D. Pujol).
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.11.006

I. Sánchez et al. / European Journal of Medicinal Chemistry 41 (2006) 340–352
341
Fig. 1. General formula of known anticancer agents.
ciently large coplanar aromatic chromophore. The compounds
showed in Fig. 1 are complex molecules, not obviously related
in any way except in the possession of coplanar chromophores
that favor the intercalation; for this reason they are designated
as DNA intercalators. Moreover, the cytotoxicity of most of the
clinically useful DNA-intercalating agents involves the inhibi-
tion of the enzyme DNA-topoisomerase I or II. Several detailed
SAR studies of acridine-based DNA-intercalating agents sug-
gest that the mode of binding is important and the chromo-
phore will locate to give maximum overlap with the DNA base
pairs. The intercalative binding appears to be a necessary but
not sufficient condition for the antiproliferative activity [10].
It is consequently of great interest to find new types of poly-
cyclic compounds possessing potent antitumor activities and
reduced side effects. Recently, Antonini et al. [11] have pre-
pared several bis functionalized acridine-4-carboxamides with
two basic side chains as important antitumor agents. Herein we
report the synthesis and biological evaluation of a series of
novel acridines with a 1,4-dioxine group fused at 2,3- or 4,5-
positions. There is only few previous reports of this ring sys-
tem in the literature [4d–g] and we give the first account here
of their biological properties.
linked to 11- position, in the size and electronic character of
the lateral chain and in the kind of the 9-substituent (com-
pounds 6–17). The introduction of N-substituent are the most
known but the O-substituents as ATP at the 11-position of the
acridine derivatives is unknown up to date, and this is the first
time that ATP was enclosed in antitumor agents (compounds
13a and 13b). It is well known that all cells need Adenosine
triphosphate (ATP) to survive. Recently, the effects of drug
binding on the ATP hydrolysis were studied [12]. Several mod-
ifications affecting ATP binding or hydrolysis have been made
and used to study the mechanism of ATP-dependent anticancer
drug transport. Now, the research is still ongoing and the re-
sults are not available. For our part, we think that the study of
compounds containing ATP framework can be assist to under-
stand the mechanism of ATP anticancer effects.
Also, the substitution of the benzene (D ring) by a pyridine
nucleus has been considered (compound 18). Both of them are
bioisostere groups. Our aim was to verify the effect on the cy-
totoxic activity and the cell cycle selectivity of the additional
dioxygenated ring of compounds compared to tricyclic deriva-
tives and if the substitution of the C-11, believed essential for
antitumor activity, could afford similar or enhanced biological
properties. In vitro cytotoxicity data against leukemic cell lines
and selectivity for the cellular cycle are described. The struc-
Other modifications at the C-11 of the acridine nucleus and
at the D ring have been made (Scheme 1). Thus, several acri-
dine derivatives have been prepared, which vary in the atom
Scheme 1.

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I. Sánchez et al. / European Journal of Medicinal Chemistry 41 (2006) 340–352
Scheme 2. Conditions: i, K2CO3/Cu/xylene ii, TFA/CH2Cl2 or H2SO4 or PPA.
ture-activity relationships should provide chemical leads to-
wards the design of novel anticancer compounds.
dine nucleus, we have synthesized different derivatives with a
nitro, bromo or methoxy group. Our first approach to the
synthesis of the nitro substituted acridine 8 was to prepare the
corresponding acridinone 26 from the 4-nitrodiphenylamine
carboxylic acid 24, obtained by Ullmann reaction between 19
and 21. In this step, the carboxylic acid was cyclised using
TFA to form a mixture of the two isomers “linear” and “non
linear” acridones in 22% (26) and 64% (28) yield, respectively.
The main step of the synthesis of this series of compounds was
the nucleophilic attack of the corresponding chloroacridines by
alkylamine, diarylamine or alcohol. The conversion of 26 in
the chloroderivative 34 by refluxing with POCl3 following by
alkylation of the corresponding N,N-diethylaminoethylamine
let us to obtain compound 8. However, the direct nitration of
the corresponding acridones would be the most convenient
way to introduce a nitro function in the acridine nucleus. Thus,
the nitro derivative 26 was obtained in 68% yield when 16
were treated with a mixture of nitric acid and acetic acid at
0 °C. Under these conditions only one isomer was obtained
and the nitro function is located at the para position of the
amino group.
2. Chemistry
Scheme 2 shows the synthetic pathway employed in the pre-
paration of compounds (16 and 17, 26–29). The N-(2,3-dihy-
dro-1,4-benzodioxin-6-yl)anthranilic acid 23 was prepared by
direct nucleophilic displacement (Ullmann conditions) [13] of
the 2-chlorobenzoic acid 20 with the 2,3-dihydro-1,4-benzo-
dioxin-6-amine. In this reaction, the substitution of the halogen
atom of the ortho-halogenobenzoic acid by aniline is promoted
by copper catalyst. The ring closure of this acid derivative was
carried out by different pathways. The standard synthesis of
11-substituted compounds from the diarylamine (23–25) using
POCl3 lead directly the 11-chloroderivatives contaminated with
side products which were difficult to remove, resulting in mod-
est yields of pure compounds. When the acid 23 was refluxed
in POCl3 the cyclisation proceeded fast, affording the chloroa-
cridines 32 and 33 [4f], corresponding to the ring closure in the
two possible positions (C7 or C5, respectively) (Scheme 3).
However, when compound 23 was treated with TFA in
CH2Cl2 the compound of cyclisation in position C7 was regio-
selectively obtained (tetracyclic ketone 16), which refluxed in
phosphorus oxychloride gave the chloroacridine 32 (Scheme
3). Another way of classical cyclisation consisted in refluxing
the carboxylic acid (23–25) in sulfuric acid under air for 4 h or
using polyphosphoric acid at 110 °C [14].
Ullmann condensation of 2-chloro-5-methoxy benzoic acid
with 2,3-dihydro-1,4-benzodioxin-6-amine afforded the diphe-
nylamine carboxylic acid 25. The intramolecular cyclisation of
25 with phosphorus oxychloride gave the two isomeric “linear”
and “non-linear” compounds 36 and 37. Attempts to obtain the
corresponding N-substituted acridine by direct alkylation of 36
and 37 were not successful and only the corresponding acri-
dones 17 and 29 together with degradation compounds were
obtained.
In order to study the role of the substituents (electron-with-
drawing versus electron-donating) in the 9 position of the acri-
Scheme 3. Conditions: i, POCl3 ii, a) R1H/K2CO3/DMF b) R1H/NaH/THF or DMF iii, Adenosin triphosphate disodium salt, Aliquat®, H2O:CH3CN.

I. Sánchez et al. / European Journal of Medicinal Chemistry 41 (2006) 340–352
343
Compound 38 which contains a bromine atom at the C-9
position was obtained from the chloro derivative 32 by treat-
ment with N-bromosuccinimide in methanol following the
standard procedure [15].
lioration in the preparation of these compounds versus the clas-
sical ways. The same procedure was applied to the double ha-
logenated acridine 38 and the corresponding ATP-O-acridine
13b was obtained in a low yield. These compounds containing
1
ATP unit were relatively stables. The spectrum of H-NMR of
The conversion of the chloroderivative intermediates in the
corresponding N- and O- substituted acridines was achieved by
applying classical procedures, following different methods of
alkylation. The acridines (6–8 and 10) possessing a diamino
chain in the C ring were prepared by nucleophilic substitution
from the corresponding chloroacridines 32–34 with the primary
alkylamine in dry dimethyl formamide (DMF) using K2CO3 as
a base [16]. These were again isolated and purified as free
bases. For the preparation of the aniline 9 and the ether 12,
the chloroacridine 32 was coupled with the appropriate aniline
or alcohol, respectively, using NaH as a base in DMF. Being
the most hindered substituent (3,4,5-trimethoxybenzyloxy) the
most difficult to couple at the C-11 position (compound 12).
For the preparation of the diaryl ether 11, K2CO3 was used as a
base or the phenoxy salt was previously formed by treatment
with Na in anhydrous methanol. This last method was prefer-
able and provides 11 in satisfactory yield. For the preparation
of 13a, which have an ATP (adenosine triphosphate) group at
the C-11 position, another synthetic method was selected. The
preparation of this compound requires very carefully controlled
conditions and preferably an inert atmosphere. The utilization
of ammonium salts as phase transfer catalysts for the introduc-
tion of an ATP group at the C-11 position of acridine deriva-
tives has not previously been reported. In this case, the treat-
ment of chloroderivative 32 with a twofold excess of the
adenosine triphosphate disodium salt with respect to the acri-
dine and a catalyst amount of Aliquat® in H2O/CH3CN (1:1)
under ultrasonic irradiation led to the formation of 13a. We
have found that ultrasonic irradiation affords a significant ame-
13a it confirms this stability on having turned out to be unal-
tered after three months. After one year the compound stored at
4 °C was hydrolysed in a 55% to the corresponding acridinone
(checked by NMR-spectra).
N-methylation of the acridinone 16 by treatment with excess
of methyl iodide in THF in presence of NaH afforded the N-
methylacridinone 30 which was reduced to 31 using LiAlH4 in
THF in 94% yield (Scheme 4).
Condensation of 32 with dimethoxyethylamine gave the
stable acetal 39, which was regioselectively cyclized by reflux
treatment with PTSA (p-toluenesulfonic acid monohydrate) in
toluene to the pentacyclic system 14 [16]. Subsequent treat-
ment of 14 with the diethylaminoethyl chloride in DMF pro-
vided 15 (Scheme 5). Pyrido[4,3,2-kl]acridine, tetracyclic ana-
logues related to the pentacyclic nucleus 14 were isolated from
marine organisms [17].
The formation of the nicotinic acid 40 was carried out by
nucleophilic substitution of the 2-chloropyridine-3-carboxilic
acid using NaH in DMF in 60% yield. Further, the cyclisation
in the presence of TFA gave only the “linear” isomer 41 in
72% yield. The cyclisation employing PPA or H2SO4 was un-
successful. For the preparation of the required 11-substituted
acridine, an alternative route via the corresponding acridone
41 appeared to be an attractive way for the direct amination.
The one pot reaction of 41 and dimethylamine hydrochloride in
hexamethylphosphoric triamide (HMPT) at 220 °C (20 h) gave
the 11-dimethylaminoacridine 18 in 40% yields without pas-
sing through the chloroacridine derivative [18].
Scheme 4. Conditions: i, NaH/CH3I/THF ii, LiAlH4/BH3/THF.
Scheme 5. Conditions: i, (CH3O)2CHCH2NH2/pyridine ii, PTSA/toluene iii, RCl/K2CO3/DMF.
Scheme 6. Conditions: i, NaH/DMF ii, TFA/CH2Cl2 iii, (CH3)2NH/HMPT.

344
I. Sánchez et al. / European Journal of Medicinal Chemistry 41 (2006) 340–352
Scheme 6 depicts the synthesis of 18, which has a pyridine
ring instead of the benzene ring in the acridine system. Both of
them in medicinal chemistry are considered bioisostere groups.
not result in any loss of potency. It is particularly interesting
to note that the ATP group afforded the most potent derivative
(compound 13a). This compound was 104- and 490-fold more
potent than compound 6 against L1210 and HT-29 cell lines,
respectively, and the only difference is the presence of the ATP
framework instead of the dialkylaminoethylamino group at the
C-11. However, the substituted analogue 13b shows a decrease
potency (13b was 157 fold less active than the acridine deriva-
tive 13a against L1210 cells and 442 fold less active than 13a
against HT-29 cells).
The in vitro data of the acridinones 16 and 17 depicted a
lower activity for these compounds compared with the corre-
sponding acridines due to they do not have a nucleofil group to
DNA binding. Also, the corresponding aminated compound 18
with a low antitumor activity suggests that the basic group on
the acridine system are significant for the enhancement of the
antitumor activity (18 have very low pKa, due to steric decon-
jugation). The decrease of activity of 16-18 is more notable
against HT-29 that against L1210. The introduction of a phe-
noxy group in the C-11 enhances a decrease of the antitumor
activity but the compounds were active. Compound 11 pos-
sesses a remarkable antitumor activity but is lower than that
displayed by 7. The substituents of the phenoxy group are im-
portant for the antitumor activity and the p-fluoro group is con-
sidered the best substituent for the antitumoral activity while
the 3,4,5-trimethoxyphenyl group showed a reduced activity
(11 was 4 fold more active than 12). Compound 9 possessing
substituted aniline showed less activity than the alkylamines 6
and 7. Substitution pattern in the C-9 position of the target
3. Results and discussion
Biological data for the compounds studied are presented in
Table 1 as IC50 values.
In vitro cytotoxic potency of these derivatives toward the
L1210 (leukemia) [18] and HT-29 (human colon carcinoma)
[19] cellular lines is described and compared to that of refer-
ence drugs, also are reported in Table 1 their effect on the
L1210 cell cycle. The structure-activity relationships in this
series were examinate by three types of structural changes: a)
Insertion of a substituent in the C-11 or C-12 position, b) the
introduction of a different substituent at the D ring of the acri-
dine system, and c) the linear or angular structure of the het-
erocyclic system.
Some of the new compounds possess an interesting cyto-
toxic activity against L1210 and HT-29 cellular lines. The
IC50 for every compound tested against each cell line are in
the micromolar range with the exception of 16–18 which are
the least effective in the series. The unsubstituted compound 6
is more active than the nitro derivative 8. The introduction of a
brome atom at C-9 position is detrimental for the activity.
When an aminoalkylamino group was appended at the C-11
position, the antitumor activity increased and reached its max-
imum value with a dimethylaminoethylamino group, but the
replacement of dimethylamino (7) by diethylamino (6) does
Table 1
Cytotoxicity of compounds 6–18
Compound
6
7
8
9
10
11
12
13-a
13-b
14
15
16
17
18
31
Structure
R1
a
b
a
c
b
d
e
R2
H
H
NO2
H
-
H
H
H
Br
H
H
H
OCH3
H
X
IC50 (μM) L1210
4.8 0.08
4.2 0.05
7.6 0.03
65.2 0.07
4.0 0.05
11.5 0.06
43.1 0.05
0.046 0.04
7.2 0.05
0.4 0.06
0.9 0.02
> 10 (83%)
> 10 (91%)
> 10 (82%)
> 10 (67%)
45.0 0.03
0.05 0.05
IC50 (μM) HT-29
5.9 0.03
4.0 0.04
4.7 0.02
37.2 1.02
3.8 0.05
16.2 0.06
64.5 0.04
0.012 0.03
5.3 0.04
0.8 0.03
0.5 0.02
N.S.
Cellular cycle
S/G2M (76%) 25 μM
G2M (85%) 10 μM
N.S. 50 μM
N.T.
G2M (88%) 5 μM
N.S.
N.T.
G1 (89%) 0.5 μM
N.S. 2 μM (apoptosis)
N.S. 2 μM
N.S. 5 μM (apoptosis)
N.S.
A
A
A
A
B
A
A
A
A
C
C
A
A
A
A
CH
CH
CH
CH
-
CH
CH
CH
CH
CH
CH
–
f
f
H
a
=O
=O
g
–
N.S.
N.S.
N.S.
37 0.05
0.08 0.09
N.S.
N.S.
N.S.
G2M (68%)
G2S (62%)
N
CH
–
–
9-Aminoacridine
m-AMSA
a) 2-(Diethyl)ethylendiamino. b) 2-(dimethyl)ethylendiamino. c) m-chloroanilino. d) p-fluorophenoxyde. e) (3,4,5-trimethoxy)benzyloxy. f) ATP. g) dimethyla-
mine. N.T. non tested. N.S. non significant.

I. Sánchez et al. / European Journal of Medicinal Chemistry 41 (2006) 340–352
345
compound was found to be of relevant interest. For these com-
pounds with electron-withdrawing or electron-donating substi-
tuents their activity were also shown in Table 1.
charged drug and nucleic acid functions difficult the DNA in-
tercalation. The SAR studies may, however, lead to the devel-
opment of new more potent compounds.
Interestingly, the fact that 13a induced a perturbation of the
cell cycle G1 different from that of classical acridines G2 sug-
gests that they act, at the molecular level, with a different me-
chanism of action. Moreover UV-visible studies of 13a show
that the cytotoxic activity is not by DNA intercalation.
Taking into account the cytotoxicity data observed above,
further modifications are underway to increase potency of the
studied acridines.
A brome atom leads to the less active compound but with
apoptosis effect (the activity decreased 157-fold, comparing
compounds 13a and 13b, but 13b showed problems of instabil-
ity) and a nitro group decreased 1.5 times the activity (see
compounds 6 and 8). The cytotoxicity was decreased for the
acridinones (16 and 17) and reduced acridines (18). Com-
pounds 13a, 14 and 15 represent a novel structural type of
antitumor agents. Removal of the alkylaminoalkyl group of
15 increases the cytotoxic activity on L1210 cell line, but in
contrast, decreases the activity against human colon carcinoma
(HT-29). It is worth noting that while 13a is a potent cytotoxic
compound the analogue cyclic 18 was poorly effective in all
biological tests. The much greater relative cytotoxicity of the
acridine series may be due to their lipophilic/hydrophilic char-
acter, which is known to contribute in vitro antitumor activity.
In order to explore further the role of the linear or angular
structure in conferring antitumoral activity the compounds 10,
14 and 15 were evaluated. The angular derivative 10 showed
similar activity to the linear analogue 7, while the pentacyclic
compound 14 was 10 fold times more potent than 7. Finally,
the N-substituted pentacyclic compound 15 showed less cyto-
toxic activity than the unsubstituted compound 14. The reason
for this is not clear; the possible correlation between hydrophi-
lic character and in vitro cytotoxic activity is not linear.
Some of these compounds could block the cell cycle pro-
gression of L1210 cells in the G2/M phase. Thus the angular
compound 10 was more specific on disturb cell cycle at G2/M
phase (> 85% at 5 μM). The linear acridine analogue 7 showed
also high selectivity for the G2/M phase of the cell cycle (in-
duced the accumulation of > 85% of cells at 10 μM). More
interesting was the cellular selectivity showed by the acridine
13a. This compound perturbs the cell cycle by accumulating
cells in the G1 phase (more of 85% in a 0.5 μM). In contrast,
the cytotoxicity of classical acridines induced a marked accu-
mulation of cells in the G2 phase. Perturbation of cell cycle
control in the G1 phase is a critical fact for the tumor progres-
sion. Therefore, compounds with the ability to arrest cells in
the G1 phase can be considered as a new class of efficient
compounds against tumors [20,21].
4. Experimental section
4.1. Chemistry
Melting points were obtained on a MFB-595010M Gallen-
kamp apparatus in open capillary tubes and are uncorrected. IR
spectra were obtained using a FTIR Perkin-Elmer 1600 Infra-
red Spectrophotometer. Only noteworthy IR absorptions are
1
listed (cm–1). H- and 13C-NMR spectra were recorded on a
Varian Gemini-200 (200 and 50.3 MHz, respectively) or Varian
Gemini-300 (300 and 75.5 MHz) Instrument using CDCl3 as
solvent with tetramethylsilane as internal standard or
1
1
(CD3)2CO. Other H-NMR spectra and heterocorrelation H-
13C (HMQC and HMBC) experiments were recorded on a Var-
ian VXR-500 (500 MHz). Chemical shifts are in parts per mil-
lion (ppm). Standard abbreviations are used. Mass spectra were
recorded on a Hewlett-Packard 5988-A using chemical ioniza-
tion (NH3 or CH4) or electronic impact. Column chromatogra-
phy was performed with silica gel (E. Merck, 70-230 mesh).
Reactions were monitored by TLC using 0.25 mm silica gel F-
254 (E. Merck). Microanalysis was determined on a Carlo
Erba–1106 analyzer. Compounds were analyzed for C, H, and
N, and analytical results obtained for these elements were with-
in 0.4% of the calculated values for the formula shown. All
reagents were of commercially quality or were purified before
use. Organic solvents were of analytical grade or were purified
by standard procedures. Commercial products were obtained
from Sigma-Aldrich. The organic extracts were dried over an-
1
hydrous Na2SO4. H-NMR and 13C-NMR data of the com-
pounds 16, 23, 30, 32 and 33 were checked with the results
reported before [4e,22].
In conclusion, we have designed and synthesized novel ac-
ridine analogues. In particular, the introduction of ATP sub-
structure enhanced the cytotoxic activity, and 13a represents
the most active compound of this series. The nature of the sub-
stituent in position C-11 is very important for cytotoxicity as
can be seen from the in vitro activity of the dialkylaminoalk-
ylamino group of 6 and 7 and the arylamino group of 9. Con-
cerning the chromophore, there are pronounced differences in
activity between class A and C, in the case compounds 14 and
15 (class C) are more effective than 6 and 7 (class A). 13a is
the most cytotoxic derivative and possesses an ATP substitu-
ent, this group is able to form hydrogen bonds with other polar
groups. On the other hand the triphosphate side substituent
with strong electrostatic repulsions between the negatively
4.1.1. General procedure to obtain N- and O- substituted
compounds
Method A. A suspension of the corresponding chloroacri-
dine (1 mmol), the appropriate amine (2.5 mmol) and anhy-
drous K2CO3 (2 mmol) in dry DMF (5 ml) was stirred at
80 °C for 3 h under an inert atmosphere. The cooled mixture
was extracted with ether (3 × 15 ml). The organic layers were
dried, filtered and concentrated under vacuum. The crude pro-
duct was subjected to column chromatography eluting with a
hexane/ethyl acetate 1:1 mixture as eluent.
Method B. A suspension formed by NaH (60%, 3 mmol),
and the corresponding aniline or alcohol (2 mmol) in 15 ml of

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I. Sánchez et al. / European Journal of Medicinal Chemistry 41 (2006) 340–352
anhydrous DMF (for the amine) or THF (for the alcohol), was
stirred at room temperature under argon atmosphere. After
40 min, the chloro-derivative 32 was drop-wise added (1 mmol)
and stirred at 100 °C for 24 h. The cooled mixture was hydro-
lyzed with water, the solvent removed and extracted with ether
(3 × 15 ml). The combined organic layers were dried, filtered
and concentrated. The crude product was purified by silica gel
column chromatography using hexane/ethyl acetate 1:1 as elu-
ent to give the corresponding N- or O- substituted compound.
150.9 (C, C-11). Anal. Calcd for C19H21N3O2; C, 70.57; H,
6.55; N, 12.99. Found: C, 70.87; H, 6.79; N, 12.83.
4.1.1.3. 9-Nitro-11-[2-(diethyl)ethylendiamine]-2,3-dihydro-
1,4-dioxino[2,3-b] acridine (8). Starting from the chloroacri-
dine 34 (0.08 g, 0.25 mmol), N,N-diethylethylendiamine
(0.073 g, 0.625 mmol) and following the general procedure
(method A), compound 8 (0.065 g, 68% yield) was obtained
1
as a colourless oil. H-NMR (CDCl3, 200 MHz) δ: 1.11 (t,
Method C. A suspension of anhydrous K2CO3 (6 mmol)
and the corresponding phenol (3 mmol) in dry DMF was stir-
red at room temperature under an inert atmosphere for 30 min.
Then, the suitable chloroacridine was added (1 mmol) and stir-
red at 100 °C for 6 h. The cooled mixture was extracted with
ether (3 × 15 ml) and the combined organic layers were dried,
filtered and concentrated under vacuum. The residue was chro-
matographed on a silica gel column using hexane/ethyl acetate
1/1 to afford the O-substituted compound.
J = 7 Hz, 6H, CH3CH2), 2.72 (m, 6H, CH2N), 3.81 (bs, 2H,
CH2N), 4.46 (m, 4H, CH2O), 6.63 (bs, 1H, NH), 7.32 (t,
J = 7.4 Hz, 1H, C7-H), 7.61 (t, J = 7.4 Hz, 1H, C8-H), 7.71
(s, 1H, C5-H), 7.98 and 8.06 (m, 2H, C10-H and C12-H). 13C
-NMR (CDCl3, 50.3 MHz) δ: 11.7 (CH3, CH3CH2), 45.9 and
46.1 (CH2, CH2N), 52.1 (CH2, CH2N), 64.0 and 65.3 (CH2,
CH2O), 108.9 (CH, C-5), 112.1 (C, C-10a), 116.2 (C, C-11a),
122.2 (CH, C-10), 122.9 (CH, C-12), 129.7 (CH, C-7), 130.0
(CH, C-8), 139.2 and 139.8 (C, C-4a, C-12a), 140.2 (C, C-9),
151.2 (C, C-6a), 152.0 (C, C-5a), 161.1 (C, C-11). MS (EI) (m/
z, %) 396 (M, 1), 293 (1), 267 (2), 86 (100). Anal. Calcd for
C21H24N4O4; C, 63.62; H, 6.10; N, 14.13. Found: C, 63.45; H,
6.34; N, 13.98.
4.1.1.1. 11-[2-(Diethyl)ethylendiamine]-2,3-dihydro-1,4-dioxi-
no[2,3-b]acridine (6). Compound 6 (0.12 g; 52% yield) was
obtained as a colourless oil following the general procedure
(method A) starting from compound 32 (0.18 g, 0.66 mmol)
and 2-(diethylamino)ethylamine (0.19 g, 1.65 mmol). IR
(KBr) υ cm–1: 3500 (N–H), 1485 (C=C), 1297 (Ar–O), 1065
4.1.1.4. 11-(m-Chlorophenylamino)-2,3-dihydro-1,4-dioxino
[2,3-b]acridine (9). Starting from 3-chloroaniline (0.14 g,
1.1 mmol), the acridine 32 (0.15 g, 0.55 mmol) and operating
via the general procedure (procedure B), compound 9 was ob-
tained as a colourless oil (0.065 g, 45%). IR (KBr) υ cm–1:
3388 (N-H), 1596 (Ar–H), 1127 (Ar–O), 1075 (C–O). 1H-
NMR (CDCl3, 200 MHz) δ: 4.25 (m, 4H, CH2O), 6.86 (m,
1H, Ar), 7.12 (m, 1H, Ar), 7.28 (m, 7H, Ar), 7.62 (m, 1H,
Ar), 7.93 (bs, 1H, NH). 13C-NMR (CDCl3, 50.3 MHz) δ:
64.3 and 64.4 (CH2, CH2O), 109.9 (CH, C-5), 113.8 (CH, C-
2′), 117.2 (C, C-10a), 117.3 (CH, C-12), 121.2 (C, C-11a),
127.1 and 127.4 (CH, C-7, C-9), 128.8, 130.2, 130.3 and
131.5 (CH, C-8, C-4′, C-5′, C-6′), 131.9 (CH, C-10), 135.2
(C, C-3′), 140.2 (C, C-6a), 143.3 and 143.4 (C, C-4a, C-12a),
143.5 (C, C-1′), 151.9 (C, C-5a), 161.9 (C, C-11). Anal. Calcd
for C21H15ClN2O2; C, 69.52; H, 4.17; N, 7.72. Found: C,
69.67; H, 4.36; N, 7.37.
1
(C–O). H-NMR (CDCl3, 200 MHz) δ: 1.12 (t, J = 7 Hz, 6H,
CH3), 2.65 (t, J = 7 Hz, 4H, CH2N), 2.69 (t, J = 7 Hz, 2H,
CH2N), 3.82 (t, J = 7 Hz, 2H, CH2N), 4.39 (m, 4H, CH2O),
7.23 (m, 2H, C8-H and C9-H), 7.58 and 7.59 (s, 2H, C5-H
and C12-H), 8.01 (d, J = 7 Hz, C10-H), 8.21 (d, J = 7 Hz,
C7-H). 13C-NMR (CDCl3, 50.3 MHz) δ: 46.1 (CH2, CH2N),
46.5 (CH2, CH2N), 52.5 (CH2, CH2N), 64.4 and 64.6 (CH2,
CH2O), 107.2 (CH, C-5), 112.5 (CH, C-12), 114.1 (C, C-
10a), 122.0 and 122.7 (CH, C-7, C-9), 124.2 (C, C-11a),
128.9 and 129.1 (CH, C-8, C-10), 141.9 and 142.0 (C, C-4a,
C-12a), 149.1 and 149.9 (C, C-9, C-5a, C-6a), 151.8 (C, C-11).
MS (EI) (m/z, %): 351 (M, 5), 293 (M-58, 2), 252 (M-99, 23),
86 (100). Anal. Calcd for C21H25N3O2; C, 71.77; H, 7.17; N,
11.96. Found: C, 72.01; H, 6.98; N, 11.67.
4.1.1.2. 11-[2-(Dimethyl)ethylendiamine]-2,3-dihydro-1,4-di-
oxino[2,3-b]acridine (7). Starting from acridine 32 (0.08 g,
0.25 mmol) and N,N-dimethylethylendiamine (0.05 g, 0.625
mmol) was obtained compound 7 as an oil (0.05 g, 68%), fol-
lowing the general procedure (method A). IR (NaCl) υ cm–1:
3395 (N–H), 1461(C=C), 1287 (Ar–O), 1121 (C–O). 1H-NMR
(CD3OD, 200 MHz) δ: 3.08 (s, 6H, CH3N), 3.40 (bs, 2H,
CH2N), 3.56 (bs, 2H, CH2N), 4.62 (m, 4H, CH2O), 7.39 (s,
1H, C5-H), 7.62 (bs, 2H, C9-H and C12-H), 7.77 (d,
J = 8 Hz, 1H, C7-H), 7.97 (bs, 1H, C8-H), 8.46 (d, J = 8 Hz,
1H, C10-H). 13C-NMR (CD3OD, 50.3 MHz) δ: 35.5 (CH3,
CH3N), 43.9 (CH2, CH2N), 55.2 (CH2, CH2N), 64.9 (CH2,
CH2O), 66.9 (CH2, CH2O), 111.7 (CH, C-5), 112.1 (C, C-
10a), 119.3 (CH, C-12), 122.0 (C, C-11a), 124.6 and 125.2
(CH, C-7, C-9), 128.9 (CH, C-8), 136.4 (CH, C-10), 138.1
and 138.5 (C, C-4a, C-12a), 148.1 and 149.2 (C, C-5a, C-6a),
4.1.1.5. 12[(2-Dimethyl)ethylendiamino]-2,3-dihydro-1,4-diox-
ino[3,4-b]acridine (10). Starting from compound 33 (0.05 g,
0.18 mmol), N,N-dimethylethylendiamine (0.04 g, 0.45 mmol)
and following the general procedure A it was obtained com-
pound 10 (0.04 g, 70% yield) as a yellow solid. m.p. (hex-
ane/ethyl acetate): 103–104 °C. 1H-NMR (CD3OD,
200 MHz) δ: 2.27 (s, 6H, CH3N), 2.47 (t, J = 8 Hz, 2H,
CH2N), 3.74 (t, J = 8 Hz, 2H, CH2-N), 7.23 (m, 2H, C10-H,
C11-H), 7.33 (d, J = 12 Hz, 1H, C5-H), 7.50 (t, J = 8 Hz, 1H,
C9-H), 6.75 (d, J = 12 Hz, 1H, C6-H), 8.15 (d, J = 8 Hz, 1H,
C8-H). 13C-NMR (CD3OD, 50.3 MHz) δ: 46.9 (CH3, CH3N),
51.7 (CH2, CH2N), 61.5 (CH2, CH2NAr), 66.4 (CH2, CH2O),
67.4 (CH2, CH2O), 102.3 (C, C-12a), 118.2 (C, C-11a), 120.5
(CH, C-5), 122.0 (CH, C-9), 124.8 (CH, C-6), 127,0 (CH, C-
10), 127.1 (CH, C-11), 131.4 (CH, C-8), 137.7 (C, C-4a and C-

I. Sánchez et al. / European Journal of Medicinal Chemistry 41 (2006) 340–352
347
12b), 139.2 (C, C-6a), 144.8 (C, C-7a), 146.8 (C, C-12). Anal.
Calcd for C19H21N3O2; C, 70.57; H, 6.55; N, 12.99. Found: C,
70.23; H, 6.88; N, 12.67.
cm–1: 3490 (OH), 2924 (NH), 1729 (C=N), 1260 (Ar–O), 1087
(C–O). H-NMR (CD3OD, 200 MHz) δ: 1.99 (bs, 5H, OH),
1
3.04 (s, 1H, CH-O), 3.24 (m, 2H, CH2O), 3.39 (s, 1H, CH-
O), 3.45 (m, 1H, CH-O), 4.23 (m, 4H, CH2-O), 7.11 (s, 1H,
C5-H), 7.18 (m, 2H, C9-H and C10-H), 7.41 (m, 1H, C7-H),
7.48 (m, 1H, C8-H), 7.87 (s, 1H, C12-H), 8.38 and 8.39 (m,
2H, C2-H and C6-H), 9.86 (bs, 2H, NH2). 13C-NMR (CD3OD,
50.3 MHz) δ: 48.1 (CH, CH-O), 61.5 (CH2, CH2O), 62.3
(CH2, CH2O), 63.1 (CH2, CH2O), 102.1 (CH, C-5), 109.8
(CH, C-9), 112.2 (CH, C-12), 116.1 (CH, C-7), 120.3 (CH,
C-8), 123.8 (CH, C-10), 126.1 (CH, C-2′), 132.3 (CH, C-6′),
134.8 (C, C-3a’), 136.1 (C, C-10a), 137.4 (C, C-11a), 138.1
(C, C-6a), 139.8 (C, C-12a), 140.3 (C, C-5a), 141.1 (C, C-
4a), 142.7 (C, C-11), 149.4 (C, C-7a), 178.1 and 178.9 (C,
C-4′). Anal.Calcd for C25H25P3N6O15; C, 40.45; H, 3.39; N,
11.32. Found: C, 40.41; H, 3.53; N, 11.11.
4.1.1.6. 11-(p-Fluorophenoxy-2,3-dihydro-1,4-dioxino[2,3-b]
acridine (11). Compound 11 (0.045 g, 64%) was obtained by
the general procedure (method C) from as the p-fluorophenol
(0.065 g, 0.6 mmol) and the acridine 32 (0.05 g, 0.19 mmol).
IR (KBr) υ cm–1: 1501 (Ar–H), 1189 (Ar–O), 1068 (C–O). 1H-
NMR (CDCl3, 200 MHz) δ: 4.39 (m, 4H, CH2O), 6.79 (m, 2H,
C2′-H and C6′-H), 7.40 (m, 2H, Ar), 7.67 (m, 2H, Ar), 7.99 (d,
J = 8.2 Hz, 1H, C10-H), 8.21 (d, J = 8.2 Hz, 1H, C7-H). 13C-
NMR (CDCl3, 50.3 MHz) δ: 64.3 and 64.4 (CH2, CH2O),
105.5 (CH, C-5), 112.8 (CH, C-7), 116.3 (C, C-10a), 116.5
(CH, d, J = 8 Hz, C-2′ and C-6′), 117.0 (CH, C-10), 119.3
(CH, C-12), 122.1 (CH, C-9), 125.1 (CH, C-8), 129.4 (CH,
d, J = 37 Hz, C-3′, C-5′), 144.8 (C, C-11a), 148.1, 148.9, and
149.5 (C, C-1′, C-11, C-4a, C-12a), 153.3 and 155.2 (C, C-5a,
C-6a), 158.6 (C, d, J = 265 Hz, C-4′). MS (m/z, %): 347 (M,
100), 252 (M-95, 14), 196 (3), 152 (6). Anal. (C21H14FNO3) C,
H, N. Anal.Calcd for C21H14FNO3; C, 72.62; H, 4.06; N, 4.03.
Found: C, 72.96; H, 4.34; N, 3.89.
4.1.1.9. 11-(Adenosin-5′-triphosphate)-9-bromo-2,3-dihydro-
1,4-dioxino[2,3-b]acridine (13b). Was prepared from the chlor-
oacridine 38 (130 mg, 0.37 mmol) as described above for 13a.
The residue was purified by silica gel column chromatography
(metanol/NH4OH) affording 75 mg (24% yield) of the com-
pound 13b as an oil. IR (KBr) υ cm–1: 3510 (OH, N–H),
2938 (NH), 1220 (Ar–O), 1082 (C–O). 1H-NMR (CD3OD,
200 MHz) δ: 2.01 (bs, 5H, OH), 3.12 (s, 1H, CH–O 3.29 (m,
2H, CH2O), 3.45 (s, 1H, CH–O), 4.30 (m, 4H, CH2–O), 7.08
(s, 1H, C5-H), 7.28 (m, 1H, Ar), 7.50 (m, 1H, C7-H), 7.56 (m,
1H, C8-H), 7.80 (s, 1H, C12-H), 8.30 (m, 2H, Ar), 8.98 (bs,
2H, NH2). This compound 13b was unstable.
4.1.1.7. 11-(3,4,5-Trimethoxybenzyloxy)-2,3-dihydro-1,4-dioxi-
no[2,3-b]acridine (12). Compound 12 (colorless oil, 0.16 g,
60% yield) was obtained using the general procedure (method
B) starting from 3,4,5-trimethoxybenzylalcohol (0.25 g, 1.24
mmol) and the chloroderivative 32 (0.17 g, 0.62 mmol). IR
(KBr) υ cm–1 (2 HCl): 3505 (N–H), 1204 (Ar–O), 1136 (C–O).
1H-NMR (CDCl3, 200 MHz) δ: 3.85 (m, 9H, CH3O), 4.25 (m,
4H, CH2O), 4.60 (s, CH2O), 6.62 (s, 2H, C2′-H and C6′-H),
7.33 (m, 5H, Ar), 8.22 (bs, 1H Ar). 13C-NMR (CDCl3,
50.3 MHz) δ: 55.6 (CH3, CH3O), 60.4 (CH3, CH3O), 63.7
(CH2, CH2O), 63.8 (CH2, CH2O), 64.7 (CH2, CH2O), 103.4
(CH, C-2′, C-6′), 112.2 (CH, C-5), 116.2 (C, C-10a), 116.6
(CH, C-12), 120.6 (CH, C-7), 124.2 (C, C-11a), 125.8 (CH,
C-9), 132.6 (CH, C-8), 137.0 (CH, C-10), 140.0 and 141.2
(C, C-4a, C-12a), 144.2 (C, C-5a, C-6a), 149.8 (C, C-11),
151,9 (C, C-1′), 152.8 (C, C-3′, C-4′, C-5′). MS (EI) (m/z, %)
4.1.1.10. 6,9-Dihydro-5H-dioxino[2,3-i]pyrido[4,3,2-kl]acri-
dine (14). A mixture of the acetal 39 (125 mg, 0.37 mmol) and
a catalytic amount of PTSA in dry toluene (20 ml) was stirred
in a 130 °C oil bath for 1 h. Then was allowed to room tem-
perature and the mixture was poured drop-wise into an aqueous
solution of ammonium hydroxide cooled to 0 °C and extracted
with ethyl acetate (3 × 20 ml). The organic layers were washed
with water, dried on sodium sulphate and the solvent was con-
centrated under reduced pressure. The residue was purified by
column chromatography using ethyl acetate 100% as eluent to
give the compound 14 as an orange solid (25 mg, 24% yield).
+
433 (M, 1), 368 (1), 252 (100), 253 (17), 197 (C10H13O4 , 59).
Anal.Calcd for C25H23NO6; C, 69.27; H, 5.35; N, 3.23. Found:
C, 69.01; H, 5.76; N, 3.56.
1
m.p. (hexane/ethyl acetate) 349–350 °C. H-NMR (CD3OD,
200 MHz) δ: 4.34 (s, 4H, CH2); 6.33 (s, 1H, C8-H); 7.01 (m,
2H, Ar); 7.16 (d, J = 6 Hz, 1H, C13-H); 7.38 (m, 1H, C9-H);
7.95 (d, J = 6 Hz, 1H, C10-H); 8.23 (d, J = 8 Hz, 1H, C3-H).
13C-NMR (CDCl3, 50.3 MHz) δ: 62.3 (CH2-O), 93.7 (CH, C-
8), 106.3 (CH, C-2), 112.5 (CH, C-10), 118.1 (CH, C-13),
123.6 (C, C-13a), 124.1 (C, Ar), 129.2 (CH, C-11 and C-12),
129.6 (CH, C-3), 136.4 (C, C-3a), 143.2 (CH, C-2), 144.3 (C,
C-7a, C-3b), 148.1 (C, C-8a and C-9a), 149.2 (C, C-13b).
Anal. Calcd for C17H12N2O2; C, 73.90; H, 4.38; N, 10.14.
Found: C, 73.21; H, 4.65; N, 9.89.
4.1.1.8. 11-(Adenosin-5′-triphosphate)-2,3-dihydro-1,4-dioxino
[2,3-b]acridine (13a).
A solution of chloroacridine 32
(120 mg, 0.44 mmol) and KI (103.5 mg, 0.62 mmol) in
CH3CN 10 ml was stirred under argon atmosphere at room
temperature for 60 minutes. After adenosin triphosphate diso-
dium salt (408 mg, 0.74 mmol) and Aliquat® (0.3 ml) in
CH3CN:H2O 1:1 (20 ml) were added and the mixture was in-
troduced in an ultrasonic irradiation bath for 24 h. Finally the
CH3CN was removed and the mixture was extracted with with
CH2Cl2. The resulting organic layers were dried, filtered and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (metanol /NH4OH) af-
fording 150 mg (46% yield) of the compound 13a. IR (KBr) υ
4.1.1.11. 6-(N,N-diethylaminoethyl)-6,9-dihydro-5H-dioxino
[2,3-i]pyrido[4,3,2-kl]acridine (15). Prepared from the com-
pound 14 (100 mg, 0.35 mmol) and 2-diethylaminoethyl chlor-

348
I. Sánchez et al. / European Journal of Medicinal Chemistry 41 (2006) 340–352
ide hydrochloride (200 mg, 1.05 mmol) as described above in
(52 mg, 0.64 mmol) in HMPT (1 ml) was stirred at 220 °C
for 20 hours. The cooled mixture was extracted with ether
(3 × 15 ml) and the combined organic layers were washed sev-
eral times with water in order to eliminate the HMPT, dried,
filtered and concentrated. The desired compound 18 was ob-
tained as yellow oil after purification by silica gel column chro-
matography using hexane / ethyl acetate 80:20 mixture as an
general procedure A, the compound 15 was obtained as a dark
1
oil (98 mg, 75% yield). H-NMR (CD3OD, 200 MHz) δ: 1.09
(t, J = 7 Hz, 6H, CH3), 2.65 (q, J = 7 Hz, 6H, CH2-N), 3.78 (t,
J = 7 Hz, 2H, CH2), 4.32 (s, 4H, CH2), 6.20 (s, 1H, C8-H),
7.02 (m, 3H, Ar), 7.39 (d, J = 8.2 Hz, 1H, C10-H), 7.97 (d,
J = 6 Hz, 1H, C13-H), 8.23 (d, J = 8.2 Hz, 1H, C2-H). 13C-
NMR (CD3OD, 50.3 MHz) δ: 9.3 (CH3), 42.9 (CH2), 45.0
(CH2), 45.9 (CH2), 61.7 (CH2-O), 62.6 (CH2-O), 93.0 (CH,
C-8), 106.9 (CH, C-12), 111.2 (CH, C-13), 117.9 (CH, C-11),
122.6 (CH, C-10), 123.1 (C, Ar), 124.5 (C, C-13a), 128.8 (CH,
C-3), 138.9 (C, C-3a), 144.6 (C, C-7a, C-3b), 141.8 (CH, C-2),
147.2 (C, C-8a, C-9a), 148.4 (C, C-13b). Anal. Calcd for
C23H25N3O2; C, 73.57; H, 6.71; N, 11.19. Found: C, 73.87;
H, 6.98; N, 10.87.
1
eluent (39 mg, 39% yield). H-NMR (CDCl3, 200 MHz) δ:
2.68 and 2.69 (s, 3H, CH3N); 4.20 (m, 4H, CH2O); 6.54 (m,
1H, C9-H); 6.71 (d, J = 1.2 Hz, 1H, C12-H); 6.97 (dd, J1 = 11,
J2 = 2.2 Hz, C9-H), 7.20 (d, J = 1.2 Hz, 1H, C5-H); 8.25 (d,
J = 2.2 Hz, 1H, C10-H); 8.48 (d, J = 11.2 Hz, C8-H). 13C-
NMR (CDCl3, 50.3 MHz) δ: 36.6 and 36.9 (CH3, CH3N);
64.38 and 64.40 (CH2, CH2O), 109.7 (CH, C-5), 113.5 (CH,
C-12), 117.1 (CH, C-9), 130.4 (C, C-10a), 131.2 (C, C-11a),
140.3 (C, C-4a and C-12a), 141.4 (C, C-11), 143.3 (C, C-5a),
144.0 (C, C-6a), 159.3 (CH, C-10), 162.8 (CH, C-8). Anal.
Calcd for C16H15N3O2; C, 68.31; H, 5.37; N, 14.94. Found:
C, 68.78; H, 5.76; N, 14.65.
4.1.2. General procedure to obtain ketones
A suspension of the corresponding 2-(2,3-dihydro-1,4-ben-
zodioxin-6-ylamino)-5-substituted benzoic acid (1 mmol) and
trifluoroacetic acid (catalyst amount) in dry CH2Cl2 was stirred
at reflux temperature for 24 h. The cooled mixture was basified
(NaOH 1 N) and extracted with ether (3 × 25 ml). The com-
bined organic layers were dried, filtered and concentrated. The
residue was purified by silica gel column chromatography
(ethyl acetate/hexane 1:1).
4.1.3. General procedure for the Ullmann reaction
A mixture of 6-amino-2,3-dihydro-1,4-benzodioxin 19
(2 mmol), the appropriate 5-substituted-2-chlorobenzoic acid
(1 mmol), anhydrous K2CO3 (3 mmol) and Cu (catalyst
amount) in xylene (40 ml) was stirred at reflux temperature
for 4 hours. The cooled mixture was acidified with HCl 2N
and the resulting product was extracted with ether (3 × 20
ml). The organic layers were dried, filtered and concentrated
under reduced pressure. The crude product was subjected to
column chromatography on silica gel eluting with ethyl acet-
ate/methanol 90:10 mixture to give the corresponding benzoic
acid derivatives.
4.1.2.1. 2,3,6,11-Tetrahydro-1,4-dioxino[2,3-b]acridin-11-
one (16). The acridone 16 was obtained as a white solid (0.32
g, 85% yield) following the general procedure to prepare acri-
dones starting from the carboxylic acid 23 (0.4 g, 1.31 mmol).
m.p. (ethyl acetate/hexane): 246–248 °C. IR (KBr) υ cm–1:
3500 (NH), 1635 (C=O), 1299 (Ar–O), 1065 (C–O). 1H-NMR
(CDCl3, 200 MHz) δ: 3.02 (bs, 1H, NH), 4.28 (s, 4H, CH2O),
6.73 (m, 2H, C5-H, C8-H), 7.06 (d, J = 8.4 Hz, 1H, C7-H),
7.34 (m, 1H, C9-H), 8.00 (m, 2H, C10-H, C12-H). 13C-NMR
(CDCl3, 50.3 MHz) δ: 63.9 and 65.0 (CH2, CH2O), 102.7 (CH,
C-5), 112.8 (CH, C-7), 114.5 (C, C-11a), 116.4 (CH, C-12),
120.7 (CH, C-9), 122.3 (C, C-10a), 126.5 (CH, C-10), 132.6
(CH, C-8), 140.1 and 141.3 (C, C-5a, C-12a), 145.1 (C, C-6a),
149.2 (C, C-4a), 178.0 (C, C=O). Anal. Calcd for C15H11NO3;
C, 71.14; H, 4.38; N, 5.53. Found: C, 71.59; H, 4.56; N, 5.67.
4.1.3.1. 2-(2,3-Dihydro-1,4-benzodioxin-6-yl)aminobenzoic
acid (23). Starting from the 2-chlorobenzoic acid (20) (2 g,
12.7 mmol) and the amine 19 (5.4 g, 35.7 mmol) and following
the general procedure of Ullmann reaction, compound 23
(2.5 g, 74% yield) was obtained as a white solid. Mp (ethyl
acetate/hexane): 126–128 °C. IR (KBr) υ cm–1: 3309 (–OH, –
NH), 1690 (C=O), 1150 (C–O). 1H-NMR (CDCl3-CD3OD,
200 MHz) δ: 4.20 (m, 4H, CH2O), 6.80–7.48 (m, 5H, Ar),
8.02 (d, J = 7.8 Hz, 1H, C3-H), 8.12 (d, J = 7.8 Hz, 1H, C6-H).
13C-NMR (CDCl3–CD3OD, 50.3 MHz) δ: 64.3 and 64.4 (CH2,
CH2O), 116.2 (C, C-1), 117.7 (CH, C-5′), 126.7 (CH, C-3),
128.4 (CH, C-7′), 130.2 (CH, C-8′), 131.4 (CH, C-5), 133.5
(CH, C-6), 133.8 (CH, C-4), 140.5 and 140.8 (C, C-4′a, C-8′
a), 141.7 (C, C-6′), 149.8 (C, C-2), 170.9 (C, COOH).
4.1.2.2. 9-Methoxy-2,3,6,11-tetrahydro-1,4-dioxino[2,3-b]acri-
din-11-one (17). The acridone 17 (0.18 g, 64% yield) was pre-
pared from the carboxylic acid 25 (0.3 g, 0.89 mmol) following
the general procedure described above. Mp (ethyl acetate / hex-
ane): > 275 °C. IR (KBr) υ cm–1: 3500 (–OH), 1630 (C=O),
1
1298 (Ar–O), 1110 (C–O). H-NMR (CDCl3, 200 MHz) δ:
3.79 (s, 3H, CH3O), 4.23 (m, 4H, CH2–O), 6.74 (s, 1H, C5-
H), 7.17 (m, 2H, C7-H, C8-H), 7.57 (m, 1H, C10-H), 7.71 (s,
1H, C12-H). Anal. Calcd for C16H13NO4; C, 67.84; H, 4.63;
N, 4.94. Found: C, 67.99; H, 4.85; N, 5.08.
4.1.3.2. 2-(2,3-Dihydro-1,4-benzodioxin-6-yl)amino-5-nitro-
benzoic acid (24). Compound 24 was obtained as a yellow so-
lid (3 g, 83% yield) from the 2-chloro-5-nitrobenzoic acid (21)
(5.33 g, 26.45 mmol) and the aniline 19 (2.08 g, 13.7 mmol)
and following the general procedure described above of Ull-
mann reaction. Mp (ethyl acetate / hexane): 135–136 °C. IR
4.1.2.3. Dimethylamino-2,3-dihydro-1,4-dioxino[2,3-g]pyrido
[2,3-b]quinoline (18). A suspension of tetracyclic ketone 41
(80 mg, 0.32 mmol) and dimethylamine hydrochloride
1
(KBr) υ cm–1: 3500 (–OH), 1656 (C=O), 1064 (C–O). H-
NMR (CDCl3–CD3OD, 200 MHz) δ: 4.31 (m, 4H, CH2O),

I. Sánchez et al. / European Journal of Medicinal Chemistry 41 (2006) 340–352
349
7.71 (m, 2H, C5′-H, C8′-H), 8.47 (m, 2H, C3-H, C7′-H), 8.94
(m, 2H, C4-H, C6-H), 9.60 (bs, 2H, OH, NH). 13C-NMR
(CDCl3-CD3OD, 50.3 MHz) δ: 64.3 and 64.4 (CH2, CH2O),
125.2 (CH, C-3), 126.9 (CH, C-6), 128.1 (CH, C-7′), 129.8
(CH, C-4), 131.1 (C, C-1), 133.4 (CH, C-5′); 135.8 (CH, C-
8′); 141.2 (C, C-8′a); 142.1 (C, C-4′a); 144.2 (C, C-5); 146.7
(C, C-6′); 148.3 (C, C-2), 172.1 (C, COOH).
Anal.Calcd for C25H10N2O5; C, 60.41; H, 3.38; N, 9.39.
Found: C, 60.65; H, 3.76; N, 9.55.
4.1.3.6. Methyl-2,3,6,11-tetrahydro-1,4-dioxino[2,3-b]acridin-
11-one (30). Under an inert atmosphere, a suspension of NaH
(60% in oil, 0.043 g, 1.78 mmol), the ketone 16 (0.15 g, 0.59
mmol) and CH3I (0.11 ml, d 2.28, 1.78 mmol) in dry THF (15
ml) was stirred at 50 °C until the starting material was comple-
tely transformed (TLC elution with 7:3 hexane/ethyl acetate
mixture). Then, the THF was removed in vacuum and the
crude residue was extracted with CH2Cl2 (3 × 10 ml). The or-
ganic layers were dried, filtered and concentrated under re-
duced pressure. The residue was purified by silica gel column
chromatography (hexane/ethyl acetate 80:20) to give 30 (0.11
g, 69%) as a solid. m.p. (hexane/ethyl acetate): 229–230 °C. IR
4.1.3.3. 2-(2,3-Dihydro-1,4-benzodioxin-6-yl)amino-5-methox-
ybenzoic acid (25). Compound 25 (0.9 g, 56% yield) was ob-
tained following the general procedure of Ullmann reaction
from 2-chloro-5-methoxybenzoic acid (22) (1 g, 5.3 mmol)
and aniline 19 (2.25 g, 14.9 mmol). Mp (ethyl acetate/hexane):
215–216 °C. IR (KBr) υ cm–1: 3460 (–OH), 1682 (C=O), 1280
1
(Ar–O), 1054 (C–O). H-NMR (CDCl3–CD3OD, 200 MHz) δ:
1
(KBr) υ cm–1: 1594 (C=O), 1241 (Ar–O), 1065 (C–O). H-
3.79 (s, 3H, CH3O), 4.27 (s, 4H, CH2O), 6.77 (m, 3H, C5′-H,
C7′-H, C8′-H), 7.00 (m, 2H, C3-H, C4-H), 7.5 (d, J = 1.2 Hz,
1H, C6-H).
NMR (CDCl3, 200 MHz) δ: 3.71 (s, 3H, CH3), 4.31 (m, 4H,
CH2O), 6.88 (s, 1H, C5-H), 7.21 (t, J = 8 Hz, 1H, C9-H), 7.40
(d, J = 8.8 Hz, 1H, C7-H), 7.62 (t, J = 8 Hz, 1H, C8-H), 7.91
(s, 1H, C12-H), 8.45 (d, J = 8 Hz, 1H, C10-H). 13C-NMR
(CDCl3, 50.3 MHz) δ: 33.7 (CH3, CH3), 64.0 and 65.1 (CH2,
CH2O), 102.0 (CH, C-5), 114.2 (CH, C-7), 114.4 (CH, C-12),
117.5 (C, C-11a), 120.7 (CH, C-9), 121.2 (C, C-10a), 127.6
(CH, C-10), 133.2 (CH, C-8), 139.0 and 139.5 (C, C-4a and
C-12a), 141.2 (C, C-6a), 149.5 (C, C-5a), 176.0 (C, C=O).
Anal. Calcd for C16H13NO3; C, 71.90; H, 4.90; N, 5.24.
Found: C, 72.01; H, 5.23; N, 5.56
4.1.3.4. Nitro-2,3,6,11-tetrahydro-1,4-dioxino[2,3-b]acridin-
11-one (26). A solution of acridone 16 (0.10 g, 0.29 mmol) in a
mixture of acetic acid (5 ml) and nitric acid (0.05 ml) was
cooled at 0 °C and stirred at room temperature overnight. The
resulting mixture was basified with 2 N NaOH and the crude
product was extracted with ether (3 × 10 ml). The organic
layers were dried, filtered and concentrated under reduced pres-
sure. The residue was purified by silica gel column chromato-
graphy (hexane/ethyl acetate 90:10) to give 26 (0.08 g, 68%)
as a solid. m.p. (ethyl acetate/hexane): 247–248 °C. IR (KBr) υ
cm–1: 1603 (C=O), 1505 (NO2), 1136 (Ar–O), 1079 (C–O). 1H
-NMR (CDCl3, 200 MHz) δ: 4.31 (s, 4H, CH2O), 6.96 (d,
J = 8.8 Hz, C7-H), 7.49 (bs, 1H, C5-H), 7.66 (s, 1H, C12-H),
8.31 (dd, J1 = 8.8, J2 = 3 Hz, 1H, C8-H), 8.85 (d, J = 3 Hz, 1H,
C10-H). 13C-NMR (CDCl3, 50.3 MHz) δ: 64.2 and 64.6 (CH2,
CH2O), 110.6 (CH, C-5), 113.4 (C, C-10a), 114.2 (C, C-11a),
117.3 and 117.9 (CH, C-8, C-12), 127.3 (CH, C-10), 130.4
(CH, C-7), 140.3 (C, C-9), 143.7 (C, C-4a, C-12a), 145.3 and
146.4 (C, C-5a, C-6a), 179.0 (C, C=O). MS (m/z, %) 298 (M,
60), 272 (17), 226 (5), 201 (53), 167 (39), 135 (78), 65 (100).
Anal. Calcd for C15H10N2O5; C, 60.41; H, 3.38; N, 9.39.
Found: C, 60.65; H, 3.67; N, 9.12.
4.1.3.7. Methyl-2,3,6,11-tetrahydro-1,4-dioxino[2,3-b]acridine
(31). Under an inert atmosphere, a suspension of acridinone 30
(0.06 g, 0.23 mmol) and LiAlH4 (0.034 g, 0.90 mmol) in dry
THF (15 ml) was stirred at room temperature for 9 h. After,
BH3 was added (1.5 ml) and the reaction mixture was heated at
50 °C for 12 h. Then, the cooled mixture was extracted with
ether (3 × 15 ml) and the combined organic layers were dried,
filtered and concentrated in vacuum. The residue was purified
by silica gel column chromatography (hexane/ethyl acetate
95:5) to give 31 (0.05 g, 94%) as a colourless oil. IR (NaCl) υ
1
cm–1: 1502, 1240 (Ar–O–C), 1064 (C–O). H-NMR (CDCl3,
200 MHz) δ: 3.30 (s, 3H, CH3N), 3.78 (s, 2H, CH2), 4.22 (m,
4H, CH2O), 6.41 (s, 1H, C5-H), 6.69 (s, 1H, C12-H), 6.87 (m,
2H, C7-H and C9-H), 7.16 (m, 2H, C8-H and C10-H). 13C-
NMR (CDCl3, 50.3 MHz) δ: 32.4 (CH2, CH2Ar), 33.1 (CH3,
CH3N) 64.3 and 64.7 (CH2, CH2O), 101.2 (CH, C-5), 111.7
(CH, C-12), 115.2 (C, C-11a), 116.5 (CH, C-9), 119.9 (CH,
C-8), 121.2 (C, C-10a), 126.7 (CH, C-10), 128.2 (CH, C-7),
138.1 and 139.0 (C, C-4a, C-12a), 141.3 and 141.7 (C, C-5a,
C-6a). MS (m/z, %): 253 (M, 5), 252 (41), 237 (100), 153 (47).
Anal. Calcd for C16H15NO2; C, 75.87; H, 5.97; N, 5.53.
Found: C, 75.98; H, 5.65; N, 5.88.
4.1.3.5. Nitro-2,3,7,12-tetrahydro-1,4-dioxino[2,3-a]acridin-
12-one (28). Following the general procedure indicated above
from the 2-(2,3-dihydro-1,4-benzodioxin-6-amino)-5-nitroben-
zoic acid (24) (0.5 g, 1.58 mmol). The residue was purified
by silica gel column chromatography (hexane/ethyl acetate
90:10) to give 26 (0.1 g, 22% yield) and 28 (0.3 g, 64%) as
a white solids. Analytical data of 28: m.p. (ethyl acetate/hex-
ane): 245–247 °C. IR (KBr) υ cm–1: 3311 (NH), 1674 (C=O),
1579 (NO2), 1128 (Ar-O), 1065 (C–O). 1H-NMR (CDCl3,
200 MHz) δ: 4.30 (m, 4H, CH2O), 6.78 (m, 2 H, C5-H and
C6-H), 6.94 (dd, J1 = 9.4, J2 = 8.4 Hz, 1H, C8-H), 8.06 (dd,
J1 = 9.4, J2 = 2.8 Hz, 1H, C9-H), 8.90 (d, J1 = 2.8, J2 = 3 Hz,
1H, C11-H). MS (m/z, %) 298 (M, 55), 272 (35), 65 (100).
4.1.4. General procedure to prepare the chloroacridine
derivatives
Method A. A solution containing the tetracyclic ketone
(1 mmol) in POCl3 (2 ml) was stirred at reflux temperature
for 3 h. The cooled mixture was extracted with CH2Cl2

350
I. Sánchez et al. / European Journal of Medicinal Chemistry 41 (2006) 340–352
(3 × 20 ml) and the combined organic layers were washed with
a saturated solution of NaHCO3, dried, filtered and concen-
trated under reduced pressure. The resulting product was pur-
ified by silica gel column chromatography (hexane/ethyl acet-
ate 50:50) giving the corresponding chlore derivative.
J = 8.8 Hz, C8-H). Anal.Calcd for C15H9ClN2O4; C, 56.89; H,
2.86; N, 8.85. Found: C, 57.15; H, 2.99; N, 8.96.
4.1.4.3. 9-Methoxy-11-chloro-2,3-dihydro-1,4-dioxino[2,3-b]
acridine (36) and 10-Methoxy-12-chloro-2,3-dihydro-1,4-diox-
ino [3,4-b]acridine (37). Compounds 36 (0.25 g, 50% yield)
and 37 (0.104 g, 21% yield) were obtained by the general pro-
cedure described above (method B) from the carboxylic acid
25 (0.5 g, 1.66 mmol).
Method B. A suspension of the corresponding substituted
benzoic acid and POCl3 (4 ml) was stirred at reflux tempera-
ture for 4 h. Then, the cooled mixture was concentrated under
vacuum, basified with NaOH 2N and extracted with CH2Cl2.
The organic layers were dried, filtered and the solvent removed
under reduced pressure. The purification of the crude product
by silica gel column chromatography (hexane/ethyl acetate 1:1)
afforded the two tetracyclic chlore-derivatives linear and angu-
lar isomers.
Compound 36. m.p. (hexane/ethyl acetate): 235–237 °C. IR
1
(NaCl) υ cm–1: 1500 (C=C), 1226 (Ar–O), 1069 (C–O). H-
NMR (CDCl3, 200 MHz) δ: 4.01 (s, 3H, CH3O); 4.44 (s, 4H,
CH2O); 7.43 (m, 2H, C5-H, C8-H); 7.63 (s, 1H, C12-H); 7.74
(s, 1H, C10-H); 8.04 (d, J = 9.2 Hz, C7-H).
Compound 37. m.p. (hexane/ethyl acetate): 173–175 °C. IR
(NaCl) υ cm–1: 1470 (C=C), 1265 (Ar–O), 1123 (C–O). H-
NMR (CDCl3, 200 MHz) δ: 4.03 (s, 3H, CH3O); 4.46 (m, 4H,
CH2O); 7.40 (m, 2H, C5-H, C9-H); 7.64 (d, J = 3 Hz, C11-H);
7.80 (d, J = 12 Hz, C6-H); 8.15 (d, J = 12 Hz, C8-H).
1
4.1.4.1. 11-Chloro-2,3-dihydro-1,4-dioxino[2,3-b]acridine (32)
and 12-chloro-2,3-dihydro-1,4-dioxino [3,4-b]acridine (33).
Compound 32 (0.45 g, 61% yield) and its isomer 33 (0.28 g,
38% yield) were obtained following the general procedure de-
scribed above (method B) starting from the carboxylic acid 23
(0.73 mg, 0.27 mmol).
4.1.4.4. 9-Bromo-11-chloro-2,3-dihydro-1,4-dioxino[2,3-b]ac-
ridine (38). N-Bromosuccinimide (0.064 g, 0.36 mmol) was
added to a cooled solution (0 °C) of compound 32 (0.1 g,
0.36 mmol) in acetonitrile (10 ml) and the mixture was stirred
at room temperature for 3 h. Then, the solvent was removed
and a solution of 5 N NaOH was added (10 ml). The suspen-
sion obtained was extracted with ether (3 × 10 ml), dried, fil-
tered and concentrated. The bromo derivative 38 (0.05 g, 40%
yield) was obtained as a yellow oil after purification of the
crude of reaction by silica gel column chromatography (hex-
ane/ethyl acetate 70:30). IR (KBr) υ cm–1: 1520 (C=N), 1282
(Ar–O), 1105 (C–O), 875 (C–Br). 1H-NMR (CDCl3,
200 MHz) δ: 4.47 (m, 2H, CH2O); 4.59 (m, 2H, CH2O); 7.62
(m, 1H, C8-H); 7.80 (m, 2H, C5-H, C12-H); 8.30 (m, 2H, C7-
H, C10-H). Anal. Calcd for C15H9BrClNO2; C, 51.89; H, 2.59;
N, 4.00. Found: C, 52.12; H, 2.67; N, 4.34.
Compound 32 (0.15 g, 75% yield) can also be obtained fol-
lowing the method A from the ketone 16 (0.2 g, 0.74 mmol).
Compound 32. m.p. (hexane): 115–117 °C. 1H-NMR
(CDCl3, 300 MHz) δ: 4.45 (s, 4H, CH2O), 7.55 (dt, J1 = 6, J2
= 0.7 Hz, 1H, C9-H), 7.63 (s, 1H, C12-H), 7.72 (dt, J1 = 6, J2
= 0.7 Hz, 1H, C8-H), 7.79 (s, 1H, C5-H), 8.12 (d, J = 6 Hz,
1H, C7-H), 8.33 (dd, J1 = 6, J2 = 0.5 Hz, 1H, C10-H). 13C-
NMR (CDCl3, 50.3 MHz) δ: 63.9 and 64.1 (CH2, CH2O),
123.3 (CH, C-5), 124.2 (CH, C-10), 124.6 (CH, C-9), 124.0
(C, C-12a), 126.8 (CH, C-7), 129.2 (C, C-10a), 129.5 (CH,
C-8), 129.7 (CH, C-7), 135.9 (C, C-11), 137.0 (C, C-4a),
138.1 (C, C-5a), 146.0 (C, C-6a). Anal. Calcd for
C15H10ClNO2; C, 66.31; H, 3.71; N, 5.16. Found: C, 66.54;
H, 3.86; N, 5.52.
Compound 33. m.p. (hexane): 189–191 °C. 1H-NMR
(CDCl3, 200 MHz) δ: 4.30 (m, 4H, CH2O), 7.40 (d,
J = 9.6 Hz, 1H, C5-H), 7.60 (m, 1H, C9-H), 7.70 (m, 1H,
C10-H), 7.75 (d, J = 9.6 Hz, 1H, C6-H), 8.17 (d, J = 8 Hz,
C8-H), 8.45 (d, J = 8 Hz, C11-H). 13C-NMR (CDCl3,
50.3 MHz) δ: 64.0 and 64.1 (CH2, CH2O), 117.7 (C, C-11a),
121.6 (CH, C-5), 124.5 (CH, C-11), 125.1 (C, C-12a), 125.7
(CH, C-9), 127.1 (CH, C-10), 127.8 (CH, C-6), 130.7 (CH, C-
8), 136.1 (C, C-12), 138.1 and 139.4 (C, C-6a, C-12b), 144.3
and 145.3 (C, C-4a, C-7a). Anal.Calcd for C15H10ClNO2; C,
66.31; H, 3.71; N, 5.16. Found: C, 66.65; H, 3.85; N, 5.37.
4.1.4.5. 11-Aminoacetaldehyde
dimethylacetal-2,3-dihydro-
1,4-dioxino[2,3-b]acridine (39). A solution of the compound
32 (0.1 g, 0.37 mmol) and aminoacetaldehyde dimethylacetal
(0.1 g, 0.92 mmol) in 2 ml of pyridine was stirred at 200 °C for
4 h into a modified Schlenk tube. After evaporation of the sol-
vent the aminoacetal 39 was obtained (0.098 g, 78%) as a yel-
low solid, which was directly used in the next reaction without
purification because of it instability. 1H-NMR (CDCl3,
200 MHz) δ: 3.45 (s, 6H, CH3), 4.17 (d, J = 5 Hz, 2H,
CH2N), 4.46 (m, 4H, CH2–O), 4.91 (m, 1H, CH), 5.48 (s,
1H, NH), 7.18 (s, 1H, C5-H), 7.41 (m, 1H, Ar), 7.68 (m, 1H,
Ar), 7.82 (m, 1H, Ar), 7.95 (s, 1H, C12-H), 8.41 (d,
J = 8.2 Hz, 1H, C7-H). 13C-NMR (CDCl3, 50.3 MHz) δ: 38.2
(CH2–N), 55.1 (CH3), 64.0 and 60.5 (CH2–O), 102.3 (CH, C-
5), 104.5 (CH, C-12), 108.2 (CH, C-7), 112.5 (C, C-10a),
119.1 (CH, C-9), 121.8 (CH, C-8), 123.0 (C, C-11a), 138.2
(C, C-4a, C-12a), 142.8 and 143.1 (C, C-5a and C-6a), 151.1
(C, C-11). Anal. Calcd for C19H21N2O4; C, 67.05; H, 5.92; N,
8.23. Found: C, 67.33; H, 6.23; N, 8.45.
4.1.4.2. 9-Nitro-11-chloro-2,3-dihydro-1,4-dioxino[2,3-b]acri-
dine (34). Compound 34 (0.9 g, 57% yield) was prepared from
the ketone 26 (0.15 g, 0.5 mmol) by following the general pro-
cedure described above (method A). IR (KBr) υ cm–1: 1530
(NO2), 1490 (C=C), 1240 (Ar–O), 1102 (C–O). 1H-NMR
(CDCl3, 200 MHz) δ: 4.53 (m, 4H, CH2O); 7.62 (t,
J = 7.8 Hz, 1H, Ar); 7.78 (dd, J1 = 7.8, J2 = 0.7 Hz, 1H, Ar);
7.91 (s, 1H, C5-H); 8.15 (d, J = 8.8 Hz, 1H, C7-H); 8.83 (d,

I. Sánchez et al. / European Journal of Medicinal Chemistry 41 (2006) 340–352
351
4.1.4.6. 2-(2,3-Dihydro-1,4-benzodioxin-6-yl)aminonicotinic
acid (40). A suspension of 6-amino-2,3-dihydro-1,4-benzo-
dioxin 19 (300 mg, 2 mmol), the 2-chloronicotinic acid
(621 mg, 4 mmol) and NaH (60%, 105 mg, 4.3 mmol) in dis-
tilled DMF (3 ml) was stirred in an argon atmosphere at 90 °C
for 24 h. The cooled mixture was neutralized with 2N HCl and
extracted with ether (3 × 20 ml). The organic layers were dried,
filtered and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (hexane/
ethyl acetate 70:30) to give the corresponding nicotinic acid
40 as a white solid. m.p. (hexane/ethyl acetate): 130–131 °C.
IR (KBr) υ cm–1: 3400 (O–H and N–H), 1682 (C=O), 1290
(O–Ar), 1064 (C–O). 1H-NMR (CDCl3, 200 MHz) δ: 4.21
(m, 4H, CH2O), 6.79 (m, 3H, C5-H, C5′-H, C8′-H), 7.18 (d,
J = 2.6 Hz, 1H, C7′-H), 8.26 (bs, 2H, NH, OH), 8.54 (d,
J = 7.4 Hz, C4-H), 8.79 (d, J = 7.6 Hz, C6-H). Anal.Calcd for
C14H12N2O4; C, 61.76; H, 4.44; N, 10.29. Found: C, 62.10; H,
4.76; N, 10.45.
4.2.2. Inhibition of cellular proliferation and cell cycle effects
[19]
L1210 leukemia cells were grown in nutrient medium RPMI
1640 supplemented with 2 mM L-glutamine, 200 IU/ml penici-
lium, 50 μg/ml streptomicyn, and 20% heat inactivated horse
serum. They were incubated in a 5% CO2 atmosphere at 37 °C
for 21 h with several drug concentrations. Cells were then fixed
by ethanol (70% v/v), then washed, and incubated with PBS
containing 100 μM RNAse and 25 μM/ml propidium iodide for
30 min at room temperature. For each concentration, 104 cells
were analyzed on an Epics XL flow cytomer (Modele Beck-
man Coulter, French). Results were expressed as a percentage
of cells accumulated in each phase of the cell cycle and are
indicated.
4.2.3. Interaction with DNA. UV-visible determinations [23]
Absorption spectra were recorded using a Kontron UVI-
KON 930 UV visible spectrophotometer. Measurements were
made in different buffers at pH 7.0 at 20 °C. Spectra were
recorded with a drug concentration of 50 μM, in the presence
of 1 mM calf thymus DNA (purchased from Sigma Chemical)
and 10 mM SDS, respectively.
4.1.4.7. 2,3,6,11-Tetrahydro-1,4-dioxino[2,3-g] pyrido[2,3-b]
quinolin-11-ona (41).
A solution of nicotinic acid 40
(300 mg, 1.1 mmol) and a catalytic amount of trifluoroacetic
acid in distilled CH2Cl2 was stirred at reflux temperature for
48 hours. The cooled mixture was basified with 2 N NaOH
and extracted with CH2Cl2. The organic layers were dried, fil-
tered and concentrated giving a crude product which was pur-
ified by silica gel column chromatography (hexane / ethyl acet-
ate 80 / 20). Compound 41 was obtained as yellow oil
(200 mg, 72% yield). IR (KBr) υ cm–1: 1592 (C=O), 1267
(O–Ar), 1180 (C–O). 1H-NMR (CDCl3, 200 MHz) δ: 4.21
(m, 4H, CH2O), 6.52 (m, 1H, Ar), 6.78 (d, J = 8.4 Hz, 1H,
Ar), 6.94 (dd, J1 = 11, J2 = 2.2 Hz, 1H, C9-H), 7.40 (bs, 1H,
NH), 8.27 (s, 1H, C10-H), 8.43 (bs, 1H, C8-H). Anal. Calcd
for C14H10N2O3; C, 66.14; H, 3.96; N, 11.02. Found: C, 66.45;
H, 4.32; N, 11.45.
Acknowledgments
The authors express their gratitude to the Generalitat de Cat-
alunya, Spain (01-SGR-00085) and to the Ministerio de Cien-
cia y Tecnología (MCYT, BQU 2002-00148) for the financial
support.
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