3,4-Dihydro-1H-[1,3]oxazino[4,5-c]acridines as a new family of cytotoxic drugs

Article abstract of DOI:10.1016/j.bmcl.2006.05.101

A series of [1,3]oxazino fused acridines has been prepared as precursors of cytotoxic 3-amino-4-hydroxymethylacridine 2. Their cytotoxic activity has been evaluated against HT29 colon carcinoma cell line and was shown to be dependent on the nature of the

Full text of DOI:10.1016/j.bmcl.2006.05.101

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4641–4643
3,4-Dihydro-1H-[1,3]oxazino[4,5-c]acridines as a new
family of cytotoxic drugs
a
a
b
b
`
Myriam Ouberai, Christian Asche, Daniele Carrez, Alain Croisy,
Pascal Dumya and Martine Demeunyncka,*
aLEDSS, CNRS UMR 5616 & ICMG-FR260, Universite´ Joseph Fourier, BP53, 38041 Grenoble ce´dex 9, France
bINSERM U579, Centre Universitaire, 91405 Orsay and Institut Curie Recherche, Laboratoire Raymond Latarjet, France
Received 23 May 2006; revised 31 May 2006; accepted 31 May 2006
Available online 13 June 2006
Abstract—A series of [1,3]oxazino fused acridines has been prepared as precursors of cytotoxic 3-amino-4-hydroxymethylacridine 2.
Their cytotoxic activity has been evaluated against HT29 colon carcinoma cell line and was shown to be dependent on the nature of
the substituent located on position 2 of the oxazine ring. Additionally, the nitrophenyl derivative 3f is activated by nitroreductase,
indicating its potency as prodrug for either gene-directed or antibody-directed enzyme prodrug therapies.
Ó 2006 Elsevier Ltd. All rights reserved.
Acridine derivatives represent a large family of potential
antitumor agents.1–3 Two compounds, amsacrine, a 9-
anilinoacridine, and nitracrine, a 1-nitroacridine, have
been used in clinic. Other compounds, such as the 4-car-
boxamido acridine DACA (XR5000),4,5 imidazoacridi-
We had observed that among the molecules tested previ-
ously,8 the oxazino[1,3]acridine derivatives were display-
ing significant cytotoxicities. We hypothesize that these
oxazino[1,3]acridines may be considered as cyclic pre-
cursors of the active amino alcohol drugs. This hypoth-
esis is based on literature data discussing the equilibrium
existing between ring and chain forms of 1,3-O,N-het-
erocyclic systems.13–15 This equilibrium, depicted in
Figure 1, is strongly dependent on the nature of the sub-
stituent located in position 2. The open-chain imine
form may hydrolyze in aqueous solution to generate
the corresponding amino alcohol. To test this hypothe-
sis, we have prepared a series of oxazino[1,3]acridines
and evaluated their cytotoxicity against HT-29 cancer
cell line.
nones,6
and
pyrazolo[3,4,5-kl]acridine
(PZA),7
underwent clinical trials. Their cytotoxicity may be
related to enzyme inhibition as topoisomerases and tel-
omerase are affected by acridines. Denny’s group work-
ing on the concept of intercalating alkylating agents has
designed a family of molecules based on the acridine
skeleton. We have previously shown that acridines
substituted by ortho-amino hydroxymethyl group were
cytotoxic at nanomolar concentrations against various
cancer cell lines.8 As the most active molecule (com-
pound 2) appears to be toxic in vivo, we are looking
for precursors of this molecule. Several approaches were
envisioned. Derivatization of 2 as para-nitrobenzyloxyc-
arbamate yielded less cytotoxic compounds that could
be activated in situ by nitroreductase according to
ADEPT or GDEPT strategies.9 In these strategies, the
exogenous enzyme has to be specifically delivered either
by an antibody–enzyme conjugate (ADEPT strategy)10
or by incorporating the gene encoding for the enzyme
into the DNA of the tumor cells (GDEPT strategy).11,12
The [1,3]oxazinoacridines 3a–f were prepared by reac-
tion of the corresponding aldehydes with the amino
alcohol 2, itself synthesized in three steps from 3-amino-
acridine 1 as previously described16 (Scheme 1).
To achieve the cyclization various conditions were tried,
ethanol, THF in the presence of catalytic amount of
N
NH
R
Keywords: Acridine; Antitumor; Prodrugs.
*
OH
2
O
R
Corresponding author. Tel.: +33 476514429; fax: +33 476514946;
e-mail: martine.demeunynck@ujf-grenoble.fr
Figure 1. Ring-chain equilibrium of [1,3]-oxazines.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.101

4642
M. Ouberai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4641–4643
3 steps
NH
i
N
N
NH
N
NH
2
2
2
3a-f
1
OH
O
R
i) 5-6 eq. RCHO, 0.12N HCl, 40˚C, 2-22h
Scheme 1. Synthesis of the drugs.
Table 1. In vitro cytotoxic activity against HT29 cell line
para-toluene sulfonic acid, and 0.12 N HCl. Only the
latter conditions gave the desired products. The reaction
failed with para-anisaldehyde and para-dimethylamino-
benzaldehyde. In both cases, the starting compound 2
remained unreacted after 6 days of stirring in the pres-
ence of excess of aldehyde. The structures of compounds
Compound
R
Cytotoxicitya IC50 (lM)
3gb
3a
H
CH3
2.25
0.6
3b
3c
CH2–Cl
CH2–Br
CH2O–CH2Ph
Ph
1.25
1.25
4
1
3dc
3a–f were determined by H NMR. The presence of an
AB system for the oxazine CH2 protons clearly confirms
a ring structure for these compounds. To prepare the
para-aminophenyl derivative, we also tried to reduce
the nitro group of 3f with hydrazine hydrate in the pres-
ence of Pd/C. The reaction proceeded quickly to give
compound 2 quantitatively (Scheme 2). The unsuccess-
ful formation of compounds substituted with electron-
donating groups, para-methoxy- or para-aminophenyl
derivatives, may be explained to the ring-chain equilibri-
um, depicted in Figure 1. This equilibrium is controlled
by the nature of the R group, electron-withdrawing
groups favoring the ring form, and electron donating
groups stabilizing the open-chain imine probably by for-
mation of hydrogen bond with the hydroxy group.
Therefore, the presence of para-methoxy or amino
groups will shift the equilibrium toward the imine form,
which in turn hydrolyzes in water to regenerate the start-
ing compound 2.
3e
3f
0.4
2.3 0.7
0.2
p-Ph–NO2
3f + nitroreductase
2d
Doxorubicine
0.025
2.37
a Concentration (lM) necessary for 50% of cell growth inhibition.
b Ref. 17.
c Isolated and tested as the para toluene sulfonate salt.
d Ref. 8.
e Ref. 18.
substituted derivative 3a. More interesting is the behav-
ior of nitro phenyl analogue 3f. It shows a six times low-
er cytotoxicity than the phenyl analogue 3e. However,
in situ reduction of the nitro group of 3f by Escherichia
coli nitroreductase has a strong impact on the cytotoxic-
ity, the molecule being 10 times more cytotoxic in these
conditions (IC50 0.2 lM).
The ability of NTR to reduce the nitrophenyl compound
3f was confirmed with an enzyme assay where phos-
phate-buffered solution (pH 7.0) of 3f (10 lM) was incu-
bated with NADH (50 lM) and E. coli nitroreductase
(2 lg/ml) at 37 °C. The reaction was followed by HPLC.
The disappearance of the peak corresponding to the
starting nitro compound correlates with the formation
of a more polar compound, which in turn slowly trans-
forms into the amino alcohol 2. We postulate that the
intermediate compound that could not be isolated was
The in vitro antiproliferative properties of the new com-
pounds were evaluated using human HT29 colon carci-
noma cell line as previously described.8 The results are
collected in Table 1.
All compounds exhibit cytotoxic activities at micromo-
lar concentrations. The presence of a bulky electron-at-
tracting group, halogen or benzyl, slightly decreases the
cytotoxicity (compare 3b–d with 3a). The unsubstituted
oxazine 3g displays a lower cytotoxicity than the methyl
NH
O
i
3h
NH
2
NH
NH
O
3f
2
ii
2
OH
NO
2
NH
O
NHOH
i) H2NNH2.H2O, Pd/C, EtOH, 70˚C ; ii) nitroreductase, NADPH, phosphate buffer, pH 7.4
3i
Scheme 2. Reductive activation of nitro compound 3f.

M. Ouberai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4641–4643
4643
in situ release of the active drug by control of the ring-
chain equilibrium. This compound 3f is also useful as
prodrug either in gene-directed enzyme prodrug therapy
(GDEPT) or antibody-directed enzyme prodrug therapy
(ADEPT).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.05.101.
References and notes
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Figure 2. Survival of HT-29 cells following treatment with compound
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either the hydroxylamino (3i) or the amino (3h) ana-
logue (Scheme 2). The transformation of the electron-at-
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ring structure and regenerates compound 2, via forma-
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the equilibrium depicted in Figure 1.
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obic or hypoxic (argon atmosphere) conditions and sur-
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As a conclusion, we have prepared a series of oxazino-
fused acridines as potential precursors of cytotoxic com-
pound 2. The biological effect is dependent on the nature
of the substituent present on position 2 of the oxazine
ring. This effect may be correlated to the ring-chain
equilibrium of [1,3]oxazines. The presence of electron-
attracting substituent stabilizes the ring form and is
associated with a lower cytotoxic effect (higher IC50).
The activation of nitro derivative 3f by nitroreductase
is in accordance with our starting hypothesis of the