A C-ring regioisomer of the marine alkaloid meridine exhibits selective in vitro cytotoxicity for solid tumours

Article abstract of DOI:10.1016/S0968-0896(01)00078-5

9-Hydroxybenzo[b]pyrido[4,3,2-de](1,10)-phenantrolin-8-one (1) a regioisomer of the marine alkaloid meridine, was synthesized from 5,8-dimethoxy-6-nitro-4(1H)-quinolinone in eight steps and 23% overall yield. A shorter route was also investigated, based o

Full text of DOI:10.1016/S0968-0896(01)00078-5

Bioorganic & Medicinal Chemistry 9 (2001) 1807–1814
A C-Ring Regioisomer of the Marine Alkaloid Meridine Exhibits
Selective In Vitro Cytotoxicity for Solid Tumours
Jesu´
s Angel de la Fuente,a Ma Jesus Martın,a Ma del Mar Blanco,b
Eva Pascual-Alfonso,b Carmen Avendano,b, and J. Carlos Menendezb,*
a
´
Instituto Biomar, S.A. Polıgono Industrial, Edificio C.E.I., 24231 Onzonilla, Leon, Spain
´
Departamento de Quımica Organica y Farmaceutica, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain
b
´
´
´
Received 4 October 2000; accepted 14 March 2001
Abstract—9-Hydroxybenzo[b]pyrido[4,3,2-de](1,10)-phenantrolin-8-one (1), a regioisomer of the marine alkaloid meridine, was
synthesized from 5,8-dimethoxy-6-nitro-4(1H)-quinolinone in eight steps and 23% overall yield. A shorter route was also investi-
gated, based on the hetero Diels–Alder reaction between o-nitrocinnamaldehyde dimethylhydrazone and 4-halogen-6-bromo-5,8-
quinolinequinones followed by reductive cyclization onto the C-5 carbonyl of the quinone. Compound 1 showed a remarkable in
vitro cytotoxicity, with a pattern of selectivity towards solid tumours that is not found in the reference alkaloid, the activity against
the human lung carcinoma (A-549) being particularly noteworthy. The activities of meridine and compound 1 as inhibitors of
topoisomerase II were also significantly different. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
based on the stepwise disconnection of rings C and D
Marine organisms are increasingly important as sources
of new natural products with interesting biological
properties, which include excellent antitumour activ-
ities.1,2 One of the most interesting groups of com-
pounds of marine origin is the family of polycyclic
aromatic alkaloids derived from the pyrido[kl]acridine
skeleton,3 such as meridine4 and amphimedine.5,6 These
compounds have been normally isolated from sponges
or tunicates,7 although they might be synthesized by
associated microorganisms.8 The marine alkaloids are
normally isolated in minute amounts and this factor has
precluded the systematic study of the structural
requirements for their biological properties; hence the
need for synthetic routes to the natural products them-
selves and to their analogues. We describe here two
efficient syntheses of compound 1, a regioisomer of
meridine.9 Compound 1 is also a derivative of iso-
ascididemin,10 which is not a natural product but can be
regarded as a structural fragment of several complex
marine alkaloids, such as eilatine11 and eudistone A12
(Fig. 1).
and in the introduction of ring E through a Stille cross-
coupling reaction. In the second approach, rings A and
E are introduced simultaneously by means of a hetero
Diels–Alder reaction between a 4-aryl-1-dimethylamino-
1-azadiene and a quinolinequinone.
Results and Discussion
Our first route is depicted in Scheme 2, and can be
related to previous work on the synthesis of iso-
ascididemin10 and meridine9a in that all of these routes
use a Stille coupling as the key step. Bromide 4 was
obtained in 76% overall yield by treatment of the
known10 5,8-dimethoxy-6-nitro-4(1H)-quinolinone
2
with trifluoromethanesulfonic anhydride to give triflate
3, followed by reaction with lithium bromide. Stille
coupling of 4 with tributyl (2-tertbutoxycarbonyl-
aminophenyl) stannane10 catalyzed by PdCl2(CH3CN)2
afforded compound 5 in quantitative yield. Hydrogena-
tion of 5 with cyclohexene in the presence of palladium
gave amine 6, which was transformed into compound 7
by treatment with Meldrum’s acid and triethyl orto-
formate. All attempts to oxidize 7 to a quinone led to
complex mixtures; fortunately, a simple change in the
N-protecting group from BOC to trifluoroacetyl (8)
allowed the oxidative demethylation and afforded the
We have developed two routes to compound 1, which
are summarized in Scheme 1. The first approach is
*Corresponding author. Tel.: +34-91-804-9230; fax: +34-91-803-
1143; e-mail: suso@pharmamar.com or josecm@eucmax.sim.ucm.es
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00078-5

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1808
rather unstable quinone 9, which by thermolysis gave 1
in 42% yield from 8. This one-pot double cyclization
from 9 to 1 is remarkable. We assume that cyclization of
the trifluoroacetamido group onto the quinone carbonyl
must involve its prior hydrolysis under the harsh reac-
tion conditions, catalyzed by the presence of traces of
acid (from cerium ammonium nitrate) in compound 9.13
The overall yield of the eight-step route to 1 was 23%.
group in the synthesis of meridine (see ref 20). In order
to increase the reactivity of the quinones as dienophiles,
we decided to use a second halogen atom rather than a
hydroxy group at C-4 of the quinoline ring. The known
6-bromo-4-chloro-5,8-quinolinequinone 12 was pre-
pared by a literature method16 involving oxidative
demethylation of the corresponding 5,8-dimethoxy-
quinoline 10 with cerium ammonium nitrate.17 4,6-
Dibromoquinolinequinone 13 was similarly obtained
from the known10 dibromoquinoline 11.
In search of a shorter route, we decided to investigate an
alternative approach based on the hetero Diels–Alder
reaction14 between a 4-aryl-1-dimethylamino-1-azadiene
bearing a nitrogenated function at the o-position and a
4-substituted derivative of 6-bromo-5,8-quinolinequi-
none, where the presence of the bromine atom at C-6
was expected to revert the ‘natural’ regiochemistry of
the Diels–Alder reactions of quinolinequinones.15 This
strategy has been previously employed by the Kubo
The Diels–Alder reactions of quinones 12 and 13 with
o-nitrocinnamaldehyde dimethylhydrazone18 gave the
results summarized in Scheme 3. Owing to the low
reactivity of 4-aryl-1-dimethylamino-1-azadienes, we
employed ultrasound irradiation19 using the neat aza-
diene as the reaction medium. Under these conditions,
quinone 12 gave the aromatic adduct 16 in 21% yield
Figure 1.
Scheme 1.

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J. Angel de la Fuente et al. / Bioorg. Med. Chem. 9 (2001) 1807–1814
1809
after 27 h;20 interruption of the reaction after 7 h
allowed detection of intermediates 14, formed by elim-
ination of HBr from the primary Diels–Alder adduct,
and 15, formed by acid-catalyzed elimination of dime-
thylamine from 14. Better results were obtained with the
more reactive dibromoquinone 13, which gave a 45%
yield of the aromatic compound 17 after 4 h of irradia-
tion. Two methods were then assayed for the transfor-
mation of 16 or 17 into the target compound 1. In the
initial approach, compound 16 was transformed into
the hydroxy derivative 18 in 62% yield by brief expo-
sure to wet trifluoroacetic acid; subsequent catalytic
hydrogenation of 18 afforded 1 in 19% yield. The order
of these steps was inverted when applied to the bromo
derivative 17, which afforded 1 in 40% yield after cata-
lytic hydrogenation followed by addition of wet tri-
fluoroacetic acid (Scheme 3). The overall yield of 1 in
this route (four steps from 11) was 11%.
drugs worldwide. The compounds under assay were
dissolved in DMSO/MeOH (1:9) and tested following
the methods described by Bergeron et al.21 and Schroeder
et al.22 The activity of quinone 9 in comparison with
that of its precursor 8 (inactive) is noteworthy. The
remarkable antitumour properties of compound 17,
which is more active (but less selective) than 1, are in
agreement with the good antitumour activity found in a
series of dialkyl 1,5-diazaanthraquinones;23 comparison
of the activities of 17 and 18 indicates the importance of
the substitution pattern in these tricyclic compounds.
Finally, the target compound 1 shows a high selectivity
for solid tumours that is not present in its regioisomer
meridine, proving that the C-ring has a significant rela-
tionship with the activity in these pyridoacridine skele-
tons, although the establishment of detailed structure–
activity relationships in the pyridoacridine family of
alkaloids is still not possible due to the lack of a suffi-
cient number of analogues. Particularly noteworthy are
the high activity and selectivity of 1 towards the human
lung carcinoma A-549. This selectivity is a very impor-
tant characteristic of compound 1 and constitutes a
distinct advantage over meridine, since a high activity
Table 1 shows the cytotoxic activities of compound 1
and some selected precursors, compared with those of
the related natural product meridine and also with
adriamycin, one of the most commonly used anticancer
Scheme 2. Reagents and conditions: (i) Tf2O, 2,6-lutidine, DMAP, CH2Cl2, rt, 20 h; (ii) LiBr, 1,4-dioxane, reflux, 2 h; (iii) PdCl2(CH3CN)2, 1,4-
dioxane, 55 ꢀC, 14 h; (iv) Cyclohexene, Pd-C (10%), EtOH, reflux, 45 min; (v) Meldrum’s acid, HC(OEt)3, reflux, 2 h, then add 6, reflux, 4 h; (vi)
(NH4)2Ce(NO3)6, CH3CN, 2 M H2SO4, rt, 3 h; (vii) TFA, TFAA, rt, 90 min; viii. Ph2O, 200–220 ꢀC, 15 min.

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J. Angel de la Fuente et al. / Bioorg. Med. Chem. 9 (2001) 1807–1814
1810
Scheme 3. Reagents and conditions: (i) (NH4)2Ce(NO3)6, CH3CN, H2O, 0 ꢀC, 3 h; (ii) o-Nitrocinnamaldehyde dimethylhydrazone (neat), ultra-
sound, 50 ꢀC; (iii) CF3CO2H-CHCl3 (3:1), rt, 5 min; (iv) H2, Pd-C, 1 atm, 1 h.
Experimental
Table 1. Cytotoxic activity in cultured cell lines (IC50 mM)
Cell linea Adriamycin Meridine
1
8
9
13
17
18
All reagents were of commercial quality (Aldrich,
Fluka, SDS, Probus) and were used as received. Sol-
vents (SDS) were dried and purified using standard
techniques. ‘‘Petroleum ether’’ refers to the fraction
boiling at 40–60 ꢀC. Reactions under ultrasound irra-
diation were performed with a Branson 450 ultrasound
probe, using the pulsed mode (pulse duration, 0.3 s),
with output values between 30 and 40 W. Reactions
were monitored by thin layer chromatography, on alu-
minium plates coated with silica gel with fluorescent
indicator (Macherey-Nagel Alugram Sil G/UV254).
Separations by flash chromatography were performed
on silica gel (SDS 60 ACC, 230–40 mesh). Melting
points were measured with a Reichert 723 hot stage
microscope, and are uncorrected. Infra-red spectra were
recorded on a Perkin-Elmer Paragon 1000 FT-IR spec-
trophotometer, with all compounds examined in KBr
pellets or as films on a NaCl disk. NMR spectra were
obtained on Varian Unity-300 (300 MHz for 1H,
75 MHz for 13C), Bruker AC-250 (250 MHz for 1H,
P-388
A-549
HT-29
MEL-28
0.03
0.01
0.09
0.03
0.08
0.08
0.84
0.08
4.18 >18 0.97
0.3 0.27 7.20
0.03 7.20
0.27 7.20
0.03 >18 0.10 >3
0.40 >18 0.97 >3
0.17 >18 0.10 NMb 0.03 7.20
aP-388D1; mouse lymphoma (ATCC CCL-46); A-549, human lung
carcinoma (ATCC CCL-185); HT-29, human colon carcinoma (ATCC
HTB-38); MEL-28, human melanoma (ATCC HTB-72).
bNM, not researched.
towards all types of tumours is normally considered as a
sign of indiscriminate cytotoxicity, and therefore is an
undesirable feature for a drug candidate.
The antitumour activity of the pyridoacridine class of
alkaloids is normally associated with their interaction
with DNA and subsequent inhibition of the activity of
toposiomerase II (TOPO II).3b,e Therefore, we have
measured the in vitro relative ability of meridine and
compound 1 to inhibit TOPO II activity. Meridine
showed some activity in this test, (IC50=3 mM) but
compound 1 was inactive at the highest concentration
assayed (33 mM). This different inhibitory activity
agrees with the different cytotoxicity patterns of both
compounds and again gives an indication of the impor-
tance of the effects of structural variations in the C-ring
within this class of pyridoacridines. It must be remarked
that the precise mechanism of action of the pyr-
idoacridine alkaloids is far from being fully under-
stood, and that several mechanisms different from
TOPO II inhibition have been described in the case of
ascididemin.24,25
1
63 MHz for 13C) and Bruker AC-500 (500 MHz for H)
spectrometers (Servicio de RMN, Universidad Com-
plutense), with CDCl3, CD3OD or CF3CO2D as sol-
vents. Mass spectra were obtained by the Servicio de
Espectroscopıa, Universidad Complutense. Combustion
elemental analyses were determined by the Servicio de
Microanalisis Elemental, Universidad Complutense.
5,8-Dimethoxy-5-nitro-4-(trifluoromethanesulfonyloxy)-
quinoline (3). To a solution of the quinolone 211 (0.2 g,
0.8 mmol), 4-dimethylaminopyridine (10 mg, 0.08
mmol) and 2,6-lutidine (0.14 mL, 1.22 mmol) in CH2Cl2
(6 mL), was slowly added trifluoromethanesulfonic
anhydride (0.15 mL, 0.88 mmol), at 0 ꢀC and under

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J. Angel de la Fuente et al. / Bioorg. Med. Chem. 9 (2001) 1807–1814
1811
argon. After the addition, the mixture was warmed to
room temperature and stirred for 20 h. The solution was
diluted with CH2Cl2 and washed with a saturated
NH4Cl aqueous solution, and the organic layer was
dried over Na2SO4. Evaporation of the solvent afforded
a crude which was chromatographed (hexane–AcOEt
4:6) to yield the triflate 3 (232 mg, 76%) as pale yellow
crystals. (Found: C, 37.51; H, 2.60; N, 7.37; S, 8.27.
C12H9F3SN2O7 requires: C, 37.70; H, 2.37; N, 7.33; S,
8.39); mp 146–148 ꢀC; nmax (KBr): 1600, 1427, 1239,
1033 cmꢁ1; dH (CDCl3, 300 MHz) 9.09 (d, J=4.6 Hz,
1H), 7.46 (s, 1H), 7.45 (d, J=4.6 Hz, 1H), 4.13 (s, 3H),
4.01 (s, 3H). dC (CDCl3, 75 MHz) 153.5, 152.3 (two sig-
nals), 144.4, 142.0, 141.5, 118.6 (q, J=321 Hz), 118.2,
116.2, 103.9, 64.5, 56.9.
(CDCl3, 300 MHz) 8.86 (br. s, 1H), 7.79 (d, J=7.5 Hz,
1H), 7.46 (m, 3H), 7.27 (m, 1H), 7.19 (t, J=6.7 Hz, 1H),
6.01 (s, 1H), 4.10 (s, 3H), 2.95 (s, 3H), 1.29 (s, 9H). dC
(CDCl3, 75 MHz) 152.8, 148.9, 147.1, 142.1, 138.2,
135.9, 131.5, 130.2, 129.4, 128.9, 126.4, 124.9, 123.3,
123.2, 121.7, 104.3, 80.5, 59.7, 56.6, 27.9.
4-(2-tertButyloxycarbonylaminophenyl)-6-(2,2-dimethyl-
4,6-dioxo-1,3-dioxan-5-ylidene-methylamino)-5,8-dimeth-
oxyquinoline (7). A solution of Meldrum’s acid (519 mg,
3.6 mmol) in triethyl ortoformate (8 mL) was refluxed
for 2 h under argon. The reaction mixture was added to
compound 6 (1.18 g, 3 mmol) and the solution thus
obtained was refluxed for 4 h. After cooling to 0 ꢀC, the
precipitated crystals were filtered and washed with
methanol to yield compound 7 as a yellow solid (750
mg, 72%). (Found: C, 63.14; H, 5.68; N, 7.58.
C29H31N3O8 requires: C, 63.38; H, 5.69; N, 7.65); mp
292–294 ꢀC; nmax (KBr): 3114, 1702, 1676, 1618, 1271
cmꢁ1; dH (CDCl3, 300 MHz) 8.95 (d, J=4.2 Hz, 1H),
8.74 (d, J=14.4 Hz, 1H), 7.90 (d, J=7.6 Hz, 1H), 7.44
(t, J=7.3 Hz, 1H), 7.37 (d, J=4.2 Hz, 1H), 7.26 (m,
1H), 7.17 (t, J=7.3 Hz, 1H), 7.07 (s, 1H), 5.91 (s, 1H),
4.19 (s, 3H), 3.09 (s, 3H), 1.75 (s, 6H), 1.30 (s, 9H); dC
(CDCl3, 75 MHz): 165.1, 163.6, 154.3, 152.8, 150.3,
148.8, 142.8, 139.6, 137.8, 135.9, 131.6, 129.4, 129.1,
128.7, 126.4, 123.2, 122.8, 121.5, 105.3, 95.7, 88.2, 80.5,
61.9, 56.8, 28.1, 27.1, 27.0.
4-Bromo-5,8-dimethoxy-5-nitroquinoline (4). A suspen-
sion of the triflate 3 (400 mg, 1.05 mmol) and lithium
bromide (273 mg, 3.15 mmol) in 1,4-dioxane (4 mL) was
refluxed for 2 h under argon. After cooling to room
temperature, the mixture was diluted with AcOEt and
washed with H2O and the organic layer was dried over
Na2SO4. Evaporation of the solvent gave the bromo
derivative 4 (328 mg, 100%), as pale yellow crystals.
(Found: C, 42.43; H, 2.93; N, 8.93. C11H9BrN2O4
requires: C, 42.20; H, 2.90; N, 8.95); mp 180–182 ꢀC;
nmax (KBr): 1602; 1492; 1360; 1242; 1005 cmꢁ1; dH
(CDCl3, 300 MHz) 8.69 (d, J=4.6 Hz, 1H), 7.85 (d,
J=4.6 Hz, 1H), 7.35 (s, 1H), 4.08 (s, 3H), 3.92 (s, 3H).
dC (CDCl3, 75 MHz) 152.2, 150.6, 143.6, 143.5, 141.3,
130.1, 123.1, 103.0, 100.5, 64.5, 56.7.
4-(2-Trifluoracetamidophenyl)-6-(2,2-dimethyl-4,6-dioxo-
1,3-dioxan-5-ylidenemethyl-amino)-5,8-dimethoxyquino-
line (8). To a solution of compound 7 (487 mg, 1.2
mmol) in trifluoroacetic acid (8 mL) was added tri-
fluoroacetic anhydride (4 mL) at 0 ꢀC and under argon.
After the addition, the reaction mixture was allowed to
warm to room temperature, stirred for 90 min and eva-
porated. The crude was dissolved in CH2Cl2, washed
with 0.1 M HNaCO3 and a saturated aqueous NaCl
solution, and dried over Na2SO4. Evaporation of the
solvent gave compound 8 (484 mg, 100%), as pale yel-
low crystals. (Found: C, 57.18; H, 4.06; N, 7.41.
C26H22N3O7F3 requires: C, 57.25; H, 4.07; N, 7.70); mp
229–231 ꢀC; nmax (KBr): 3418, 1734, 1684, 1618, 1275
cmꢁ1; dH (CDCl3, 300 MHz) 8.92 (d, J=4.3 Hz, 1H),
8.75 (d, J=14.0 Hz, 1H), 7.83 (d, J=7.5 Hz, 1H), 7.72
(s, 1H), 7.53 (td, J=6.7 and 2.0 Hz, 1H), 7.44 (m, 2H),
7.37 (d, J=4.3 Hz, 1H), 7.10 (s, 1H), 4.16 (s, 3H), 3.06
(s, 3H), 1.73 (s, 3H), 1.72 (s, 3H). dC (CDCl3, 75 MHz):
165.2, 163.4, 155.0, 154.5, 150.3, 148.7, 141.4, 139.1,
136.6, 134.4, 132.9, 129.9, 129.5, 129.0, 126.7, 126.5,
123.6, 121.9, 115.3 (q, J=289 Hz), 105.3, 95.8, 88.5,
62.0, 56.8, 27.1, 26.9.
4-(2-tertButyloxycarbonylaminophenyl)-5,8-dimethoxy-
6-nitroquinoline (5). A suspension of compound 4 (498
mg, 1.6 mmol), tributyl (o-tertbutyloxycarbonyl-
aminophenyl) stannane10 (1.02 g, 2.86 mmol) and
bis(acetonitrile)dichloropalladium (II) (54 mg, 0.21
mmol) in 1,4-dioxane (13 mL) was heated at 55 ꢀC for
14 h, under an argon atmosphere. The cooled reaction
mixture was diluted with AcOEt and washed with H2O,
and the organic layer was dried over Na2SO4. The
resulting crude was chromatographed (CH2Cl2/AcOEt
8:2) to afford compound 5 (680 mg, 100%), as pale yel-
low crystals. (Found: C, 62.11; H, 5.46; N, 9.75.
C22H23N3O6 requires C, 62.11; H, 5.45; N, 9.88); mp
194–196 ꢀC; nmax (KBr): 3222; 1724; 1532; 1508; 1236;
1162 cmꢁ1; dH (CDCl3, 300 MHz) 9.04 (d, J=4.1 Hz,
1H), 7.88 (d, J=7.8 Hz, 1H), 7.41 (m, 3H), 7.19 (m,
2H), 5.95 (s, 1H), 4.13 (s, 3H), 3.17 (s, 3H), 1.30 (s, 9H).
dC (CDCl3, 75 MHz) 152.7, 152.4, 151.5, 145.8, 145.6,
143.0, 140.3, 135.6, 131.9, 129.0, 128.9, 126.7, 126.3,
123.1, 121.6, 102.7, 80.6, 63.1, 56.6, 28.0.
6-Amino-4-(2-tertbutyloxycarbonylaminophenyl)-5,8-di-
methoxyquinoline (6). To a solution of the nitro deriva-
tive 5 (67 mg, 0.16 mmol) in ethanol (5 mL) was added
10% Pd/C (100 mg) and cyclohexene (0.081 mL, 0.8
mmol). After being refluxed for 45 min, the solution was
cooled to room temperature, filtered and evaporated to
yield the amine 6 (62 mg, 100%), as red crystals.
(Found: C, 66.54; H, 6.09; N, 10.42. C22H25N3O4
requires C, 66.82; H, 6.37; N, 10.63); mp 170–172 ꢀC;
nmax (KBr): 3388, 1707, 1618, 1241, 1161 cmꢁ1. dH
4-(2-Trifluoracetamidophenyl)-6-(2,2-dimethyl-4,6-dioxo-
1,3-dioxan-5-ylidenemethylamino)-5,8-quinolinequinone
(9). To a solution of compound 8 (150 mg, 0.27 mmol)
in CH3CN (2.1 mL) was added a solution of ammonium
cerium (IV) nitrate (636 mg, 1.16 mmol) in 2M H2SO4
(3 mL). After stirring for 3 h, the reaction mixture was
partitioned between AcOEt and saturated aqueous
NH4Cl solution. The organic layer was dried over
Na2SO4, and evaporation of the solvent gave a residue
which was purified by flash column chromatography

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1812
(AcOEt) to afford 9 as a red solid (75 mg, 53%). dH
(CD3OD, 300 MHz): 8.96 (d, J=4.9 Hz, 1H), 8.78 (s,
1H), 7.58 (m, 2H), 7.50 (td, J=7.2 and 1.4 Hz, 1H), 7.43
(dd, J=7.5 and 2.2 Hz, 1H), 7.32 (dd, J=7.5 and 1.4
Hz, 1H), 7.22 (s, 1H), 1.72 (s, 6H); dC (CDCl3, 75 MHz):
180.9, 179.3, 164.0, 161.9, 154.8, 154.6 (d, J=111.5 Hz),
148.3, 148.1, 141.2, 134.1, 131.2, 130.5, 130.1, 129.0,
128.8, 128.2, 126.1, 124.5, 115.5 (q, J=288 Hz), 113.1,
106.1, 105.1, 94.3, 27.3. MS (FAB, CH3CN/NaOAc):
538.1 (M++23).
neat ethyl acetate, yielding 50 mg (45%) of compound
17, as a brown solid. (Found: C, 52.45; H, 2.21; N,
10.30. C18H8BrN3O4 requires: C, 52.71; H, 1.97; N,
10.24); mp 98-101 ꢀC; nmax (KBr): 1674, 1523, 1346, 757
cmꢁ1; dH (CDCl3, 250 MHz): 9.15 (d, 1H, J=4.9 Hz),
8.68 (d, 1H, J=5.0 Hz); 8.33 (dd, 1H, J=8.0 and 1.5
Hz), 7.95 (d, 1H, J=5.0 Hz), 7.76–7.62 (m, 2H), 7.49 (d,
1H, J=4.9 Hz), 7.23 (m, 1H).
4-Hydroxy-8-(2-nitrophenyl)-1,5-diazaanthracene-9,10-
dione (18). A solution of 17 (80 mg, 0.21 mmol) in 10
mL of a 3:1 mixture of CF3CO2H/CHCl3 containing
water (0.1 mL) was stirred at room temperature and
evaporated. The residue was chromatographed on silica
gel eluting with 9:1 ethyl acetate/methanol, yielding 45
mg (62%) of compound 18. (Found: C, 62.02; H, 2.49;
N, 12.10. C18H9N3O5 requires: C, 62.25; H, 2.61; N,
12.10); mp >250 ꢀC; nmax (KBr): 3422, 1684, 1522,
1350, 1310; dH (CDCl3, 250 MHz): 12.36 (s, 1H); 9.16
(d, 1H, J=4.8 Hz); 8.73 (d, 1H, J=5.7 Hz); 8.34 (dd,
1H, J=7.5 and 1.4 Hz); 7.74 (td, 1H, J=7.5 and 1.4);
7.66 (td, 1H, J=7.5 and 1.4 Hz); 7.53 (d, 1H, J=4.8
Hz); 7.21 (dd, 1H, J=7.5 and 1.4 Hz); 7.19 (d, 1H,
J=5.7 Hz); dC (CDCl3, 63 MHz): 181.4, 179.4, 159.9,
153.8, 151.6, 149.3, 134.0, 133.7, 129.8, 129.6, 129.4,
128.4, 128.2, 124.8, 124.2; owing to low solubility, the
signals due to C-20, C-9a and C-10a could not be detec-
ted.
9-Hydroxybenzo[b]pyrido[4,3,2-de](1,10)-phenantrolin-8-
one (1). A solution of compound 9 in diphenyl ether (22
mL) was heated between 200 and 220 ꢀC for 15 min. The
reaction mixture was cooled to room temperature and
added to hexane at 0 ꢀC. Filtration of the precipitated
solid afforded compound 1, as orange crystals (35 mg,
80%). (Found: C, 71.96; H, 2.92; N, 13.85. C18H9N3O2
requires: C, 72.24; H, 3.03; N, 14.04); mp 245 ꢀC; nmax
3426, 1683 cmꢁ1; dH (CF3CO2D, 300 MHz): 9.53
(d,J=6.3 Hz, 1H), 9.35 (d, J=6.3 Hz, 1H), 8.91 (m,
2H), 8.54 (d, J=8.1 Hz, 1H), 8.29 (t, J=8.1 Hz, 1H),
8.18 (t, J=8.1 Hz, 1H), 7.65 (d, J=7.2 Hz, 1H); dC
(CF3CO2D, 75 MHz): 177.9, 174.1, 146.5, 146.5, 145.8,
144.7, 140.7, 138.6, 137.0, 136.2, 134.5, 133.1, 126.2,
125.3, 120.5, 117.6, 116.9, 113.4. MS (CI, methane): 300
(M++1). MS (FAB, CH3CN/NaOAc): 322 (M++23).
4,6-Dibromo-5,8-quinolinequinone (13). To a cooled
(0 ꢀC) solution of the dibromoquinoline 1110 (110 mg,
0.32 mmol) in acetonitrile (5 mL) was added a solution
of ammonium cerium (IV) nitrate (348 mg, 0.63 mmol)
in water (5 mL). The solution was stirred at 0 ꢀC for 3 h,
diluted with water (10 mL) and extracted with CHCl3
(3ꢂ30 mL). The residue was chromatographed on silica
gel, eluting with 1:1 petroleum ether/ethyl acetate,
yielding quinone 13 (40 mg, 63%) as a yellow solid.
(Found: C, 33.96; H, 1.12; N, 4.26. C9H3Br2NO2
requires: C, 34.11; H, 0.95; N, 4.42); mp 147 ꢀC; nmax
(KBr): 3056, 2918, 1681, 1595, 1545, 1298, 1092, 641
cmꢁ1; dH (CDCl3, 250 MHz): 8.73 (d, 1H, J=5.2 Hz),
7.94 (d, 1H, J=5.2 Hz), 7.65 (s, 1H); dC (CDCl3,
63 MHz): 179.4, 176.0, 153.6, 149.2, 141.0, 139.2, 134.6,
134.4, 126.6.
Reductive cyclization of 18. To a solution of compound
18 (25 mg, 0.7 mmol) in methanol (20 mL) was added
10% Pd-C (8 mg). The suspension was stirred under a
hydrogen atmosphere for 60 min, filtered through Celite
and evaporated. The residue was chromatographed on
silica gel, eluting with 9:1 ethyl acetate–methanol,
yielding 6 mg (19%) of compound 1.
Reductive cyclization-hydrolysis of 17. To a solution of
compound 17 (30 mg, 0.073 mmol) in methanol (7 mL)
was added 10% Pd-C (10 mg). The suspension was
stirred under a hydrogen atmosphere for 1 h at room
temperature and filtered through Celite, which was
washed with 15 mL of a 3:1 mixture of CF3CO2H/
CHCl3 containing water (0.1 mL). The combined wash-
ings were evaporated under reduced pressure and
washed repeatedly with ethyl ether, yielding 8.5 mg
(40%) of compound 1.
4-Chloro-8-(2-nitrophenyl)-1,5-diazaanthracene-9,10-dione
(16). A mixture of quinone 12 (185 mg, 0.68 mmol)
and o-nitrocinnamaldehyde dimethylhydrazone10 (438
mg, 2 mmol) was heated at 50 ꢀC until dissolution. The
solution was irradiated with ultrasound at 50 ꢀC for 27
h and then it was chromatographed on silica gel, eluting
with ethyl acetate, to yield 52 mg (21%) of 169b as a
brown solid. When the reaction was interrupted after 7
h, two intermediates were isolated, namely compounds
1424 (18%)26 and 1527 (4%).
Cytotoxicity assays
The compounds were tested for cytotoxic activity
against the following cell lines: P-388 (ATCC CCL-46,
suspension culture of a lymphoid neoplasm from a
DBA/2 mouse); A-549 (ATCC CCL-185, monolayer
culture of a human lung carcinoma); HT-29 (ATCC
HTB-38, monolayer culture of a human colon carci-
noma); MEL-28 (ATCC HTB-72, monolayer culture of
a human melanoma). Cells were maintained in loga-
rithmic growth in Eagle’s Minimum Essential Medium,
with Earle’s Balanced Salts, with 2.0 mM l-glutamine,
with non-essential amino acids, without sodium bicar-
bonate (EMEM/neaa), supplemented with 10% Fetal
Calf Serum (FCS), 10ꢁ2 M sodium bicarbonate, 0.1 g/L
4-Bromo-8-(2-nitrophenyl)-1,5-diazaanthracene-9,10-dione
(17). A mixture of quinone 13 (90 mg, 0.28 mmol) and
o-nitrocinnamaldehyde dimethylhydrazone (415 mg, 1.9
mmol) was heated at 50 ꢀC until dissolution and was
irradiated with ultrasound at 50 ꢀC for 4 h. The dark red
solution was chromatographed on silica gel, eluting with
a gradient from 2:1 petroleum ether/ethyl acetate to

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J. Angel de la Fuente et al. / Bioorg. Med. Chem. 9 (2001) 1807–1814
1813
penicillin G and 0.1 g/L streptomycin sulfate. The com-
pounds under assay were dissolved in DMSO/MeOH (1:9).
G. P.; Kelly-Borges, M.; Pomponi, S. A. J. Nat. Prod. 1992,
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P-388 cells were seeded into 16 mm wells at 1ꢂ10ꢁ4 cells
per well in 1 mL aliquots of MEM 5FCS containing the
indicated concentration of drug. A separate set of cul-
tures without drug was seeded as control growth to
ensure that cells remained in the exponential phase of
growth. All determinations were carried out in dupli-
cate. After 3 days of incubation at 37 ꢀC under a 98%
humidity atmosphere containing 10% CO2, IC50 values
were determined by comparing the growth in wells with
drug to the growth in control wells.
A-549, HT-29 and MEL-28 were seeded into 16 mm
wells at 2ꢂ10ꢁ4 cells per well in 1 mL aliquots of MEM
10FCS containing the indicated concentration of drug.
A separate set of cultures without drug was seeded as
control growth to ensure that cells remained in the
exponential phase of growth. All determinations were
carried out in duplicate. After 3 days of incubation at
37 ꢀC under a 98% humidity atmosphere containing
10% CO2, the wells were stained with 0.1% crystal vio-
let. IC50 values were determined by comparing the
growth in wells with drug to the growth in control wells.
3. (a) Alvarez, M.; Salas, M.; Joule, J. A. Heterocycles 1991,
32, 759. (b) Molinski, T. F. Chem. Rev. 1993, 93, 1825. (c)
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Helm, D. J. Org. Chem. 1991, 56, 1657.
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P. G.; Shoolery, J. N. J. Am. Chem. Soc. 1983, 105, 4835.
6. Other representative examples of the group are the cysto-
dytins (Kobayashi, J., Cheng, J.-F., Walchi, M. R., Naka-
mura, Y., Hirata, Y., Sasaki, T., Ohizumi, H. J. Am. Chem.
Soc. 1988, 53, 1800) and ascididemin (Kobayashi, J., Cheng,
J., Nakamura, H., Ohizumi, Y., Hirata, Y., Sasaki, T., Ohta,
T., Nozoe, S. Tetrahedron Lett. 1988, 29, 1177.
Assay for DNA TOPO II catalytic activity. The in vitro
inhibition of TOPO II decatenation assay was carried
out as described by Hsiang and et al.,28 with small
modifications. Topoisomerase II reaction mixtures (20
mL) containing 10 mM Tris–ClH, pH 7.9, 50 mM NaCl,
50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 15 mg/mL
bovine serum albumin, 1 mM ATP, 150 ng supercoiled
pBR322 DNA (Gibco) and 2 U of calf thymus topoi-
somerase II (TopoGen) were prepared. Compounds
under assay were added in DMSO (1% final DMSO
concentration) at 10–0.01 mg/mL. After 30 min at 37 ꢀC,
the reaction was stopped with 2 mL 10% SDS. Then 1
mL of 1 mg/mL proteinase K was added and incubated
another h at 37 ꢀC. Then 2 mL of 10ꢂ gel loading buffer
(0.25% bromophenol blue, 50% glycerol) were added to
each sample. The samples were submitted to electro-
phoresis using 1% agarose gel in 40 mM Tris base, 1
mM EDTA and acetic acid to pH 8.0. Gels were run at
1.5–2 V/cm, stained with 0.5 mg/mL ethidium bromide,
destained with water and photographed under UV light.
7. Davidson, B. S. Chem. Rev. 1993, 93, 1771.
8. Kobayashi, J.; Ishibashi, M. Chem. Rev. 1993, 93, 1753.
9. Previous total syntheses of meridine: (a) Bontemps, N.,
Delfourne, E., Bastide, J., Francisco, C., Bracher, F. Tetra-
hedron 1997, 53, 1743. (b) Kitahara, Y.; Tamura; F.; Nishi-
mura, M.; Kubo, A. Tetrahedron 1998, 54, 8421.
10. Gomez-Bengoa, E.; Echavarren, A. M. J. Org. Chem.
1991, 56, 3497.
11. Rudi, A.; Kashman, Y. J. Org. Chem. 1989, 54, 5331.
12. He, H.; Faulkner, D. J. J. Org. Chem. 1991, 56, 5369.
13. This cyclization is not restricted to 9, since it was also
observed in the thermolysis in the presence of a trace of cerium
ammonium nitrate of the related 3-methyl-5-(o-tri-
fluoroacetamido)-1,8-diazaanthracene-2,9,10-trione,
which
gave the corresponding pyridoacridine derivative in 40% yield.
14. (a) Boger, D. L. Tetrahedron 1983, 39, 2869. (b) Boger,
D. L. Chem. Rev. 1986, 86, 781. (c) Boger, D. L., Weinreb, S.
M. Hetero Diels–Alder Methodology in Organic Synthesis.
Academic Press: San Diego, 1987. (d) Boger, D. L. In
Paquette. L. A., Ed. Comprehensive Organic Synthesis; Trost,
B. M., Fleming, I., General Eds. Pergamon Press, 1991, Vol. 5,
p. 451. (e) Barluenga, J.; Tomas, M. Adv. Heterocycl. Chem.
1993, 57, 1. (f) Tietze, L. F.; Kettschau, G. Topics Curr. Chem.
1997, 189, 1.
Acknowledgements
The authors thank Dr. Dolores Garcıa-Gravalos and
Dr. Teresa Garcıa de Quesada (PharmaMar, Madrid)
for the biological studies. We thank Professor Francis
Schmitz (University of Oklahoma) for providing spectra
and a sample of meridine. Financial support from CICYT
(project PTR-95-0230) is also gratefully acknowledged.
15. These reactions give the 1,8-diazaanthraquinone regioi-
somer as the sole or major product: (a) Potts, K. T., Walsh, E.
B., Bhattacharjee, D. J. Org. Chem. 1987, 52, 2285. (b) See
also ref. 19a.
16. (a) Kitahara, Y.; Nakahara, S.; Yonezawa, T.; Nagatsu,
T. M.; Kubo, A. Heterocycles 1993, 36, 943–946. (b) Kitahara,
Y.; Nakahara, S.; Yonezawa, T.; Nagatsu, T. M.; Shibano, Y.;
Kubo, A. Tetrahedron 1997, 53, 17029.
17. Addition of pyridine-2,6-dicarboxylic acid N-oxide
(PDANO), as often recommended in the literature for related
oxidations with cerium ammonium nitrate, led to 58% of 12,
together with 18% of a side product which was identified as
4-chloro-8-methoxyquinoline-5,6-dione.
References and Notes
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19. For precedent of the ultrasound-promoted Diels–Alder
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(ddd, 1H, J=7.7, 7.7 and 1.2 Hz); 7.67 (ddd, 1H, J=7.7, 7.7
and 1.4 Hz); 7.27 (d, 1H, J=5.2 Hz); 7.26 (dd, 1H, J=7.7 and
1,2 Hz); 7.06 (dd, 1H, J=7.7 and 1.4 Hz); 6.52 (d, 1H, J=7.9
Hz); 5.60 (d, 1H, J=4.5 Hz); 5.37 (dd, 1H, J=7.9 and 4.5 Hz);
2.79 (s, 6H).
27. dH (250 MHz, d5-pyridine): 10.56 (br. s, 1H); 7.75 (ddd,
1H, J=7.7, 7.7 and 1.2 Hz); 7.67 (ddd, 1H, J=7.7, 7.7 and 1.4
Hz); 7.36 (dd, 1H, J=7.7 and 1.2 Hz); 7.35 (dd, 1H, J=5.2
Hz); 7.08 (dd, 1H, J=7.7 and 1.4 Hz); 6.50 (dd, 1H, J=7.7
and 1.0 Hz); 5.74 (d, 1H, J=4.4 Hz); 5.25 (ddd, 1H, J=7.7,
4.4 and 1.2 Hz); the H-6 signal was hidden by the solvent. dH
(250 MHz, d6-DMSO): 9.48 (d, 1H, J=3.8 Hz); 8.73 (d, 1H,
J=5.1 Hz); 7.85 (m, 1H); 7.83 (d, 1H, J=5.1 Hz); 7.71–7.63
(m, 2H); 7.42 (m); 6.39 (dd, 1H, J=7.9 and 3.8 Hz); 5.22 (d,
1H, J=4.8 Hz); 5.17 (m, 1H).
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