Synthesis and characterization of the antitumor activities of analogues of meridine, a marine pyridoacridine alkaloid

Article abstract of DOI:10.1021/jm0108496

Marine compounds with pyridoacridine skeletons are known to exhibit interesting antitumor activities. Among these compounds, meridine has already been reported as having significant antitumor activities in vitro. We synthesized 24 analogues of meridine su

Full text of DOI:10.1021/jm0108496

J . Med. Chem. 2001, 44, 3275-3282
3275
Syn th esis a n d Ch a r a cter iza tion of th e An titu m or Activities of An a logu es of
Mer id in e, a Ma r in e P yr id oa cr id in e Alk a loid
Evelyne Delfourne,*,† Francis Darro,‡ Nataly Bontemps-Subielos,† Christine Decaestecker,§ J ean Bastide,†
Armand Frydman,‡ and Robert Kiss§
Centre de Phytopharmacie, UMR-CNRS 5054, Universite´ de Perpignan, 52 Avenue de Villeneuve,
66860 Perpignan Cedex, France, Laboratoire Louis Lafon, Centre de Recherches, 19 Avenue du Pr Cadiot,
B.P. 22, 94701 Maisons-Alfort Cedex, France, and Laboratoire d’Histopathologie, Faculte´ de Me´decine, Universite´ Libre de
Bruxelles, 808 Route de Lennik, 1070 Bruxelles, Belgique
Received February 12, 2001
Marine compounds with pyridoacridine skeletons are known to exhibit interesting antitumor
activities. Among these compounds, meridine has already been reported as having significant
antitumor activities in vitro. We synthesized 24 analogues of meridine substituted on ring A
with the aim of obtaining compounds that display significantly higher in vitro antitumor
activities than meridine. The 24 compounds and meridine used as a control compound were
tested at 6 different concentrations on 12 different human cancer cell lines including various
histopathological types (glioblastomas and breast, colon, lung, prostate, and bladder cancers).
The IC50 value (i.e., the drug concentration inhibiting the mean growth value of the 12 cell
lines by 50%) of these 25 compounds ranged over 5 log concentrations, i.e., between 10 and
0.0001 µM, with four of the compounds exhibiting a significantly higher in vitro antitumor
activity than meridine. These compounds will now be subjected to further pharmacological
investigation including in vivo testing on both conventional murine tumors and human tumors
grafted onto nude mice.
In tr od u ction
In the search for new anticancer drugs, metabolites
from marine organisms (sponges, tunicates, bryozoa,
and algae) have attracted considerable interest in the
past 15 years because these organisms contain cytotoxic
compounds with unique molecular structures.1 The
largest family of marine alkaloids characterized to date
is based on the pyrido[k,l]acridine skeleton.2 These
polycyclic aromatic compounds have been reported as
exhibiting very interesting biological properties, includ-
ing the inhibition of a variety of cultured cell clones at
micromolar concentrations.3 From the few published
studies on the mechanism of action of these structures,
two general properties of pyridoacridine alkaloids have
emerged so far: (a) They are DNA intercalating agents,
and (b) nucleic acid intercalation is further modulated
by binding to other receptors (topoisomerases, transition
metals, etc.).4 Recently, other original mechanisms have
been reported that involve cytotoxicity action. Matsu-
moto et al.5 have shown that ascididemin 1 exhibits a
thiol-dependent oxidative DNA cleavage. De Guzman
et al.6 have shown that neoamphimedine 2 has the novel
ability to stimulate topoisomerase II to catenate DNA
to a high molecular weight complex. From all these
results, it appears that pyridoacridine derivatives are
good candidates for the discovery of compounds with
unique mechanisms and selectivity associated with
antitumor activity. Among these, we are especially
interested in meridine 3, which was first isolated by
Schmitz et al.7 from the ascidian Amphicarpa meridi-
ana, and which has been described as having antifun-
gal8 and antitumoral3 properties. To our knowledge,
only two papers have been published to date on the
cytotoxic characteristics of meridine.3,8
We set out to prepare a series of analogues of 3
substituted on ring A with a view to investigating in
vitro the effect of the substituents on antitumor activity.
The synthesis and assessment of the in vitro antitumor
activity of these substances are described in the present
report.
Ch em istr y
The different meridine-like structures were synthe-
sized on the basis of Kubo’s strategy for the synthesis
of meridine.9 The hetero-Diels-Alder-reactions were
carried out between different quinoline-5,8-dione de-
rivatives and o-nitro- or o-trifluoroacetamidocinnamal-
dehyde-dimethylhydrazone (Scheme 1).
* To whom correspondence should be addressed. Phone: 33 468 66
22 51. Fax: 33 468 66 22 23. E-mail: delfourn@univ-perp.fr.
† Universite´ de Perpignan.
‡ Laboratoire Louis Lafon.
§ Universite´ Libre de Bruxelles.
10.1021/jm0108496 CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/29/2001

3276 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Delfourne et al.
Sch em e 1a
Ta ble 1
trifluro-
acetamido pentacyclic
derivatives derivatives derivatives
nitro
R1
R2
R3
12
13
14
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
12a
12b
12c
13a
13b
13c
13d
13e
13f
13g
13h
14a
14b
14c
14d
14e
14f
14g
OMe
NH2
Br
H
NO2
NMe2
NHCOMe
OH
H
H
H
H
OH
12i
12j
3
14j
14k
CO2Et
H
13j
13k
the absence of water. Compound 13c was prepared from
13a by the action of sodium azide in DMF, with the
reduction of the azido intermediate in amine occurring
spontaneously in the reaction medium as described by
Kitahara et al.15 in the case of 12c. The o-trifluoro-
acetamido adducts 13a , 13b, 13d -f, 13h , 13j, and 13k
were also obtained by a Diels-Alder addition, which
proceeded regioselectively but with low yields. The 13g
derivative was prepared from chloro analogue 13a by
the action of dimethylamine. The aromatization of the
Diels-Alder adducts was realized by the treatment of
the crude product with 10% Pd/C in toluene. The
catalytic hydrogenation of derivatives 12a -c,i,j or the
acidic or alkaline hydrolysis of the trifluoroacetamido
group of compounds 13a -g,j,k gave the amino com-
pounds, which cyclized in situ to yield pentacyclic
pyridoacridines 14a -g,j,k . Compound 13h was the only
one that could not be transformed into its pentacyclic
parent. The different compounds obtained are reported
in Table 1.
a
(a) CH3CN, Ac2O, reflux; 10% Pd/C, toluene, reflux. (b) H2,
10% Pd/C. (c) CF3COOH or NaOH, CH2Cl2, reflux.
Sch em e 2a
a
(a) NaNO2, tetrabutylammoniun chloride, CH2Cl2/H2O, room
temp, 3 days. (b) CAN, CH3CN/H2O, room temp, 15 min. (c) NaN3,
DMF/H2O, 90 °C, 2h and 30 min. (d) Ph3P, THF/H2O, room temp,
2 h. (e) Ac2O, DMAP, room temp, 24 h.
P h a r m a cology: In Vitr o Deter m in a tion of th e
Dr u g-In d u ced In h ibition of Hu m a n Ca n cer Cell
Lin e Gr ow th
Quinoline-5,8-dione10 (4), 4-chloro, 4-methoxyquino-
line-5,8-dione (respectively 511 and 612), and 5,8-dioxo-
carbostyril (713) were obtained according to procedures
previously described. 4-Bromoquinoline-5,8-dione (8)
was prepared in two steps from 5,8-dimethoxyquinolin-
4-ol triflate, i.e., bromination and the subsequent oxida-
tion by cerium ammonium nitrate. The addition of
sodium nitrite to 4-chloro-5,8-dimethoxyquinoline and
oxidation by cerium ammonium nitrate led to 9. 4-Chloro-
5,8-dimethoxyquinoline was first transformed by the
action of sodium azide into the azido compound 10a ,
which was then reduced to amine 10b by triphenylphos-
phine; this amine was acetylated by acetic anhydride,
leading to the acetamido derivative 10c, which was
oxidized by cerium ammonium nitrate in compound 10
(Scheme 2).
3-Ethyl-5,8-dimethoxyquinolinecarboxylate14 was also
oxidized by cerium ammonium nitrate to give the
corresponding quinolinedione 11.
The nitro derivatives 12a ,15 12b,9 and 12c15 were
synthesized according to methods previously described.
12j was synthesized by a similar Diels-Alder reaction.
12i was obtained by the same procedure as 12c, i.e.,
the treatment of 12a by sodium azide in DMF but in
Six concentrations of each of the 25 compounds under
study were tested on 12 different human cancer cell
lines including various histopathological types (glio-
blastomas and breast, colon, lung, prostate, and bladder
cancers). This experimental approach enabled us to
determine the IC50 value for each drug, i.e., the con-
centration that reduced the mean growth value of the
12 cell lines by 50% compared to the mean control
growth value. Table 2 illustrates the individual IC50
values obtained for each of the 12 cell lines under study.
These data show that IC50 values ranged between 10
and 0.0001 µM. They thus ranged over 5 logarithmic
concentrations. The compounds that exhibited the high-
est in vitro antitumor activities included 13b, 13e, 14b,
and 14e. These newly synthesized compounds therefore
appeared markedly more efficient in vitro in terms of
antitumoral activity than the parent drug from which
they were derived, i.e., meridine (Table 2). Indeed, the
meridine-associated IC50 values ranged between 10 and
0.01 µM over the 12 cell lines under study (Table 2).
The 12 cell lines exhibited differences in terms of drug
sensitivity, which varied over more than 3 logarithmic
concentrations for a given compound. This is the case
for compound 13e, for example.

Analogues of Meridine
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3277

3278 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Delfourne et al.
Discu ssion
mediated decatenation of kinetoplast DNA correlates
with their cytotoxic potency against human colon cancer
cells. These authors4 thus concluded that the disruption
of the function of topoisomerase-II after DNA intercala-
tion is a probable mechanism by which pyridoacridines
inhibit the proliferation of human colon tumor cells.
In conclusion, we have now succeeded in synthesizing
meridine analogues. Among these compounds 13b, 14b,
13e, and 14e have levels of in vitro cytotoxic antitumor
activity up to 10 000 times greater than the parent
meridine when directed against a panel of 12 human
cancer cell lines. Whether the antitumor activities of the
original meridine analogues that we synthesized are
exerted through anti-topoisomerase action or through
other mechanisms is not yet known. Experiments are
under way to characterize the mechanisms of action of
these drugs and to discover further antitumor activity
on in vivo models.
Longley et al.3 studied 24 metabolites (including
pyridoacridine structures and others) from marine
sponges and evaluated them for their cytotoxicity
against two human tumor cell lines, i.e., the A549
nonsmall cell lung carcinoma (also used in our present
study) and the HT-29 colon adenocarcinoma, and also
against one murine leukemia cell line. Of these 24
compounds, meridine had a weaker level of antitumor
activity than other compounds, including latrunculin A,
batzelline A, and chondrillin.3 We synthesized new
meridine analogues and investigated whether any of
them displayed any greater antitumor activity than the
parent meridine. In the present work we were exclu-
sively interested in substitutions on ring A, with R1
being the most modulated position. The same strategy
based on the Diels-Alder reaction and involving diverse
substituted quinoline-5,8-diones enabled the different
meridine derivatives to be synthesized. The yield (low
in all cases) and the regioselectivity of the Diels-Alder
reaction was not altered by the nature of the dienophile
substituent. It should be noted that the aromatic dienes
led to a unique regioisomer unlike the same reaction
with aliphatic dienes,16 in which the formation of two
isomers was generally observed.
We made use of the colorimetric MTT assay, which
indirectly assesses the effect of potentially anticancer
compounds on the overall growth of adherent cell lines.17
The data that we obtained in vitro showed that almost
all the compounds have cytotoxic properties and that
some of them had levels of antitumor activity up to
10 000 times higher than the parent meridine (see Table
2). These results could be in accordance with the
mechanism proposed by Matsumoto et al. for another
pyridoacridine: ascididemine.5 These authors have
demonstrated that this alkaloid can act to oxidatively
damage DNA, probably via its iminoquinone moiety.
Compounds 13 and 14 respectively possess a quinone
or an iminoquinone function that could involve a redox
reaction. The substituent effect is rather the same from
one structure (13) to another (14), with the most active
compounds being the unsubstituted ones and those with
a methoxyl group in R1.
To explain the results of the R1 position of pentacyclic
compounds, we investigated a number of PM318-derived
parameters such as the geometry of minimum energy
and charge parameters. The most active compounds
were those with two concomitant properties, i.e., first,
PM3-optimized conformations close to planar structures,
which is the case for compounds 14a -c and 14j and,
second, the N(13) charge above -0.06; compounds 3 and
14c exhibit a strong hydrogen bond between the hydro-
gen of the substituent and the N(13), leading to a high
negative charge on N(13). The in vitro activity of these
two compounds was lower than that of 14b and 14e.
Another hypothesis from the point of view of the mode
of action is that the antitumor activity observed could
relate, at least partly, to the inhibition of topoisomerase-
II activity. Indeed, McDonald et al.4 characterized the
antitumor activities of several newly isolated pyri-
doacridine alkaloids (including dehydrokuanoniamine
B, shermilamine C, cystodytin K, etc.) from a Fijian
Cystodytes sp. ascidian. They pointed out that the
pyridoacridines’ ability to inhibit the topoisomerase-II-
Exp er im en ta l Section
1. Ch em istr y. Ch em ica l Syn th esis. 1H and 13C NMR
spectra were obtained with a J EOL 400 MHz spectrometer
with the chemical shifts of the remaining protons of the
deuterated solvents serving as internal standards. IR spectra
were obtained with a Perkin-Elmer (1600 series FTIR) spec-
trometer. Mass spectra (MS) were recorded on an automass
Unicam spectrometer. Reagents were purchased from com-
mercial sources and used as received. Chromatography was
performed on silica gel (15-40 µm) by means of the solvent
systems indicated below. The purity of the different meridine
analogues was evaluated on two analytical chromatographic
systems. System I consisted of an ultrabase UB 235, 5 µm
column (250 mm × 4.6 mm), H2O/CH3CN 50:50 at 1 mL/min
flow rate, 200 nm, and the system II consisted of a Luna
phenylhexyl, 5 µm column (150 mm × 4.6 mm), H2O/CH3CN
60:40 at 1.5 mL/min flow rate, 200 nm.
4-Br om o-5,8-d im et h oxyq u in olin e. A mixture of 5,8-
dimethoxyquinolin-4-ol triflate9 (3 g, 9.35 mmol) and LiBr (8.2
g, 94.2 mmol) in dioxane (60 mL) was refluxed for 30 min. The
mixture was cooled and H2O (200 mL) added. The mixture was
extracted with AcOEt (3 × 200 mL), and the extract was dried
over MgSO4 and concentrated to dryness to give the bromo
derivative (3 g, 91%) as a yellow solid: mp, 86 °C. 1H NMR
(CDCl3): δ 3.90 (s, 3H), 4.02 (s, 3H), 6.89 (d, 1H, J ) 8.8 Hz),
6.97 (d, 1H, J ) 8.8 Hz), 7.71 (d, 1H, J ) 4.4 Hz), 8.57(d,1H,
J ) 4.4 Hz). 13C NMR (CDCl3): δ 56.10, 56.26, 107.54, 107.89,
120.49, 128.08, 128.79, 141.81, 148.18, 148.85, 149.44.
4-Br om oqu in olin e-5,8-d ion e (8). Cerium ammonium ni-
trate (CAN) (0.6 g, 1.11 mmol) was added to a solution of
4-bromo-5,8-dimethoxyquinoline (0.1 g, 0,373 mmol), CH3CN
(8 mL), and H2O (4 mL). The mixture was stirred at room
temperature for 30 min. The CH3CN was evaporated, and H2O
(100 mL) was added to the residue. The mixture was extracted
with HCCl3 (3 × 100 mL). The extract was dried over MgSO4,
and the solvent was evaporated to yield dione (83 mg, 93%)
1
as a pink solid: mp, 190 °C. H NMR (CDCl3): δ 7.06 (d, 1H,
J ) 10 Hz), 7.12 (d, 1H, J ) 10 Hz), 7.93 (d, 1H, J ) 5.2 Hz),
8.73 (d, 1H, J ) 5.2). 13C NMR (CDCl3): δ 126.71, 132.83,
134.13, 136.89, 139.16, 148.95, 152.88, 181.53, 182.6.
4-Nitr o-5,8-dim eth oxyqu in olin e (9a). A solution of 4-chlo-
ro-5,8-dimethoxyquinoline11 (1.89 g, 8.47 mmol), NaNO2 (0.69
g, 10.06 mmol), and tetrabutylammonium chloride, (2.9 g,
10.59 mmol) in CH2Cl2 (50 mL) and H2O (50 mL) was stirred
at room temperature for 3 days. The organic layer was
recovered, and the aqueous layer was extracted with CH2Cl2
(3 × 50 mL). The combined organic layers were dried over
MgSO4 and concentrated to dryness. Purification of the crude
product by flash chromatography (CH2Cl2/MeOH, 98:2) gave
the nitro compound (1.2 g, 60%) as a bright-yellow solid: mp,
1
169 °C. H NMR (CDCl3): δ 3.85 (s, 3H), 4.05 (s, 3H), 6.92 (d,
1H, J ) 8.8 Hz), 7.07 (d, 1H, J ) 8.8 Hz), 7.36 (d, 1H, J ) 4.4

Analogues of Meridine
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3279
1
Hz), 9.01 (d, 1H, J ) 4.4 Hz). 13C NMR (CDCl3): δ 56.41, 56.52,
107.61, 108.99, 111.02, 113.69, 141.99, 146.48, 149.31, 149.53,
152.68.
mp, 124 °C. H NMR (CDCl3): δ 1.45 (t, 3H, J ) 7.3 Hz), 4.49
(q, 2H, J ) 7.3 Hz), 7.13 (d, 1H, J ) 10.4 Hz), 7.22 (d, 1H,
J ) 10.4 Hz), 8.99 (d, 1H, J ) 2.2 Hz), 9.58 (d, 1H, J ) 2.2
Hz). 13C NMR (CDCl3): δ 14.22, 62.48, 128.62, 129.86, 135.98,
138.29, 139.33, 149.14, 155.17, 163.44, 182.40, 183.63.
4-Nitr oqu in olin e-5,8-d ion e (9). 9 was prepared as de-
scribed for 8, from 4-nitro-5,8-dimethoxyquinoline (0.4 g, 1.71
mmol), CAN (2.8 g, 5.2 mmol), CH3CN (10 mL), and H2O (5
mL). After the mixture was stirred for 15 min, an orange solid
was obtained (290 mg, 83%): mp, 180 °C. 1H NMR (CDCl3):
δ 7.10 (d, 1H, J ) 10.4 Hz), 7.23 (d, 1H, J ) 10.4 Hz), 7.64 (d,
1H, J ) 5.6 Hz), 9.24 (d, 1H, J ) 5.6 Hz). 13C NMR (DMSO-
d6): δ 118.49, 120.00, 137.89, 139.26, 148.83, 152.83, 156.38,
181.07, 182.04.
4-Azid o-5,8-d im eth oxyqu in olin e (10a ). A solution of
4-chloro-5,8-dimethoxyquinoline (10 g, 44.7 mmol) and NaN3
(16.7 g, mmoles) in a mixture DMF/H2O (160 mL/60 mL) was
heated at 90 °C for 2 h and 30 min. After the solution was
cooled, a saturated solution of NH4Cl (500 mL) was added and
the mixture extracted by CHCl3 (3 × 150 mL). The extract
was dried over MgSO4 and concentrated to dryness to give the
azido compound (8.7 g, 85%) as a brown solid: mp, 106 °C. 1H
NMR (CDCl3): δ 3.86 (s, 3H), 3.95 (s, 3H), 6.75 (d, 1H, J ) 8.6
Hz), 6.89 (d, 1H, J ) 8.6 Hz), 7.10 (d, 1H, J ) 4.8 Hz), 8.72 (d,
1H, J ) 4.8 Hz). 13C NMR (CDCl3): δ 55.77, 56.50, 106.44,
107.49, 110.76, 114.20, 142.25, 145.45, 148.82, 148.96, 149.13.
4-Am in o-5,8-d im eth oxyqu in olin e (10b). A solution of
4-azido-5,8-dimethoxyquinoline (8.7 g, 37.8 mmol) and triphen-
ylphosphine (12 g, 45.8 mmol) was stirred at room temperature
for 2 h in a THF/H2O mixture (110 mL/110 mL). After
concentration, 1 N HCl (200 mL) was added and the mixture
extracted with ether (3 × 500 mL). The aqueous layer was
then made alkaline by 1 N NaOH (250 mL) and extracted by
CHCl3 (3 × 250 mL). The extract was dried over MgSO4 and
concentrated to dryness to give quantitatively the amino
derivative as a brown solid (which decomposes before melting).
1H NMR (CDCl3): δ 3.86 (s, 3H), 3.94 (s, 3H), 5.93 (br.s, 2H),
6.39 (d, 1H, J ) 5.2 Hz), 6.55 (d, 1H, J ) 8.8 Hz), 7.77 (d, 1H,
J ) 8.8 Hz), 8.36 (d, 1H, J ) 5.2 Hz). 13C NMR (CDCl3): δ
55.77, 55.80, 102.22, 104.73, 106.32, 111.14, 142.45, 149.48,
149.55, 150.81, 152.09.
4-Aceta m id o-5,8-d im eth oxyqu in olin e (10c). A suspen-
sion of 4-amino-5,8-dimethoxyquinoline (3 g, 14.7 mmol) and
DMAP (0.72 g, 5.88 mmol) was stirred at room temperature
for 24 h in acetic anhydride (30 mL). A saturated solution of
NaHCO3 (40 mL) was added after concentration, and the
mixture was extracted with CHCl3 (3 × 50 mL). The extract
was dried over MgSO4 and concentrated (benzene was added
and the mixture concentrated to remove the acetic anhydride)
to give the acetamido derivative (2.7 g, 74%) as a yellow solid:
mp, 152 °C. 1H NMR (CDCl3): δ 2.26 (s, 3H), 4.03 (s, 6H), 6.81
(d, 1H, J ) 8.4 Hz), 6.90 (d, 1H, J ) 8.4 Hz), 8.62 (d, 1H, J )
4.8 Hz), 8.78 (d, 1H, J ) 4.8 Hz), 10.86 (br s, 1H). 13C NMR
(CDCl3): δ 14.15, 44.29, 44.91, 93.65, 94.89, 98.22, 99.96,
129.73, 131.69, 137.16, 138.35, 138.57, 157.42.
4-Aceta m id oqu in olin e-5,8-d ion e (10). A solution of 4-ac-
etamido-5,8-dimethoxyquinoline 10c (5 g, 20.3 mmol) and CAN
(22 g, 40.1 mmol) in CH3CN (50 mL) and H2O (50 mL) was
stirred for 1 h and 45 min at 0 °C. The CH3CN was evaporated,
and a solution of saturated NaHCO3 (500 mL) and H2O (200
mL) was added and the mixture extracted with CHCl3 (3 ×
500 mL). The extract was dried over MgSO4 and concentrated
to give the expected product (3.4 g, 77%) as a yellow-brown
solid, which was quickly used in the next step. 1H NMR
(CDCl3): δ 2.28 (s, 3H), 6.97 (d, 1H, J ) 10.4 Hz), 7.08 (d, 1H,
J ) 10.4 Hz), 8.83 (d, 1H, J ) 5.6 Hz), 8.88 (d, 1H, J ) 5.6
Hz), 11.75 (br s, 1H). 13C NMR (CDCl3): δ 26.00, 114.40,
117.75, 138.41, 139.31, 147.34, 148.60, 155.73, 170.53, 182.96,
189.75.
3-E t h ylqu in olin eca r b oxyla t e-5,8-d ion e (11). 11 was
prepared as described for 10 from 3-ethyl-5,8-dimethoxyquino-
linecarboxylate13 (1 g, 3.83 mmol) and CAN (7.4 g, 13.5 mmol)
in CH3CN/H2O (45 mL/23 mL), and the mixture was stirred
for 1 h at room temperature. A saturated solution of NaHCO3
(17 mL) and H2O (60 mL) was added and the extraction was
with CH2Cl2 (3 × 100 mL), giving a yellow solid (0.8 g, 91%):
6-Hyd r oxy-4-(2-n itr op h en yl)p yr id o[3,2-g]qu in olin e-5,-
10-d ion e (12i). A solution of chloro adduct 12a 15 (1.3 g, 3.56
mmol) and sodium azide (1.3 g, 20.03 mmol) in DMF (11 mL)
was heated for 1 h at 90 °C. After the mixture was cooled and
concentrated to dryness, a saturated solution of NH4Cl (20 mL)
was added. The mixture was extracted with 5% MeOH/CH2-
Cl2 (5 × 50 mL). The extracts were dried over MgSO4 and
concentrated to give 12i (0.26 g, 21%) as a yellow solid: mp,
180 °C. 1H NMR (CDCl3): δ 7.14 (d, 1H, J ) 5.6 Hz), 7.26 (dd,
1H, J ) 1.6 and 8.4 Hz), 7.48 (d, 1H, J ) 4.8 Hz), 7.70 (ddd,
1H, J ) 1.6, 8.4 and 7.2 Hz), 7.78 (ddd, 1H, J ) 0.8, 7.2 and
8.0 Hz), 8.36 (dd, 1H, J ) 1.2 and 8.0 Hz), 8.81 (d, 1H, J ) 5.6
Hz), 9.18 (d, 1H, J ) 4.8 Hz), 11.85 (s, 1H). 13C NMR (CDCl3):
δ 115.83, 117.56, 124.99, 126.63, 128.92, 129.71 (2C), 134.17,
134.53, 146.59, 149.24, 149.73, 155.29, 155.38, 156.32, 167.78,
189.23. IR (CHCl3): 3376, 1701, 1649 cm-1. MS (EI mode): m/z
301 (100), 273 (8). tR is 2.9 min (98% purity), using system I,
and tR is 1.57 min (97% purity), using system II.
7-E t h yl-4-(2-n it r op h en yl)p yr id o[3,2-g]q u in olin eca r -
boxyla te-5,10-d ion e (12j). A solution of 3-ethylquinolinecar-
boxylate-5,8-dione (0.3 g, 1.29 mmol), 4-(2-nitrophenyl)-1-
(dimethylamino)-1-aza-1,3-butadiene (0.33 g, 1.47 mmol), and
acetic anhydride (1.4 mL) in CH3CN (50 mL) was refluxed
under nitrogen for 64 h. After the mixture was cooled, the
mixture was concentrated to dryness and the residue purified
by flash chromatography (CH2Cl2/MeOH, 98:2) to give 12j (53
1
mg, 10%) as a decomposing brown solid. H NMR (CDCl3): δ
1.41 (t, 3H, J ) 7 Hz), 4.44 (q, 2H, J ) 7 Hz), 7.28 (dd, 1H,
J ) 7.7 and 1.5 Hz), 7.54 (d, 1H, J ) 4.8 Hz), 7.72 (dd, 1H,
J ) 8.0 and 1.5 Hz), 7.80 (ddd, 1H, J ) 7.7, 8.0 and 1.5 Hz),
8.37 (dd, 1H, J ) 8.0 and 1.5 Hz), 8.97 (d, 1H, J ) 2 Hz), 9.21
(d, 1H, J ) 4.8 Hz), 9.65 (d, 1H, J ) 2 Hz). 13C NMR (CDCl3):
δ 14.19, 62.49, 124.91, 127.08, 128.93, 129.74, 129.78, 129.82,
130.23, 134.13, 134.45, 137.05, 146.80, 149.40, 149.85, 149.88,
155.11, 155.99, 163.26, 179.28, 181.96. IR (CHCl3): 1728, 1706,
1680 cm-1. MS (EI mode): m/z 357 (100), 356 (85), 329 (17),
328 (8). tR is 6.62 min (98% purity), using system I, and tR is
6.0 min (96% purity), using system II.
Diels-Ald er Rea ction s of Su bstitu ted Qu in olin e-5,8-
d ion es w ith 4-(2-Tr iflu or oa ceta m id op h en yl)-1-d im eth yl-
1-a za -1,3-bu ta d ien e. Gen er a l Meth od A. A solution of
substituted quinoline-5,8-dione, 4-(2-trifluoroacetamidophen-
yl)-1-(dimethylamino)-1-aza-1,3-butadiene, and acetic anhy-
dride in CH3CN was refluxed under nitrogen. After cooling,
the mixture was concentrated to dryness and the unreacted
starting materials were filtered off over silica gel. Pd/C (10%)
and toluene were added to the crude product, and the suspen-
sion was refluxed. After cooling, the mixture was concentrated
and purified by flash chromatography to give the expected
product.
6-Ch lor o-4-(2-tr iflu or oa ceta m id op h en yl)p yr id o[3,2-g]-
qu in olin e-5,10-d ion e (13a ). Method A was used and involved
4-chloroquinoline-5,8-dione 5 (0.7 g, 3.63 mmol), 4-(2-trifluo-
roacetamidophenyl)-1-(dimethylamino)-1-aza-1,3-butadiene (1.2
g, 4 mmol), acetic anhydride (1 mL), CH3CN (50 mL), and
reflux of 6 h. Filtration was done on silica gel (CH2Cl2/MeOH
99.5:0.5). Pd/C (10%, 4 g) and toluene (100 mL) were added,
and the solution was refluxed for 2 h. Flash chromatography
(CH2Cl2/MeOH 98:2) gave 13a (87 mg, 6%) as a bright-yellow
solid: mp, 152 °C. 1H NMR (CDCl3): δ 7.21 (d, 1H, J ) 9 Hz),
7.46 (dd, 1H, J ) 7.5 and 7.5 Hz), 7.58 (m, 2H), 7.72 (m, 2H),
7.81 (s, 1H), 8.94 (d, 1H, J ) 5.1 Hz), 9.13 (d, 1H, J ) 4.7 Hz).
13C NMR (CDCl3): δ 115.49 (q, J ) 287.2 Hz), 125.55, 128.11,
128.27, 129.20, 129.35, 130.16, 131.02, 131.32, 131.52, 133.58,
145.13, 147.57, 148.24, 149.85, 154.35, 154.86, 155.34 (q, J )
37 Hz), 179.28, 182.40. IR (CHCl3): 3410, 1737, 1707, 1648,
1602 cm-1. MS (EI mode): m/z 357 (100), 356 (85), 329 (17),
328 (8). tR is 4.99 min (97% purity), using system I, and tR is
3.34 min (95% purity), using system II.

3280 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Delfourne et al.
6-Meth oxy-4-(2-tr iflu or oa ceta m id op h en yl)p yr id o[3,2-
g]qu in olin e-5,10-d ion e (13b). Method A was used and
involved 4-methoxyquinoline-5,8-dione 6 (3.5 g, 16 mmol), 4-(2-
trifluoroacetamidophenyl)-1-(dimethylamino)-1-aza-1,3-buta-
diene (7 g, 24 mmol), acetic anhydride (15 mL), CH3CN (200
mL), and reflux of 18 h. Filtration was done on silica gel (CH2-
Cl2/MeOH 99.5:0.5). Pd/C (10%, 6 g) and toluene (150 mL) were
added, and the solution was refluxed for 2 h. Flash chroma-
tography (CH2Cl2/MeOH 95:5) gave 13b (0.6 g, 9%) as a pale-
green solid: mp, 158 °C. 1H NMR (CDCl3): δ 4.04 (s, 3H), 7.22
(d, 1H, J ) 6 Hz), 7.24 (dd, 1H, J ) 1.6 and 7.6 Hz), 7.46 (dd,
1H, J ) 7.6 and 7.2 Hz), 7.56 (d, 1H, J ) 4.8 Hz), 7.60 (dd,
1H, J ) 7.6 and 7.2 Hz), 7.79 (d, 1H, J ) 8.4 Hz), 7.97 (s, 1H),
8.95 (d, 1H, J ) 6 Hz), 9.13 (d, 1H, J ) 5.2 Hz). 13C NMR
(CDCl3): δ 65.84, 111.53, 115.34 (q, J ) 305 Hz), 120.60,
125.44, 127.82, 129.09, 129.46, 129.80, 131.28, 131.70, 133.83,
147.15, 148.22, 150.08, 154.29, 155.09 (q, 42 Hz), 156.07,
165.95, 180.26, 183.00. IR (CHCl3): 3399, 1734, 1706, 1675,
1584 cm-1. MS (EI mode): m/z 427 (100), 358 (36), 315 (71).
tR is 3.87 min (98% purity), using system I, and tR is 2.21 min
(90% purity), using system II.
4.4 Hz), 7.77 (d, 1H, J ) 4.4 Hz), 8.04 (s, 1H), 9.18 (d, 1H,
J ) 4.4 Hz), 9.32 (d, 1H, J ) 4.4 Hz). 13C NMR (CDCl3): δ
115.41 (q, J ) 287 Hz), 120.85, 122.58, 126.51, 128.16, 129.25,
129.33, 130.39, 130.64, 131.42, 134.24, 148.11, 148.18, 149.11,
154.51, 155.21 (q, 37 Hz), 155.77, 157.13, 177.79, 180.54. IR
(CHCl3): 3400, 1740, 1707, 1648, 1601 cm-1. MS (EI mode):
m/z 442 (7), 330 (12), 284 (40). tR is 5.46 min (99% purity),
using system I, and tR is 4.31 min (96% purity), using system
II.
6-(Aceta m id o)-4-(2-tr iflu or oa ceta m id op h en yl)p yr id o-
[3,2-g]qu in olin e-5,10-d ion e (13h ). Method A was used and
involved 4-acetamidoquinoline-5,8-dione 10 (3.4 g, 15.7 mmol),
4-(2-trifluoroacetamidophenyl)-1-(dimethylamino)-1-aza-1,3-
butadiene (5 g, 17.5 mmol), acetic anhydride (6 mL), CH3CN
(270 mL), 10% Pd/C (4.4 g), and reflux of 15 h. Flash
chromatography (CHCl3/MeOH 98:2) gave 13h (80 mg, 1%) as
a brown solid: mp, >260 °C. 1H NMR (CDCl3): δ 2.23 (s, 3H),
7.25 (dd, 1H, J ) 7.7 and 1.5 Hz), 7.48 (ddd, 1H, J ) 7.3, 7.7,
and 1.1 Hz), 7.53 (dd, 1H, J ) 4.8 Hz), 7.60 (ddd, 1H, J ) 7.7,
7.3, and 1.5 Hz), 7.78 (d, 1H, J ) 7.3 Hz), 7.93 (br s, 1H), 8.86
(d, 1H, J ) 5.7 Hz), 8.93 (d, 1H, J ) 5.7 Hz), 9.08 (d, 1H, J )
4.8 Hz). 13C NMR (CDCl3): δ 25.20, 115.63 (q, J ) 288 Hz),
115.93, 116.66, 126.61, 127.86, 128.75, 129.11, 129.28, 131.31,
131.62, 134.87, 146.17, 146.82, 148.33, 149.60, 154.30, 154.82,
154.94 (q, J ) 36 Hz), 170.13, 179.34, 186.80. IR (CHCl3):
3410, 3277, 1719, 1707, 1702 cm-1. MS (EI mode): m/z 454
(9), 412 (7), 343 (100), 300 (17). tR is 3.83 min (96% purity),
using system I, and tR is 2.56 min (93% purity), using system
II.
6-Br om o-4-(2-tr iflu or oa ceta m id op h en yl)p yr id o[3,2-g]-
qu in olin e-5,10-d ion e (13d ). Method A was used and involved
4-bromoquinoline-5,8-dione 8 (2 g, 8.4 mmol), 4-(2-trifluoro-
acetamidophenyl)-1-(dimethylamino)-1-aza-1,3-butadiene (3.6
g, 12.6 mmol), silica gel (5 g) instead of acetic anhydride, CH3-
CN (220 mL), and reflux of 10 h. Filtration was done on silica
gel (CH2Cl2/MeOH 99.5:0.5). Pd/C (10%, 2.1 g) and toluene (21
mL) were added, and the solution was refluxed for 4 h. Flash
chromatography (CH2Cl2/MeOH 98:2) gave 13d (0.19 g, 5%)
as a beige solid: mp, 145 °C. 1H NMR (CDCl3): δ 7.21 (dd,
1H, J ) 7.2 and 1.2 Hz), 7.44 (ddd, 1H, J ) 7.2, 7.2, and 1.2
Hz), 7.55 (ddd, 1H, J ) 7.2, 7.2, and 1.2 Hz), 7.57 (d, 1H, J )
4.8 Hz), 7.69 (dd, 1H, J ) 7.2 and 1.2 Hz), 7.91 (d, 1H, J ) 4.8
Hz), 8.20 (br s, 1H), 8.73 (d, 1H, J ) 4.8 Hz), 9.05 (d, 1H, J )
4.8 Hz). 13C NMR (CDCl3): δ 115.12 (q, J ) 287.1 Hz), 125.41,
127.71, 128.94, 129.30, 129.30, 129,72, 130.91, 131.10, 132.89,
133.31, 134.26, 147.35, 147.79, 149.24, 153.44, 154.30, 154.78
(q, J ) 36.8 Hz), 178.86, 182.05. IR (CHCl3): 3401, 1735, 1706,
1684, 1603 cm-1. MS (EI mode): m/z 477 (34), 474 (35), 408
(21), 406 (25), 365 (76), 362 (100). tR is 5.21 min (92% purity),
using system I, and tR is 3.66 min (95% purity), using system
II.
4-(2-Tr iflu or oacetam idoph en yl)pyr ido[3,2-g]qu in olin e-
5,10-d ion e (13e). Method A was used and involved quinoline-
5,8-dione 4 (1 g, 6.3 mmol), 4-(2-trifluoroacetamidophenyl)-1-
(dimethylamino)-1-aza-1,3-butadiene (3.59 g, 12.6 mmol), acetic
anhydride (7.5 mL), CH3CN (175 mL), and reflux of 24 h.
Filtration on silica gel (CH2Cl2/MeOH 95:5). Pd/C (10%, 6.2 g)
and toluene (150 mL) were added, and the solution was
refluxed for 12 h. Flash chromatography (CH2Cl2/MeOH 95:
5) gave 13e (125 mg, 5%) as a yellow solid: mp, 205 °C. 1H
NMR (CDCl3): δ 7.20 (dd, 1H, J ) 8 and 1.2 Hz), 7.46 (ddd,
1H, J ) 8.0, 8.0, and 1.2 Hz), 7.58 (d, 1H, J ) 4.4 Hz), 7.59
(ddd, 1H, J ) 8.0, 8.0, and 1.2 Hz), 7.74 (m, 3H), 8.42 (dd, 1H,
J ) 8.0 and 1.6 Hz), 9.14 (dd, 1H, J ) 4.4 and 1.6 Hz), 9.16 (d,
1H, J ) 4.4 Hz). 13C NMR (CDCl3): δ 115.46 (q, J ) 291 Hz),
125.26, 127.45, 127.94, 128.54, 129.01, 129.99, 130.49, 131.15,
131.50, 133.90, 135.57, 147.84, 147.99, 149.68, 154.93, 155.39
(q, J ) 40 Hz), 155.86, 179.66, 183.19. IR (CHCl3): 3420, 1740,
1706, 1679, 1587 cm-1. MS (EI mode): m/z 397 (97), 382 (27),
328 (48), 285 (100). tR is 4.20 min (92% purity), using system
I, and tR is 2.35 min (99% purity), using system II.
7-E t h yl-4-(2-t r iflu or oa cet a m id op h en yl)p yr id o[3,2-g]-
qu in olin eca r boxyla te-5,10-d ion e (13j). Method A was used
and involved 3-ethylquinolinecarboxylate-5,8-dione 11 (0.45 g,
1.94 mmol), 4-(2-trifluoroacetamidophenyl)-1-(dimethylamino)-
1-aza-1,3-butadiene (0.61 g, 2.14 mmol), acetic anhydride (1.4
mL), CH3CN (50 mL), and reflux of 24 h. Filtration was done
on silica gel (CH2Cl2/ MeOH 98:2), MnO2 (0.63 g), CHCl3 (20
mL), room temperature, 3 h. Flash chromatography (CH2Cl2)
1
gave 13j (27 mg, 3%) as a brown solid: mp, 124 °C. H NMR
(CDCl3): δ 1.42 (t, 3H, J ) 7 Hz), 4.45 (q, 2H, J ) 7 Hz), 7.22
(dd, 1H, J ) 7.7 and 1.5 Hz), 7.47 (ddd, 1H, J ) 7.7, 7.7, and
0.8 Hz), 7.61 (ddd, 1H, J ) 7.7, 7.7, and 1.5 Hz), 7.63 (d, 1H,
J ) 4.8 Hz), 7.68 (br s, 1H), 7.74 (d, 1H, J ) 8.1 Hz), 9.00 (d,
1H, J ) 1.9 Hz), 9.20 (d, 1H, J ) 4.8 Hz), 9.65 (d, 1H, J ) 1.9
Hz). 13C NMR (CDCl3): δ 14.11, 62.56, 115.46 (q, J ) 287 Hz),
125.73, 127.50, 127.99, 129.01, 129.97 (2), 130.23, 131.27,
131.43, 134.16, 136.91 (2), 148.32, 149.53, 154.88, 155.57 (q,
J ) 38 Hz), 155.76, 163.11, 179.05, 182.10. IR (CHCl3): 3401,
1730, 1709, 1681 cm-1. MS (EI mode): m/z 469 (17), 468 (6),
357 (100), 356 (81), 329 (17), 328 (6). tR is 6.62 min (98%
purity), using system I, and tR is 6.00 min (96% purity), using
system II.
8-Oxo-4-(2-tr iflu or oacetam idoph en yl)pyr ido[3,2-g]qu in -
olin e-5,10-d ion e (13k ). Method A was used and involved 5,8-
dioxocarbostyril 7 (1.04 g, 5.9 mmol), 4-(2-trifluoroacetami-
dophenyl)-1-(dimethylamino)-1-aza-1,3-butadiene (1.9 g, 6.5
mmol), acetic anhydride (2.2 mL), 10% Pd/C (1.6 g), CH3CN
(500 mL), and reflux of 15 h. Flash chromatography (CH2Cl2/
MeOH 95:5) gave 13k (0.4 g, 16%) as a yellow solid: mp, >260
°C. 1H NMR (CDCl3): δ 6.90 (dd, 1H, J ) 9.9 Hz), 7.19 (dd,
1H, J ) 7.8 and 1.5 Hz), 7.47 (ddd, 1H, J ) 7.8, 7.8, and 1.1
Hz), 7.60 (m, 2H), 7.68 (m, 2H), 7.93 (d, 1H, J ) 9.9 Hz), 9.09
(d, 1H, J ) 4.8 Hz). 13C NMR (CDCl3): δ 115.5 (q, J ) 288
Hz), 126.34, 126.90, 127.08, 128.69, 129.12, 130.89, 134.61,
135.63, 146.85, 148.02, 150.56, 151.70, 152.83, 154.92, 155.57
(q, J ) 38 Hz), 161.92, 169.63, 179.50, 181.13. IR (CHCl3):
3401, 3334, 1735, 1685, 1664 cm-1. MS (EI mode): m/z 413
(33), 344 (17), 301 (100), 177 (44). tR is 3.56 min (92% purity),
using system I, and tR is 2.02 min (97% purity), using system
II.
6-Nit r o-4-(2-t r iflu or oa cet a m id op h en yl)p yr id o[3,2-g]-
qu in olin e-5,10-d ion e (13f). Method A was used and involved
4-nitroquinoline-5,8-dione 9 (1.5 g, 7.35 mmol), 4-(2-trifluoro-
acetamidophenyl)-1-(dimethylamino)-1-aza-1,3-butadiene (4.2
g, 14.7 mmol), acetic anhydride (7.5 mL), CH3CN (100 mL),
and reflux of 18 h. Filtration was done on silica gel (CH2Cl2/
MeOH 95:5). Pd/C (10%, 2.5 g) and toluene (30 mL) were
added, and the solution was refluxed for 5 h. Flash chroma-
tography (CH2Cl2/MeOH 95:5) gave 13f (0.4 g, 13%) as a beige
solid: mp, 158 °C. 1H NMR (CDCl3): δ 7.34 (d, 1H, J ) 7.2
Hz), 7.45 (d, 1H, J ) 7.2 Hz), 7.54 (m, 2H), 7.69 (d, 1H, J )
6-Am in o-4-(2-tr iflu or oa ceta m id op h en yl)p yr id o[3,2-g]-
qu in olin e-5,10-d ion e (13c). A solution of chloro adduct 13a
(0.64 g, 1.48 mmol) and sodium azide (0.27 g, 8.4 mmol) in
DMF (12 mL) was heated for 4 h at 90 °C. After the mixture
was cooled and concentrated to dryness, CHCl3 (1 L) was added

Analogues of Meridine
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3281
and the mixture was stirred for 12 h. The mixture was
concentrated and the residue purified by flash chromatography
(CH2Cl2/MeOH, 95:5) to give 13c (37 mg, 6%) as a yellow
cm-1. MS (EI mode): m/z 319 (100), 317(28), 291 (100), 289
(32), 254 (26). tR is 6.8 min (98% purity), using system I, and
tR is 5.54 min (98% purity), using system II.
1
solid: mp, >260 °C. H NMR (DMSO-d6): δ 5.75 (s, 2H), 7.24
12-Br om oben zo[b]p yr id o[4,3,2-d e][1,7]p h en a n th r olin -
8-on e (14d ). Method C was used and involved 13d (20 mg,
0.045 mmol) and 1 N NaOH (0.6 mL) in CHCl3 (13 mL). Flash
chromatography on silica gel (CH2Cl2/MeOH 98:2) gave 14d
(8.1 mg, 55%) as a yellow solid: mp >260 °C. 1H NMR
(CDCl3): δ 7.26 (d, 1H, J ) 5.6 Hz), 7.87 (ddd, 1H, J ) 8.4,
8.4, and 1.2 Hz), 7.97 (ddd, 1H, J ) 8.4, 8.4, and 1.2 Hz), 8.23
(dd, 1H, J ) 8.4 and 1.2 Hz), 8.64 (dd, 1H, J ) 8.4 and 1.2
Hz), 8.67 (d, 1H, J ) 5.6 Hz), 8.79 (d, 1H, J ) 5.6 Hz), 9.39 (d,
1H, J ) 5.6 Hz). 13C NMR (CDCl3): δ 118.09, 120.19, 121.01,
122.77, 129.93, 130.48, 131.50, 131.87, 132.44, 135.62, 138.01,
144.62, 146.04, 146.70, 149.72, 150.37, 151.05, 180.06. IR
(CHCl3): 1686, 1601 cm-1. MS (EI mode): m/z 328 (4), 327
(14), 312 (82), 283 (28), 255 (100). tR is 7.37 min (98% purity),
using system I, and tR is 6.41 min (95% purity), using system
II.
(d, 1H, J ) 5.8 Hz), 7.72-7.82 (m, 4H), 7.82 (d, 1H, J ) 5.0
Hz), 8.61 (d, 1H, J ) 5.8 Hz), 9.31 (d, 1H, J ) 5.0 Hz), 10.78
(br s, 1H). 13C NMR (DMSO-d6): δ 112.65, 114.61, 115.86 (q,
J ) 286 Hz), 118.30, 126.37, 127.37, 128.60, 129.20, 131.18,
131.51, 136.10, 146.89, 148.50, 150.09, 151.48, 153.18, 154.93
(q, J ) 42.8 Hz), 155.25, 180.39, 184.89. IR (CHCl3): 3410,
3351, 1711, 1652, 1606 cm-1. MS (EI mode): m/z 412 (10), 343
(66), 300 (100). tR is 3.78 min (95% purity), using system I,
and tR is 2.05 min (95% purity), using system II.
6-(Dim et h yla m in o)-4-(2-t r iflu or oa cet a m id op h en yl)-
pyr ido[3,2-g]qu in olin e-5,10-dion e (13g) an d 12-(Dim eth yl-
a m in o)b en zo[b]p yr id o[4,3,2-d e][1,7]p h en a n t h r olin e-8-
on e (14g). Dimethylamine hydrochloride (0.37 g, 4.6 mmol)
and NaOH (0.18 g, 4.6 mmol) were successively added to a
solution of chloro adduct 13a (0.5 g, 1.15 mmol) in a mixture
H2O/THF (12 mL/25 mL). The mixture was heated at 60 °C
for 3 h and concentrated to dryness. The crude product was
purified by flash chromatography (CH2Cl2/MeOH, 98:2) to give
successively 13g and 14g.
13g: yield of 70 mg, 14%, orange solid, mp 150 °C. 1H NMR
(CDCl3): δ 2.75 (s, 6H), 6.94 (d, 1H, J ) 6 Hz), 7.07 (dd, 1H,
J ) 7.7 and 1.5 Hz), 7.32 (ddd, 1H, J ) 7.7, 7.7, and 0.7 Hz),
7.49 (d, 1H, J ) 5 Hz), 7.55 (ddd, 1H, J ) 7.7, 7.7, and 1.7
Hz), 7.85 (d, 1H, J ) 7.7 Hz), 8.29 (br s, 1H), 8.55 (d, 1H, J )
6 Hz), 9.05 (d, 1H, J ) 5 Hz). 13C NMR (CDCl3): δ 43.21,
112.58, 115.58 (q, J ) 287 Hz), 119.13, 125.29, 126.72, 129.03,
129.60, 130.30, 131.13, 132.77, 133.28, 146.18, 147.82, 149.71,
151.05, 153.28, 154.76, 155.76 (q, J ) 37 Hz), 180.48, 183.96.
IR (KBr): 3175, 1724, 1701, 1654 cm-1. MS (EI mode): m/z
440 (100), 353 (15), 69 (84). tR is 4.85 min (98% purity), using
system I, and tR is 2.78 min (95% purity), using system II.
14 g: yield of 40 mg, 11%, red solid, mp 238 °C. 1H NMR
(CDCl3): δ 3.10 (s, 6H), 7.10 (d, 1H, J ) 5.5 Hz), 7.73 (dd, 1H,
J ) 6.8 and 6.8 Hz), 7.87 (dd, 1H, J ) 6.8 and 6.8 Hz), 8.22 (d,
1H, J ) 6.8 Hz), 8.54 (m, 2H), 8.59 (d, 1H, J ) 5.5 Hz), 9.26
(d, 1H, J ) 5.2 Hz). 13C NMR (CDCl3): δ 44.07, 113.72, 117.93,
119.68, 120.41, 122.89, 128.44, 130.47, 131.74, 137.82, 145.29,
146.53, 149.38, 150.06, 150.32, 151.18, 156.85, 181.72. IR
(KBr): 1690 cm-1. MS (EI mode): m/z 326 (44), 311 (100), 254
(14). tR is 6.45 min (91% purity), using system I, and tR is 2.91
min (92% purity), using system II.
F or m a tion of th e P en ta cyclic Com p ou n d s. Gen er a l
Meth od B. Compound 13 was dissolved in a mixture of CH2-
Cl2-CF3COOH, and the mixture was refluxed for different
periods. After the mixture was cooled and concentrated to
dryness, 1 N NaOH and CHCl3 were added and the mixture
was stirred overnight. The organic layer was separated, and
the aqueous layer was extracted with CHCl3. The combined
organic layers were dried over MgSO4 and concentrated in
vacuo. Purification of the residue on a flash chromatography
column gave the expected product.
Gen er a l Meth od C. Compound 13 was dissolved in a
mixture of CHCl3 and 1 N NaOH, and the mixture was stirred
at room temperature for 1 h. The organic layer was separated
and the aqueous layer extracted with CHCl3. The combined
organic layers were dried over MgSO4 and concentrated in
vacuo. Purification of the residue on a flash chromatography
column gave the expected product.
12-Ch lor oben zo[b]p yr id o[4,3,2-d e][1,7]p h en a n th r olin -
8-on e (14a ). Method B was used and involved 13a (100 mg,
0.232 mmol), TFA (1 mL) in CH2Cl2 (10 mL), reflux of 30 min,
and 1 N NaOH (6 mL) in CHCl3 (6 mL). Flash chromatography
(CH2Cl2/MeOH 97:3) gave 14a (64 mg, 87%) as a yellow solid:
mp >260 °C. 1H NMR (CDCl3): δ 7.77 (d, 1H, J ) 4.8 Hz),
7.80 (dd, 1H, J ) 8 and 8 Hz), 7.89 (dd, 1H, J ) 8 and 8 Hz),
8.25 (d, 1H, J ) 8 Hz), 8.51 (d, 1H, J ) 8 Hz), 8.61 (d, 1H,
J ) 5.6 Hz), 8.84 (d, 1H, J ) 4.8 Hz), 9.28 (d, 1H, J ) 5.6 Hz).
13C NMR (CDCl3): δ 118.06, 120.19, 120.79, 122.65, 129.42,
129.95, 131.66, 131.77, 132.01, 137.88, 144.71, 144.73, 145.78,
147.01, 149.77, 150.27, 151.22, 179.88. IR (CHCl3): 1694, 1601
Ben zo[b]pyr ido[4,3,2-de][1,7]ph en an th r olin -8-on e (14e).
Method B was used and involved 13e (30 mg, 0.076 mmol),
TFA (1 mL) in CH2Cl2 (2 mL), reflux of 3 h, and 1 N NaOH (2
mL) in CHCl3 (2 mL). 14e was obtained (17 mg, 80%) as a
1
yellow solid: mp >260 °C. H NMR (CDCl3): δ 7.94 (dd, 1H,
J ) 4.4 and 8 Hz), 7.97 (dd, 1H, J ) 8.2 and 8.2 Hz), 8.10 (dd,
1H, J ) 8.2 and 8.2 Hz), 8.48 (d, 1H, J ) 8.2 Hz), 8.76 (d, 1H,
J ) 8.2 Hz), 8.80 (d, 1H, J ) 5.2 Hz), 9.23 (dd, 1H, J ) 1.6
and 4.4 Hz), 9.49 (d, 1H, J ) 5.2 Hz), 9.50 (dd, 1H, J ) 1.6
and 8 Hz). 13C NMR (CDCl3): δ 118.06, 119.82, 121.88, 123.02,
128.07, 129.33, 131.44, 131.98, 132.98, 134.17, 138.08, 145.62,
147.27, 147.50, 147.76, 150.46, 153.18, 180.94. IR (CHCl3):
1690, 1604 cm-1. MS (EI mode): m/z 283 (100), 255 (51). tR is
5.75 min (98% purity), using system I, and tR is 2.92 min (98%
purity), using system II.
12-Nitr oben zo[b]p yr id o[4,3,2-d e][1,7]p h en a n th r olin -8-
on e (14f). Method C was used and involved 13f (20 mg, 0.045
mmol) and 1 N NaOH (0.6 mL) in CHCl3 (13 mL). Flash
chromatography on silica gel (CH2Cl2/MeOH 98:2) gave 14f
(8.1 mg, 55%) as a yellow solid: mp >260 °C. 1H NMR (DMSO-
d6): δ 7.56 (d, 1H, J ) 6.0 Hz), 7.89 (dd, 1H, J ) 8.1 and 6.0
Hz), 8.02 (dd, 1H, J ) 8.1 and 6.4 Hz), 8.20 (d, 1H, J ) 8.0
Hz), 8.79 (d, 1H, J ) 6.4 Hz), 8.94 (d, 1H, J ) 8.0 Hz), 9.10 (d,
1H, J ) 5.1 Hz), 9.28 (d, 1H, J ) 5.1 Hz). 13C NMR (DMSO-
d6): δ 120.45, 120.74, 123.65, 123.78, 125.42, 129.14, 130.79,
131.82, 137.04, 144.88, 149.62, 151.83, 153.57, 153.68, 156.85,
162.28, 164.75, 178.09. IR (CHCl3): 1688, 1601 cm-1. tR is 5.33
min (97% purity), using system I, and tR is 3.54 min (92%
purity), using system II.
11-Eth ylben zo[b]p yr id o[4,3,2-d e][1,7]p h en a n th r olin e-
ca r boxyla te-8-on e (14j). A suspension of nitro derivative 12j
(60 mg, 0.15 mmol) and 10% Pd/C (48 mg) in MeOH (10 mL)
was fitted into a hydrogenation apparatus (1 atm) and stirred
for 2 h under a hydrogen atmosphere. The mixture was
concentrated and the residue purified by flash chromatography
(CH2Cl2/MeOH 98:2) to give 14j (33 mg, 63%) as a yellow
1
solid: mp >260 °C. H NMR (CDCl3): δ 1.53 (t, 3H, J ) 7.3
Hz), 4.57 (q, 2H, J ) 7.3 Hz), 7.86 (ddd, 1H, J ) 8.0, 8.0, and
1.1 Hz), 7.99 (ddd, 1H, J ) 8.0, 8.4, and 1.1 Hz), 8.42 (d, 1H,
J ) 8.4 Hz), 8.64 (d, 1H, J ) 8.0 Hz), 8.70 (d, 1H, J ) 5.5 Hz),
9.37 (d, 1H, J ) 5.5 Hz), 9.59 (d, 1H, J ) 2.2 Hz), 9.90 (d, 1H,
2.2 Hz). 13C NMR (CDCl3): δ 13.01, 60.97, 116.85, 118.69,
120.56, 121.68, 128.30, 128.49, 130.31, 130.79, 131.29, 134.52,
136.82, 144.27, 145.31, 145.83, 148.13, 149.29, 151.91, 162.90,
178.99. IR (CHCl3): 1726, 1693 cm-1. MS (EI mode): m/z 355
(100). tR is 8.19 min (94% purity), using system I, and tR is
8.94 min (98% purity), using system II.
10-Hyd r oxyben zo[b]p yr id o[4,3,2-d e][1,7]p h en a n th r o-
lin e-8-on e (14k ). Method C was used and involved a solution
of 13k (180 mg, 0.44 mmol) and 1 N NaOH (22 mL) in CHCl3
(70 mL) that was stirred overnight. CH3COOH was added until
neutralization, and the organic layer was separated and
extracted with CHCl3/MeOH 95:5 (3 × 50 mL). The combined
organic extracts were dried over MgSO4 and concentrated to
give 14k (110 mg, 85%) as a purple solid: mp >260 °C. 1H

3282 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Delfourne et al.
(2) Molinski, T. F. Marine pyridoacridine alkaloids: structure,
synthesis, and biological chemistry. Chem. Rev. 1993, 93, 1825-
1838.
NMR (DMSO-d6): δ 6.91 (d, 1H, J ) 9.2 Hz), 7.84 (d, 1H, J )
7.8 and 8.8 Hz), 7.97 (dd, 1H, J ) 7.8 and 7.8 Hz), 8.21 (d, 1H,
J ) 7.8 Hz), 8.71 (d, 1H, J ) 9.2 Hz), 8.87 (d, 1H, J ) 8.8 Hz),
9.04 (d, 1H, J ) 5.5 Hz), 9.27 (d, 1H, J ) 5.5 Hz). IR (KBr):
1664, 1608 cm-1. MS (EI mode): m/z 299 (100), 298 (66), 271
(17), 270 (13), 243 (39), 242 (29). tR is 4.16 min (96% purity),
using system I, and tR is 2.66 min (93% purity), using system
II.
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2. P h a r m a cology. 2.1. In Vitr o Ch a r a cter iza tion of
Dr u g-In d u ced Effects w ith Resp ect to Hu m a n Ca n cer
Cell Lin e Gr ow th . Twelve human tumor cell lines were
obtained from the American Type Culture Collection (ATCC,
Manassas, VA). These included three glioblastomas (A-172,
U-373 MG, and U-87 MG), two colon (HCT-15 and LoVo), two
non-small-cell-lung (A549 and A-427), two bladder (J 82 and
T24), one prostate (PC-3), and two breast (T-47D and MCF7)
cancer models. The ATCC numbers of these cell lines were
CRL1620 (A-172), HTB 14 (U-87 MG), HTB 17 (U-373 MG),
CCL225 (HCT-15), CCL229 (LoVo), CCL 185 (A549), HBT 53
(A-427), HTB1 (J 82), HTB4 (T24), HTB133 (T-47D), HTB22
(MCF7), and CRL1435 (PC-3). The cells were cultured at 37
°C in sealed (airtight) Falcon plastic dishes (Nunc, Gibco,
Belgium) containing Eagle’s minimal essential medium (MEM,
Gibco) supplemented with 5% fetal calf serum (FCS). All the
media were supplemented with a mixture of 0.6 mg/mL
glutamine (Gibco), 200 IU/mL penicillin (Gibco), 200 IU/mL
streptomycin (Gibco), and 0.1 mg/mL gentamycin (Gibco). The
FCS was heat-inactivated for 1 h at 56 °C.
The 12 cell lines were incubated for 24 h in 96-microwell
plates (at
a concentration of 40 000 cells/mL of culture
medium) to ensure adequate plating prior to cell growth
determination, which was carried out by means of the colori-
metric MTT assay, as detailed previously.19,20 This assessment
of cell population growth is based on the capability of living
cells to reduce the yellow reagent MTT (3-(4,5)-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis,
MO) to a blue product, formazan, by a reduction reaction
occurring in the mitochondria. The number of living cells is
directly proportional to the intensity of the blue, which is
quantitatively measured by spectrophotometry on a DIAS
microplate reader (Dynatech Laboratories, Guyancourt, France)
at a wavelength of 570 nm (with a reference at 630 nm). Each
experiment was carried out in sextuplicate. We validated the
MTT-related data using two alternative techniques, namely,
direct cell counting and the genomic incorporation of tritiated
thymidine (data not shown).
Six concentrations ranging from 10-5 to 10-10 M were
assayed for each of the 25 drugs under study (see Table 2).
2.2. Sta tistica l An a lysis. The statistical comparisons of
the data were carried out by means of Student t (for two
groups) tests after a check of the equality of variance by means
of the Levene test and by means of the normal distribution
fitting of the data with the ø2 test of goodness-of-fit. When
these parametric conditions were not satisfied, the nonpara-
metric Mann-Whitney (for two groups) tests were carried out.
All the statistical analyses were carried out using Statistica
(Statsoft, Tulsa, OK).
Refer en ces
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Martinez, J .; Kiss, R. Influence of gastrin on human astrocytic
tumor cell proliferation. J . Nat. Cancer Inst. 1996, 88, 594-600.
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