Synthesis and biological evaluation of new 1,5-diazaanthraquinones with cytotoxic activity

Article abstract of DOI:10.1016/j.bmc.2004.09.021

A series of 1,5-diazaanthraquinone derivatives was synthesized and their in vitro cytotoxic activities were evaluated against several human cancer cell lines. The 1,5-diazaanthraquinone chromophore has been synthesized either on the basis of hetero Diels-

Full text of DOI:10.1016/j.bmc.2004.09.021

Bioorganic & Medicinal Chemistry 12 (2004) 6505–6515
Synthesis and biological evaluation of
new 1,5-diazaanthraquinones with cytotoxic activity
a
Sonia Manzanaro, Marıa Jesu´s Vicent, Marıa Jesu´s Martın, Nelida Salvador-Tormo,
a
a
a
´
´
´
´
Jose Marıa Perez, Marıa del Mar Blanco, Carmen Avendan˜o,
b
b
b
´
´
´
´
Jose Carlos Menendez and Jesu´s Angel de la Fuente
b
a,
*
´
´
´
a
´
´
Instituto Biomar, S.A., Polıgono Industrial, Edificio CEEI, 24231 Onzonilla, Leon, Spain
´ ´ ´
Departamento de Quımica Organica y Farmaceutica, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain
b
Received 3 August 2004; revised 10 September 2004; accepted 14 September 2004
Available online 6 October 2004
Abstract—A series of 1,5-diazaanthraquinone derivatives was synthesized and their in vitro cytotoxic activities were evaluated
against several human cancer cell lines. The 1,5-diazaanthraquinone chromophore has been synthesized either on the basis of hetero
Diels–Alder reactions involving different quinoline-5,8-diones and a,b-unsaturated aldehyde N,N-dimethylhydrazones or by ther-
molysis of different arylaminomethylene Meldrumꢀs acid derivatives. Some of these compounds showed cytotoxic activity compa-
rable to that of mitoxantrone against most of the cell lines tested. Compounds 20, 30, 31 and 37 were 4–54 times more potent
that mitoxantrone against A549, H116, PSN1 and T98G cancer cell lines but, interestingly, they were 3–16 times less potent against
the human breast carcinoma SKBR3. Some structure–activity relationships are described, the most significant one being the increase
in cytotoxicity resulting from the introduction of a halogen atom at the C-4 position.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
H
N
NH2
O
O
HN
HN
OH
OH
O
O
HN
HN
OH
OH
Products containing an anthracene-9,10-dione substruc-
ture are an important class of antitumour compounds.
They include the anthracyclines,1 as mitoxantrone
(Fig. 1), the pluramycins2 and some of the enediyne anti-
biotics.3 At least in the case of the anthracyclines, the
antitumour activity is mainly attributed to two mecha-
nisms of action. Because of their planar chromophore,
they are able to insert between two base pairs in the
DNA helix4 causing a local untwisting of the helix that
results in topoisomerase II inhibition,5 miscoding and
possible cell death.6 Additionally, their antitumour
activity may be associated to the formation of DNA
damaging anion-radical intermediates by reduction of
the quinone unit.7 The generation of reactive oxygen
species also has a role in modulation of angiogenesis
by the anthracyclines.8
COOH
2 HCl
2
N
COOH
NH2
N
H
Mitoxantrone (dihydrochloride)
Pixantrone (dimaleate)
Figure 1.
Isosteric substitution of one or more carbons of the benz-
ene rings by nitrogen atoms should afford compounds
with geometries similar to those of the parent structures,
but with increased affinity for DNA due to the presence
of sites suitable for hydrogen bonding or ionic interac-
tions. Also, the electron-withdrawing properties of the
heterocyclic rings would facilitate the formation of an-
ion radicals. Based on this idea, some aza bioisosteres
related to the anthracene-9,10-diones have been synthe-
sized and screened in vitro and in vivo against a wide
spectrum of tumour cell lines.9 Among them, pixantrone
(Fig. 1) shows promise for development as anticancer
agent, currently being in phase III trials for the
Keywords: Antitumour compounds; Cytotoxicity; 1,5-Diazaanthraqui-
nones; Heterocyclic quinones.
*
Corresponding author. Tel.: +34 918466060; fax: +34 918466001;
e-mail: jdelafuente@pharmamar.com
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.09.021

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S. Manzanaro et al. / Bioorg. Med. Chem. 12 (2004) 6505–6515
the behaviour of quinone 2 towards 4-substituted 1-
O
R8
N
R7
dimethylamino-1-azadienes was different to the one
observed for 2,6-dibromobenzoquinone, where the
Diels–Alder products were compounds 5 and their aro-
matization to 6 had to be forced by pyrolysis (Scheme
1b).16a,b This difference could be attributed to steric
compression of compounds 6, which does not exist in
their regioisomers 4.
R3
N
R4
O
1
Figure 2.
treatment of non-Hodgkingꢀs lymphoma.10 Interest-
ingly, this compound showed an antitumour activity
comparable to that of mitoxantrone and doxorubicin
but it showed no measurable cardiotoxicity compared
to them at equi-effective doses in animal models.11
A Stille coupling was used to obtain some 4-aryl substi-
tuted derivatives (compounds 16–18, Scheme 2) by a
route similar to that used in the synthesis of a regio-
isomer of the marine alkaloid meridine.14 Thus, Stille
coupling of the previously reported triflate 717 with the
known 2-nitrophenyltrimethylstannane18 catalyzed by
Pd(PPh3)4 afforded compound 8 in 84% yield. Oxidative
demethylation of 8 with cerium(IV) ammonium nitrate
in acetonitrile–sulfuric acid afforded the quinoline-5,8-
dione 10 in 52% yield. The known quinoline 9 and quin-
oline-5,8-dione 1119 were obtained by the same strategy.
Aminoquinoline-5,8-diones 12 and 13 were obtained by
conjugate addition of hydrazoic acid to 10 and 11,
respectively, in the conditions reported by Couladouros
et al.20 The addition was regiospecific, as expected, be-
cause it is controlled by the position of the nitrogen
atom of the quinoline-5,8-dione.21 Amines 12 and 13
were transformed into compounds 14 and 15 by treat-
ment with Meldrumꢀs acid and triethyl orthoformate.
The thermolysis of 15 gave quantitatively 16, which by
treatment with diazomethane gave 17 in 19% yield.
The known 1813b was obtained in 17% overall yield by
the same two-step procedure.
Although the considerations outlined above regarding
bioisosteric replacement of carbon by nitrogen would
apply particularly well to diaza derivatives, these com-
pounds have received little attention and most of the
published studies are focused on 1,8-diazaanthraqui-
nones.12 1,5-Diazaanthraquinones have been mainly
used as intermediates in the synthesis of pyridoacri-
dines13 but their potential as antitumour agents is al-
most unexplored.12a,14 Recently we described in a
preliminary communication15 the synthesis and the
activity of a small series of 1,5-diazaanthraquinones.
Herein, we report in full the synthesis and biological
evaluation of 1,5-diazaanthraquinone derivatives,
with general structure 1 (Fig. 2), bearing different
substituents to explore their influence on the cytotoxic
activity of this type of diazaanthraquinone chromo-
phore.
Treatment of quinoline-5,8-dione derivative 1913c with
3-methyl-1-dimethylamino-1-azadiene at room tempera-
ture afforded the fully aromatic derivative 20 in 80%
yield (Scheme 3), while a similar reaction of 19 with 4-
2. Results and discussion
2.1. Chemistry
(2-nitrophenyl)-1-dimethylamino-1-azadiene
afforded
Symmetrically substituted derivatives of the 1,5-diaza-
anthraquinone system (Scheme 1a) were prepared in a
single step using a strategy based on the double regiose-
lective hetero Diels–Alder reaction.16 Thus, 2,5-di-
the known aromatic derivative 2113b,14 only when the
mixture was sonicated at 50ꢁC for 125h. On the other
hand, use of 4-methyl-1-dimethylamino-1-azadiene led
to compound 22, arising from elimination of hydrobro-
mic acid from the primary Diels–Alder adduct, which
was aromatized by elimination of dimethylamine under
thermal conditions to give compound 23. Treatment of
bromobenzoquinone
2
was treated with the
corresponding 1-dimethylamino-1-azadienes 3 to give
compounds 4, in 68–89% yields. It is worthy of note that
R4
R4
N
O
O
4a R3=Et, R4=H
4b R3=Me, R4=H
4c R3=H, R4=Me
4d R3=Me, R4=Et
4e R3=H, R4=H
R3
Br
N
R3
2x
2x
+
+
(a)
(b)
R3
N
NMe2
Br
Br
R4
O
O
2
3
4
R4
R4
N
O
R4
R4
N
O
R4
N
O
R3
R3
R3
R3
R3
N
Br
N
NMe2
O
Me2N
O
NMe2
O
5
6
Scheme 1.

S. Manzanaro et al. / Bioorg. Med. Chem. 12 (2004) 6505–6515
6507
MeO
R
O
R
O
O
R
OMe OTf
H2N
i
ii
iii
+
N
N
N
R
N
OMe
O
OMe
SnMe3
R=H, NO2
8 R=NO2
9 R=H
10 R=NO2
11 R=H
12 R=NO2
13 R=H
7
iv
O
N
O
R
O
NO2
H
N
N
v
R=H
v-vi
O
O
R=NO2
N
N
N
OR
O
O
O
O
OMe O
14 R=NO2
15 R=H
18
16 R=H
vi
17 R=Me
Scheme 2. Reagents and conditions: (i) Pd(PPh3)4, CuBr, 1,4-dioxane, 90ꢁC, 1h 30min; (ii) (NH4)2Ce(NO3)6, CH3CN/H2SO4 (2M), rt, 3h; (iii)
NaN3, MeOH/HCl, rt, 3h 30min; (iv) Meldrumꢀs acid, HC(OEt)3, reflux, 6h; (v) Ph2O, 200–220ꢁC, 15min; (vi) diazomethane, CH2Cl2/MeOH, rt,
10min.
afforded the quinoline-5,8-diones 2813b and 29. Treat-
ment of 28 or 29 with 3-methyl-1-dimethylamino-1-
azadiene in ClCH2CH2Cl at 80ꢁC afforded the fully
aromatic derivatives 30 and 31, respectively. The reac-
tion of bromoquinoline-5,10-dione 30 with (N,N-
dimethylamino)trimethyltin in toluene in the presence
O
O
N
N
N
NMe2
Br
i
N
Cl
O
Cl
O
19
20
NO2
23
of 5mol% of PdCl2(CN)2 afforded 32 after 6h in
N
NMe2
86% yield, and this compound was transformed
into the hydrochloride 33 by treatment with HCl in
dioxane.
ii
iii
N
O
O
NO2
NMe2
N
N
The bromoquinoline-5,8-dione 34 was obtained (Scheme
5) as previously described.24 A hetero Diels–Alder reac-
tion between 34 and crotonaldehyde dimethylhydrazone
gave the 1,5-diazaanthraquinone 35. Oxidation of 35
O
Cl
N
21
iv
N
N
O
Cl
O
25
N
with CrO3/H2SO4 gave the dicarboxylic derivative
36. When the oxidation of 35 was carried out with
SeO2, the reaction was regioselective and gave only com-
pound 37, with an aldehyde group at the C-4 position.26
Compound 37 was oxidized to the monocarboxylic
derivative 38 by treatment with NaClO2/H2O2 in
92% yield. An alkene derivative (compound 39) was pre-
pared from the aldehyde 37 by reaction with the stabi-
22
N
v
Cl
O
O
23
N
N
27
N
O
lized ylide Ph3P@CHCN, which afforded
a 10:1
24
mixture of Z and E isomers but only the Z isomer was
isolated as a pure compound. The unusual stereochemi-
cal outcome of the Wittig reaction may be ascribed to
steric compression by the neighbouring C10@O group.
Finally, compounds 40 and 41 (Fig. 3) were obtained
as previously described.28
Scheme 3. Reagents and conditions: (i) Et2O/CH3CN, rt, 16h; (ii)
CHCl3, ultrasound, 50ꢁC, 125h; (iii) Et2O/CH3CN, rt, 22h; (iv)
110ꢁC, 0.1Torr, 2h; (v) THF/HCl (6N), 80ꢁC, 1h.
compound 22 with dilute HCl led to aromatization with
concomitant reaction of the liberated dimethylamine at
the C-8 position, affording compound 24.
2.2. Antiproliferative activity
The antiproliferative activity of the 1,5-diazaanthraqui-
none compounds was evaluated using a panel of five hu-
man cell lines. The results are shown in Tables 1 and 2.
Data for mitoxantrone are included for comparison.
The nucleophilic substitution of triflate 25, which was
obtained as previously described,22 with excess of LiBr
or LiI in dioxane under reflux gave the corresponding
bromide 2622 or iodide 27, respectively (Scheme 4).
Oxidative demethylation of these compounds with
cerium(IV) ammonium nitrate in aqueous acetonitrile
The IC50 values (the drug concentration inhibiting the
growth value of cell lines by 50%) show that the

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S. Manzanaro et al. / Bioorg. Med. Chem. 12 (2004) 6505–6515
OMe
OMe
O
N
N
R
N
i
ii
Br
Br
Br
OTf OMe
OMe
R
O
26 R=Br
27 R=I
28 R=Br
29 R=I
25
iii
N
NMe2
O
O
O
O
N
N
N
N
N
N
R
SnMe3
v
iv
N
O
N
O
N
HCl
30 R=Br
31 R=I
33
32
Scheme 4. Reagents and conditions: (i) LiBr or LiI, 1,4-dioxane, reflux, 96h; (ii) (NH4)2Ce(NO3)6, CH3CN/H2O, rt, 2h; (iii) Cl2CHCHCl2, 80ꢁC,
30min; (iv) PdCl2(CN)2, toluene, reflux, 6h; (v) HCl(1,4-dioxane), 1,4-dioxane, rt, 15min.
O
O
O
O
COOH
N
N
N
N
i
iii
ii
2
Br
N
HOOC
O
O
34
35
36
iv
O
COOH
N
O
CHO
N
O
CN
N
N
N
v
vi
N
O
O
O
38
37
39
Scheme 5. Reagents and conditions: (i) ClCH2CH2Cl, methacrolein dimethylhydrazone, 80ꢁC, 2h; (ii) a. CHCl3, Et3N, rt, 16h; b. 110ꢁC, 0.1Torr,
2h; (iii) CrO3, H2SO4, 80ꢁC, 4h; (iv) SeO2, 1,4-dioxane, 80ꢁC, 24h; (v) NaClO2, H2O2, Na2HPO4, CH3CN/H2O, rt, 4h; (vi) Ph3P@CHCN, CH2Cl2,
reflux, 1h.
present, such as in the case of derivatives 32, 33 and 35,
respectively, a clear loss of activity is observed.
O
N
Y
X
When one or both methyl groups in 35 are transformed
into a carboxylic acid group (compounds 38 and 36,
respectively) a total loss of the activity was observed.
On the other hand, when the C-8 methyl group of 35
was transformed into an aldehyde function (compound
37), an increase in the cytotoxic activity was observed.
However, the transformation of the aldehyde group in
37 into the conjugated nitrile, present in derivative 39,
was accompanied by a slight decrease in the activity.
The activities of the 1,5-diazaanthraquinone 40 and its
1,8-diaza analogue 41 were similar in the cell lines
assayed.
O
40 X=N, Y=C
41 X=C, Y=N
Figure 3.
halogenated derivatives 20, 30 and 31 were the most
cytotoxic compounds, exhibiting an activity about 30-
fold higher than mitoxantrone for the cell lines A549,
(lung), H116 (colon) and PSN1 (pancreas) and about
6-fold higher for T98G (glioblastoma), but interestingly
these compounds are about 5-fold less active than
mitoxantrone for SKBR3 (breast), showing that this
breast cell line is substantially less sensitive to these
1,5-diazaanthraquinone derivatives than to mitoxan-
trone. Activity for these three halogenated derivatives
is almost identical, showing that the nature of the halo-
gen (Cl for 20, Br for 30 and I for 31) is unimportant for
the activity. When instead of the halogen atom an elec-
tron-donating group such as dimethylamino or methyl is
None of the quinoline-5,8-dione derivatives showed sig-
nificant activity (Table 2), showing that the full 1,5-dia-
zaanthraquinone chromophore is necessary for having
high cytotoxic activity.
Cytotoxic activities for compounds 4a–e and 22–24 were
previously reported against a different panel of cell lines
but they have not been re-evaluated in the panel used in
this work.15

S. Manzanaro et al. / Bioorg. Med. Chem. 12 (2004) 6505–6515
6509
Table 1. In vitro cytotoxic activity (IC50, lM) of 1,5-diazaanthraquinone derivatives against human cancer cell linesa
O
O
R8
N
R7
R3
N
R4
Compound
R3
R4
R7
R8
A549
H116
PSN1
T98G
SKBR3
16
17
H
OH
OMe
H
Phenyl
Phenyl
2-Nitrophenyl
14.49
10.55
>14
7.73
10.55
>14
0.39
6.39
0.12
0.15
9.36
6.58
14.00
>17
0.20
>19
1.82
2.97
2.23
6.44
5.96
4.74
>14
11.26
>16
>14
1.93
>14
1.21
1.05
7.49
8.23
6.30
>17
7.94
>19
9.27
8.92
2.23
8.06
7.45
H
H
15.82
>14
3.86
18
20
H
OMe
Cl
H
H
Me
H
H
2-Nitrophenyl
0.32
13.68
0.18
0.14
3.12
3.29
>21
0.26
9.12
0.12
0.14
15.61
12.07
21.00
>17
21
30
H
Cl
Br
10.26
3.30
2.38
H
Me
Me
Me
Me
H
H
31
32
H
I
NMe2
H
H
H
12.48
13.72
21.01
>17
33
35
H
NMe2ÆHCl
H
Me
Me
COOH
Me
Me
Me
Me
H
H
H
H
H
H
36
37
H
COOH
CHO
>17
H
0.33
>19
5.95
>19
2.64
>19
38
39
H
COOH
CH@CH–CN
H
H
7.88
3.72
2.23
7.25
3.64
8.18
2.97
4.51
10.30
14.87
11.16
0.70
40
41
H
Mitoxantrone
a A549, human lung carcinoma; H116, human colon carcinoma; PSN1, human pancreas adenocarcinoma; T98G, caucasian human glioblastoma;
SKBR3, human breast carcinoma. All values are the mean of at least three experiments.
Table 2. In vitro cytotoxic activity (IC50, lM) of quinoline-5,8-dione derivatives against human cancer cell linesa
O
N
R3
R6
R4
O
Compound
R3
R4
R6
H
A549
H116
PSN1
T98G
SKBR3
11
12
13
14
15
28
29
34
H
H
H
H
H
H
H
Me
Phenyl
2-Nitrophenyl
14.19
3.95
18.00
>11
8.51
8.47
9.67
>11
>12
2.12
13.78
1.98
10.64
14.12
14.00
>11
21.28
4.52
17.50
>11
10.64
12.71
15.00
>11
NH2
NH2
A
Phenyl
2-Nitrophenyl
Phenyl
A
Br
>12
>12
4.77
>12
>12
3.71
Br
I
5.56
13.78
3.97
5.56
>14
Br
Br
10.34
1.98
13.78
9.92
H
2.64
O
O
H
N
CH
A represents:
.
O
O
a A549, human lung carcinoma; H116, human colon carcinoma; PSN1, human pancreas adenocarcinoma; T98G, caucasian human glioblastoma;
SKBR3, human breast carcinoma. All values are the mean of at least three experiments.
3. Conclusion
mitoxantrone. Some structure–activity relationships
have been described. Further studies on this interesting
class of antitumour compounds are in progress in our
laboratories.
In conclusion, a series of 1,5-diazaanthraquinone deriv-
atives have been synthesized and their in vitro cytotoxic
activities were evaluated against several human cancer
cell lines. The 1,5-diazaanthraquinone chromophore
has been synthesized either on the basis of hetero
Diels–Alder reactions involving different quinoline-5,
8-diones and a,b-unsaturated aldehyde N,N-dimethyl-
hydrazones or by thermolysis of different arylamino-
methylene Meldrumꢀs acid derivatives. Some of these
compounds showed cytotoxic activity higher than
4. Experimental
The reagents used were of commercial origin (Aldrich,
Fluka) and were employed without further purification.
Solvents (SDS, Scharlau) were purified and dried by
standard procedures. Reactions were monitored by

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S. Manzanaro et al. / Bioorg. Med. Chem. 12 (2004) 6505–6515
thin-layer chromatography, using Macherey-Nagel or
Merck plates with fluorescent indicator. Separations
by flash liquid chromatography were performed using
silica gel from SDS 60 ACC (230–400mesh) or Merck
(60, 40–63lm).
4.1.4. 9-Ethyl-3-methylpyrido[2,3-g]quinoline-5,10-dione
(4d, R3 = Me, R4 = Et). Yield, 70%. Found: C, 67.71;
H, 3.15; N, 13.08. C12H6N2O2 requires: C, 68.57; H,
2.88; N, 13.33; mp 180–182ꢁC; t (KBr): 1680.0
(C@O)cmꢀ1. dH (CDCl3, 300MHz): 8.81 (s, 2H), 3.19
(q, 4H, J = 7.9Hz), 2.53 (s, 6H), 1.31 (t, 6H,
J = 7.9Hz); dC (CDCl3, 75MHz): 181.3, 156.01,
152.78, 141.9, 137.60, 129.30, 23.08, 16.30, 14.21; MS
(ESI-positive) m/z: 295 [M+H]+.
Melting points are uncorrected, and were determined
using a Hoffler hot stage microscope. Spectroscopic data
were obtained with the following instruments: IR, Per-
kin Elmer Paragon 1000 FT-IR; NMR, Bruker AC-
250 (250MHz for 1H and 63MHz for 13C), Varian
Unity 300 (300MHz for 1H and 75MHz for 13C) or Var-
4.1.5. Pyrido[2,3-g]quinoline-5,10-dione (4e, R3 = R4 =
H). Yield, 79%. Found: C, 69.70; H, 4.59; N, 11.54.
C14H10N2O2 requires: C, 70.58; H, 4.20; N, 11.76;
mp > 300ꢁC; t (KBr): 1681.3 (C@O)cmꢀ1. dH (CDCl3,
300MHz): 9.03 (dd, 2H, J = 4.8 and 2.0Hz), 8.51 (dd,
2H, J = 8.2 and 2.0Hz), 7.59 (dd, 2H, J = 8.2 and
4.8Hz,); dC (CDCl3, 75MHz): 183.91, 151.03, 144.07,
124.2, 129.14, 125.20.
1
ian Unity Inova 500 (500MHz for H and 125MHz for
13C). Combustion elemental analyses were obtained by
´
the Servicio de Microanalisis Elemental, Universidad
Complutense, using Perkin Elmer 2400 CHN and Leco
CHNS 932 microanalyzers.
4.1. Double hetero Diels–Alder reactions for the synthesis
of symmetrically substituted 1,5-diazaanthraquinones.
General procedure
4.1.6. 5,8-Dimethoxy-4-(2-nitrophenyl)quinoline (8). A
mixture of 7 (1.99g, 5.88mmol), 2-nitrophenyltrimethyl-
stannane (2.86g, 10mmol), Pd(PPh3)4 (580mg,
0.5mmol), CuBr (422mg, 2.9mmol), in 1,4-dioxane
(64mL) was heated at 90ꢁC, under argon for 90min.
After cooling to room temperature, the reaction mixture
was diluted with AcOEt, washed with H2O and the or-
ganic layer dried over Na2SO4. The resulting crude
was chromatographed (AcOEt) to afford 8 (1.53g,
84%). Found: C, 66.05; H, 4.56; N, 9.06. C17H14N2O4
requires: C, 65.80; H, 4.55; N, 9.03; dH (CDCl3,
300MHz): 8.96 (d, 1H, J = 4.1Hz), 8.17 (dd, 1H,
J = 1.5 and 8.0Hz), 7.54 (m, 1H), 7.45 (m, 1H), 7.30
(dd, 1H, J = 1.5 and 8.0Hz), 7.20 (d, 1H, J = 4.1Hz),
6.96 (d, 1H, J = 8.5Hz), 6.68 (d, 1H, J = 8.5Hz), 4.06
(s, 3H) 3.39 (s, 3H); dC (CDCl3, 75MHz): 149.92,
148.81, 144.21, 140.74, 132.48, 132.10, 131.96, 131.87,
130.93, 128.53, 128.37, 128.08, 123.41, 107.15, 105.40,
56.13, 55.52.
To a solution of 2,5-dibromobenzoquinone29 (100–
200mg, 0.375–0.750mmol) in chloroform (10–15mL)
was added the suitable azadiene (2equiv), and in the
case of 4-substituted azadienes, triethylamine (2equiv).
After stirring at room temperature for 1min, the solu-
tion was evaporated. In the reactions using triethylam-
ine, the residue was washed with H2O. In the other
reactions, the residue was washed with ethyl ether,
affording the desired 1,5-diazaanthraquinones.
4.1.1. 3,8-Diethylpyrido[2,3-g]quinoline-5,10-dione (4a,
R3 = Et, R4 = H). Yield, 68%. Found: C, 71.86; H,
5.50; N, 10.31. C16H14N2O2 requires: C, 72.18; H,
5.26; N, 10.53; mp 218–220ꢁC; t (KBr): 1682.6
(C@O)cmꢀ1. dH (CDCl3, 300MHz): 8.93 (d, 2H,
J = 2.1Hz), 8.50 (d, 2H, J = 2.1Hz), 2,85 (q, 4H,
J = 7.5Hz), 1.35 (t, 6H, J = 7.5Hz); dC (CDCl3,
75MHz): 181.65, 155.68, 146.50, 145.57, 135.54,
130.58, 26.56, 14.83; MS (APCI-positive) m/z: 267
[M+H]+.
4.1.7. 4-(2-Nitrophenyl)quinoline-5,8-dione (10). To a
mixture of 8 (1.53g, 4.96mmol) and CH3CN (28mL)
was added a solution of ammonium cerium(IV) nitrate
(11.16g, 20.25mmol) in H2SO4 (2M) (41mL). After stir-
ring for 3h, the reaction mixture was partitioned be-
tween AcOEt and saturated NH4Cl aqueous solution.
The organic layer was dried over Na2SO4, and evapora-
tion of the solvent gave compound 10 (720mg, 52%).
Found: C, 64.19; H, 2.90; N, 10.03. C15H8NO4 requires:
C, 64.29; H, 2.88; N, 10.00; dH (CDCl3, 300MHz): 9.08
(d, 1H, J = 4.9Hz), 8.32 (dd, 1H, J = 1.3 and 7.8Hz),
7.75 (td, 1H, J = 1.3 and 7.8Hz), 7.67 (td, 1H, J = 1.5
and 7.8Hz), 7.44 (d, 1H, J = 4.9Hz), 7.22 (dd, 1H,
J = 1.5 and 7.8Hz), 7.14 (d, 1H, J = 10.5Hz), 6.87 (d,
1H, J = 10.5Hz); dC (CDCl3, 75MHz): 184.71, 182.74,
153.72, 148.63, 147.52, 138.82, 137.73, 133.87, 132.11,
131.87, 131.74, 129.79, 129.13, 128.55, 124.55.
4.1.2. 3,8-Dimethylpyrido[2,3-g]quinoline-5,10-dione (4b,
R3 = Me, R4 = H). Yield, 89%. Found: C, 69.70; H,
4.59; N, 11.54. C14H10N2O2 requires: C, 70.58; H,
4.20; N, 11.76; mp > 300ꢁC;
t
(KBr): 1682.6
(C@O)cmꢀ1. dH (CDCl3, 300MHz): 8.95 (d, 2H,
J = 1.9Hz), 8.51 (d, 2H, J = 1.9Hz), 2.58 (s, 6H); dC
(CDCl3, 75MHz): 181.42, 156.07, 146.15, 139.55,
135.54, 130.22, 18.95; MS (APCI-positive) m/z: 239
[M+H]+.
4.1.3. 4,9-Dimethylpyrido[2,3-g]quinoline-5,10-dione (4c,
R3 = H, R4 = Me). Yield, 88%. Found: C, 69.16; H,
4.49; N, 11.43. C14H10N2O2 requires: C, 70.58; H,
4.20; N, 11.76; mp 253–254ꢁC; t (KBr): 1682.5
(C@O)cmꢀ1. dH (CDCl3, 300MHz): 8.85 (d, 2H,
J = 5.1Hz), 7.3 (d, 2H, J = 5.1Hz), 2.53 (s, 6H); dC
(CDCl3, 75MHz): 185.03, 154.23, 150.10, 145.71,
134.21, 128.17, 21.08; MS (APCI-positive) m/z: 239
[M+H]+.
4.1.8. 6-Amino-4-(2-nitrophenyl)quinoline-5,8-dione (12).
To a degassed solution of 10 (720mg, 2.57mmol) in
methanol (42mL) was added NaN3 (1g, 15.4mmol) in
6.8mL of degassed H2O. Then, HCl (1M, 10mL) was
added to pH = 4, and the mixture was stirred for 3h
30min. The solution was diluted with H2O and ex-

S. Manzanaro et al. / Bioorg. Med. Chem. 12 (2004) 6505–6515
6511
tracted with AcOEt. The organic layers were dried over
Na2SO4 and the resulting crude was chromatographed
(CH2Cl2/MeOH; 250:5 ! 100:3 ! 100:10) to afford 12
(556mg, 73%). Found: C, 61.16; H, 3.08; N, 14.28.
C15H9N3O4 requires: C, 61.02; H, 3.07; N, 14.23; t
(KBr): 1686, 1604, 1569, 1525cmꢀ1. dH (CDCl3,
300MHz): 8.92 (d, 1H, J = 4.9Hz), 8.27 (dd, 1H,
J = 1.2 and 8.3Hz), 7.74 (td, 1H, J = 1.2 and 7.4Hz),
7.65 (ddd, 1H, J=1.6, 7.4 and 8.3Hz), 7.34 (d, 1H,
J = 4.9Hz), 7.23 (dd, 1H, J = 1.6 and 7.4Hz), 6.07 (s,
1H); dC (CDCl3, 75MHz): 189.04, 181.91, 154.01,
150.49, 149.97, 148.9, 147.17, 134.70, 134.41, 130.36,
129.84, 127.62, 124.91, 124.55, 103.37; MS (ESI-nega-
tive) m/z: 294 [MꢀH]ꢀ.
and 220ꢁC for 15min. The reaction mixture was cooled
to room temperature and added to hexane at 0ꢁC. Fil-
tration of the reaction afforded 16 (172mg, 100%) as a
brown solid. Found: C, 71.43; H, 3.35; N, 9.29.
C18H10N2O3 requires: C, 71.52; H, 3.33; N, 9.27;
mp > 280ꢁC; t (KBr): 1695 (C@O), 1676 (C@O)cmꢀ1
.
dH (trifluoroacetic acid-d4 500MHz): 9.28 (d, 1H,
J = 5.6Hz), 8.90 (d, 1H, J = 6.6Hz), 8.42 (d, 1H,
J = 5.6Hz), 7.82 (d, 1H, J = 6.6Hz), 7.68 (t, 1H,
J = 7.3Hz), 7.58 (dd, 2H, J = 7.3, 7.8Hz), 7.46 (d, 2H,
J = 7.8Hz); dC (trifluoroacetic acid-d4 500MHz):
177.89, 174.23, 171.65, 165.54, 147.29, 146.75, 142.88,
141.44, 135.79, 133.62, 132.95, 129.49 (2C), 128.38
(2C), 127.01, 121.15, 114.62; MS (ESI-negative) m/z:
301 [MꢀH]ꢀ.
4.1.9. 6-Amino-4-phenylquinoline-5,8-dione (13). Same
procedure as for 12 involving 1119 (295mg, 1.25mmol).
The reaction mixture was stirred for 4h. The crude was
chromatographed (CH2Cl2/MeOH; 100:5) to afford 13
(200mg, 64%). Found: C, 71.99; H, 4.02; N, 11.18.
C15H10N2O2 requires: C, 71.99; H, 4.03; N, 11.19; mp
276–279ꢁC; dH (CDCl3 + methanol-d4, 500MHz): 8.84
(d, 1H, J = 4.9Hz), 7.41 (m, 3H), 7.33 (d, 1H,
J = 4.9Hz), 7.21 (m, 2H), 6.06 (s, 1H); dC (CDCl3 +
methanol-d4, 125MHz): 181.73, 181.11, 153.07, 151.64,
150.20, 149.99, 138.32, 129.08, 128.38, 128.11 (2C),
127.52 (2C), 124.49, 103.06; MS (ESI-negative) m/z:
249 [MꢀH]ꢀ.
4.1.13. 4-Methoxy-9-phenylpyrido[2,3-g]quinoline-5,10-
dione (17). To a solution of 16 (20mg, 0.066mmol) in
3mL of CH2Cl2/MeOH (1:1) was added 20mL of a solu-
tion of diazomethane in ethyl ether. After 10min, the
resulting mixture was evaporated to dryness and the
crude was purified under flash chromatography
(CH2Cl2/MeOH; 95:5) to afford 17 (4mg, 19%). Found:
C, 72.21; H, 3.83; N, 8.84. C19H12N2O3 requires: C,
72.15; H, 3.82; N, 8.86; dH (CDCl3, 500MHz): 9.06 (d,
1H, J = 4.9Hz), 8.85 (d, 1H, J = 5.8Hz), 7.54 (d, 1H,
J = 4.9Hz), 7.46 (m, 3H), 7.31 (m, 2H), 7.20 (d, 1H,
J = 5.8Hz), 4.13 (s, 3H); dC (CDCl3, 125MHz):
182.03, 180.24, 166.29, 155.83, 154.00, 152.40, 151.71,
150.30, 137.98, 130.06, 128.58, 128.41 (2C), 128.05
(2C), 127.33, 119.53, 111.05, 56.98; MS (ESI-positive)
m/z: 317 [M+H]+, 339 [M+Na]+.
4.1.10. 6-[(2,2-Dimethyl-4,6-dioxo-[1,3]dioxan-5-ylidene-
methyl)amino]-4-(2-nitrophenyl)quinoline-5,8-dione (14).
A solution of Meldrumꢀs acid (318mg, 22mmol) and tri-
ethylorthoformate (6.4mL) was refluxed for 2h under ar-
gon. The reaction mixture was added to 12 (480mg,
1.62mmol) and refluxed for 4h. After being cooled to
0ꢁC, the solution was filtered and washed with cold meth-
anol to yield 14 (615mg, 84%). Found: C, 58.91; H, 3.22;
N, 9.28. C22H15N3O8 requires: C, 58.80; H, 3.36; N, 9.35;
dH (CDCl3, 300MHz): 9.10 (d, 1H, J = 4.9Hz), 8.47 (d,
1H, J = 13.9Hz), 8.35 (d, 1H, J = 7.9Hz), 7.77 (t, 1H,
J = 7.9Hz), 7.70 (td, 1H, J = 1.4 and 7.9Hz), 7.43 (d,
1H, J = 4.9Hz), 7.21 (dd, 1H, J = 1.4 and 7.9Hz), 6.91
(s, 1H), 1.73 (s, 3H), 1.72 (s, 3H); dC (CDCl3, 75MHz):
181.01, 179.16, 163.93, 161.98, 154.95, 149.39, 148.19,
148.08, 146.46, 141.12, 134.19, 133.72, 129.90, 129.72,
128.08, 125.15, 124.37, 113.14, 105.92, 94.44, 27.34.
4.1.14. 4-Methoxy-9-(20-nitrophenyl)pyrido[2,3-g]quino-
line-5,10-dione (18).
A
solution of 14 (100mg,
0.22mmol) in degassed diphenyl ether (14mL), was
heated between 200 and 220ꢁC for 15min. The reaction
mixture was cooled to room temperature and added to
hexane at 0ꢁC. Filtration of the reaction afforded a res-
idue that was used without purification. To a solution of
the former residue in 10mL of CH2Cl2/MeOH (1:1) was
added 50mL of a solution of diazomethane in ethyl
ether. After 10min, the resulting mixture was evapo-
rated to dryness and the crude was purified under flash
chromatography (CH2Cl2/MeOH; 95:5) to afford the
known 1813b (14mg, 17%). Spectral data for this com-
pound were identical to those previously described.
4.1.11. 6-[(2,2-Dimethyl-4,6-dioxo-[1,3]dioxan-5-ylidene-
methyl)amino]-4-phenylquinoline-5,8-dione (15). The
same procedure as for 14 involving 13 (200mg, 0.8mmol)
was used to yield 15 (245mg, 76%). Found: C, 65.39; H,
3.92; N, 7.02. C22H16N2O6 requires: C, 65.34; H, 3.99; N,
6.93; mp 257.8–259.4ꢁC; dH (CDCl3 + methanol-d4,
500MHz): 8.91 (d, 1H, J = 4.9Hz), 8.51 (s, 1H), 7.45
(d, 1H, J = 4.9Hz), 7.39 (m, 3H), 7.19 (m, 2H), 6.93 (s,
1H), 1.65 (s, 6H); dC (CDCl3 + methanol-d4, 125MHz):
181.36, 178.62, 163.80, 162.46, 153.83, 152.80, 148.71,
148.50, 141.54, 137.40, 130.59, 128.84, 128.34 (2C),
127.60 (2C), 124.43, 112.92, 105.90, 93.95, 27.10 (2C).
4.1.15.
9-Chloro-3-methylpyrido[2,3-g]quinoline-5,10-
dione (20). To a solution of quinone 1913c (318mg,
1.17mmol) in CH3CN (10mL) was added a solution
of methacrolein dimethylhydrazone (224mg, 2mmol)
in ethyl ether (2mL). The violet solution was stirred at
room temperature for 16h and evaporated to dryness.
The residue was chromatographed on silica gel
(AcOEt/CH2Cl2; 4:1) to yield 20 (241mg, 80%) as a
brown pale solid. Found: C, 60.20; H, 2.58; N, 10.99.
C13H7ClN2O2 requires: 60.38; H, 2.71; N, 10.83; mp
208–210ꢁC; t (KBr): 1691 (C@O)cmꢀ1; dH (CDCl3,
250MHz): 8.93 (d, 1H, J = 2Hz), 8.90 (d, 1H,
J = 5.1Hz), 8.43 (d, 1H, J = 2Hz), 7.75 (d, 1H,
J = 5.1Hz), 2.55 (s, 3H); dC (CDCl3, 63MHz): 180.88,
179.59, 156.97, 154.06, 150.53, 146.71, 146.50, 139.70,
4.1.12. 4-Hydroxy-9-phenylpyrido[2,3-g]quinoline-5,10-
dione (16). A solution of 15 (230mg, 0.57mmol) in de-
gassed diphenyl ether (36mL), was heated between 200

6512
S. Manzanaro et al. / Bioorg. Med. Chem. 12 (2004) 6505–6515
135.35, 131.53, 129.02, 127.88, 19.13; MS (ESI-positive)
259 [M+H]+, 281 [M+H]+.
bined organic phases were dried over Na2SO4 and evap-
orated. The resulting crude was purified by silica gel
column chromatography (hexane/AcOEt; 1:1) affording
27 (931mg, 66%) as a beige solid. Found: C, 33.64; H,
2.33; N, 3.56. C11H9BrINO2 requires: C, 33.53; H,
2.30; N, 3.55; dH (CDCl3, 300MHz): 8.32 (d, 2H,
J = 4.8Hz), 8.14 (d, 2H, J = 4.8Hz), 7.16 (s, 1H), 4.01
(s, 3H), 3.81 (s, 3H); dC (CDCl3, 75MHz): 152.31,
148.40, 145.30, 140.45, 137.68, 125.21, 116.18, 112.81,
98.46, 62.65, 56.67; MS (ESI positive) m/z: 396 [M+2H]+.
4.1.16. 4-Chloro-9-(2-nitrophenyl)pyrido[2,3-g]quinoline-
5,10-dione (21). A solution of quinone 1913c (185mg,
0.68mmol) and o-nitrocinnammaldehyde dimethylhy-
drazone (438mg, 2mmol) in chloroform (1mL) was
sonicated at 50ꢁC for 125h in an ultrasound cleaning
bath. The solvent was evaporated and the residue was
chromatographed on silica gel (AcOEt) to afford the
known compound 2113b,14 (52mg, 20%). Found: C,
59.89; H, 1.99; N, 11.17. C18H8ClN3O4 requires: 59.13;
H, 2.18; N, 11.48.
4.1.21. 6-Bromo-4-iodoquinoline-5,8-dione (29). To a
solution of 27 (250mg, 0.63mmol) in AcCN/H2O
(42:10mL) at 0ꢁC, a solution of ammonium cerium(IV)
nitrate (1.74g, 3.17mmol) in H2O (2.2mL) was dropwise
added. The mixture was stirred at room temperature for
2h and partitioned between H2O and CH2Cl2. The com-
bined organic phases were washed with brine, dried over
Na2SO4 and evaporated. The crude was triturated with
ethyl ether affording 29 (230mg, 98%) as orange solid.
Found: C, 29.78; H, 0.85; N, 3.86. C9H3BrINO2 re-
quires: C, 29.70; H, 0.83; N, 3.85; mp 182.3–185.0ꢁC;
dH (CDCl3, 300MHz): 8.38 (d, 1H, J = 5.0Hz), 8.34
(d, 1H, J = 5.0Hz), 7.66 (s, 1H); dC (CDCl3, 75MHz):
179.15, 176.00, 152.82, 148.33, 141.85, 140.41, 139.32,
128.20, 106.27; MS (APCI-positive) m/z: 364 [M+H]+.
4.1.17. 9-Chloro-1-dimethylamino-4-methyl-1,4-dihydro-
pyrido[2,3-g]quinoline-5,10-dione (22). To a solution of
quinone 1913c (77mg, 0.29mmol) in CH3CN (5mL) was
added a solution of crotonaldehyde dimethylhydrazone
(52mg, 0.46mmol) in ethyl ether (1mL). The violet solu-
tion was stirred at room temperature for 22h. The solvent
and excess azadiene were evaporated under reduced pres-
sure, and the residue was chromatographed on silica gel
(AcOEt) to afford 22 (76mg, 89%). Found: C, 59.81; H,
4.23; N, 14.02. C15H14ClN3O2 requires: 59.34; H, 4.61;
N, 13.83; t (KBr): 1667, 1640 (C@O)cmꢀ1; dH (CDCl3,
250MHz): 8.73 (d, 1H, J = 5.3Hz), 7.53 (d, 1H,
J = 5.3Hz), 6.25 (d, 1H, J = 7.9Hz), 5.20 (dd, 1H,
J = 7.9 and 5.1Hz), 3.76 (m, 1H), 2.69 (s, 6H), 1.17 (d,
3H, J = 6.6Hz); dC (CDCl3, 63MHz): 180.20, 177.92,
152.74, 152.39, 149.48, 146.37, 143.09, 128.75, 121.46,
120.30, 120.16, 113.34, 44.85, 26.04, 23.95.
4.1.22.
9-Bromo-3-methylpyrido[2,3-g]quinoline-5,10-
dione (30). To a solution of 2814 (500mg, 1.57mmol)
in 1,2-dichloroethane (13mL) at 80ꢁC, methacrolein
dimethylhydrazone (235mg, 2.01mmol) in 1,2-dichloro-
ethane (4mL) was dropwise added. The mixture was
kept at 80ꢁC for 30min, then cooled to room tempera-
ture and the solvent was evaporated. The crude was
purified by silica gel column chromatography (AcOEt)
affording 30 (332mg, 68%) as a brown solid. Found:
C, 51.53; H, 2.30; N, 9.27. C13H7BrN2O2 requires: C,
51.51; H, 2.33; N, 9.24; mp > 200ꢁC (decomp.); t
(KBr) 1687cmꢀ1; dH (CDCl3, 300MHz): 8.96 (d, 1H,
J = 2.2Hz), 8.76 (d, 1H, J = 5.0Hz), 8.45 (d, 1H,
J = 2.2Hz), 8.00 (d, 1H, J = 5.0Hz), 2.57 (s, 3H); dC
(CDCl3, 75MHz): 180.72, 179.71, 156.97, 153.65,
150.39, 146.58, 139.73, 135.31, 135.25, 134.61, 129.09,
129.02, 19.15; MS (APCI-positive) m/z: 304 [M+H]+.
4.1.18.
4-Chloro-9-methylpyrido[2,3-g]quinoline-5,10-
dione (23). A sample of compound 22 (43mg, 0.14mmol)
was heated at 110ꢁC and 0.1Torr during 2h and then
washed with ethyl ether and chloroform. The residue
obtained (22mg, 60%) was identified as compound 23.
Found: C, 60.79; H, 2.23; N, 11.11. C13H7ClN2O2 re-
quires: C, 60.38; H, 2.71; N, 10.83; mp > 300ꢁC; t
(KBr): 1689 (C@O)cmꢀ1; dH (DMSO-d6, 250MHz,):
8.92 (d, 1H, J = 5.1Hz), 8.87 (d, 1H, J = 4.8Hz), 7.97
(d, 1H, J = 5.1Hz), 7.70 (d, 1H, J = 4.8Hz), 2.77 (s, 3H).
4.1.19. 4-Chloro-9-dimethylaminopyrido[2,3-g]quinoline-
5,10-dione (24). A solution of compound 22 (21mg,
0.11mmol) in THF (2mL) and aqueous HCl (6M)
(2mL) was heated at 80ꢁC for 1h. The reaction mixture
was saturated with solid sodium carbonate and ex-
tracted with CHCl3 and AcOEt. The combined organic
layers were dried over Na2SO4 and evaporated, yielding
24 (16mg, 88%). Found: C, 67.52; H, 4.48; N, 15.43.
C15H13N3O2: 67.40; H, 4.90; N, 15.72; mp 97–100ꢁC; t
4.1.23. 9-Iodo-3-methylpyrido[2,3-g]quinoline-5,10-dione
(31). Same procedure as for 30 involving 29 (50mg,
0.13mmol). The crude was purified by silica gel column
chromatography (AcOEt) affording mainly impure frac-
tions but only few pure 31 (7mg, 15%) was isolated as a
brown solid. Found: C, 44.78; H, 2.03; N, 8.02.
C13H7IN2O2 requires: C, 44.60; H, 2.02; N, 8.00;
mp > 215ꢁC (decomp.); t (KBr) 1683cmꢀ1 (C@O); dH
(CDCl3, 300MHz): 8.98 (d, 1H, J = 2.0Hz), 8.57 (d,
1H, J = 4.9Hz), 8.47 (d, 1H, J = 2.0Hz), 8.40 (d, 1H,
J = 4.9Hz), 2.59 (s, 3H); dC (CDCl3, 75MHz): 180.37,
179.55, 156.75, 152.86, 149.37, 146.11, 142.44, 139.58,
135.08, 130.37, 129.02, 106.07, 18.94; MS (APCI-posi-
tive) m/z: 351 [M+H]+.
(KBr): 1683 and 1654 (C@O)cmꢀ1
; dH (CDCl3,
250MHz): 8.84 (d, 1H, J = 4.9Hz), 8.53 (d, 1H,
J = 5.1Hz), 7.44 (d, 1H, J = 4.9Hz), 6.99 (d, 1H,
J = 6.1Hz), 3.11 (s, 6H), 2.86 (s, 3H).
4.1.20. 6-Bromo-4-iodo-5,8-dimethoxyquinoline (27). A
suspension of 2522 (1.5g, 3.6mmol) and lithium iodide
(1.4g, 10.8mmol) in 1,4-dioxane (150mL) was refluxed
for 96h. The mixture was cooled to room temperature
and diluted with AcOEt (250mL), washed with H2O.
The aqueous phase was extracted to AcOEt. The com-
4.1.24. 9-Dimethylamino-3-methylpyrido[2,3-g]quinoline-
5,10-dione (32). To a solution of 30 (50mg, 0.16mmol)
and Pd(CH3CN)2Cl2 (2mg, 0.008mmol) in anhydrous

S. Manzanaro et al. / Bioorg. Med. Chem. 12 (2004) 6505–6515
6513
toluene (3.5mL) was added dimethylaminotrimethyl-
stannane. The mixture was refluxed for 6h and cooled
to room temperature. It was diluted with CH2Cl2 and
washed with H2O. The aqueous phase was extracted to
CH2Cl2. The combined organic phases were washed
with brine, dried over Na2SO4 and evaporated. The
crude was purified by silica gel column chromatography
(CH2Cl2/MeOH; 100:1) affording 32 (37mg, 86%) as a
red solid. Found: C, 67.38; H, 4.93; N, 15.76.
C15H13N3O2 requires: C, 67.40; H, 4.90; N, 15.72; mp
241.7–243.1ꢁC; dH (CDCl3, 300MHz): 8.88 (d, 1H,
J = 2.6Hz), 8.51 (d, 1H, J = 6.1Hz), 8.40 (d, 1H,
J = 2.6Hz), 7.00 (d, 1H, J = 6.1Hz), 3.10 (s, 6H), 2.53
(s, 3H); dC (CDCl3, 75MHz): 182.73, 180.73, 156.25,
156.17, 151.90, 151.52, 148.20, 138.44, 135.17, 128.52,
118.59, 113.33, 43.92, 18.93; MS (APCI-positive) m/z:
268 [M+H]+.
C, 56.39; H, 2.03; N, 9.39; dH (DMSO-d6, 300MHz): d
9.55 (d, 1H, J = 1.8Hz), 9.22 (d, 1H, J = 4.8Hz), 8.95
(d, 1H, J = 1.8Hz), 7.97 (d, 1H, J = 4.8Hz); dC
(DMSO-d6, 300MHz): 180.66, 169.15, 165.65, 155.49,
154.90, 150.88, 149.54, 143.69, 139.11, 136.13, 130.96,
130.72, 125.99, 120.00; MS (ESI-positive) m/z: 299
[M+H]+.
4.1.28.
8-Methyl-5,10-dioxo-5,10-dihydropyrido[2,3-
g]quinoline-4-carbaldehyde (37). To a solution of 35
(231mg, 0.97mmol) in 1,4-dioxane (30mL) at 80ꢁC,
SeO2 (215mg, 1.94mmol) in 1,4-dioxane (12mL) and
H2O (1mL) was dropwise added. The mixture was kept
at 80ꢁC for 8h. The reaction was cooled to room tem-
perature, filtered over Celite and the solvent was evapo-
rated. The residue was again dissolved in 1,4-dioxane
(30mL), heated at 80ꢁC and SeO2 (215mg, 1.94mmol)
in 1,4-dioxane (12mL) and H2O (1mL) was dropwise
added. The mixture was kept at 80ꢁC overnight and
cooled to room temperature, filtered over Celite, washed
with a mixture of CH2Cl2/MeOH (1:1) and the solvent
evaporated. The crude was purified by silica gel column
4.1.25. Dimethyl-(8-methyl-5,10-dioxo-5,10-dihydropyr-
ido[2,3-g]quinolin-4-yl)-ammonium chloride (33). To a
solution of 32 (15mg, 0.06mmol) in the minimum
amount necessary of 1,4-dioxane was added HCl
(5.9M in 1,4-dioxane) (20lL, 0.12mmol). The mixture
was stirred at room temperature for 15min and the
resulting solid was filtered affording 33 (13mg, 72%) as
an orange solid. Found: C, 59.54; H, 4.19; N, 13.80.
C15H14ClN3O2 requires: C, 59.31; H, 4.65; N, 13.83;
mp > 270ꢁC (decomp.); dH (methanol-d4, 300MHz):
8.92 (br, 1H), 8.46 (br, 1H), 8.28 (d, 1H, J = 6.6Hz),
7.51 (d, 1H, J = 6.6Hz), 3.30 (s, 6H), 2.59 (s, 3H); dC
(methanol-d4, 75MHz): 180.11, 179.14, 158.50, 157.26,
148.35, 146.37, 141.50, 141.25, 135.94, 128.92, 117.48,
114.37, 44.91, 18.66; MS (APCI-positive) m/z: 268
[MꢀCl]+, 290 [MꢀCl+Na]+.
chromatography
(CH2Cl2/AcOEt/MeOH;
1:1:0.1)
affording 37 (177mg, 77%) as a brown solid. Found:
C, 66.72; H, 3.19; N, 11.12. C14H8N2O3 requires: C,
66.67; H, 3.20; N, 11.11; mp > 215ꢁC (decomp.); dH
(CDCl3, 300MHz): 10.84 (s, 1H), 9.26 (d, 1H,
J = 4.67Hz), 9.46 (d, 1H, J = 2.19Hz), 8.49 (d, 1H,
J = 2.19Hz), 7.91 (d, 2H, J = 4.67Hz), 2.59 (s, 3H); dC
(CDCl3, 300MHz): 191.05, 182.52, 180.66, 156.76,
156.08, 149.12, 145.96, 145.61, 140.29, 135.58, 129.80,
128.88, 125.93, 19.06; MS (APCI-positive) m/z: 225.0
[MꢀCHO+H]+, 253 [M+H]+.
4.1.29.
8-Methyl-5,10-dioxo-5,10-dihydropyrido[2,3-
4.1.26.
3,9-Dimethylpyrido[2,3-g]quinoline-5,10-dione
g]quinoline-4-carboxylic acid (38). To a solution of 37
(100mg, 0.40mmol) in CH3CN (4mL), NaH2PO4
(12.7mg, 0.1mmol) in H2O (0.5mL), H2O2 (45lL,
0.41mmol) was added and then NaClO2 (64mg,
0.55mmol) was dropwise added in H2O (1mL). The
mixture was stirred at room temperature for 4h. A cat-
alytic amount of Na2S2O5 and HCl (2M) to pH = 1 was
consecutively added to the reaction. The precipitated
obtained was filtered affording 38 (78mg, 73%). The
mother liquors were extracted with AcOEt. The com-
bined organic phases was washed with brine, dried over
Na2SO4, and evaporated affording a new amount of 38
(20mg, 19%) as a pale orange solid. Found: C, 62.80;
H, 3.03; N, 10.40. C14H8N2O4 requires: C, 62.69; H,
3.01; N, 10.44; mp > 300ꢁC (decomp.); dH (methanol-
d4, 300MHz): 9.12 (d, 1H, J = 4.8Hz), 8.94 (d, 1H,
J = 2.1Hz), 8.39 (d, 1H, J = 2.1Hz), 7.86 (d, 1H,
J = 4.8Hz), 2.54 (s, 3H); dC (methanol-d4, 300MHz):
180.76, 180.42, 168.60, 155.33, 154.58, 148.87, 146.04,
143.02, 139.21, 134.50, 130.14, 126.50, 125.17,
18.27; MS (ESI-positive) m/z: 269 [M+H]+, 559
[2M+Na]+.
(35). To a solution of 3424 (480mg, 1.90mmol) in CHCl3
(9mL), crotonaldehyde dimethylhydrazone (235mg,
2.09mmol) and Et3N (260lL, 1.90mmol) were added.
The mixture was stirred to room temperature for 16h.
The solvent was evaporated and the residue was heated
at 110ꢁC and 0.1Torr for 2h. The crude was purified by
silica gel column chromatography (CH2Cl2/AcOEt;
8:2 ! 1:1) affording 35 (385mg, 84%) as a brown solid.
Found: C, 70.47; H, 4.28; N, 11.78. C14H10N2O2 re-
quires: C, 70.58; H, 4.20; N, 11.76; mp 255.8–258.0ꢁC;
dH (CDCl3, 300MHz): 8.92 (d, 1H, J = 2.6Hz), 8.90
(d, 1H, J = 5.3Hz), 8.43 (d, 1H, J = 2.6Hz), 7.56 (d,
1H, J = 5.3Hz), 2.94 (s, 3H), 2.59 (s, 3H); dC (CDCl3,
300MHz): 182.56, 181.44, 155.99, 153.25, 151.71,
149.21, 146.52, 138.80, 134.67, 131.24, 129.05, 128.73,
22.38, 18.54; MS (APCI-positive) m/z: 239.0 [M+H]+.
4.1.27. 5,10-Dioxo-5,10-dihydropyrido[2,3-g]quinoline-
3,9-dicarboxylic acid (36). To a solution of 35 (200mg,
0.84mmol) in H2SO4 (6mL) was added CrO3 (504mg,
5.04mmol) during 1h in portions. The mixture was
heated at 80ꢁC for 4h and then stirred at room temper-
ature overnight. The crude was diluted with H2O and
extracted to AcOEt. The combined organic phases was
washed with brine, dried over Na2SO4 and evaporated
affording 36 (100mg, 40%) as a pale yellow solid.
Found: C, 56.52; H, 2.04; N, 9.36. C14H6N2O6 requires:
4.1.30. 3-(8-Methyl-5,10-dioxo-5,10-dihydropyrido[2,3-
g]quinolin-4-yl)acrylonitrile (39). A solution of 37
(118mg, 0.47mmol) and (triphenylphosphoranylid-
ene)acetonitrile (211mg, 0.70mmol) in anhydrous
CH2Cl2 (118mL) was refluxed for 1h. The mixture

6514
S. Manzanaro et al. / Bioorg. Med. Chem. 12 (2004) 6505–6515
was cooled to room temperature and the solvent was
evaporated. H NMR spectra of the crude showed a
support of this research. S.M. is grateful to the MCyT
´
1
for a predoctoral fellowship (accion MIT-Becas del
Programa PACTI del plan nacional de I+D). M.J.M.
is grateful to the MCyT for a postdoctoral fellowship
10:1 mixture of Z:E isomers. The resulting crude was
purified by silica gel column chromatography
(CH2Cl2/AcOEt/MeOH; 1:1:0.1) affording the Z iso-
mer 39 (76mg, 59%) as a pale green solid. Found: C,
69.73; H, 3.31; N, 15.30. C16H9N3O2 requires: C,
69.81; H, 3.30; N, 15.27; mp > 250ꢁC (decomp.); dH
(CDCl3, 300MHz): 9.22 (d, 1H, J = 4.9Hz), 8.97 (br,
1H), 8.52 (br, 1H), 8.17 (d, 1H, J = 11.8Hz), 7.95 (d,
1H, J = 4.9Hz), 5.88 (d, 1H, J = 11.8Hz), 2.60 (s,
3H); dC (CDCl3, 300MHz): 182.17, 180.94, 156.64,
155.26, 149.37, 147.89, 146.11, 144.15, 139.94, 135.40,
129.22, 128.26, 126.40, 115.53, 101.31, 18.98; MS
(ESI-positive) m/z: 276 [M+H]+, 298 [M+Na]+, 573
[2M+Na]+. The E isomer was not isolated as pure
compound.
´
(accion IDE-Becas del Programa del Plan Nacional de
I+D). Financial support of this research through grant
PTR95-0623.OP is also gratefully acknowledged. The
´
authors thank Marta Martınez for the MS studies.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2004.09.021.
References and notes
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