Straightforward palladium-mediated synthesis and biological evaluation of benzo[j]phenanthridine-7,12-diones as anti-tuberculosis agents

Article abstract of DOI:10.1016/j.ejmech.2011.11.033

In 1991, WHO recognized the resurgence of tuberculosis as a global health problem. Although modern chemotherapy is effective against the causative pathogen Mycobacterium tuberculosis, the current drug regimens have failed to eradicate the disease. The suc

Full text of DOI:10.1016/j.ejmech.2011.11.033

European Journal of Medicinal Chemistry 48 (2012) 57e68
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journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Straightforward palladium-mediated synthesis and biological evaluation of benzo
[j]phenanthridine-7,12-diones as anti-tuberculosis agents
,
,
Davie Cappoen a 1, Jan Jacobs b 1, Tuyen Nguyen Van b, Sven Claessens b, Gaston Diels c, Roel Anthonissen d,
**
Thorbjorg Einarsdottir a, Maryse Fauville d, Luc Verschaeve e, Kris Huygen a , Norbert De Kimpe
,
b,
*
a Service Immunology, O.D. Communicable & Infectious Diseases, Scientific Institute of Public Health (Site Ukkel), Engelandstraat 642, B-1180 Ukkel, Belgium
b Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium
c Johnson & Johnson Pharmaceutical Research & Development, Synthesis Enabling Technologies, Turnhoutseweg 30, B-2340 Beerse, Belgium
d ServiceBacterial Diseases, O.D. Communicable & Infectious Diseases, Scientific Institute of Public Health (Site Ukkel), Engelandstraat 642, B-1180 Ukkel, Belgium
e O.D. Public Health and Surveillance, Scientific Institute of Public Health (Site Elsene), J. Wytsmanstraat 14, B-1050 Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
In 1991, WHO recognized the resurgence of tuberculosis as a global health problem. Although modern
chemotherapy is effective against the causative pathogen Mycobacterium tuberculosis, the current drug
regimens have failed to eradicate the disease. The success of the pathogen, partially attributed to drug
resistance, necessitates the development of novel anti-tuberculosis drugs. Benzo[j]phenanthridine-7,12-
diones, tetracyclic derivatives of the natural product benz[g]isoquinoline-5,10-dione, were conveniently
synthesized via palladium-catalyzed intramolecular cyclization of N-methanesulfonyl-3-bromo-2-(ary-
lamino)methyl-1,4-naphthoquinones. Here we report on the bioactivity of eight benzo[j]phenanthridine-
7,12-dione derivatives as candidate drug molecules against M. tuberculosis and on their cytotoxicity on
C3A human hepatocytes. The strongest antimicrobial activity (as detected by growth inhibition of
bacteria, using luminometry and BACTEC 460-TB) and lowest cytotoxicity was found for 3-methylbenzo
[j]phenanthridine-7,12-dione 5e, which was also effective in targeting intracellular M. tuberculosis (in
murine J774 macrophages) and was not genotoxic for C3A hepatocytes.
Received 21 September 2011
Received in revised form
18 November 2011
Accepted 21 November 2011
Available online 1 December 2011
Keywords:
Benz[g]isoquinoline-5,10-dione
Benzo[j]phenanthridine-7,12-dione
Heck reaction
Mycobacterium tuberculosis
Anti-mycobacterial activity
Cytotoxicity
Ó 2011 Elsevier Masson SAS. All rights reserved.
Intracellular pathogen
1. Introduction
metabolically inactive, dormant bacteria [4]. The treatment of MDR
and XDR M.tb. cases necessitates very expensive and often more
Tuberculosis (TB) is an infectious pulmonary disease, spread by
aerosolized droplets generated by a person with active disease.
Caused by the weakly gram positive bacterium Mycobacterium
tuberculosis (M.tb.) it presents a serious challenge to modern
chemotherapy [1e3]. For starters, M.tb. has a thick, lipid rich cell
wall, highly impermeable for antimicrobial agents, secondly, M.tb.
has developed bacillary resistance against the current drug treat-
ments, giving rise to Multi-Drug Resistant (MDR) and even Exten-
sively Drug Resistant (XDR) M.tb. Moreover, this intracellular
parasite has also developed mechanisms to persist in a non-
replicating, dormant stage, leading to a so-called latent infection,
estimated to affect about 2 billion of people worldwide. Currently
known anti-mycobacterial agents are ineffective against these
toxic compounds, and it is clear that the development of novel anti-
tuberculosis drugs is urgently needed [5]. The negative effects of
the current drug regimens are further exacerbated by the global
human immunodeficiency virus (HIV) pandemic. The anti-
tuberculosis therapy is prone for drugedrug interactions and side
effects when combined with anti-retroviral compounds. As a result,
antibiotic treatment of people co-infected with both HIV and TB is
even more problematic [6]. Although a few decades ago TB was
announced to be eradicated before the end of the 20th century, at
present, it is still the most severe infectious disease caused by
a single pathogen [7]. According to the latest WHO reports, more
than 9 million people developed TB in 2008 and 1.7 million people
died of the disease. Thus, TB seems far from eliminated [8,9].
Poverty, imperfect diagnostic assays, poor access to health care,
limited vaccine efficacy and lack of new drugs remain obstacles
[10]. The development of new anti-tuberculosis drugs is far from
a trivial matter and although some promising candidates are in the
pipeline, not one new TB drug has been developed during the last
* Corresponding author. Tel.: þ32 9 264 59 51; fax: þ32 9 264 62 43.
** Corresponding author. Tel.: þ32 2 373 33 70; fax: þ32 2 373 33 67.
E-mail address: norbert.dekimpe@UGent.be (N. De Kimpe).
1
Both authors contributed equally to the paper.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.11.033

58
D. Cappoen et al. / European Journal of Medicinal Chemistry 48 (2012) 57e68
40 years [11]. Identification of new classes of possible drug candi-
dates would therefore be extremely valuable in the battle against
TB [12]. The naturally occurring 2-azaanthraquinones such as
bostrycoidin 1, 9-O-methylbostrycoidin 2, tolypocladin 3 and benz
[g]isoquinoline-5,10-dione 4 are of considerable interest in organic
synthesis due to their important biological activities (Fig. 1) [13].
For instance, bostrycoidin 1 shows in vitro antibiotic activity against
M. tuberculosis. 9-O-Methylbostrycoidin 2, an antibiotic effective
against Gram positive bacteria, has been isolated from cultures of
Fusarium moniliforme [14]. Other members of the class of natural 2-
azaanthraquinones are known as intercalating DNA binding agents
or possess metal-chelating properties, e.g. tolypocladin 3 [15]. Benz
[g]isoquinoline-5,10-dione 4, isolated from a.o. Psychotria campo-
nutans [16] and Mitracarpus scaber Zucc. [17] was found to be active
against multi-drug resistant Plasmodium falciparum [16] and
Leishmania [17]. In the course of our research on the synthesis of
biologically active 2-azaanthraquinones [18], we became interested
in benzo[j]phenanthridine-7,12-diones 5, which are considered as
tetracyclic derivatives of the natural product benz[g]isoquinoline-
5,10-dione 4 and which are of considerable interest as potential
antitumor agents. Here, we describe a new and convenient method
for the synthesis of substituted benzo[j]phenanthridine-7,12-
diones 5 and we report on the antimicrobial activity against M.tb.
and the cytotoxicity on C3A hepatocytes of these derivatives.
In the literature, only three reports for the synthesis of benzo[j]
phenanthridine-7,12-diones 5 have been described so far. The first
method is based on the oxidative coupling of 1-(1,4-
dihydroxynaphth-2-yl)ethanone with different anilines in the
presence of sodium iodate [19]. However, under these reaction
conditions a mixture of different coupling products was obtained,
amongst which benzo[j]phenanthridine-7,12-diones 5. In a second
report, the phthalide annulation reaction of the carbanion derived
from 3-cyano-1(3H)-isobenzofuranone and the corresponding het-
eroaryne derivative of 4-bromoquinoline to benzo[j]phenan-
thridine-7,12-dione 5a was reported [20]. Finally, a more recent
paper describes the heteroatom-directed tandem lithiation meth-
odology for the synthesis of benzo[j]phenanthridine-7,12-dione 5a
[21]. Because of the fact that the above methods have disadvantages
suchas lowyields and harshreaction conditions, thedevelopmentof
a new reliable method for the synthesis of benzo[j]phenanthridine-
7,12-diones 5 is needed. Furthermore, the synthesis of benzo[j]
phenanthridine-7,12-diones 5 with a different substitution pattern
is hampered in the above presented literature methods since
different regioisomers would be formed. Therefore, it was decided to
investigate a new synthesis of different benzo[j]phenanthridine-
7,12-diones based on an intramolecular Heck reaction, which was
studied earlier at our department for the synthesis of different
heterocyclic quinones [22]. In this way, the synthesis started from 2-
bromo-3-bromomethyl-1,4-dimethoxynaphthalene 7, which was
believed to give the key intermediates 6 after substitution of the
benzylic bromide with a proper aniline, N-protection and subse-
quent oxidation of the 1,4-dimethoxynaphthalene moiety
(Scheme 1).
2. Results and discussion
The total synthesis of benzo[j]phenanthridine-7,12-diones 5
started with 2-bromo-3-bromomethyl-1,4-dimethoxynaphthalene
7, which was synthesized using the commercially available mena-
dione [23]. Treatment of the functionalized naphthalene 7 with two
equivalents of a suitable aniline in ethanol at room temperature for
16
phthalenes 8aei in 32e80% yield. Then, the amino group of 2-
(arylamino)methyl-3-bromo-1,4-dimethoxynaphthalenes 8aei
h afforded 2-(arylamino)methyl-3-bromo-1,4-dimethoxyna-
was protected by treatment with methanesulfonyl chloride (1.3
equiv.) in dichloromethane in the presence of pyridine at reflux for
12 h, resulting in the corresponding N-protected compounds 9aei
in 44e91% yield. Subsequent oxidative demethylation of N-meth-
anesulfonyl-2-(arylamino)methyl-3-bromo-1,4-dimethoxynaphth-
alenes 9aei by treatment with 2.3 equivalents of cerium(IV)
ammonium nitrate in aqueous acetonitrile at room temperature for
30 min gave N-methanesulfonyl-2-(arylamino)methyl-3-bromo-
1,4-naphthoquinones 6aei in 50e95% yield (Scheme 2 and Table 1).
Finally, the intramolecular cyclization of N-methanesulfonyl-2-
(arylamino)methyl-3-bromo-1,4-naphthoquinones 6 using a palla-
dium(0)-catalyzed reaction was investigated as a method to ach-
ieve the synthesis of tetracyclic benzo[j]phenanthridine-7,12-
diones 5. In our attempts to induce the palladium(0)-catalyzed
ring closure of compounds 6aei, the aryl coupling to vinyl
bromides using palladium(II) acetate in the presence of triphenyl-
phosphine as a catalyst was applied (Scheme 2, Table 1). However,
using these standard reaction conditions during the initial
attempts, the formation of complex reaction mixtures was noticed,
from which benzo[j]phenanthridine-7,12-diones 5 could not be
isolated. Therefore, the Heck reaction was investigated further and
it was found that degassing the solvent prior to use was important
in order to remove residual air oxygen, which can reoxidize the
generated Pd(0) to Pd(þII) and which can oxidize the triphenyl-
phosphine making a Heck reaction impossible. The intramolecular
cyclization could then be achieved with 20 mol% of palladium(II)
acetate in the presence of 40 mol% of triphenylphosphine and 3
equivalents of potassium carbonate in boiling toluene for 1e3 h.
Under the above mentioned reaction conditions, tetracyclic benzo
[j]phenanthridine-7,12-diones 5aeh were obtained in moderate to
fair yield (27e62%). These moderate yields can possibly be ascribed
to the presence of a naphthoquinone moiety in compounds 6,
which can reoxidize the necessary Pd(0) to Pd(þII) and thus
limiting the overall efficiency of the Heck reaction. Furthermore,
upon the presence of electron-donating substituents at the ortho-
position of the arylaminogroup of the intermediates 6, a lower
isolated yield of the corresponding benzo[j]phenanthridine-7,12-
diones, i.e. derivatives 5d and 5g, was witnessed. Moreover, the
introduction of N-methanesulfonyl-3-bromo-2-(2,4-dimethoxyph-
enylamino)methyl-1,4-naphtho-quinone 6i, which bears two
methoxy substituents at the ortho- and para-position of the aryla-
minogroup, in the Heck reaction furnished a complex reaction
mixture from which the pure benzo[j]phenanthridine-7,12-dione 5i
R1
O
O
O
N
N
N
R2
R
R3
O
O
O
1 R1 = R3 = OH, R2 = OMe (Bostrycoidin)
2 R1 = R2 = OMe, R3 = OH (9-O-Methylbostrycoidin)
3 R1 = R2 = R3 = OH (Tolypocladin)
5
4 (Benz[g]isoquinoline-5,10-dione)
Fig. 1. Natural 2-azaanthraquinones, benz[g]isoquinoline-5,10-dione and its synthetic analogs.

D. Cappoen et al. / European Journal of Medicinal Chemistry 48 (2012) 57e68
59
R2
R1
R3
OMe
O
O
Br
N
N
Mes
R1
R2
Br
Br
O
OMe
O
R3
7
6
5
Scheme 1. Retrosynthetic analysis of benzo[j]phenanthridine-7,12-diones 5 from 2-bromo-3-bromomethyl-1,4-dimethoxynaphthalene.
could not be obtained. These observations can most probably be
ascribed to electronic as well as steric effects of the electron-
donating substituents of the arylaminogroup in compounds 6.
The next step was to test the benzo[j]phenanthridine-7,12-
diones 5aeh for cytotoxicity against C3A hepatocytes and antimi-
crobial activity against M.tb. Acute toxicity was analyzed using
a neutral red dye uptake assay on a human cell line of C3A hepa-
tocytes. Hepatocytes were chosen for this test, as drug toxicity can
result in necrosis of liver tissue. Hepatotoxicity is a problem often
encountered with the currently used second line drugs. The anti-
tuberculosis activity of the benzo[j]phenantridine-7,12-dione
derivatives was evaluated in vitro by luminometry using a lumi-
nescent M.tb. H37Rv strain. This luminescent M.tb. H37Rv is
transformed with pSMT1 plasmid encoding the luxAB genes from
V. harveyi driven by the constitutive hsp60 promoter [24] and can
be used for the rapid luminometric determination of the Minimal
Inhibitory Concentration which reduces the bacterial growth by
50% (MIC50). This method allows a cheap and rapid screening of
novel drug candidates and as shown below is as sensitive as the
BACTEC 460-TB method, which is more cumbersome, more
expensive and more hazardous for the environment, because of its
use of the 14C isotope [25].
(MIC50 ¼ 0.21
m
M) and a lower cytotoxicity (IC50 ¼ 3.21
m
M)
resulting in a more than tenfold increase of SI to 15.29.
As shown in Table 2, the place and nature of the substituents
profoundly affected the antimicrobial activity of the candidate drug
molecules. Thus, in 3-methylbenzo[j]phenanthridine-7,12-dione
5e, methylation at the R2 meta-position was found to increase the
antimicrobial potency and to decrease twofold the cytotoxicity (as
compared to the parental compound 5a), whereas R1 ortho-meth-
ylation in derivative 5d showed decreased antimicrobial activity
albeit that cytotoxicity (IC50 ¼ 3.96
mM) was also lower. On the
other hand, methylation of the R3 para-position of 5f increased both
potency (MIC50 ¼ 0.88 M) and cytotoxicity (IC50 ¼ 1.48) resulting
m
in a comparable selectivity index (SI ¼ 1.69) as for the parental
compound 5a. The same trend was observed for benzo[j]phenan-
thridine-7,12-diones 5g and 5h. While R1 substitution with OMe
gave a decreased cytotoxicity (IC50 ¼ 2.40
mM), it also decreased
the potency (MIC50 ¼ 1.66 M) compared to 5a. Derivative 5h
m
followed the same pattern as 5f with a substitution of a OMe group
at R3 resulting in a lower MIC (MIC50 ¼ 0.43
mM), but an increased
cytotoxicity (IC50 ¼ 0.95) affording only a slight rise in SI. Substi-
tution with OMe seemed to be more potent than regular methyl-
ation, as indicated by results obtained for 5d (MIC50 ¼ 3.68;
IC50 ¼ 3.96; S ¼ 1.08) and 5g (MIC50 ¼ 1.66; IC50 ¼ 2.40; SI ¼ 1.45)
and those of 5f (MIC50 ¼ 0.88; IC50 ¼ 1.48; SI ¼ 1.69) and 5h
(MIC50 ¼ 0.43; IC50 ¼ 0.95; SI ¼ 2.22), respectively.
The benzo[j]phenanthridine-7,12-diones 5aeh all showed
a high bioactivity against the M.tb. H37Rv and significantly inhibi-
ted the growth at micromolar to submicromolar levels (Table 2).
The parental benzo[j]phenanthridine-7,12-dione 5a showed a high
For the various derivatives, the Polar Surface Area values (PSA)
and the lipophility (MiLogP) were calculated using molinspir-
ationplugin (Table 2).
The Minimal Inhibitory Concentration of 99% (MIC99) of M.tb.
H37Rv was measured in a BACTEC 460-TB assay, which is consid-
ered to be the golden standard in susceptibility testing of M.tb. This
tool was used to verify the data obtained in the luminescence assay
potency (MIC50 ¼ 1.20
mM), but its cytotoxicity on C3A hepatocytes
(IC50 ¼ 1.51 M, detected in the neutral red dye exclusion assay
m
[26]) was unacceptable when compared to its activity, resulting in
a low Selectivity Index (SI) of only 1.26. On the other hand, the
methylated analog 5e with a methyl substitution at the 3rd position
(R2
¼
Me) showed
a
significant increase in potency
NH2
R1
R2
R2
R1
R3
R1
R3
2 equiv.
OMe
OMe
OMe
OMe
OMe
R2
1.3 equiv. MesCl
R3
Br
N
H
N
Mes
Br
Pyr/CH2Cl2 (1:2),
EtOH, rt, 16 h
Br
Br
Δ
, 12 h
OMe
7
8a-i (32-83%)
9a-i (44-91%)
2.3 equiv. CAN
CH3CN/H2O (1:1),
rt, 30 min
R2
R1
R3
0.2 equiv. Pd(OAc)2
0.4 equiv. PPh3
3 equiv. K2CO3
O
O
O
O
N
N
Mes
R1
toluene, 80°C, 1 h
then Δ, 1-3 h
Br
R2
R3
6a-i (50-95%)
5a-h (27-62%)
Scheme 2. Total synthesis of benzo[j]phenanthridine-7,12-diones 5.

60
D. Cappoen et al. / European Journal of Medicinal Chemistry 48 (2012) 57e68
Table 1
Total synthesis of benzo[j]phenanthridine-7,12-diones
5
from 2-bromo-3-
bromomethyl-1,4-dimethoxy-naphthalene.
Entry
R1
R2
R3
Yield (%)
8
9
6
5
a
b
c
d
e
f
g
h
i
H
H
H
Me
H
H
OMe
H
H
H
H
H
Me
H
H
H
H
H
Cl
F
H
H
Me
H
OMe
OMe
80
73
74
34
83
57
44
47
32
62
83
91
44
67
76
84
84
82
85
71
90
50
91
95
87
94
70
42
53
53
27
62
59
28
39
/
OMe
Fig. 2. 5e screened against a MDR M. tuberculosis LAM-1 strain. Results obtained for
compound T represents the growth curve of the negative control and T/100 represents
the growth curve of a 100 times diluted inoculum used as a reference to determine the
and served both as a control and a tool to accurately determine the
MIC99. This system is a semi-automatic radiometric assay that
relies on the unique ability of mycobacteria to metabolize 14C-
labeled palmitic acid [26]. Palmitic acid is converted to 14CO2 which
is expelled in the gaseous phase and can in turn be measured in
a Beta counter. Results are expressed as Growth Index (GI) values
and reduction in 14CO2 (as compared to production by untreated
control cultures) indicates growth inhibition. We used BACTEC 460-
TB to determine at what concentration the derivatives were able to
inhibit 99% growth of the M.tb. H37Rv culture by comparing the GI
in cultures treated with compound dilutions, to untreated M.tb.
H37Rv cultures which were 100 times diluted upon inoculation. All
compounds, except for derivative 5h, were tested. As shown in
Table 2, results obtained with the BACTEC 460-TB were very similar
to the luminometry results and showed the same trend. Thus, the
most promising compound in luminometry, i.e. derivative 5e, with
the meta substitution, also showed the highest potency against the
M.tb. strain H37Rv in BACTEC 460-TB with a submicromolar MIC99
MIC99 value
¼ T/100
¼ T
¼(0.5
mM)
¼(1
m
M) ¼(3
m
M)
¼ INH.
shows resistance to isoniazid, rifampicin, rifabutin and prothiona-
mid in a susceptibility assay. Susceptibility screening of the LAM-1
strain for compound 5e was performed with the BACTEC 460-TB
system.
As a negative control isoniazid was tested in parallel to ensure
the bacillary resistance. The MIC99 of 5e against this clinical isolate
was determined and was found to be 0.71 mM, very close to the
potency found against the H37Rv M.tb. laboratory strain. This
finding indicates that the mechanism of action of compound 5e
differs from that of the four other drugs, suggesting that its target or
the place on the target is different (Fig. 2).
New drug candidates cannot have cytotoxic and genotoxic
effects at concentrations at which they have to be used to exert
their antimicrobial effects. Possible mutagenicity of the various
compounds was detected using the VITOTOXÔ kit from Gentaur
established by the Flemish institute for Technological Research. The
model is regarded as rapid and highly sensitive for genotoxic
screening of newly synthesized chemicals and is closely correlated
to the Ames test [27]. The VITOTOXÔ system makes use of two
genetically engineered Salmonella typhimurium strains lacking the
necessary oxidative enzymes. Addition of post-mitochondrial
supernatant (S9), prepared from the liver of aroclor-treated rats
enables the bacilli to metabolize foreign compounds to electro-
philic metabolites. Genotoxicity is reported by the strain which
carries a bacterial luciferase luxCDABE operon placed under tran-
scriptional control of the RecN promoter. As shown in Table 3, none
of the derivatives promoted transcription of the SOS DNA repair
concentration of 0.64
mM. From the most to the least active
compounds, the trend of activity was the same in function of place
and nature of substitution except for compound 5b and 5c in which
the nature of the halogen substitution showed less importance in
the BACTEC system. Again derivative 5d with the methyl substitu-
tion in ortho was singled out as the least potent derivative followed
by 5g which has the same substitution place. This pointed out that
substitution of this position makes the derivative less potent than
the parental molecule 5a. Substitutions in para 5f, 5b and 5c
showed increased activity as compared to 5a, but not to the same
extent as substitution in meta-position in the 5e compound.
Next, compound 5e was screened against a multi-drug resistant
M. tuberculosis LAM-1 isolate which has been spolygotyped and
Table 2
Bioactivity and drug-like characteristics of benzo[j]phenanthridine-7,12-diones 5aeh.
Compd
R1
R2
R3
MIC50 (
m
M)a
MIC99 (
m
M)b
Toxicity IC50 (
m
M)c
SId
1.26
PSA (Å)e
MiLogPf
5a
5b
5c
5d
5e
5f
H
H
H
Me
H
H
OMe
H
H
H
H
H
Me
H
H
H
Cl
F
H
H
Me
H
1.20
1.03
0.74
3.68
0.21
0.88
1.66
0.43
0.54
ND
1.90
1.35
1.38
4.30
0.64
1.53
2.74
1.51
1.56
1.09
3.96
3.21
1.48
2.4
0.95
ND
0.222 nM
47.03
47.03
47.03
47.03
47.03
47.03
56.27
56.27
ND
4.10
4.75
3.27
4.50
4.52
4.52
4.11
4.13
ND
1.50
1.46
1.08
15.29
1.69
1.45
2.22
ND
5g
5h
INH
SDS
H
OMe
ND
ND
ND
a
MIC50 minimal concentration that inhibits the growth by 50% of M.tb. H37Rv determined by luminescent assay.
MIC99 minimal concentration that inhibits the growth by 99% of M.tb. H37Rv determined by BACTEC 460-TB.
Concentration showing 50% neutral red uptake of C3A hepatocytes.
SI, selectivity index: ratio of cytotoxicity to in vitro activity against H37Rv (IC50/M.tb. MIC50).
Polar surface area (PSA) calculated using www.molinspiration.com.
b
c
d
e
f
MiLogP calculated using www.molinspiration.com.

D. Cappoen et al. / European Journal of Medicinal Chemistry 48 (2012) 57e68
61
Table 3
VITOTOXÔ results benzo[j]phenanthridine-7,12-diones 5aeh.
Compd
Lowest concentration
showing Genotoxicitya
Lowest concentration
showing Cytotoxicitya
ꢀS9b
þS9c
ꢀS9b
þS9c
5a
5b
5c
5d
5e
5f
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
1
1
1
10
10
10
10
10
5g
5h
a
Concentrations tested were 10 mM, 1 mM and 0.1 mM.
Effects observed in absence of post-mitochondrial supernatant (S9).
Effects observed after addition of post-mitochondrial supernatant (S9).
b
c
genes in the presence or absence of S9 in the S. typhimurium strain.
It was interesting to observe that addition of the solution of
compounds 5 in the absence of S9 did not cause mutagenic effects
and only upon addition of S9, cytotoxicity of the compounds was
observed in case of the S. typhimurium carrying an episomal lux
operon. Concentrations at which toxicity occurs, shown in Table 3
are similar to those found in the neutral red uptake assay. It
seems that only when the compounds are processed by the cell to
reactive electrophilic metabolites they become active and enable to
react with various targets.
Genomic DNA damage such as DNA breakage or fragmentation
caused by the benzo[j]phenanthridine-7,12-diones could be ruled
out by the Comet assay [28]. All compounds were tested at 3
mM,
1
m
M, 0.3 M and 0.1 M, but none of the concentrations caused
m
m
increased DNA fragmentation. The results obtained for compound
5e are shown in Table 4. The percentage of DNA observed in the tail
did not exceed 4% and showed no significant increase (Man-
neWhitney U test, P > 0.05) as compared to the negative control.
Significantly lower DNA damage than in the control sample was
Fig. 3. Macrophage infection assay and neutral red uptake assay for compound 5e. (a)
J774 macrophage cell cultures after 7 days of exposure to the different concentrations
observed at 1 mM concentration, possibly as a result of DNA stabi-
of 5e. (b) Neutral red uptake assay to test macrophage viability with -B- 0
-*- ¼ 0.3 M, - - 1 M, -:-3 M. (c) Macrophage infection assay expressed in relative
light units measured in the lysate of infected macrophages cultures at different
mM,
lization by intercalation of 5e into the genome. This stabilization of
the compounds would decrease the fragmentation and DNA
migration under electrophoresis could be slowed down. Further
elucidation of this effect is needed to confirm this hypothesis.
As M.tb. is an intracellular pathogen, we also examined whether
the most promising derivative 5e was able to reach and kill the
bacilli residing inside J774 macrophages [29]. In this assay we
infected a murine J774.A1 macrophage cell line with luminescent
M.tb. H37Rv at a multiplicity of infection (MOI) of 0.1. After infec-
tion, the infected adherent cell layer was treated with a series of
m
m
m
;
concentrations of 5e. The values obtained for 10 mM were regarded as unreliable due
the high cytotoxicity and are not shown.
and seven days, the cell monolayer was lysed and the number of
intracellular bacteria was determined by luminometry of the lysate.
A neutral red dye uptake (RNU) assay was performed in parallel, to
ascertain that the reduction in luminescent signal was related to
the specific antimicrobial activity of 5e against M.tb. and not to
cytotoxicity. Uninfected J774 macrophages were treated with the
compound and their viability was measured by the NRU assay at
dilutions (10 mM, 3 mM, 1 mM, 0.3 mM and 0.1 mM) of the 5e deriv-
ative. Streptomycin was used as positive control. After one, three
Table 4
Comet assay using 3-methylbenzo[j]phenanthridine-7,12-dione 5e.
0
mM
0.1
mM
0.3
m
M
1
m
M
3
m
M
EMS 0.7 mM
% DNA
# cells
SD
Median
P-Level
3.69
104
6.7
3.47
73
5.44
0.23
2.52
31
4.12
0
0.16709
2.15
147
3.8
3.31
62
4.67
0.98
62.34
49
11.86
65.83
1.12
0
0.434663
0.00509
0.744523
Nucleus from lysed C3A cells, pre-incubated for 24 h in compound 5e, immobilized and stained with gel red.

62
D. Cappoen et al. / European Journal of Medicinal Chemistry 48 (2012) 57e68
the same time points used for testing effects on bacterial replica-
4. Experimental section
tion. Compound 5e could successfully inhibit intracellular myco-
bacterial growth (Fig. 3). At a concentration of 3
the macrophages dropped to 6.5% on day 7, so the luminometric
results obtained for 10 M and 3 M were regarded as unreliable. At
M however, 92.9% of the macrophages were viable and at this
concentration a 83.5% reduction in intracellular mycobacterial
growth was detected. At the same concentration of 1 M, the
inhibition after 1 day and 3 days of exposure was respectively 47.6%
and 74.2%. At a concentration of 0.3 M of compound 5e no intra-
m
M, the viability of
4.1. General experimental methods
m
m
4.1.1. Biological data
1
m
4.1.1.1. Monitoring mycobacterial growth by luminometry. The
minimal inhibitory concentration (MIC) against M.tb. H37Rv of all
synthesized compounds was evaluated by testing serial dilutions.
The in vitro assay was based on a method in which luminescent
M.tb. H37Rv transformed with pSMT1 luciferase reporter plasmid is
used [30]. The compounds were solubilized in DMSO (Sigma-
eAldrich) at stock concentrations of 1 mM. Serial dilutions of each
compound were made in liquid 7H9 medium [Middlebrook 7H9
m
m
cellular growth inhibition could be measured.
Because the NRU assay relies on the uptake and exclusion of
a neutral red dye, the NRU assay in parallel indicated not only that
the J774 macrophages where viable but more importantly, that the
cell membrane was fully intact and functional. By showing that the
5e derivate could lower the bacterial burden inside the cells, it was
shown that this compound can cross the natural barrier made up by
the cell membrane while leaving it intact and reach the bacteria
residing in the cytosol or vacuole of the cell. Because the reported
toxicity of the compound class it is a favorable that the compound
has a fast antimicrobial effect within the cells. This was shown for
analog 5e and activity was already noticeable after 24 h of treat-
ment of the infected macrophages.
broth based (Difco)] þ 10% FCS (Gibco). Volumes of 20
mL of the
serial dilutions were added in triplicate to 96 well, flat-bottomed
microwell plates. The bacterial suspension was made by thawing
and diluting a frozen M.tb. H37Rv aliquot in 7H9-10% FCS. The
diluted bacteria were passed through a 5.0 mM filter (Millipore) to
eliminate clumps and left for 1 h to recover at 37 ꢁC, 5% CO2. Next,
the bacterial suspension was diluted in 7H9-10% FCS to obtain
50,000 Relative Light Units (RLU)/ml (corresponding to
5 ꢂ 104 CFU) and a volume of 180
m
L of bacteria was added to each
well. The final concentration of the compounds was 10
m
M, 3 M,
m
1 mM, 0.3 mM and 0.1 mM. Bacterial replication were analyzed by
luminometry after 1, 3 and 7 days of culture. The bacterial
suspension from each well was collected, and brought in a 2.5 ml
3. Conclusion
Eppendorf tube. Each well was washed four times with 200
mL PBS
In conclusion, an efficient intramolecular palladium(0)-
catalyzed cyclization of tetracyclic benzo[j]phenanthridine-7,12-
diones 5aeh from N-methanesulfonyl-2-(arylamino)methyl-3-
bromo-1,4-naphthoquinones 6aeh was achieved. All derivatives
showed antimicrobial activities against M.tb. However, due to their
high cytotoxicity for C3A hepatocytes, the resulting selectivity
index of the tested derivatives was unacceptably low for most of
them. Nevertheless, 3-methylbenzo[j]phenanthridine-7,12-dione
5e showed a SI of 15.3 and a MIC50 value lower than the first line
drug isoniazid. Although the 5e SI is still too low for immediate
application, our results showing the importance of the place and
nature of substitution encourages the further exploration and
synthesis of new derivatives with other side chains at the same
position as 5e. Although the calculated MiLogP value is 4.52 for the
5e component, it may be possible to increase the efficiency of this
compound by further increasing its lipophility. Because M.tb. is
localized initially inside infected alveolar macrophages, it was
important that these compounds would also be able to affect the
intracellular growth of the bacilli, and this was confirmed for
compound 5e, which demonstrated a fast bacteriostatic effect
(Difco). To measure the luminescence, 100 L of 1% n-decanal in
m
ethanol (SigmaeAldrich) was added to the Eppendorf tube and
light emission was measured over 10 s using a Turner Modulus
Single Tube Luminometer from Biosystems.
4.1.1.2. Monitoring M.tb. growth by BACTEC 460-TB. Antimicrobial
effects of the compounds were also monitored using BACTEC 460-
TB cultures. The compounds were solubilized in DMSO (Sigma-
eAldrich) at stock concentrations of 1 mM. Serial dilutions of each
compound were made in 7H9 þ 10% FCS, at 40 times the final
concentrations. A bacterial pre-culture of M.tb. H37Rv was made in
a 4 ml BACTEC vial containing 7H9-based medium until a Growth
index (GI) of 300 was reached. When the pre-culture was fully
grown, 100
mL of bacteria were inoculated into a new 4 ml BACTEC
vial together with 100
mL of the serial dilutions of the compounds.
Each day the GI was measured and compared to untreated inocula.
Each measurement was repeated at least twice.
4.1.1.3. Inhibition of intracellular M.tb. growth. Inhibition of intra-
cellular M.tb. growth was measured in a macrophage infection
assay. The efficiency of the compounds was tested in the murine
J774 cells infected with luminescent M.tb. H37Rv transformed with
pSMT1 luciferase reporter plasmid. The J774 macrophages were
grown at 37 ꢁC 5% CO2 in complete DMEM medium [DMEM
(Gibco) þ 10% FCS (Gibco) þ 1% Non-essential amino acids
against M.tb. growing in macrophages at a 1
mM concentration.
Benzo[j]phenanthridine-7,12-diones are a new family of anti-
tuberculosis candidates and their mechanism of action is novel
and unknown. We showed the antimicrobial effect against a MDR
clinical isolate of M.tb. LAM-1. The MIC99 against this MDR isolate
was similar to the MIC99 against the susceptible M. tuberculosis
H37Rv laboratory strain, confirming the hypothesis of a novel
mechanism of action. Besides more rapid diagnosis and more
effective vaccines, new drugs for anti-tuberculous therapy are
urgently needed to control tuberculosis. Testing the activity of our
molecules against dormant M.tb. as well as testing their potency
against other mycobacterial species are the next steps taken by our
research teams. Creation of bifunctional molecules, e.g., by fixing an
isoniazid moiety as a sidechain on benzo[j]phenanthridine-7,12-
diones might also increase their efficiency. Further optimization
studies and development of new derivatives of benzo[j]phenan-
thridine-7,12-diones as well as investigating the mode of action are
both ongoing projects.
(Gibco)
þ
1% Sodium Pyruvate (Gibco)
þ
1% Glutamax
(Gibco) þ 0.1% 2-mercaptoethanol 5 ꢂ 10ꢀ5 M (Gibco) þ 1% gen-
tamycin (Gibco)] until a semi confluent layer was formed. The
macrophages were washed and seeded in a 96 well flat-bottomed
microwell plate at a cell density of 100,000 cells per well. The
cells were left to recover overnight and after recovery washed three
times. M.tb. H37Rv was grown at 37 ꢁC in 7H9 þ 10% FCS þ 0.2%
hygromycin (Roche) to an O. D between 0.6 and 1.0. The fully grown
bacterial suspension was measured and brought into complete
DMEMPen/Fung [DMEM medium þ 0.1% penicillin (Roche) þ 0.8%
Fungizone (Gibco) without gentamycin]. Compounds 5 were solu-
bilized in DMSO at stock concentrations of 1 mM. Serial dilutions of
each compound were made in DMEMPen/Fung in which 2 times

D. Cappoen et al. / European Journal of Medicinal Chemistry 48 (2012) 57e68
63
the final concentration of each compound was prepared. A volume
of 100 L of the bacterial suspension in DMEMPen/Fung containing
10,000 RLU (corresponding to 104 CFU) of bacteria (multiplicity of
infection of 0.1.) and 100 L of the serial compound dilutions was
added to the macrophage cultures, resulting in final concentrations
of 10 M, 3 M, 1 M, 0.3 M, 0.1 M All tests were performed in
triplicate.
the nuclei (comet head þ tail) by appropriate imager software from
m
Metasystems Altlussheim Germany.
m
4.1.2. Chemical data
Spectroscopic data were recorded as follows: 1H NMR spectra
were recorded at 300 MHz, 13C NMR spectra were recorded at
75 MHz and 19F NMR spectra were recorded at 282 MHz using a Jeol
NMR spectrometer. Peak assignments were performed with the aid
of 2D-COSY and HSQC spectra. Melting points (mp) were deter-
mined on a Büchi B540 Melting Point Apparatus with a tempera-
ture gradient of 1 ꢁC/min. The reported melting points are not
corrected. Infrared spectra were recorded with a Perkin Elmer
Spectrum BX FT-IR apparatus using the attenuated total reflection
(ATR) technology. Mass spectra were recorded using a direct inlet
system (70 eV) with a VL detector (ES, 4000 V). Elemental analyses
were analyzed with Perkin Elmer Series II CHNS/O Analyzer 2400.
All tested compounds were found to possess a purity of at least 95%.
Flash chromatography was carried out using a glass column with
silica gel (particle size 0.035e0.07 mm, pore diameter ca. 6 nm).
Solvent systems were determined via initial TLC analysis.
m
m
m
m
m
To measure the effect of the compounds on intracellular growth
of M.tb., the infected macrophages were washed to eliminate
extracellular bacteria and lysed on day 1, day 3 or day 7 with 200
1% Triton X-100 (SigmaeAldrich) and the wells washed four times
with 200 L PBS. The lysate was transferred in a 2.5 ml Eppendorf
tube together with the 4 PBS washings of 200 L. A volume of 100
mL
m
m
mL
of 1% n-decanal in ethanol was added to the Eppendorf tube and
emission of luminescence was measured over a time span of 10 s
with a Turner Modulus Single Tube Luminometer from Biosystems.
Mean RLU values obtained from triplicate cultures are shown. Cell
viability of uninfected J774 cells cultured in the presence of the
various compounds was examined by neutral red uptake assay.
4.1.1.4. Assessment of cytotoxicity. The 50% inhibitory concentration
(IC50) toward C3A human hepatocytes was determined for the 8
derivatives 5 by a neutral red uptake assay as described before [26].
The C3A cells were grown in DMEM þ 10% FCS until a semi
confluent layer of cells was obtained. The cells were trypsinized,
washed and 40,000 cells were seeded per well of a 96 well plate
and left for recovery at 37 ꢁC, 5% CO2. The following days, the
compounds were solubilized in DMSO (SigmaeAldrich) to stock
concentrations of 1 mM. A serial dilution of each compound was
made in DMEM þ 10% FCS to achieve the final concentrations
4.2. Synthesis of 2-(arylamino)methyl-3-bromo-1,4-
dimethoxynaphthalenes 8
4.2.1. General procedure
To a stirred solution of an aniline (4 mmol), which was distilled
previously, in 10 ml of ethanol was added dropwise a solution of 2-
bromo-3-bromomethyl-1,4-dimethoxynaphthalene 7 [23] (720 mg,
2 mmol) in 20 ml of ethanol. The reaction mixture was stirred
overnight at room temperature. Most of the solvent was evaporated
in vacuo and the residue was dissolved in dichloromethane (100 ml),
washed twice with water, and dried (MgSO4). Flash chromatography
on silica gel using dichloromethane as eluent gave 2-(arylamino)
methyl-3-bromo-1,4-dimethoxynaphthalenes 8, which were
further purified by crystallization in diethyl ether through dropwise
addition of petroleum ether.
(10 mM, 3 mM, 1 mM, 0.3 mM, 0.1 mM). The C3A cells were washed and
exposed to the derivatives by adding the serial dilutions of the
compounds to the wells. The plates were left for incubation at 37 ꢁC,
5% CO2 for 24 h. After exposure, the cells were washed with 200
PBS and 200 L neutral red working solution (Sigma) was added per
well. Subsequently the plates were incubated for 3 h at 37 ꢁC, 5%
CO2. The wells were washed with 200 L PBS and 200 L of an
mL
m
m
m
ethanol/acetic acid (50%) mixture. The plates were left on the
shaker until the color became homogeneous purple and the optical
density was measured at 530 nm (NR max) and 620 nM (reference
wavelength) with the Paradigm detection platform.
4.2.1.1. 3-Bromo-1,4-dimethoxy-2-(phenylamino)methylnaphthalene
8a. Yield 80%, white crystals, mp 98.2e99.5 ꢁC. 1H NMR (CDCl3):
d
3.98 (3H, s, OMe), 3.99 (3H, s, OMe), 4.63 (2H, s, CH2), 4.01 (1H, br.
s, NH), 6.66e6.78 (2H, m, 2ꢂ]CH), 6.82 (1H, d, J ¼ 8.3 Hz, C-20 or C-
60), 7.13e7.26 (2H, m, 2ꢂ]CH), 7.54e7.60 (2H, m, H-6 and H-7),
4.1.1.5. DNA breakage. Possible DNA breakage effects of the deriv-
atives on C3A cells were investigated by the alkaline comet assay
[28]. The C3A cells were grown in DMEM þ 10% FCS until a semi
confluent layer of cells was obtained. The cells were trypsinized,
washed and seeded at 100,000 cells per well of a 24 well plate and
left for recovery at 37 ꢁC, 5% CO2. The following day, compounds 5
were dissolved in DMSO as a stock concentration of 1 mM. Serial
dilutions of each compound were made in DMEM þ 10% FCS to
8.08e8.14 (2H, m, H-5 and H-8). 13C NMR (CDCl3):
d 43.12 (CH2),
61.47 (OMe), 63.68 (OMe), 113.37 (2ꢂ]CH), 115.12 (]Cquat), 117.85
(]CH),122.56 and 122.97 (C-5 and C-8),126.89 and 127.24 (C-6 and
C-7), 127.41 (]Cquat), 127.94 (]Cquat), 128.80 (]Cquat), 129.26
(2ꢂ]CH), 147.99 (C-10), 150.36 (]CeO), 151.90 (]CeO). IR (ATR):
nmax 3420,1601, 1514,1354, 1262, 1078, 1069, 1002 cm-1. MS m/z (%):
372/374 (M þ Hþ, 100). Anal. Calcd for C19H18BrNO2: C 61.30%, H
4.87%, N 3.76%. Found: C 61.69%, H 5.012%, N 3.55%.
obtain the final concentrations (10 mM, 3 mM, 1 mM, 0.3 mM, 0.1 mM).
The C3A cells were washed and exposed to the derivatives by adding
1 ml of the serial dilutions to each well. The plates were incubated at
37 ꢁC, 5% CO2 for 24 h. After incubation, the cells were trypsinized,
4.2.1.2. 3-Bromo-2-(4-chlorophenylamino)methyl-1,4-dimethoxynaph-
thalene 8b. Yield 73%, white crystals, mp 134.8e135.3 ꢁC. 1H NMR
(CDCl3):
d 3.96 (3H, s, OMe), 3.97 (3H, s, OMe), 4.20 (1H, br. s, NH),
washed with PBS, and 10
mL of cell suspension was dissolved in
4.59 (2H, s, CH2), 6.73 (2H, d, J ¼ 8.8 Hz, H-30 and H-50), 7.13 (2H, d,
300 L low melting point agarose. The dissolved cell suspensionwas
m
J ¼ 8.8 Hz, H-20 and H-60), 7.53e7.60 (2H, m, H-6 and H-7),
then placed onto a frosted microscope slide and left on ice for 5 min.
The slide was subsequently placed in a jar containing lysing solution
for 1 h. After lysis, the agarose cell suspension was subjected to
electrophoresis for 20 min at 300 mA. The slides were washed with
neutralization buffer for 5 min and dried in ice cold ethanol for
10 min. Staining of the DNA was done with gel red (SigmaeAldrich).
For the quantification of the DNA migration a fluorescence micro-
scope was used and the percentage DNA in the comet tail of the cells
nuclei core was calculated in proportion to the total DNA present in
8.07e8.12 (2H, m, H-5 and H-8). 13C NMR (CDCl3):
d 43.15 (CH2),
61.48 (OMe), 63.67 (OMe), 114.43 (C-30 and C-50), 115.55 (]Cquat),
122.39 (]Cquat),122.60 and 122.97 (C-5 and C-8),126.98 and 127.33
(C-6 and C-7), 126.98 (]Cquat), 127.88 (]Cquat), 128.88 (]Cquat),
129.04 (C-20 and C-60), 146.43 (C-10), 150.45 (]CeO), 151.88 (]
CeO). IR (ATR): nmax 3397, 1599, 1577, 1570, 1497, 1352, 1080,
1002 cm-1. MS m/z (%): 406/408/410 (M þ Hþ, 20), 279/281 (100).
Anal. Calcd for C19H17BrClNO2: C 56.11%, H 4.21% N 3.44%. Found: C
56.03%, H 4.30% N 3.42%.

64
D. Cappoen et al. / European Journal of Medicinal Chemistry 48 (2012) 57e68
4.2.1.3. 3-Bromo-1,4-dimethoxy-2-(4-fluorophenylamino)methyl-
naphthalene 8c. Yield 74%, white crystals, mp 135.7e136.5 ꢁC. 1H
6.75e6.77 (1H, m, ]CH), 6.89e6.98 (1H, m, ]CH), 7.51e7.56 (2H, m,
H-6 and H-7), 8.07e8.12 (2H, m, H-5 and H-8). 13C NMR (CDCl3):
NMR (CDCl3):
d
3.97 (3H, s, OMe), 3.98 (3H, s, OMe), 4.07 (1H, br. s,
d 42.96 (CH2), 55.65 (OMe), 61.52 (OMe), 63.80 (OMe),109.76 (]CH),
110.60 (]CH), 116.18 (]Cquat), 116.93 (]CH), 121.48 (]CH), 122.64
and 123.12 (C-5 and C-8),126.89 and 127.25 (C-6 and C-7),127.79 (]
NH), 4.58 (2H, s, CH2), 6.72e6.76 (2H, m, H-30 and H-50), 6.87e6.93
(2H, m, H-20 and H-60), 7.53e7.59 (2H, m, H-6 and H-7), 8.08e8.11
(2H, m, H-5 and H-8). 13C NMR (CDCl3):
d
43.79 (CH2), 61.47 (OMe),
Cquat), 128.11 (]Cquat), 128.95 (]Cquat), 138.21 (]Cquat), 147.30 (C-
63.65 (OMe), 114.28 and 114.37 (C-30 and C-50), 115.49 and 115.78
(C-20 and C-60), 115.64 (]Cquat), 122.59 and 122.97 (C-5 and C-8),
126.95 and 127.27 (C-6 and C-7), 127.27 (]Cquat), 127.91 (]Cquat),
128.83 (]Cquat), 144.31 (C-10), 150.41 (]CeO), 151.89 (]CeO),
10), 150.46 (]CeO), 152.11 (]CeO). IR (ATR): nmax 3416, 1600, 1510,
1356, 1220, 1083, 1024, 1000 cm-1. MS m/z (%): 402/404 (M þ Hþ,
100). Anal. Calcd for C20H20BrNO3: C 59.71%, H 5.01%, N 3.48%.
Found: C 60.00%, H 5.27%, N 3.44%.
156.12 (d, J ¼ 235.4 Hz, C-40). 19F NMR (CDCl3):
ꢀ127.42. IR (ATR):
d
nmax 3391, 1509, 1354, 1213, 1081, 1004 cm-1. MS m/z (%): 390/392
(M þ Hþ, 60), 279/281 (100). Anal. Calcd for C19H17BrFNO2: C
58.48%, H 4.39%, N 3.59%. Found: C 58.54%, H 4.52%, N 3.63%.
4.2.1.8. 3-Bromo-1,4-dimethoxy-2-(4-methoxyphenylamino)methyl-
naphthalene 8h. Yield 47%, white crystals, mp 139.5e140.1 ꢁC. 1H
NMR (CDCl3):
d 3.73 (3H, s, OMe), 3.91 (1H, br. s, NH), 3.97 (6H, s,
2 ꢂ OMe), 4.57 (2H, s, CH2), 6.78e6.81 (4H, m, 4ꢂ]CH), 7.52e7.57
4.2.1.4. 3-Bromo-1,4-dimethoxy-2-(2-methylphenylamino)methyl-
(2H, m, H-6 and H-7), 8.07e8.12 (2H, m, H-5 and H-8). 13C NMR
naphthalene 8d. Yield 34%, white crystals, mp 98.0e99.0 ꢁC. 1H
(CDCl3): d 44.32 (CH2), 55.88 (OMe), 61.55 (OMe), 63.74 (OMe),
NMR (CDCl3):
d
2.12 (3H, s, Me), 3.98 (3H, s, OMe), 3.99 (3H, s, OMe),
114.96 (4ꢂ]CH), 115.91 (]Cquat), 122.66 and 123.07 (C-5 and C-8),
126.96 and 127.28 (C-6 and C-7), 127.77 (]Cquat), 128.06 (]Cquat),
128.88 (]Cquat), 142.37 (C-10), 150.44 (]Cquat), 151.99 (]Cquat),
152.61 (]Cquat). IR (ATR): nmax 3397, 1513, 1352, 1233, 1080, 1036,
1004 cm-1. MS m/z (%): 402/404 (M þ Hþ, 100). Anal. Calcd for
4.00 (1H, br. s, NH), 4.66 (2H, s, CH2), 6.66e6.71 (1H, m, ]CH), 6.97
(1H, d, J ¼ 8.3 Hz, ]CH), 7.05 (1H, d, J ¼ 7.7 Hz, ]CH), 7.16e7.21 (1H,
m, ]CH), 7.52e7.58 (2H, m, H-6 and H-7), 8.09e8.13 (2H, m, H-5
and H-8). 13C NMR (CDCl3):
d 17.61 (Me), 43.32 (CH2), 61.55 (OMe),
63.80 (OMe), 110.62 (]CH), 115.94 (]Cquat), 117.53 (¼CH), 122.67
and 123.13 (C-5 and C-8), 122.67 (]Cquat), 126.99 (C-6 or C-7),
127.33 (]CH and C-6 or C-7), 127.66 (]Cquat), 128.14 (]Cquat),
128.98 (]Cquat), 130.17 (¼CH), 146.22 (C-10), 150.54 (]CeO), 152.16
(]CeO). IR (ATR): nmax 3429, 1513, 1449, 1356, 1257, 1247, 1082 cm-
1. MS m/z (%): 386/388 (M þ Hþ, 100), 279/281 (10). Anal. Calcd for
C20H20BrNO3: C 59.71%, H 5.01%, N 3.48%. Found: C 60.09%, H 5.35%,
N 3.38%.
4.2.1.9. 3-Bromo-1,4-dimethoxy-2-(2,4-dimethoxyphenylamino)
methylnaphthalene 8i. Yield 32%, red crystals, mp 123.0e123.6 ꢁC.
1H NMR (CDCl3):
d 3.75 (3H, s, OMe), 3.77 (3H, s, OMe), 3.97 (3H, s,
C
20H20BrNO2: C 62.19%, H 5.22%, N 3.63%. Found: C 62.48%, H 5.36%,
OMe), 3.98 (3H, s, OMe), 4.58 (2H, s, CH2), 6.45 (1H, s, H-30),
6.44e6.49 (1H, m, H-50), 6.88 (1H, d, J ¼ 8.3 Hz, H-60), 7.51e7.57 (2H,
m, H-6 and H-7), 8.07e8.12 (2H, m, H-5 and H-8). 13C NMR (CDCl3):
N 3.57%.
4.2.1.5. 3-Bromo-1,4-dimethoxy-2-(3-methylphenylamino)methyl-
d 43.77 (CH2), 55.66 (OMe), 55.89 (OMe), 61.49 (OMe), 63.77 (OMe),
naphthalene 8e. Yield 83% white crystals, mp 104.5e105.5 ꢁC. 1H
99.45 (C-30), 104.03 (C-50), 111.14 (C-60), 116.18 (]Cquat), 122.63 and
123.09 (C-5 and C-8), 126.84 and 127.19 (C-6 and C-7), 128.00 (]
NMR (CDCl3): d 2.30 (3H, s, Me), 3.97 (3H, s, OMe), 3.98 (3H, s, OMe),
4.11 (1H, br. s, NH), 4.61 (2H, s, CH2), 6.57 (1H, d, J ¼ 7.7 Hz, ]CH),
6.64e6.65 (2H, m, 2ꢂ]CH), 7.07e7.14 (1H, m, ]CH), 7.53e7.59
(2H, m, H6 and H-7), 8.10 (2H, ddd, J ¼ 1.7, 3.0, 6.9 Hz, H-5 and H-8).
C
C
quat), 128.09 (]Cquat), 128.89 (]Cquat), 132.57 (]Cquat), 148.49 (]
quat), 150.40 (]Cquat), 152.05 (]Cquat), 152.23 (]Cquat). IR (ATR):
nmax 3412, 1514, 1358, 1201, 1084, 1031, 999 cmꢀ1. MS m/z (%): 432/
444 (M þ Hþ, 100). Anal. Calcd for C21H22BrNO4: C 58.34%, H 5.13%,
N 3.24%. Found: C 58.19%, H 5.32%, N 3.18%.
13C NMR (CDCl3):
d 21.63 (Me), 43.24 (CH2), 61.44 (OMe), 63.64
(OMe), 110.50 (]CH), 114.27 (]CH), 115.82 (]Cquat), 118.87 (]CH),
122.55 and 122.97 (C-5 and C-8), 126.87 and 127.22 (C-6 and C-7),
127.48 (]Cquat), 128.81 (]Cquat), 129.13 (]CH and ]Cquat), 139.00
(]Cquat), 148.01 (C-10), 150.34 (]CeO), 151.95 (]CeO). IR (ATR):
nmax 3426, 1605, 1355, 1077, 1001 cm-1. MS m/z (%):386/388
(M þ Hþ, 100), 279/281 (7). Anal. Calcd for C20H20BrNO2: C 62.19%,
H 5.22%, N 3.63%. Found: C 62.48%, H 5.55%, N 3.42%.
4.3. Synthesis of N-methanesulfonyl-2-(arylamino)methyl-3-bromo-
1,4-dimethoxynaphthalenes 9
4.3.1. General procedure
To a stirred solution of a 2-(arylamino)methyl-3-bromo-1,4-
dimethoxynaphthalene 8 (1 mmol) in 10 ml of pyridine was
added dropwise a solution of methanesulfonyl chloride (1.3 mmol)
in 10 ml of dichloromethane. The reaction mixture was boiled
under reflux for 12 h and then poured in water. The reaction
mixture was extracted with dichloromethane and the combined
organic extracts were washed twice with water and dried (MgSO4).
Flash chromatography on silica gel using dichloromethane as
eluent gave N-methanesulfonyl-2-(arylamino)methyl-3-bromo-
1,4-dimethoxynaphthalenes 9, which were further purified by
recrystallization in from methanol. For some derivatives, the N-
methanesulfonyl-2-(arylamino)methyl-3-bromo-1,4-
4.2.1.6. 3-Bromo-1,4-dimethoxy-2-(4-methylphenylamino)methyl-
naphthalene 8f. Yield 57%, white crystals, mp 128.2e129.8 ꢁC. 1H
NMR (CDCl3): d 2.24 (3H, s, Me), 3.97 (3H, s, OMe), 3.98 (3H, s, OMe),
4.00 (1H, br. s, NH), 4.60 (2H, s, CH2), 6.74 (2H, d, J ¼ 8.6 Hz, H-30 and
H-50), 7.02 (2H, d, J ¼ 8.6 Hz, H-20 and H-60), 7.52e7.58 (2H, m, H-6
and H-7), 8.07e8.12 (2H, m, H-5 and H-8). 13C NMR (CDCl3):
d 20.42
(Me), 43.50 (CH2), 61.41 (OMe), 63.63 (OMe), 113.57 (C-30 and C-50),
115.81 (]Cquat), 122.53 and 122.96 (C-5 and C-8), 126.83 and 127.16
(C-6 and C-7), 127.06 (]Cquat), 127.60 (]Cquat), 127.95 (]Cquat),
128.76 (]Cquat), 129.72 (C-20 and C-60), 145.74 (C-10), 150.31 (]
CeO), 151.89 (]CeO). IR (ATR): nmax 3419, 1611, 1522, 1354, 1253,
1071, 995 cmꢀ1. MS m/z (%): 386/388 (M þ Hþ, 100). Anal. Calcd for
dimethoxynaphthalenes 9 could be obtained pure directly from the
reaction crude by recrystallization from methanol.
C
20H20BrNO2: C 62.19%, H 5.22%, N 3.63%. Found: C 61.98%, H 5.30%,
N 3.66%.
4.3.1.1. N-methanesulfonyl-3-bromo-1,4-dimethoxy-2-(phenylamino)
methylnaphthalene 9a. Yield 62%, light-brown crystals, mp
4.2.1.7. 3-Bromo-1,4-dimethoxy-2-(2-methoxyphenylamino)methyl-
156.0e157.4 ꢁC. 1H NMR (CDCl3):
d 3.06 (3H, s, MeSO2), 3.85 (3H, s,
naphthalene 8g. Yield 44%, white crystals, mp 128.4e129.1 ꢁC. 1H
OMe), 3.95 (3H, s, OMe), 5.25 (2H, s, CH2), 7.15e7.17 (5H, m, 5ꢂ]
NMR(CDCl3):
d 3.79(3H, s, OMe), 3.97 (3H, s, OMe), 3.98(3H, s, OMe),
CH), 7.51e7.57 (2H, m, H-6 and H-7), 7.98e8.07 (2H, m, H-5 and H-
4.63 (2H, s, CH2), 4.66 (1H, br.s, NH), 6.65e6.71 (1H, m, ]CH),
8). 13C NMR (CDCl3):
d 38.31 (MeSO2), 48.66 (CH2), 61.30 (OMe),

D. Cappoen et al. / European Journal of Medicinal Chemistry 48 (2012) 57e68
65
63.16 (OMe), 116.19 (]Cquat), 122.63 and 122.95 (C-5 and C-8),
124.33 (]Cquat), 126.80 and 127.49 (C-6 and C-7), 128.35 (]CH),
128.85 (2ꢂ]CH), 129.05 (]Cquat), 129.98 (2ꢂ]CH and ]Cquat),
137.76 (C-10), 150.16 (]CeO), 152.88 (]CeO). IR (ATR): nmax 1360,
1347,1330,11316,1302,1148,1086,1076,1002 cm-1. MS m/z (%): 472/
474 (M þ Na, 2), 279/281 (M-[N(SO2Me)C6H5], 100). Anal. Calcd for
and H-7), 7.98e8.07 (2H, m, H-5 and H-8). 13C NMR (CDCl3):
d
21.07
(Me), 38.24 (MeSO2), 48.67 (CH2), 61.25 (OMe), 63.13 (OMe), 116.19
(]Cquat), 122.60 and 125.95 (C-5 and C-8), 124.43 (]Cquat), 126.63
and 127.44 (H-6 and H-7), 126.76 (]CH), 127.57 (]Cquat), 128.48
(¼CH), 129.05 (]CH and ]Cquat), 130.56 (]CH), 137.72 (]Cquat),
138.67 (]Cquat), 150.16 (]CeO), 152.93 (]CeO). IR (ATR): nmax
1578, 1569, 1491, 1452, 1358, 1326, 1284, 1258, 1168, 1148, 1086,
1070, 1003 cm-1. MS m/z (%): 486/488 (M þ Na, 3), 279/281 (M-
[N(SO2Me)C6H4Me], 100). Anal. Calcd for C21H22BrNO4S: C 54.32%,
H 4.78%, N 3.02%. Found: C 54.40%, H 4.81%, N 2.99%.
C
20H20BrNO4S: C 53.34%, H 4.48%, N 3.11%. Found: C 53.60%, H 4.67%,
N 3.02%.
4.3.1.2. N-methanesulfonyl-3-bromo-2-(4-chlororophenylamino)
methyl-1,4-dimethoxynaphthalene 9b. Yield 83%, yellow crystals,
mp 176.6e178.4 ꢁC. 1H NMR (CDCl3):
d
3.04 (3H, s, MeSO2), 3.87
4.3.1.6. N-methanesulfonyl-3-bromo-1,4-dimethoxy-2-(4-methylph-
(3H, s, OMe), 3.95 (3H, s, OMe), 5.22 (2H, s, CH2), 7.12 (4H, br. s, 4ꢂ]
enylamino)methylnaphthalene 9f. Yield 76%, light-yellow crystals,
CH), 7.55 (2H, ddd, J ¼ 3.7, 6.6, 6.6 Hz, H-6 and H-7), 7.98e8.08 (2H,
mp 134.4e134.8 ꢁC. 1H NMR (CDCl3):
d 2.19 (3H, s, Me), 3.04 (3H, s,
m, H-5 and H-8). 13C NMR (CDCl3):
d
38.27 (MeSO2), 48.54 (CH2),
MeSO2), 3.86 (3H, s, OMe), 3.93 (3H, s, OMe), 5.23 (2H, s, CH2), 6.94
(2H, br. d, J ¼ 8.3 Hz, 2ꢂ]CH), 7.05 (2H, br. d, J ¼ 8.3 Hz, 2ꢂ]CH),
7.53 (2H, ddd, J ¼ 3.3, 6.6, 6.6 Hz, H-6 and H-7), 7.98e8.07 (2H, m,
61.33 (OMe), 63.21 (OMe), 115.82 (]Cquat), 122.69 and 122.93 (C-5
and C-8), 123.85 (]Cquat), 126.97 and 127.67 (C-6 and C-7), 127.49
(]Cquat),129.05 (2ꢂ]CH),129.15 (]Cquat), 131.11 (2ꢂ]CH), 134.22
(]Cquat), 136.34 (]Cquat), 150.34 (]CeO), 152.85 (]CeO). IR
(ATR): nmax 1359, 1337, 1159, 1150, 1080 cm-1. MS m/z (%): 506/508
(M þ Na, 9), 279/281 (M-[N(SO2Me)C6H4Cl], 100). Anal. Calcd for
H-5 and H-8). 13C NMR (CDCl3):
d 21.03 (Me), 38.18 (MeSO2), 48.59
(CH2), 61.26 (OMe), 63.13 (OMe), 116.24 (]Cquat), 122.64 and 123.01
(C-5 and C-8), 124.47 (]Cquat), 126.74 and 127.44 (C-6 and C-7),
127.56 (]Cquat), 129.05 (]Cquat), 129.51 (2ꢂ]CH), 129.64 (2ꢂ]
CH), 135.12 (]Cquat), 138.24 (]Cquat), 150.17 (]CeO), 152.90 (]
CeO). IR (ATR): nmax 1508, 1451, 1358, 1334, 1158, 1149, 1079, 1048,
1007 cm-1. MS m/z (%): 486/488 (M þ Na,1), 279/281 (M-[N(SO2Me)
C6H4Me], 100). Anal. Calcd for C21H22BrNO4S: C 54.32%, H 4.78%, N
3.02%. Found: C 54.27%, H 4.86%, N 3.05%.
C
20H19BrClNO4S: C 49.55%, H 3.95%, N 2.89%. Found: C 49.86%, H
4.23%, N 2.75%.
4.3.1.3. N-methanesulfonyl-3-bromo-1,4-dimethoxy-2-(4-fluorophe-
nylamino)methylnaphthalene 9c. Yield 91%, white crystals, mp
193.0e193.4 ꢁC. 1H NMR (CDCl3):
d 3.07 (3H, s, MeSO2), 3.86 (3H, s,
OMe), 3.95 (3H, s, OMe), 5.22 (2H, s, CH2), 6.79e6.86 (2H, m, 2ꢂ]
4.3.1.7. N-methanesulfonyl-3-bromo-1,4-dimethoxy-2-(2-methoxy-
CH), 7.09e7.16 (2H, m, 2ꢂ]CH), 7.52e7.58 (2H, m, H-6 and H-7),
phenylamino)methylnaphthalene 9g. Yield 84%, yellow crystals, mp
7.97e8.08 (2H, m, H-5 and H-8). 13C NMR (CDCl3):
d
38.31 (MeSO2),
169.8e170.1 ꢁC. 1H NMR (CDCl3):
d 3.11 (3H, s, MeSO2), 3.72 (3H, s,
48.69 (CH2), 61.32 (OMe), 63.15 (OMe), 115.61 (]CH), 115.92 (]
CH), 122.68 and 122.92 (C-5 and C-8), 124.03 (]Cquat), 126.94 and
127.64 (C-6 and C-7), 127.50 (]Cquat), 129.12 (]Cquat), 131.75
(¼CH), 131.86 (]CH), 133.63 (]Cquat), 150.28 (]CeO), 152.86 (]
OMe), 3.81 (3H, s, OMe), 3.94 (3H, s, OMe), 5.27 (2H, s, CH2), 6.62
(1H, ddd, J ¼ 1.7, 7.7, 7.7 Hz, ]CH), 6.77 (1H, br. d, J ¼ 8.3 Hz, ]CH),
6.93 (1H, dd, J ¼ 1.7, 7.7 Hz, ]CH), 7.13 (1H, ddd, J ¼ 1.7, 7.7, 8.3 Hz,
]CH), 7.48e7.54 (2H, m, H-6 and H-7), 7.98e8.05 (2H, m, H-5 and
CeO), 162.17 (d, J ¼ 248.1 Hz, C-40). 19F NMR (CDCl3):
d
ꢀ112.42. IR
H-8). 13C NMR (CDCl3):
d 40.27 (MeSO2), 47.86 (CH2), 55.34
(ATR): nmax 1506, 1361, 1341, 1322, 1208, 1159, 1149, 1082, 1050 cm-1.
MS m/z (%): 490/492 (M þ Na, 13), 279/281 (M-[N(SO2Me)C6H4F],
100). Anal. Calcd for C20H19BrFNO4S: C 51.29%, H 4.09%, N 2.99%.
Found: C 50.99%, H 4.41%, N 2.90%.
(OMe), 61.33 (OMe), 62.99 (OMe), 111.58 (¼CH), 116.61 (]Cquat),
120.57 (]CH), 122.61 and 123.09 (C-5 and C-8), 125.09 (]Cquat),
125.93 (]Cquat), 126.70 and 127.41 (C-6 and C-7), 127.71 (]Cquat),
128.99 (]Cquat), 130.11 (]CH), 133.54 (]CH), 149.93 (]Cquat),
153.30 (]CeO), 157.47 (]CeO). IR (ATR): nmax 1494, 1357, 1324,
1253,1146,1086,1070,1003 cm-1. MS m/z (%): 502/504 (M þ Na,10),
279/281 (M-[N(SO2Me)C6H4OMe], 100). Anal. Calcd for
4.3.1.4. N-methanesulfonyl-3-bromo-1,4-dimethoxy-2-(2-methyl-
phenylamino)methylnaphthalene 9d. Yield 44%, light-yellow
crystals, mp 147.0e148.4 ꢁC. 1H NMR (CDCl3):
d
2.37 (3H, s, Me),
C21H22BrNO5S: C 52.51%, H 4.62%, N 2.92%. Found: C 52.77%, H
3.14 (3H, s, MeSO2), 3.80 (3H, s, OMe), 3.97 (3H, s, OMe), 5.04 (1H,
d, J ¼ 13.8 Hz, CHaHbN), 5.42 (1H, d, J ¼ 13.8 Hz, CHaHbN), 6.64
(1H, br. d, J ¼ 7.7 Hz, H-30 or H-60), 6.85 (1H, ddd, J ¼ 1.7, 7.4,
7.7 Hz, H-40 or H-50), 7.10 (1H, ddd, J ¼ 1.1, 7.4, 7.7 Hz, H-40 or H-50),
7.15 (1H, dd, J ¼ 1.7, 7.7 Hz, H-30 or H-60), 7.55 (2H, ddd, J ¼ 3.5, 6.7,
6.7 Hz, H-6 and H-7), 8.01e8.09 (2H, m, H-5 and H-8). 13C NMR
4.81%, N 2.90%.
4.3.1.8. N-methanesulfonyl-3-bromo-1,4-dimethoxy-2-(4-methoxy-
phenylamino)methylnaphthalene 9h. Yield 84%, light-yellow crys-
tals, mp 140.8e141.1 ꢁC. 1H NMR (CDCl3):
d 3.04 (3H, s, MeSO2), 3.66
(3H, s, OMe), 3.86 (3H, s, OMe), 3.93 (3H, s, OMe), 5.21 (2H, s, CH2),
6.64 (2H, br. d, J ¼ 8.8 Hz, 2ꢂ]CH), 7.06 (2H, br. d, J ¼ 8.8 Hz, 2ꢂ]
CH), 7.53 (2H, ddd, J ¼ 3.3, 6.6, 6.6 Hz, H-6 and H-7), 7.98e8.07 (2H,
(CDCl3):
d 18.36 (Me), 39.43 (MeSO2), 48.53 (CH2), 61.33 (OMe),
62.90 (OMe), 117.16 (]Cquat), 122.80 and 123.10 (C-5 and C-8),
124.99 (]Cquat), 126.11 (C-40 or C-50), 127.70 and 130.23 (C-6 and
C-7), 127.74 (]Cquat), 128.84 (C-40 or C-50), 129.22 (]Cquat),
130.23 and 131.44 (C-30 and C-60), 136.36 (]Cquat), 140.67 (]
m, H-5 and H-8). 13NMR (CDCl3):
d 38.18 (MeSO2), 48.64 (CH2),
55.27 (OMe), 61.30 (OMe), 63.12 (OMe), 114.05 (2ꢂ]CH), 114.05 (]
quat), 122.65 and 123.00 (C-5 and C-8), 124.48 (]Cquat), 126.79 and
127.47 (C-6 and C-7), 127.58 (]Cquat), 129.08 (]Cquat), 130.24 (]
C
C
quat), 150.14 (]CeO), 153.00 (]CeO). IR (ATR): nmax 1513, 1504,
1450, 1358, 1323, 1142, 1083, 1064, 1000 cm-1. MS m/z (%): 486/
488 (M þ Na, 10), 279/281 (M-[N(SO2Me)C6H4Me], 100). Anal.
Calcd for C21H22BrNO4S: C 54.32%, H 4.78%, N 3.02%. Found: C
54.60%, H 4.97%, N 3.11%.
C
quat), 131.17 (2ꢂ]CH), 150.19 (]Cquat), 152.91 (]Cquat), 159.32 (]
C
quat). IR (ATR): nmax 1508, 1357, 1329, 1247, 1148, 1077, 1047, 1035,
1006 cm-1. MS m/z (%): 502/504 (M þ Na, 2), 279/281 (M-
[N(SO2Me)C6H4OMe], 100). Anal. Calcd for C21H22BrNO5S: C 52.51%,
H 4.62%, N 2.92%. Found: C 52.29%, H 4.95%, N 2.84%.
4.3.1.5. N-methanesulfonyl-3-bromo-1,4-dimethoxy-2-(3-methylph-
enylamino)methylnaphthalene 9e. Yield 67%, light-yellow crystals,
4.3.1.9. N-methanesulfonyl-3-bromo-1,4-dimethoxy-2-(2,4-dimetho-
mp 111.0e111.4 ꢁC. 1H NMR (CDCl3):
d
2.16 (3H, s, Me), 3.05 (3H, s,
xyphenylamino)methylnaphthalene 9i. Yield 82%, orange crystals,
MeSO2), 3.84 (3H, s, OMe), 3.94 (3H, s, OMe), 5.23 (2H, s, CH2),
mp 137.3e138.4 ꢁC. 1H NMR (CDCl3):
d
3.01 (1H, s, MeSO2), 3.58 (3H,
6.91e7.04 (4H, m, 4ꢂ]CH), 7.52 (2H, ddd, J ¼ 3.5, 6.7, 6.7 Hz, H-6
s, OMe), 3.61 (3H, s, OMe), 3.76 (3H, s, OMe), 3.87 (3H, s, OMe), 5.16

66
D. Cappoen et al. / European Journal of Medicinal Chemistry 48 (2012) 57e68
(2H, s, CH2), 6.06 (1H, dd, J ¼ 2.5, 8.4 Hz, H-50), 6.24 (1H, d,
J ¼ 2.5 Hz, H-30), 6.76 (1H, d, J ¼ 8.4 Hz, H-60), 7.44 (2H, ddd, J ¼ 3.3,
6.6, 6.6 Hz, H-6 and H-7), 7.91e7.99 (2H, m, H-5 and H-8). 13NMR
1338, 1282, 1145 cm-1. MS m/z (%): 438/440 (M þ Hþ, 100). Anal.
Calcd for C18H13BrFNO4S: C 49.33%, H 2.99%, N 3.20%. Found: C
49.20%, H 3.11%.
(CDCl3):
d 40.06 (MeSO2), 47.84 (CH2), 55.34 (OMe), 55.43 (OMe),
61.34 (OMe), 62.97 (OMe), 99.19 (C-30), 104.28 (C-50), 116.70 (]
4.4.1.4. N-methanesulfonyl-3-bromo-2-(2-methylphenylamino)methyl-
C
C
C
C
quat), 118.83 (]Cquat), 122.60 and 123.15 (C-5 and C-8), 125.19 (]
quat), 126.67 and 127.38 (C-6 and C-7), 127.74 (]Cquat), 128.98 (]
quat), 134.00 (C-60), 149.88 (]Cquat), 153.30 (]Cquat), 158.25 (]
quat), 161.04 (]Cquat). IR (ATR): nmax 1508, 1357, 1324, 1309, 1212,
1,4-naphthoquinone 6d. Yield 50%, yellow-brown crystals, mp
135.9e136.8 ꢁC. 1H NMR (CDCl3):
d 2.40 (3H, s, Me), 3.19 (3H, s,
MeSO2), 4.95 (1H, d, J ¼ 13.2 Hz, CHaHbN), 5.14 (1H, d, J ¼ 13.2 Hz,
CHaHbN), 6.99e7.09 (2H, m, 2ꢂ]CH), 7.16e7.23 (2H, m, 2ꢂ]CH),
1162, 1142, 1085, 1067, 1002 cm-1. MS m/z (%): 431/433 ([M-meth-
anesulfinate]þHþ, 100), 279/281 (M-[N(SO2Me)C6H3(OMe)2], 25).
Anal. Calcd for C22H24BrNO6S: C 51.77%, H 4.74%, N 2.74%. Found: C
51.93%, H 4.89%, N 2.77%.
7.73e7.82 (2H, m, H-6 and H-7), 8.09e8.15 (2H, m, H-5 and H-8). 13
C
NMR (CDCl3):
d 18.47 (Me), 39.07 (MeSO2), 49.70 (CH2), 126.63 (]
CH), 127.24 and 127.82 (C-5 and C-8), 129.20 (]CH), 129.32 (]CH),
130.86 (]Cquat), 131.20 (]Cquat), 131.93 (]CH), 134.33 and 134.65
(C-6 and C-7), 136.89 (]Cquat), 139.92 (]Cquat), 143.18 (]Cquat),
144.18 (]Cquat), 177.26 (C]O), 181.35 (C]O). IR (ATR): nmax 1677,
1657, 1335, 1278, 1148 cm-1. MS m/z (%): 451/453 (M þ NHþ4 , 100).
Anal. Calcd for C19H16BrNO4S: C 52.54%, H 3.71%, N 3.23%. Found: C
52.38%, H 3.80%, N 3.21%.
4.4. Synthesis of N-methanesulfonyl-2-(arylamino)methyl-3-
bromo-1,4-naphthoquinones 6
4.4.1. General procedure
To a stirred solution of a N-methanesulfonyl-2-(arylamino)
methyl-3-bromo-1,4-dimethoxy-naphthalene 9 (1 mmol) in 10 ml
of acetonitrile was added dropwise a solution of cerium(IV)
ammonium nitrate (CAN) (2.3 mmol) in 10 ml of water. The reaction
mixture was stirred for 30 min at room temperature and then
poured in water. The reaction mixture was extracted with three
small portions of ethyl acetate. The combined organic extracts were
washed with brine and dried (MgSO4). Recrystallization from
methanol afforded pure N-methanesulfonyl-2-(arylamino)methyl-
3-bromo-1,4-naphthoquinones 6.
4.4.1.5. N-methanesulfonyl-3-bromo-2-(3-methylphenylamino)methyl-
1,4-naphthoquinone 6e. Yield 91%, yellow crystals, mp 175.0
e175.4 ꢁC. 1H NMR (CDCl3):
d
2.29 (3H, s, Me), 3.06 (3H, s, MeSO2),
5.03 (2H, s, CH2), 7.05e7.22 (4H, m, 4ꢂ]CH), 7.70e7.79 (2H, m, H-6
and H-7), 8.07e8.14 (2H, m, H-5 and H-8). 13C NMR (CDCl3):
21.25
d
(Me), 37.36 (MeSO2), 49.41 (CH2), 125.47 (]CH), 127.23 and 127.67
(C-5 and C-8), 129.08 (]CH), 129.43 (2ꢂ]CH), 130.86 (]Cquat),
131.25 (]Cquat), 134.16 and 134.50 (C-6 and C-7), 139.04 (]Cquat),
139.41 (]Cquat), 142.38 (]Cquat), 144.62 (]Cquat), 177.37 (C]O),
181.03 (C]O). IR (ATR): nmax 1661, 1601, 1588, 1332, 1306, 1271,
1146, 1070 cm-1. MS m/z (%): 434/436 (M þ Hþ, 100). Anal. Calcd for
4.4.1.1. N-methanesulfonyl-3-bromo-2-(phenylamino)methyl-1,4-
naphthoquinone 6a. Yield 85%, yellow crystals, mp 176.8e179.1 ꢁC.
C19H16BrNO4S: C 52.54%, H 3.71%, N 3.23%. Found: C 52.81%, H
1H NMR (CDCl3):
d
3.06 (3H, s, MeSO2), 5.05 (2H, s, CH2), 7.26e7.35
(5H, m, 5ꢂ]CH), 7.71e7.80 (2H, m, H-6 and H-7), 8.08e8.14 (2H, m,
H-5 and H-8). 13C NMR (CDCl3):
37.48 (MeSO2), 49.48 (CH2),
3.89%, N 3.16%.
d
4.4.1.6. N-methanesulfonyl-3-bromo-2-(4-methylphenylamino)methyl-
127.38 and 127.82 (C-5 and C-8), 128.80 (]CH), 128.92 (2ꢂ]CH),
129.53 (2ꢂ]CH), 130.96 (]Cquat), 131.27 (]Cquat), 134.32 and
134.63 (C-6 and C-7), 139.16 (]Cquat), 142.70 (]Cquat), 144.55 (]
1,4-naphthoquinone 6f. Yield 95%, yellow needles, mp 189.3
e190.0 ꢁC. 1H NMR (CDCl3):
d 2.29 (3H, s, Me), 3.05 (3H, s, MeSO2),
5.02 (2H, s, CH2), 7.11 (2H, br. d, J ¼ 8.0 Hz, 2ꢂ]CH), 7.20 (2H, br. d,
C
quat), 177.46 (C]O), 181.17 (C]O). IR (ATR): nmax 1676, 1660, 1602,
J ¼ 8.0 Hz, 2ꢂ]CH), 7.71e7.79 (2H, m, H-6 and H-7), 8.07e8.13 (2H,
1588, 1339, 1334, 1279, 1152, 1146, 1066 cm-1. MS m/z (%): 420/422
(M þ Hþ, 100). Anal. Calcd for C18H14BrNO4S: C 51.44%, H 3.36%, N
3.33%. Found: C 51.32%, H 3.43%, N 3.37%.
m, H-5 and H-8).13C NMR (CDCl3):
d 21.10 (Me), 37.28 (MeSO2), 49.42
(CH2), 127.26 and 127.67 (C-5 and C-8), 128.60 (2ꢂ]CH), 130.05
(2ꢂ]CH), 130.89 (]Cquat), 131.23 (]Cquat), 134.18 and 134.50 (C-6
and C-7), 136.34 (]Cquat), 138.74 (]Cquat), 142.53 (]Cquat), 144.57
(]Cquat), 177.37 (C]O), 181.05 (C]O). IR (ATR): nmax 1660, 1600,
1588,1331,1270,1144,1065 cm-1. MS m/z (%): 434/436 (M þ Hþ,100),
355/357 (M-methanesulfinate, 80). Anal. Calcd for C19H16BrNO4S: C
52.54%, H 3.71%, N 3.23%. Found: C 52.93%, H 3.95%, N 3.37%.
4.4.1.2. N-methanesulfonyl-3-bromo-2-(4-chlorophenylamino)methyl-
1,4-naphthoquinone 6b. Yield 71%, yellow crystals, mp 170.8
e172.5 ꢁC. 1H NMR (CDCl3):
d
3.06 (3H, s, MeSO2), 5.02 (2H, s, CH2),
7.28e7.30 (4H, m, 4ꢂ]CH), 7.72e7.81 (2H, m, H-6 and H-7),
8.08e8.17 (2H, m, H-5 and H-8). 13C NMR (CDCl3):
37.48 (MeSO2),
d
49.30 (CH2), 127.34 and 127.81 (C-5 and C-8), 129.66 (2ꢂ]CH),
130.05 (2ꢂ]CH), 130.88 (]Cquat), 131.11 (]Cquat), 134.35 and
134.62 (C-6 and C-7), 134.62 (]Cquat), 137.60 (]Cquat), 142.85 (]
4.4.1.7. N-methanesulfonyl-3-bromo-2-(2-methoxyphenylamino)
methyl-1,4-naphthoquinone 6g. Yield 87%, yellow crystals, mp
206.3e206.9 ꢁC. 1H NMR (CDCl3):
d 3.10 (3H, s, MeSO2), 3.63 (3H, s,
C
quat), 144.10 (]Cquat), 177.22 (C]O), 181.07 (C]O). IR (ATR): nmax
OMe), 5.06 (2H, s, CH2), 6.83 (1H, br. d, J ¼ 8.3 Hz, ]CH), 6.91 (1H,
ddd, J ¼ 1.1, 7.6, 7.6 Hz), 7.25e7.32 (2H, m, 2ꢂ]CH), 7.71e7.82 (2H,
m, H-6 and H-7), 8.11e8.15 (2H, m, H-5 and H-8). 13C NMR (CDCl3):
1660, 1338, 1282, 1270, 1146 cm-1. MS m/z (%): 454/456 (M þ Hþ,
100). Anal. Calcd for C18H13BrClNO4S: C 47.54%, H 2.88%, N 3.08%.
Found: C 47.63%, H 2.99%, N 3.02%.
d
39.74 (MeSO2), 49.47 (CH2), 55.20 (OMe), 116.61 (]CH), 121.34 (]
CH), 126.45 (]Cquat), 127.08 and 127.66 (C-5 and C-8), 130.60 (]
CH), 130.80 (]Cquat), 131.46 (]Cquat), 133.64 (]CH), 134.07 and
134.50 (C-6 and C-7), 141.29 (]Cquat), 145.06 (]Cquat), 156.45 (]
4.4.1.3. N-methanesulfonyl-3-bromo-2-(4-fluorophenylamino)methyl-
1,4-naphthoquinone 6c. Yield 90%, yellow needles, mp 133.2
e133.9 ꢁC. 1H NMR (CDCl3):
d
3.07 (3H, s, MeSO2), 5.02 (2H, s, CH2),
Cquat), 177.55 (C]O), 181.08 (C]O). IR (ATR): nmax 1676, 1662, 1598,
6.97e7.05 (2H, m, 2ꢂ]CH), 7.26e7.34 (2H, m, 2ꢂ]CH), 7.72e7.81
1592, 1495, 1337, 1272, 1178, 1150 cm-1. MS m/z (%): 472/474
(M þ Na, 10), 450/452 (M þ Hþ, 10), 371/373 (M-methanesulfinate,
100). Anal. Calcd for C19H16BrNO5S: C 50.68%, H 3.58%, N 3.11%.
Found: C 50.46%, H 3.73%, N 3.19%.
(2H, m, H-6 and H-7), 8.08e8.15 (2H, m, H-5 and H-8). 13C NMR
(CDCl3): d 37.56 (MeSO2), 49.48 (CH2), 116.26 (]CH), 116.57 (]CH),
127.30 and 127.79 (C-5 and C-8), 130.78 (]CH), 130.86 (]Cquat),
130.91 (]CH), 131.09 (]Cquat), 134.34 and 134.62 (C-6 and C-7),
134.80 (]Cquat), 142.88 (]Cquat), 144.16 (]Cquat), 162.29 (d,
J ¼ 249.2 Hz, C-40),177.23 (C]O),181.05 (C]O). IR (ATR): nmax 1660,
4.4.1.8. N-methanesulfonyl-3-bromo-2-(4-methoxyphenylamino)
methyl-1,4-naphthoquinone 6h. Yield 94%, yellow crystals, mp

D. Cappoen et al. / European Journal of Medicinal Chemistry 48 (2012) 57e68
67
120.8e122.6 ꢁC. 1H NMR (CDCl3):
d
3.05 (3H, s, MeSO2), 3.74 (3H, s,
4.5.1.2. 2-Chlorobenzo[j]phenanthridine-7,12-dione 5b. Yield 53%,
yellow needles, mp 183.8e184.1 ꢁC. 1H NMR (CDCl3):
7.80 (1H, dd,
OMe), 5.01 (2H, s, CH2), 6.80 (2H, dd, J ¼ 2.2, 6.7 Hz, 2ꢂ]CH), 7.23
d
(2H, dd, J ¼ 2.2, 6.7 Hz, 2ꢂ]CH), 7.70e7.80 (2H, m, H-6 and H-7),
J ¼ 2.2, 8.6 Hz, H-3), 7.84e7.89 (2H, m, H-9 and H-10), 8.13 (1H, d,
8.07e8.13 (2H, m, H-5 and H-8). 13C NMR (CDCl3):
d 37.57 (MeSO2),
J ¼ 8.6 Hz, H-4), 8.24e8.31 (2H, m, H-8 and H-11), 9.61 (1H, d,
49.48 (CH2), 55.41 (OMe), 114.56 (2ꢂ]CH), 127.26 and 127.69 (C-5
and C-8), 130.24 (2ꢂ]CH), 131.19 (]Cquat), 131.25 (]Cquat), 134.21
and 134.53 (C-6 and C-7), 142.68 (]Cquat), 144.48 (]Cquat), 159.55
(]Cquat), 177.34 (C]O), 181.03 (C]O). IR (ATR): nmax 1681, 1658,
1508, 1330, 1273, 1251, 1145 cm-1. MS m/z (%): 472/474 (M þ Na, 18),
371/373 (M-methanesulfinate, 100). Anal. Calcd for C19H16BrNO5S:
C 50.68%, H 3.58%, N 3.11%. Found: C 50.77%, H 3.64%, N 3.08%.
J ¼ 2.2 Hz, H-1), 9.77 (1H, s, H-6). 13C NMR (CDCl3):
d 123.20 (]
C
quat), 124.91 (]Cquat), 126.70 and 127.09 (C-8 and C-11), 127.40 (C-
1), 131.69 (C-4), 131.77 (]Cquat), 132.61 (]Cquat), 132.96 (C-3),
134.04 (]Cquat), 134.67 and 134.83 (C-9 and C-10), 136.94 (]Cquat),
148.63 (C-6), 150.22 (]Cquat), 182.96 (C]O), 185.63 (C]O). IR
(ATR): nmax 1668, 1586, 1567, 1336, 1284, 1006 cm-1. MS m/z (%):
294/296 (M þ Hþ, 100). Anal. Calcd for C17H8ClNO2: C 69.52%, H
2.75%, N 4.77%. Found: C 69.89%, H 3.14%, N 4.56%.
4.4.1.9. N-methanesulfonyl-3-bromo-2-(2,4-dimethoxyphenylamino)
methyl-1,4-naphthoquinone 6i. Yield 70%, yellow crystals, mp
4.5.1.3. 2-Fluorobenzo[j]phenanthridine-7,12-dione 5c. Yield 53%,
233e235 ꢁC. 1H NMR (CDCl3):
d
3.08 (3H, s, MeSO2), 3.57 (3H, s,
yellow needles, mp 182.4e182.8 ꢁC.1H NMR (CDCl3):
d 7.69 (1H, ddd,
OMe), 3.75 (3H, s, OMe), 5.03 (2H, br. d, J ¼ 12.7 Hz, CH2), 6.34 (1H,
d, J ¼ 2.8 Hz, H-30), 6.41 (1H, dd, J ¼ 2.8, 8.3 Hz, H-50), 7.21 (1H, d,
J ¼ 8.3 Hz, H-60), 7.74 (1H, ddd, J ¼ 1.7, 7.2, 7.2 Hz, H-6 or H-7), 7.79
(1H, ddd, J ¼ 1.7, 7.2, 7.2 Hz, H-6 or H-7), 8.11e8.16 (2H, m, H-5 and
J ¼ 2.8, 7.3, 9.3 Hz, H-3), 7.82e7.89 (2H, m, H-9 and H-10), 8.21 (1H,
dd, J ¼ 6.1, 9.3 Hz, H-4), 8.25e8.31 (2H, m, H-8 and H-11), 9.30 (1H, d,
J ¼ 2.8, 11.0 Hz, H-1), 9.76 (1H, s, H-6). 13C NMR (CDCl3):
d 112.11 (d,
J ¼ 26.5 Hz, C-1),122.32 (d, J ¼ 26.5 Hz, C-3),123.69 (d, J ¼ 11.7 Hz, ]
H-8). 13C NMR (CDCl3):
d
39.77 (MeSO2), 49.60 (OMe), 55.25 (OMe),
Cquat), 124.93 (]Cquat), 126.70 and 127.40 (C-8 and C-11), 131.84 (]
quat),132.74 (d, J ¼ 9.2 Hz, C-4),133.03 (d, J ¼ 6.9 Hz, ]Cquat),134.08
55.57 (OMe), 99.31 (C-30), 105.01 (C-50), 119.34 (]Cquat), 127.16 and
127.73 (C-5 and C-8), 130.93 (]Cquat), 131.62 (]Cquat), 134.11 and
134.25 (C-6 and C-7),134.54 (C-60), 141.41 (]Cquat), 145.21 (]Cquat),
157.44 (]Cquat), 161.39 (]Cquat), 177.67 (C]O), 181.15 (C]O). IR
(ATR): nmax 1677, 1660, 1606, 1590, 1582, 1336, 1308, 1275, 1207,
1148, 1132 cm-1. MS m/z (%): 502/504 (M þ Na, 10), 401/403 (M-
methanesulfinate, 100). Anal. Calcd for C20H18BrNO6S: C 50.01%, H
3.78%, N 2.92%. Found: C 50.33%, H 3.95%, N 2.82%.
C
(]Cquat),134.62 and 134.80 (C-9 and C-10),147.72 (d, J ¼ 2.3 Hz, H-6),
149.19 (]Cquat), 163.26 (d, J ¼ 251.5 Hz, C-2), 183.11 (C]O), 185.74
(C]O). IR (ATR): nmax 1668, 1424, 1286, 1272, 1250, 1209, 1014 cm-1.
MS m/z (%): 278 (M þ Hþ, 100). Anal. Calcd for C17H8FNO2: C 73.65%,
H 2.91%, N 5.05%. Found: C 73.48%, H 3.04%, N 5.01%.
4.5.1.4. 4-Methylbenzo[j]phenanthridine-7,12-dione 5d. Yield 27%,
yellow needles, mp 178.8e181.2 ꢁC. 1H NMR (CDCl3):
d 2.87 (3H, s,
4.5. Synthesis of benzo[j]phenanthridine-7,12-diones 5
Me), 7.67e7.76 (2H, m, H-2 and H-3), 7.82e7.89 (2H, m, H-9 and H-
10), 8.27e8.34 (2H, m, H-8 and H-11), 9.44 (1H, d, J ¼ 1.7, 8.3 Hz, H-
4.5.1. General procedure
1), 9.85 (1H, s, H-6). 13C NMR (CDCl3):
d 18.76 (Me), 122.83 (]Cquat),
A
mixture of N-methanesulfonyl-2-(arylamino)methyl-3-
124.31 (]Cquat), 126.15 (C-1), 126.61 and 127.45 (C-8 and C-11),
130.29 and 132.48 (C-2 and C-3), 132.08 (]Cquat), 134.08 (]Cquat),
134.46 and 134.67 (C-9 and C-10), 134.61 (]Cquat), 138.17 (]Cquat),
147.16 (C-6), 151.10 (]Cquat), 183.67 (C]O), 186.43 (C]O). IR (ATR):
nmax 1664, 1588, 1570, 1459, 1343, 1287, 1274 cm-1. MS m/z (%): 274
(M þ Hþ,100). Anal. Calcd for C18H11NO2: C 79.11%, H 4.06%, N 5.13%.
Found: C 78.83%, H 4.30%, N 5.01%.
bromo-1,4-naphthoquinone 6 (1 mmol) and palladium(II) acetate
(0.2 mmol) (Acros Organics), triphenylphosphine (0.4 mmol) and
potassium carbonate (3 mmol) in toluene (15 ml), which was
previously degassed, was heated at 80 ꢁC for 1 h under N2 atmo-
sphere, after which the reaction mixture was boiled under reflux
for 1e3 h. The reaction mixture was poured in water, extracted with
ethyl acetate, washed with water and dried (MgSO4). Flash chro-
matography on silica gel with an appropriate mixture of petroleum
ether/ethyl acetate, which was determined via initial TLC analysis,
followed by recrystallization from methanol gave pure benzo[j]
phentanthridine-7,12-diones 5. Although the 13C NMR of benzo[j]
phenanthridine-7,12-dione 5a is corresponding to data in the
literature [21], a difference of more than 10 ꢁC in melting point was
witnessed, which could be ascribed to the use of a different solvent
for the recrystallization, along with a different chemical shift for H-
3 in the 1H NMR in comparison with the first reported synthesis of
benzo[j]phenanthridine-7,12-dione 5a [20]. Therefore, all the
spectroscopic data, which were recorded for benzo[j]phenan-
thridine-7,12-dione 5a, are listed below.
4.5.1.5. 3-Methylbenzo[j]phenanthridine-7,12-dione 5e. Yield 62%,
yellow needles, mp 186.1e187.3 ꢁC. 1H NMR (CDCl3):
d 2.61 (3H, s,
Me), 7.62 (1H, d, J ¼ 8.8 Hz, H-2), 7.81e7.85 (2H, m, H-9 and H-10),
7.97 (1H, s, H-4), 8.25e8.30 (2H, m, H-8 and H-11), 9.44 (1H, d,
J ¼ 8.8 Hz, H-1), 9.76 (1H, s, H-6). 13C NMR (CDCl3):
d 21.96 (Me),
120.66 (]Cquat), 124.06 (]Cquat), 126.66 (C-1), 127.36 and 127.91
(C-8 and C-11), 129.51 (C-4), 132.06 (]Cquat), 132.86 (C-2), 133.71
(]Cquat), 134.40 (]Cquat), 134.51 and 134.61 (C-9 and C-10), 143.24
(]Cquat), 148.64 (C-6), 152.28 (]Cquat), 183.41 (C]O), 186.37 (C]
O). IR (ATR): nmax 1672, 1657, 1568, 1293, 1274 cm-1. MS m/z (%): 274
(M þ Hþ,100). Anal. Calcd for C18H11NO2: C 79.11%, H 4.06%, N 5.13%.
Found: C 78.78%, H 4.39%, N 4.99%.
4.5.1.1. Benzo[j]phenanthridine-7,12-dione 5a. Yield 42%, yellow
needles, mp 188.3e188.9 ꢁC (methanol), (Lit.: 200e202 ꢁC, benzene
4.5.1.6. 2-Methylbenzo[j]phenanthridine-7,12-dione 5f. Yield 59%,
yellow needles, mp 174.1e174.8 ꢁC. 1H NMR (CDCl3):
d 2.61 (3H, s,
[20]). 1H NMR (CDCl3):
d
7.78e7.93 (4H, m, H-2, H-3, H-9 and H-10),
Me), 7.67 (1H, dd, J ¼ 1.5, 8.7 Hz, H-3), 7.79e7.87 (2H, m, H-9 and H-
10), 8.04 (1H, d, J ¼ 8.7 Hz, H-4), 8.22e8.27 (2H, m, H-8 and H-11),
9.29 (1H, d, J ¼ 1.5 Hz, H-1), 9.66 (1H, s, H-6). 13C NMR (CDCl3):
8.23 (1H, dd, J ¼ 1.1, 8.3 Hz, H-4), 8.27e8.34 (2H, m, H-8 and H-11),
9.60 (1H, dd, J ¼ 1.1, 8.6 Hz, H-1), 9.84 (1H, s, H-6). 13C NMR (CDCl3):
d
122.68 (]Cquat), 124.59 (]Cquat), 126.63 and 127.35 (C-8 and C-
d 22.31 (Me), 122.63 (]Cquat), 124.50 (]Cquat), 126.51 (C-1), 126.91
11), 128.28 (C-1), 130.40 and 130.44 (C-2 and C-4), 131.92 (]Cquat),
132.11 (C-3),133.82 (]Cquat),134.33 (]Cquat),134.50 and 134.66 (C-
9 and C-10), 148.50 (C-6), 151.92 (]Cquat), 183.37 (C]O), 186.22
(C]O). IR (ATR): nmax 1676,1664,1589,1567,1290,1276 cm-1. MS m/
z (%): 260 (M þ Hþ, 100). Anal. Calcd for C17H9NO2: C 78.76%, H
3.50%, N 5.40%. Found: C 79.01%, H 3.78%, N 5.53%.
and 127.20 (C-8 and C-11), 129.98 (C-4), 131.86 (]Cquat), 132.89 (]
quat), 134.25 (C-3), 134.34 and 134.54 (C-9 and C-10, ]Cquat),
141.00 (]Cquat), 147.40 (C-6), 150.62 (]Cquat), 183.35 (C]O), 186.20
(C]O). IR (ATR): nmax 1665, 1589, 1565, 1358, 1287, 1275 cm-1. MS
m/z (%): 274 (M þ Hþ, 100). Anal. Calcd for C18H11NO2: C 79.11%, H
4.06%, N 5.13%. Found: C 78.92%, H 4.33%, N 5.09%.
C

68
D. Cappoen et al. / European Journal of Medicinal Chemistry 48 (2012) 57e68
[14] P.S. Steyn, P.L. Wessels, W.O.F. Marasas, Pigments form Fusarium moniliforme
Sheldon e structure and C-13 nuclear magnetic resonance assignments of an
4.5.1.7. 4-Methoxybenzo[j]phenanthridine-7,12-dione 5g. Yield %,
orange crystals, mp 209.9e210.7 ꢁC. 1H NMR (CDCl3):
4.15 (3H, s,
d
azaanthraquinone and
3
naphthoquinones, Tetrahedron 35 (1979)
OMe), 7.25e7.29 (1H, m, H-3), 7.76 (1H, dd, J ¼ 8.3, 8.8 Hz, H-2),
7.83e7.89 (2H, m, H-9 and H-10), 8.29e8.34 (2H, m, H-8 and H-11),
9.18 (1H, d, J ¼ 8.8 Hz, H-1), 9.85 (1H, s, H-6). 13C NMR (CDCl3):
1551e1555.
[15] U. Gräfe, W. Ihn, D. Tresselt, N. Miosga, U. Kaden, B. Schlegel, E.-J. Bormann,
P. Sedmera, J. Novak, Tolypocladin new metal-chelating 2-
azaanthraquinone from Tolypocladium inflatum, Biol. Met. 3 (1990) 39e44.
[16] P.N. Solis, C. Lang’at, M.P. Gupta, G.C. Kirby, D.C. Warhurst, J.D. Phillipson,
Bioactive compounds from Psychotria camponutans, Planta Med. 61 (1995)
62e65.
[17] A.L. Okunade, A.M. Clark, C.D. Hufford, B.O. Oguntimein, Azaanthraquinone:
an antimicrobial alkaloid from Mitracarpus scaber, Planta Med. 65 (1999)
447e448.
[18] (a) B. Kesteleyn, N. De Kimpe, Synthesis of two naphthoquinone antibiotics:
dehydroherbarin and 6-deoxybostrycoidin, J. Org. Chem. 65 (2000) 640e644;
(b) T. Nguyen Van, G. Verniest, S. Claessens, N. De Kimpe, Total synthesis of four
naturally occurring 2-azaanthraquinone antibiotics: 6-deoxy-8-methylbos-
trycoidin, 6-deoxybostrycoidin, 7-O-demethyl-6-deoxybostrycoidin and scor-
pinone, Tetrahedron 61 (2005) 2295e2300;
e
a
d
56.39 (OMe), 110.40 (C-3), 119.65 (C-1), 124.03 (]Cquat), 125.18 (]
quat), 126.69 and 127.48 (C-8 and C-11), 128.41 (]Cquat), 128.55 (]
quat), 130.98 (C-2), 129.67 (]Cquat), 134.54 and 134.78 (C-9 and C-
C
C
10), 143.91 (]Cquat), 147.16 (C-6), 155.64 (]Cquat), 183.50 (C]O),
186.19 (C]O). IR (ATR): nmax 1664, 1438, 1288, 1266 cm-1. MS m/z
(%): 290 (M þ Hþ, 100). Anal. Calcd for C18H11NO3: C 74.73%, H
3.83%, N 4.84%. Found: C 75.00%, H 4.12%, N 4.59%.
4.5.1.8. 2-Methoxybenzo[j]phenanthridine-7,12-dione 5h. Yield 39%,
orange needles, mp 185.9e186.7 ꢁC. 1H NMR (CDCl3):
d 4.03 (3H, s,
(c) J. Jacobs, S. Claessens, B. Kesteleyn, K. Huygen, N. De Kimpe, New synthesis of
N-substituted benz[g]isoquinoline-3,5,10(2H)-triones, Tetrahedron 63 (2007)
2503e2510;
(d) S. Claessens, J. Jacobs, N. De Kimpe, Synthesis of 2-aza-1-cyano-4-
hydroxyanthraquinones, Synlett (2007) 741e744;
(e) J. Jacobs, B. Kesteleyn, N. De Kimpe, Synthesis of N-substituted 1,2-
dihydrobenz[g]isoquinoline-5,10-diones, Tetrahedron 64 (2008) 4985e4992;
(f) J. Jacobs, J. Deblander, B. Kesteleyn, K. Abbaspour Tehrani, N. De Kimpe,
Synthesis of N-substituted 1,2,3,4-tetrahydrobenz[g]isoquinoline-5,10-diones,
Tetrahedron 64 (2008) 5345e5353;
(g) J. Jacobs, B. Mavinga Mbala, B. Kesteleyn, G. Diels, N. De Kimpe, Straightfor-
ward palladium-mediated synthesis of N-substituted 1,2-dihydrobenz[g]iso-
quinoline-5,10-diones, Tetrahedron 64 (2008) 6364e6371;
(h) J. Jacobs, B. Kesteleyn, N. De Kimpe, First straightforward synthesis of 2,4-
disubstituted benz[g]isoquinoline-3,5,10(2H)-triones, 1,2,3,5-substituted naph-
tho[3,2,1-de]isoquinoline-4,7-diones, and 6-substituted benzo[h]pyrido[3,4,5-
kl]-1,2,3,4-tetrahydroacridine-5,8-diones, Tetrahedron 64 (2008) 7545e7554;
(i) J. Jacobs, S. Claessens, B. Mavinga Mbala, K. Huygen, N. De Kimpe, New and
highly efficient synthesis of 3-substituted 1-hydroxybenz[g]isoquinoline-5,10-
diones, Tetrahedron 65 (2009) 1193e1199.
OMe), 7.47 (1H, dd, J ¼ 2.8, 9.1 Hz, H-3), 7.78e7.85 (2H, m, H-9 and
H-10), 8.04 (1H, d, J ¼ 9.1 Hz, H-4), 8.22e8.28 (2H, m, H-8 and H-
11), 8.96 (1H, d, J ¼ 2.8 Hz, H-1), 9.61 (1H, s, H-6). 13C NMR (CDCl3):
d
55.89 (OMe), 105.25 (C-1), 124.43 (]Cquat), 124.84 (]Cquat),
125.16 (C-3), 126.61 and 127.22 (C-8 and C-11), 131.74 (C-4), 132.08
(]Cquat), 134.37 and 134.58 (C-9 and C-10, ]Cquat), 134.41 (]Cquat),
145.70 (C-6), 148.86 (]Cquat),161.35 (]Cquat), 183.53 (C]O),186.39
(C]O). IR (ATR): nmax 1665, 1288, 1275, 1230 cm-1. MS m/z (%): 290
(M þ Hþ, 100). Anal. Calcd for C18H11NO3: C 74.73%, H 3.83%, N
4.84%. Found: C 74.85%, H 3.92%, N 4.77%.
Acknowledgments
The authors are indebted to Janssen Research Foundation and the
“Research Foundatione Flanders (FWO-Vlaanderen)” for financial
support of this research. We thank Prof. D. Young (Imperial College,
London, UK) for providing us with the luminescent M.tb. H37Rv
strain. Dr. V. Mathys and Dr. R. Vanhoof (Scientific Institute of Public
Health, Brussels, Belgium) are thanked for the tedious revision.
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